Mitochondrial Medicine 2019: Washington DC€¦ · Mitochondrial Medicine 2019: Washington DC 6FLHQWLÀF DQG &OLQLFDO 0HHWLQJV June 26-29, 2019 Hilton Alexandria Mark Center Alexandria,

Mitochondrial Medicine 2019:Washington DC

June 26-29, 2019Hilton Alexandria Mark Center

Alexandria, VA

2019 Course Chairs: Amel Karaa, MD and Carla Koehler, PhD 2019 CME Chair: Bruce H. Cohen, MD

Mitochondrial Medicine 2019: Washington DC

June 26-29, 2019

Course Description The United Mitochondrial Disease Foundation and PeerPoint Medical Education Institute have joined efforts to sponsor and organize a CME-accredited symposium. Mitochondrial diseases are more common than previously recognized and mitochondrial pathophysiology is now a recognized part of many disease processes, including heart disease, cancer, AIDS and diabetes. There have been significant advances in the molecular genetics, proteomics, epidemiology and clinical aspects of mitochondrial pathophysiology. This conference is directed toward the scientist and clinician interested in all aspects of mitochondrial science. The content of this educational program was determined by rigorous assessment of educational needs and includes surveys, program feedback, expert faculty assessment, literature review, medical practice, chart review and new medical knowledge. The format will include didactic lectures from invited experts intermixed with peer-reviewed platform presentations. There will be ample time for professional discussion both in and out of the meeting room, and peer-reviewed poster presentations will be given throughout the meeting. This will be a four-day scientific meeting aimed at those with scientific and clinical interests.

TARGET AUDIENCE

Neurologists, Geneticists, Researchers/Scientists, Pediatrics, Internal Medicine, Nephrologists, Cardiologists, Endocrinologists, Genetic Counselors, Advanced Practice Nurses, Physicians Assistants, RNs, Occupational Therapy, Physical Therapy and Speech-Language Pathology, Nutritional Therapy, and Residents/Fellows/Students.

LEARNING OBJECTIVES

• Learn about new clinical trials investigating treatments for mitochondrial diseases in children and adults

• Become familiar with new insights the technology of mitochondrial transfer as it relates to possible treatment for mitochondrial disease.

• Learn how the use of true Whole Genome Sequencing can be applied to diagnose mitochondrial diseases

• Summarize the development of consortium-based discovery and care networks are changing the face of clinical practice as it relates to mitochondrial medicine.

• Discover the role of the US Food and Drug Administration in the approval process for treatments of rare diseases including mitochondrial disease.

• Understand the application of iPS cells for Leigh syndrome diagnostics and possible therapy

• Describe the interactions between mitochondrial and the microbiota. • Learn about innate immunity and the role of the mitochondria in innate

immunity and immunity in those with known mitochondrial diseases.

• Learn of the advances in the understanding of organelle cross-talk and mitochondrial dynamics

• Discover the role of mitochondrial epigenetics and the process of DNA methylation of the mtDNA

ACCREDITATION STATEMENT

This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of PeerPoint Medical Education Institute, LLC and United Mitochondrial Disease Foundation. The PeerPoint Medical Education Institute, LLC is accredited by the ACCME to provide continuing medical education for physicians.

DESIGNATION STATEMENT

The PeerPoint Medical Education Institute, LLC designates this live activity for a maximum of 18.50 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

Special Announcements

Abstract Cash Awards will be presented on Friday, June 28 at 4:45pm in Plaza A.

CME Certificate Instructions Attendees for this year’s meeting will generate their own CME Certificate through the PeerPoint CME Vault online system. Post meeting, you will receive an email within a week with instructions on how to access the CME Vault and secure your certificate.

NOTE: Peerpoint will set up your login identities based on the information you confirmed at registration upon check-in. If you did not confirm and initial your information, please do so before you leave the meeting this week.

Each attendee must have a unique email address; groups or partners cannot share an email address to access the CME Vault. Make sure you check your spam folders for emails coming from peerpt.com. For general information about PeerPoint, visit http://www.peerpt.com.

Special Announcements Name Badges All attendees must wear a name badge to all course functions.

Scientific Sessions Majority of scientific sessions will be held in Plaza A. On Friday morning, sessions will begin in Plaza C through 10:00am break and will resume in Plaza A. On Saturday morning, the MSeqDR Workshop session will be in Beech from 7:15am to 7:45am but the general scientific session will begin at 8:00am in Plaza A.

Refreshment Breaks/Exhibits/Posters Exhibits will be open in the Plaza Foyer during all breaks and lunches. Posters will be in Arbors Room. All posters are assigned numbers in the back of this syllabus. Presenters will station themselves at their poster to field questions according to those numbers as follows: even numbers on Wednesday from 5:30 pm to 7:30 pm and odd numbers on Thursday from 5:30 pm to 7:30 pm. If a Lightning Round person speaks on Wednesday, they will station themselves at their poster on Wednesday evening (regardless of poster number).

Meals Continental Breakfasts and Lunches will be held in Plaza C.

2019 Scientific/Clinician Meeting – Faculty

• 2019 Scientific Meeting – Confirmed Faculty o Dario C. Altieri, MD, Wistar Institute Cancer Center, Philadelphia, PA o Ana Andreazza, PhD, University of Toronto, Toronto, Canada o Brendan Battersby, PhD, University of Helsinki, Institute of Biotechnology,

Finland o William Copeland, PhD, NIEHS, Research Triangle Park, NC o Patrick Chinnery, PhD, FRCP, University of Cambridge, Cambridge,

United Kingdom o David Dimmock, MD, Rady Children’s Institute for Genomic Medicine, San

Diego, CA o Amy Goldstein, MD, Children’s Hospital of Philadelphia, Philadelphia, PA o Michio Hirano, MD, Columbia University, New York, NY o Adam Hughes, PhD, University of Utah School of Medicine, Salt Lake

City, UT o Shilpa Iyer, PhD, University of Arkansas, Fayetteville, AR o Amel Karaa, MD, Massachusetts General Hospital, Boston, MA o Carla Koehler, PhD, University of California Los Angeles, Los Angeles, CA o Mary Kay Koenig, MD, University of Texas McGovern Medical School,

Houston, TX o Danuta Krotoski, PhD, Eunice Kennedy Shriver National Institute of Child

Health and Human Development, NIH, Bethesda, MD o Austin Larson, MD, Children’s Hospital Colorado, Denver, CO o David J. Livingston, PhD, Brown University, Providence, RI o David Lynch, MD, PhD, Children’s Hospital of Philadelphia, Philadelphia,

PA o Peter McGuire, MS, MBBCh, Metabolism, Infection and Immunity Section,

National Human Genome Research Institute, NIH, Bethesda, MD o Vamsi K. Mootha, MD, Harvard Medical School, Boston, MA o Sumit Parikh, MD, The Cleveland Clinic, Cleveland, OH o Anne Pariser, MD, NIH/National Center for Advancing Translational

Sciences (NCATS), Bethesda, MD o William Prinz, PhD, NIH/National Institute of Diabetes and Digestive and

Kidney Disease, Bethesda, MD o Shamima Rahman, MA, BMBCh, PhD, FRCP, FRCPCH, Great Ormond

Street Hospital and the National Hospital for Neurology, London, UK o David Shackelford, PhD, University of California Los Angeles

(UCLA) David Geffen School of Medicine, CA o Keshav K. Singh, PhD, University of Alabama at Birmingham, AL o Michael Teitell, MD, PhD, University of California, Los Angeles, Jonsson

Comprehensive Cancer Center, CA

o David Thorburn, PhD, Murdoch Children’s Research Institute, Melbourne, Australia

o Douglas C. Wallace, PhD, Children’s Hospital of Philadelphia, Philadelphia, PA

o A. Phillip West, PhD, Texas A&M University College of Medicine, College Station, TX

o Takehiro Yasukawa, PhD, Department of Clinical Chemistry and Laboratory Medicine Graduate School of Medical Sciences, Kyushu University

o Philip Yeske, PhD, UMDF, Science & Alliance Officer, Pittsburgh, PA

2019 Scientific/Clinician Meeting – Planning Committee

• 2019 Scientific Planning Committee o Amel Karaa, MD, Course Co-Chair, Massachusetts General Hospital,

Boston, MA o Carla Koehler, PhD, Course Co-Chair, University of California Los

Angeles, Los Angeles, CA o Bruce H. Cohen, MD, CME Chair, Akron Children’s Hospital, Akron, OH o William Copeland, PhD, NIEHS, Research Triangle Park, NC o Amy Goldstein, MD, Children’s Hospital of Philadelphia, Philadelphia, PA o Larry Grossman, PhD, Wayne State University School of Medicine,

Detroit, MI o Adam Hartman, MD, NINDS/NIH, Rockville, MD o Gene Kelly, UMDF Industry Advisor Council, Chair, Stealth

BioTherapeutics, Boston, MA o Giovanni Manfredi, MD, PhD, Weill Cornell Medicine, New York, NY o Robert K. Naviaux, MD, PhD, UCSD, San Diego, CA o Sumit Parikh, MD, The Cleveland Clinic, Cleveland, OH o Russell Saneto, DO, PhD, Course Chair, Seattle Children’s Research

Institute, Seattle, WA o Peter Stacpoole, PhD, MD, University of Florida, Gainesville, FL o Keshav K. Singh, PhD, University of Alabama at Birmingham, AL o Philip Yeske, PhD, UMDF Science & Alliance Officer o Kara Strittmatter, CMM, MA, UMDF Meeting Event Director

2019 Abstract Presenters o Jirair Bedoyan, MD, PhD, Case Western Research University/University Hospitals Cleveland

Medical Center, Cleveland, OH, United Stateso Yan Burelle, PhD, University of Ottawa, Ottawa, Ontario, Canadao Patrick Chinnery, PhD, FRCP, University of Cambridge, Cambridge, Cambridgeshire, United

Kingdomo Bruce H. Cohen, MD, Akron Children's Hospital, Akron, Ohio, United Stateso Liam Coyne, MD/PhD Student, State University of New York Upstate Medical University,

Syracuse, New York, United Stateso Kirsten Hoff, PhD, NIEHS, Research Triangle Park, NC, United Stateso Amel Karaa, MD, Massachusetts General Hospital, Boston, MAo Rustum Karanjia, MD, PhD, o Nam Chul Kim, PhD, University of Minnesota, Duluth, Minnesota, United Stateso Matthew Klein, MD, BioElectron, Mountain View, CA, United Stateso Piotr Kopinski, MD, PhD Student, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania,

United Stateso Samantha Lewis, PhD, University of California, Davis, Davis, CA, United Stateso Robert K. Naviaux, PhD, University of California, San Diego, San Diego, CA, United Stateso Xavier Lloria, MD, Santhera Pharmaceuticals, Pratteln, Switzerlando Charles McCall, MD, Wake Forest University Medical Center, Winston Salem, NC, United Stateso Cameron McKnight, PhD Student, University of Melbourne, Parkville, Victoria, Australiao Claudia Pereira, PhD, University of Miami, Miller School of Medicine, Miami, Florida, United

Stateso Peter Stacpoole, PhD, MD, University of Florida, Gainesville, Florida, United Stateso Stephen Thomas, PhD, Cerecor Inc., Rockville, Maryland, United Stateso Hilary Vernon, MD, PhD, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins

University School of Medicine and at the Kennedy Krieger Institute; Director, Barth SyndromeClinic at Kennedy Krieger Institute, Baltimore, Maryland, United States

o Natalie Yivgi Ohana, PhD, Minovia Therapeutics Ltd, Tirat Hacarmel, Israel

UMDF Mitochondrial Medicine 2019 Accreditation and Designation Statements and Disclosure Report

This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of PeerPoint Medical Education Institute, LLC and United Mitochondrial Disease Foundation. The PeerPoint Medical Education Institute, LLC is accredited by the ACCME to provide continuing medical education for physicians.

The PeerPoint Medical Education Institute, LLC designates this live activity for a maximum of 18.50 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

Financial Disclosures

The following presenters, planners, editors, or staff have relevant financial relationships to disclose:

“I, or an immediate family member, have at present or have had within the last 12 months, the following affiliation with one or more organizations that could be perceived as a real or apparent conflict of interest in context to the design, implementation, presentation, evaluation, etc. of CME Activities.”

All other presenters, planners, editors, or staff reported no relevant relationships with the following statement:

“I, or an immediate family member, do not have at present, or within the last 12 months, nor anticipate having financial interest, arrangement or affiliation with any organizations that could be perceived as a real or apparent conflict of interest in context to the design, implementation, presentation, evaluation, etc. of CME Activities.”

Commercial Interest For what role What was receivedDr. Peter Stacpoole Research grant from Saol Therapeutics investigator-initiated research research grant

Praesidio Pharma, LLC Chief Scientific Officer No monetary or other compensationDr. Russell Saneto Stealth Biotherapeutics Principal Investigator Study GrantDr. Adam Hartman BestDoctors, Inc. Consultant Consulting feesDr. Michael Teitell NanoCav, LLC co-founder, Board Member, consultant non-voting shares of stock, consulting feeDr. Amel Karaa Stealth Biotherapeutic Scientific presentations Travel support

Sanofi Genzyme Advisory board Consulting feeAmicus Therapeutic Advisory board Consulting feeTakeda Speaking Honoraria

Dr. Bruce Cohen Stealth Biotherapeutics Consulting, Principal Investigator Consulting fees, research pmt to employerBioElectron Technologies Principal Investigator Payment to Employer for Contracted ResearchReata Principal Investigator FeesModis Consulting Consulting FeesHorizon Pharma Principal Investigator Fees

Dr. David Thorburn Murdoch Children's Research Institute Medical researcher SalaryVictorian Clinical Genetics Services Executive no compensation

Dr. Austin Larson Stealth Biotherapeutics Site investigator for clinical trial Salary supportRetrophin Site investigator for clinical trial Salary supportMallinckrodt Site investigator for clinical trial Salary supportOrphan Technologies Site investigator for clinical trial Salary support

Dr. Shamima Rahman Bioinsight Consulting Consulting feeNeurovive Consulting Consulting feePartners4Access Consulting Consulting fee

Dr. Patrick Chinnery Wellcome Trust research fellow grant/research supportMitochondrial Research Center Clinical Director grant/research support

Dr. David Livingston Metro International Biotech, LLC Employment, management Salary, ownership interestDr. David Dimmock Biomarin Consultant Consulting fees

Audentes Therapeutics Scientific Advisory Board Consulting feesIchorion Therapeutics Consultant Consulting feesDemeter Therapeutics Consultant Consulting feesComplete Genomics Scientific Advisory Board Consulting feesGenzyme/Sanofi Clinical trial support Clinical trial support to host institutionHyperion Clinical trial support Clinical trial support to host institutionAlexion Clinical trial support Clinical trial support to host institution

Dr. Vamsi Mootha Janssen Pharmaceuticals Scientific advisory board consulting fee5am Ventures Scientific advisory board consulting fee, optionsRaze Therpeutics Scientific advisory board options

Stephen Thomas Cerecor Inc. Employment Salary, Ownership Interest

Join Us for…

5:30 Reception and Cash Bar – Plaza Foyer

6:30 An Evening of Energy Banquet Celebration – Plaza C Join us for this special awards ceremony celebrating our volunteer leaders, research prize winners and community of supporters. Hosted by Patti Mercer of WRHI 100.1, along with Jr. Hosts, Cavan McGovern and Olivia Kallaos, and Teen Host, Katie Parsons.

SAVE THE DATE!

Mitochondrial Medicine 2020: Phoenix, Arizona

Scientific Program: June 24 - 27, 2020Family & LHON Program: June 26 - 27, 2020

JW Marriott Phoenix Desert Ridge Resort & Spa

5350 East Marriott Drive Phoenix, AZ 85054

www.umdf.org/symposium

Mitochondrial Medicine 2019: Washington DCSpecial Thanks to Our Supporters and Supporter Exhibitors!

The United Mitochondrial Disease Foundation gratefully acknowledges the following organizations for their support of Mitochondrial

Medicine 2019:

The Edith L. Trees Charitable Trust

Stealth BioTherapeutics

Minovia Therapeutics

Tishcon Corp

Solace Nutrition

Delta Gamma Foundation

Entrada Therapeutics

GenSight Biologics

Modis Therapeutics

GeneDx

Perkin Elmer Genomics

Santhera Pharmaceuticals

Saol Therapeutics

Summit Health Rx

Biolog

Neurovive Pharmaceuticals

Reneo Pharmaceuticals

The Hunt Michael Hollis Fund

Mitochondrial Medicine 2019: Washington DCExhibitors

Thank you to the following exhibitors for attending:

Baylor Genetics

Casimir Trials

Chemistry Rx

Fulgent Genetics

Miracles for Mito

MitoAction

Mitochondrial Research Guild

MNG Labs

NAMDC

National Human Genome Research Institute

Novogene

PALS

PhenoVista Biosciences

Quten Research “Qunol”


Wednesday, June 26, 2019

Welcome

Philip Yeske, PhD, UMDF Science and Alliance Officer Amel Karaa, MD and Carla Koehler, PhD, Course Co-Chairs

Primary Mitochondrial Disease Patient Testimony – My Diagnostic Odyssey, What My Diagnosis Means to

Me, and My Hopes for the Future

Stacy Taylor Stacy lives in Baltimore, MD, with her husband David and their four boys: Lucas, Benjamin, Marshall and Sam. Marshall and Sam have genetically

confirmed mitochondrial disease caused by a mutation in their nuclear DNA. Stacy is a UMDF Ambassador and is trying to get an active group of

affected adults and children together for regular events in the Baltimore metropolitan area. Stacy enjoys spending time with her kids.... and

spending time away from her kids.

Rachel Schanzenbach

Rachel has been married to her best friend Daniel for 16 years, and together they are raising their two children while also fighting against

mitochondrial disease. Rachel’s life verse is, “My flesh and my heart may fail, but God is the strength of my heart and my portion forever.” She and

her family make their home in the DC/MD/VA region.

Morning Session Opening and Organelle Cross-Talk and Mitochondrial

Dynamics

Keynote Address: Mitochondrial Genetic Medicine: Past, Present and Future

Douglas Wallace, PhD

Presenter: Douglas C. Wallace, PhD Institution: Center for Mitochondrial and Epigenomic Medicine (CMEM) and the

Mitochondrial Medicine Frontier Program (MMFP), Children’s Hospital of Philadelphia, Department of Pediatrics, Division of Human Genetics, University of Pennsylvania, Philadelphia, PA 19104 USA

Title: Mitochondrial Genetic Medicine: Past, Present and Future That mitochondria are cellular organelles was established by Richard Altmann in 1890, the term “mitochondria” being coined in 1898 by Carl Benda. Early on, the mitochondrial structure was suggestive of a bacterial origin, but the symbiotic origin of mitochondria and chloroplasts, championed by Lynn Margulis, came to the fore with the discovery of chloroplast DNA by Alexander Rich and animal mitochondrial DNA (mtDNA) by Margit Nass in 1963. In 1949 Boris Ephrussi discovered yeast petite cells, which in the early 1960s were found to be due to mitochondrial DNA (mtDNA) deletions by Gottfried Schatz and Giorgio Bernardi. Yeast mtDNA genetics was then elucidated by Piotr Slonimski, Antony Linneane, and associates in the 1960s through the 1980s. The mitochondrial role in oxidative phosphorylation (OXPHOS) was elucidated by Otto Warburg, Hans Krebs, Albert Lehninger, Britton Chance, and Youssef Hatefi, culminating in the chemiosmotic theory of ATP synthesis by Peter Mitchell published in 1961. That mitochondrial dysfunction could be associated with disease was first reported by Rolf Luft and associates in 1962 and extended by the Salvatore Di Mauro and John Morgan-Hughes programs. That the human mtDNA could code for phenotypic manifestations was demonstrated by Douglas Wallace and collaborators in cultured cells in 1975. They then demonstrated the maternal inheritance of the human mtDNA and the intercontinental radiation of human mtDNAs starting in 1980-1981. The nucleotide sequence of the mtDNA was published by the Fred Sanger group in 1981 and the mtDNA transcription map was subsequently defined by the Giuseppe Attardi team. In 1988, the first inherited mtDNA mutations were reported by Wallace and collaborators and the first spontaneous mtDNA deletion mutations were reported by Ian Holt, Anita Harding, and Morgan-Hughes. The importance of nuclear DNA (nDNA) mutations in mitochondrial disease was then shown in 1990 by Massimo Zeviani in DiMauro’s laboratory and has since been advanced by numerous investigators including Anu Suomalainen, Jan Smeitink, Vamsi Mootha, and Marni Falk. Mouse models of mitochondrial disease have been generated by the Wallace, Jun-Ichi Hayashi, Nils-Göran Larsson, and Tomas Prolla laboratories and permitted the verification that mtDNA mutations can be sufficient to cause common diseases, cancer, and aging. A number of nutraceutical and pharmacological treatments are being developed for mitochondrial disease and both somatic and germline gene therapies have been reported. Somatic mtDNA gene therapy has focused on allotopic protein complementation advanced by the research of John Guy, Marisol Corral-Debrinski, & Bin Li and germline gene therapy via nuclear transplantation by Mary Herbert, Douglass Turnbull, Shoukhrat Mitalipov, and others. Given that mtDNA diseases were thought not to exist 31 years ago, mitochondrial genetic medicine has come a long in a short time.


Dynamics

Endoplasmic Reticulum (ER) and Mitochondria and the Role of Close Contacts between these

Organelles in Lipid Trafficking

William Prinz, PhD

Presenter: Will Prinz

Institution: Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Title: Endoplasmic Reticulum (ER) and Mitochondria and the Role of Close Contacts

between these Organelles in Lipid Trafficking

Mitochondrial membrane biogenesis requires lipid transport to mitochondria since most lipids in mitochondrial membranes are synthesized outside mitochondria. This transport is thought to be nonvesicular and occur at regions of close contact between the mitochondria and the endoplasmic reticulum (ER), where most lipid synthesis occurs. Mitochondria form close contacts with other organelles as well and these contacts may also facilitate lipid exchange between mitochondria and the rest of the cell. The mechanism and regulation of lipid transport between the ER and mitochondria remains poorly understood. Some proteins that maintain ER-mitochondria contacts and transport lipids have been identified but many questions about the mechanism and regulation of lipid transport remain. One is that it is not clear how the lipid transporters are regulated or how they facilitate sufficient lipid movement to mitochondria since, in vitro, they do not seem to transport lipids at high enough rate to facilitate membrane biogenesis. My lab studies the mechanisms of lipid transport to mitochondria in the yeast S. cerevisiae. It has long been known that the parts of the ER in contact with mitochondria, often referred to as mitochondrial associated membranes (MAMs), are enriched in lipid biosynthesis enzymes. This is true of yeast and mammalian cells. We wanted to determine whether lipid synthesis in MAM promotes lipid transport to mitochondria. We focused on phosphatidylserine (PS) synthase, which is enriched in MAM. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Additional evidence for a connection between lipid synthesis and transport was provided by in vitro studies that showed newly synthesized lipids are more readily transferred between membranes. Together, these findings suggest that phospholipid production at membrane contacts sites may be a general mechanism of channeling lipids to mitochondria and other compartments.


Dynamics

The Mitochondria and Lysosome – Two Organelles with Important Roles in Aging and

Metabolism

Adam Hughes, PhD

Presenter: Adam Hughes, PhD

Institution: University of Utah School of Medicine, Salt Lake City, UT Title: The Mitochondria and Lysosome – Two Organelles with Important Roles in Aging

and Metabolism

Mitochondrial dysfunction is widely associated with the aging process, and functions as a catalyst for many age-associated diseases. Our lab has been interested in understanding how aging drives mitochondrial impairment, and how cells protect mitochondria from stress. Along these lines, we recently found that mitochondrial health is tightly coupled to lysosome function, and that loss of lysosomal acidity in aged cells drives age-related changes in mitochondria. In addition to their prominent role in protein degradation, lysosomes in play a central role in cellular metabolism through sequestration of amino acids through membrane-bound transporters. Interestingly, it appears that lysosome-mediated mitochondrial impairment in aged cells is not driven by a collapse in lysosome-mediated degradation systems, but rather, by alterations in subcellular localization of amino acids upon lysosome failure. These results suggest that lysosomes compartmentalize nutrients at least in part to maintain optimal mitochondrial function. We are currently exploring how changes in nutrient compartmentation impairs mitochondrial function in lysosome-deficient cells, and how mitochondria protect themselves from lysosome-induced nutrient stress. Here, I will discuss our recent insights in these areas, including identification of potential mechanisms linking mis-compartmentation of amino acids to mitochondrial collapse, and our discovery of a protein degradation system called the Mitochondrial-Derived Compartment (MDC) pathway that selectively destroys mitochondrial nutrient transporters in response elevated cellular nutrient stress.


Dynamics

The Role of Mitochondrial Chaperones in Cellular Adaptation and Tumor Metabolism

Dario C. Altieri, MD

Presenter: Dario C. Altieri, MD

Institution: Wistar Institute Cancer Center, Philadelphia, PA

Title: The Role of Mitochondrial Chaperones in Cellular Adaptation and Tumor Metabolism

Changes in cellular metabolism are now a recognized hallmark of cancer, contributing to the acquisition of malignant traits and disease progression. Although much effort has been devoted to the increased glycolytic rate of tumors even when oxygen is present, the so-called “Warburg effect”, recent data has underscored an important role of mitochondria as a multifaceted tumor driver. Mechanistically, this involves oxidative bioenergetics and other metabolic pathways, signaling by reactive oxygen species, and regulation of multiple forms of cell death. In addition, fresh experimental evidence as highlighted how mitochondrial dynamics, an adaptive process that controls organelle size, shape and subcellular distribution is broadly exploited in genetically heterogeneous malignancies and directly contributes to increase tumor cell movements, chemotaxis and invasion across basement membranes, altogether promoting greater metastatic dissemination in preclinical models, in vivo. In this context, recent studies from our group have demonstrated that dynamic redistribution of energetically active mitochondria to the peripheral, or cortical cytoskeleton of tumor cells plays an important role in this process, providing an efficient, concentrated energy source to fuel focal adhesion complex dynamics, increased membrane lamellipodia formation and heightened tumor cell migration, invasion and metastatic spreading. The molecular requirements of subcellular mitochondrial trafficking in cancer are beginning to emerge and new findings have highlighted how the cellular machinery that supports mitochondrial motility in neurons, including atypical mitochondrial outer membrane GTPases, RHOT1 and RHOT2, adapter proteins, TRAK1 and TRAK2, anterograde kinesin, Kif5B and negative regulator of mitochondrial movements, Syntaphlin (SNPH), becomes prominently hijacked in disparate tumor cells to support increased cell motility and metastatic propensity, in vivo. Ongoing research efforts focus on the characterization of mitochondrial dynamics and subcellular organelle trafficking as novel metastasis regulators, the modulation of this mitochondrial motility network by oncogenic signaling and transcriptional responses, and how cellular stress conditions typical of the tumor microenvironment, including hypoxia and nutrient deprivation, affect this process. The elucidation of this pathway will uncover new, actionable therapeutic targets to limit metastatic dissemination in patients with advanced cancer, a currently incurable and often fatal condition with only palliative treatment options.

Morning Abstracts

11:15am PA-0530 Nam Chul Kim The PINK1/Parkin pathway mediates dominant mitochondrial toxicity in CHCHD10-induced ALS-FTD

11:30am PA-0582 Liam Coyne Protein Misfolding on the Inner Mitochondrial Membrane is Toxic and has Pathological Consequences

11:45am PA-0539 Piotr Kopinski Regulation of nuclear epigenome by mitochondrial DNA heteroplasmy

12:00pm PA-0625 Cameron McKnight Using human pluripotent stem cell models of mitochondrial disease to identify candidate drug treatments

Morning Lightning Rounds

Epigenetic Control and Mitochondrial DNA Methylation

Takehiro Yasukawa, PhD

Afternoon Session Mitochondrial Architecture: Regulation and Maintenance

Presenter: Takehiro Yasukawa, PhD

Authors: Takehiro Yasukawa1, Shigeru Matsuda1, Yuriko Sakaguchi2, Kenji Ichiyanagi3, Motoko Unoki3, Kazuhito Gotoh1, Kei Fukuda3, Hiroyuki Sasaki3, Tsutomu Suzuki2, Dongchon Kang1

Institution: 1Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; 2Department of Chemistry and Biotechnology, GraduateSchool of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,Tokyo 113-8656, Japan; 3Division of Epigenomics and Development, MedicalInstitute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku,Fukuoka 812-8582, Japan

Title: Accurate estimation of 5-methylcytosine in mammalian mitochondrial DNA

Cytosine methylation in mammalian mitochondrial DNA (mtDNA) is one of the hottest topics in mitochondrial biology and medicine in recent years. It could have profound impact on gene expression, replication and maintenance of mtDNA, and abnormalities in mtDNA methylation might well be a cause of disease with mitochondrial dysfunction. Elucidation of whether mtDNA is methylated or not is thus of pivotal importance. Methylation of mtDNA has been argued for by several recent papers, but after careful reading of them it may be difficult to draw a clear picture of the methylation partly because of conflicting data presented among them. Here, we set out on investigation with a hope of resolving the confusing situation. This study was conducted through collaboration of three research groups who are experts on mtDNA, DNA methylation and mass spectrometry. We carefully prepared and investigated mouse mtDNA using three methods that are based on different principles for detecting 5-methylcytosine; next generation bisulfite-sequencing, a cleavage assay with a methylation-dependent restriction enzyme and mass spectrometric nucleoside analysis. Data from these analyses led us to propose that the 5-methylcytosine is not present at any specific position(s) in mtDNA and levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in the gene expression or metabolism of mtDNA.


In vivo Imaging of Mitochondrial Membrane Potential in Non-Small Cell Lung Cancer

David Shackelford, PhD

Presenter: David Shackelford, PhD

Authors: Milica Momcilovic1, Anthony Jones3, Sean T. Bailey12, Christopher M. Waldmann3, Rui Li1, Jason T. Lee3,8,9, Gihad Abdelhady1, Adrian Gomez5, Travis Holloway3, Ernst Schmid4, David Stout11, Michael C. Fishbein2, Linsey Stiles6, Deepa V. Dabir10, Steven M. Dubinett1,2,3,9, 13, Heather Christofk3,4,7,9, Orian Shirihai6,9, Carla M. Koehler5, Saman Sadeghi3 and David B. Shackelford1,9§

Institution: 1Division of Pulmonary and Critical Care Medicine, Department of Medicine; 2Department of Pathology and Laboratory Medicine; 3Department of Molecular and Medical Pharmacology; 4Department of Biological Chemistry; 5Department of Chemistry and Biochemistry; 6Department of Endocrinology; 7UCLA Metabolomics Center; 8Crump Institute for Molecular Imaging; 9Jonsson Comprehensive Cancer Center; David Geffen School of Medicine at the University of California, Los Angeles, CA 90095. 10Department of Biology, Loyola Marymount University, Los Angeles, CA 90045. 11Regis College, Weston, MA 02493. 12University of North Carolina Chapel Hill, Chapel Hill, NC 27599, 13VA Greater Los Angeles Healthcare System, Los Angeles CA 90073. §corresponding author

Title: In vivo imaging of mitochondrial membrane potential in non-small cell lung cancer

Key words: mitochondria, 18FBnTP, membrane potential, bioenergetics, OXPHOS, non-small cell lung cancer, Kras, Lkb1, tumor heterogeneity

The mitochondria are essential regulators of cellular energy and metabolism and they play a critical role in sustaining growth and survival of cancer cells. A central process of the mitochondria is the synthesis of ATP through oxidative phosphorylation (OXPHOS) known as bioenergetics. The mitochondria maintain OXPHOS by creating a membrane potential gradient ( ) that is generated by the electron transport chain (ETC) in order to drive ATP synthesis1,2. Mitochondria are essential for tumor initiation and maintenance as seminal experiments identified that loss of mtDNA inhibited mitochondrial bioenergetics and suppressed tumor cell growth in cell culture and xenografts3-5. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because the majority of studies have been performed in vitro in cell culture models. This has left a large gap in our knowledge of how oxidative mitochondrial metabolism supports tumor growth and highlights a need for in vivo studies. Therefore we sought to measure mitochondrial in vivo in non-small cell lung cancer (NSCLC) using a voltage sensitive, positron emission tomography (PET) tracer known as 4-[18F]fluorobenzyl triphenylphosphonium (18FBnTP)6. We used 18FBnTP PET imaging to profile mitochondrial in autochthonous mouse models of lung cancer and discovered distinct functional mitochondrial heterogeneity within NSCLC tumor subtypes. The use of 18FBnTP PET imaging enabled us to functionally profile mitochondrial in live tumors. We anticipate 18FBnTP PET imaging will have dynamic applications beyond that of cancer and benefit a broad range of fields focused on understanding how mitochondria impact human disease.

References: 1 Mitchell, P. & Moyle, J. Evidence discriminating between the chemical and the

chemiosmotic mechanisms of electron transport phosphorylation. Nature 208, 1205-1206 (1965).

2 Mitchell, P. & Moyle, J. Stoichiometry of proton translocation through the respiratory chain and adenosine triphosphatase systems of rat liver mitochondria. Nature 208, 147-151 (1965).

3 Morais, R. et al. Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA. Cancer Res 54, 3889-3896 (1994).

4 Desjardins, P., Frost, E. & Morais, R. Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts. Mol Cell Biol 5, 1163-1169 (1985).

5 Cavalli, L. R., Varella-Garcia, M. & Liang, B. C. Diminished tumorigenic phenotype after depletion of mitochondrial DNA. Cell Growth Differ 8, 1189-1198 (1997).

6 Madar, I. et al. Characterization of uptake of the new PET imaging compound 18F-fluorobenzyl triphenyl phosphonium in dog myocardium. J Nucl Med 47, 1359-1366 (2006).


Mitochondria and Psychiatric Disorders

Ana C. Andreazza, Pharm PhD

Presenter: Ana C. Andreazza, Pharm PhD

Authors: Angela Duong1, Liliana Attisano2, Martin Beaulieu1, Ana Andreazza1

Institution: 1 Department of Pharmacology and Toxicology, University of Toronto, ON, Toronto, Canada; 2 Department of Biochemistry, University of Toronto, ON, Toronto, Canada

Title: Energy Metabolism and Bipolar Disorder: Where we are and what is coming

Mitochondria are the powerhouse of our cells. A crucial consequence of impaired mitochondrial function is damage to highly metabolically active tissues in the body. The highest energy user in the body is the brain, accounting for 20% of the total energy budget to power neuronal function, including neuronal electrical activity and neurotransmission. Continuous mitochondrial dysfunction can have profound effects on neurotransmission and may contribute substantially to changes in neuronal circuits in the brain that underlie cognition, memory and other forms of neuronal plasticity. One plausible hypothesis is that bipolar disorder is due in part to the failure of mitochondrial function to support adequate neurotransmission and synaptic plasticity, potentially affecting mood regulation, memory, and executive function. This hypothesis is supported by several studies in neuropsychiatric conditions showing higher (1) frequency of mtDNA mutations; (2) polymorphisms in autosomal mitochondrial complex I genes; (3) lactate levels; (4) reactive oxygen species (ROS) production; and downregulation of mitochondrial electron transport chain subunits. One of the most intriguing clinical observations is that individuals with mitochondrial disease commonly present with psychiatric symptoms. In this lecture Dr. Andreazza will provide an overview of where we are and where we re going on the study of energy metabolism in the diagnosis and treatment of neuropsychiatric ocnditions. She will focus on a simple question “: Is mitochondrial function the cause of neurotransmission changes in mood regulation?” and finish her presentation with novel models including the use of 3D brain organoids to understand the involvement of the energy metabolism on neurotransmission.


David Lynch, MD, PhD

Presenter: David R Lynch, MD PhD

Institution: Division of Neurology, Children’s Hospital of Philadelphia

Title:

Friedreich ataxia (FRDA) is a slowly progressive multisystem degenerative disease characterized by ataxia, dysarthria, loss of coordination, cardiomyopathy, scoliosis and other features. The disorder is caused by mutations on both alleles of the FXN gene, leading to decreased production of the protein frataxin. Frataxin is a small nuclear encoded mitochondrial protein involved in synthesis of iron sulfur clusters for enzymes of oxidative phosphorylation, the Krebs cycle, and many other pathways. This leads to downstream events such as diminished function of Aconitase, deficiency of ATP production, and other evidence of mitochondrial dysfunction. Theoretically, FRDA could be treated by reversal of mitochondrial dysfunction or restoration of functional frataxin.

A variety of tools have been created to facilitate clinical trials in FRDA. Two natural history studies now follow approximately 1000 subjects in North America/Australia, and a similar number in Europe. This has facilitated design of outcome measures and provided a road map for relative change in different subjects. In addition, patient registries have facilitated rapid recruitment of clinical trials.

In spite of such well-developed tools, no agents have successfully met their endpoints in later stage trials. In some situations, insufficient preliminary data has led to inaccurate powering calculations. In other situations, trials were clearly too short to allow the placebo and active drug arms to separate. In still other situations, the outcome measures or the agents did not match the biological properties of the cohort.

A crucial component of future trials will be the use of biomarkers, such as monitoring of frataxin levels and assessment of event downstream such as intermediary metabolism. In platelets, which are readily measured in trials ex vivo, FRDA patients have decreased glycolysis compared to controls and increased oxidation of palmitate. This provides a biomarker of the events immediately downstream from frataxin deficiency. One agent, RT 408 (an NRF2 activator) has shown promise in reversing metabolic biomarkers, improving exercise function, and improving exam findings in a double blind trial for 3 months (MOXIE). Part 2 of MOXIE is underway and will be analyzed late in 2019.

Even as agents based on reversal of mitochondrial dysfunction reach potential success, newer agents will address frataxin deficiency. Such approaches include protein replacement and gene therapy, both of which show substantial benefit in mouse models. It is expected that in spite of obstacles these will reach clinical trials in the next 2 years.

Afternoon Abstracts

4:30pm PA-0551 Samantha Lewis Twinkle helicase selectively interacts with the inner mitochondrial membrane lipid cardiolipin to scaffold mtDNA replisome recruitment at ER-mitochondria contact sites

4:45pm PA-0552 Yan Burelle Coping with respiratory chain deficiency through adaptive optimization of the OXPHOS assembly line: Insights from the hepatic LRPPRC knockout mouse model.

5:00pm PA-0548 Kirsten Hoff A novel function for the Polymerase accessory subunit in regulating mtDNA copy number

Afternoon Lightning Rounds


Thursday, June 27, 2019

Morning Session Mitochondrial Communication and Quality Control

Mitochondria and Microbiota Inter-Talk

Keshav Singh, PhD

Presenter: Keshav K. Singh, PhD

Authors: Bhupendra Singh1, Keshav K. Singh1* Institution: Department of Genetics, School of Medicine, The University of Alabama

at Birmingham, Kaul Genetics Building, Suite 620, 720 20th St. South, Birmingham, AL, USA 35294

Title: Mitochondria and Microbe Cross Talk in MIce The MDSs (Mitochondrial DNA depletion) are a heterogeneous group of disorders, characterized by low mtDNA levels in specific tissues. Mitochondrial DNA (mtDNA) depletion impairs mitochondrial function that leads to mtDNA depletion syndrome (MDS). In different target organs, mtDNA depletion presents specific phenotype. Furthermore, mtDNA copy number declines with age, and such changes increase the risk of age-associated skin diseases. However, an association between the decline in mtDNA copy number changes in skin aging and microbiome is not addressed. To evaluate the consequences of depletion of mtDNA in the whole animal, we created a doxycycline inducible mouse (mtDNA-depleter) expressing, in the polymerase domain of POLG1, a dominant-negative mutation to induce depletion of mtDNA in different tissues. These mice showed reduced mtDNA content, changes in mitochondrial protein expression, and reduced stability of mitochondrial oxidative phosphorylation complexes. We demonstrate that ubiquitous depletion of mtDNA in mice has profound effects on the skin resulting in wrinkles and hair loss. Microbiome studies in these mice showed significant changes in the composition and/or function of the skin microbiome. Skin microbiome studies in this mouse model provide clues about the role mitochondria on the microflora of the skin. This mouse model provides an opportunity to expand our knowledge of how mitochondria contribute to the pathogenesis of MDS and other human diseases. The mtDNA-depleter mouse should provide an impetus to the research about the development of preventative and therapeutic strategies for skin related maladies associated with mitochondrial diseases.


Phillip West, PhD

Presenter: A. Phillip West, PhD

Authors: Yuanjiu Lei, Sylvia Torres-Odio, Camila Guerra Martinez, & A. Phillip West

Institution: Dept. of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M University, College Station, TX USA

Title: Mitochondrial control of innate immune responses: implications for mitochondrial disease

It is increasingly well appreciated that mitochondria are key modulators of mammalian innate immunity. Mitochondrial dysfunction has emerged as an important driver of deleterious inflammatory and interferon responses in numerous human diseases; however, it remains unclear whether alterations in mitochondria-innate immune crosstalk contribute to the pathobiology of mitochondrial diseases. Several clinical reports have noted that patients with mitochondrial disease exhibit recurrent infections and are more at risk for developing systemic inflammatory response syndrome (SIRS) compared to the general population. Moreover, intercurrent bacterial and viral infections have been noted to unmask and/or accelerate the progression of mitochondrial disease. Together, these findings suggest immune system imbalances in these patients, yet a mechanistic examination of innate immune function and its influence on mitochondrial disease progression has not been completed. To examine these questions in a physiologically relevant and genetically tractable setting, we are employing a murine model of Polymerase gamma (POLG)-related mitochondrial disease. We have uncovered that mitochondrial DNA (mtDNA) instability in POLG-mutant mice engages DNA sensors of the innate immune system, leading to the sustained and systemic expression of type I interferon (IFN-I) responses that increase with age. Furthermore, we show that chronic IFN-I signaling dramatically augments myeloid cell populations in the bone marrow and blood, heightening secretion of inflammatory cytokines after stimulation and markedly increasing susceptibility to SIRS and lethal septic shock. Mechanistically, basal IFN-I signaling in POLG-mutants suppresses the activation and nuclear localization of the transcription factor Nrf2, a key antioxidant and anti-inflammatory protein, leading to elevated oxidative damage, sustained inflammatory cytokine secretion, and accelerating mitochondrial dysfunction. Finally, we report that genetic ablation of IFN-I signaling attenuates some aspects of tissue pathology in POLG-mutant mice by boosting Nrf2-mediated antioxidant responses and attenuating the hyper-inflammatory myeloid phenotype of these animals. These results provide new insight into the molecular and cellular innate immune rewiring in a model of mitochondrial dysfunction, and may have implications for managing recurrent infections, SIRS, and immunopathology in patients with POLG-related diseases or other mitochondrial disorders.


Peter McGuire, MS, MBBCh

Presenter: Peter J. McGuire MS, MD

Institution: Metabolism, Infection and Immunity Section, National Human Genome Research Institute, NIH, Bethesda, MD

Title: The Immune Paradox in Mitochondrial Disease

In the field of mitochondrial medicine, one of the most challenging issues for patients and providers is metabolic decompensation. Metabolic decompensation in mitochondrial disease is a life-threatening deterioration in mitochondrial function which can lead to hastened disease progression and further disability. Treatment during metabolic decompensation in mitochondrial disease is symptomatic and focuses on infection as a bioenergetic stress. As a result, treatment involves: 1) maintaining hydration, 2) providing calories, 3) correcting metabolic derangements, 4) providing antioxidant/cofactor therapies, and 5) avoiding mitochondrial toxins, mostly drugs. Although definitive data on precipitants of metabolic decompensation is absent from the medical literature, it is generally acknowledged that physiological stressors like infection play a major role. Complicating matters further, many children with mitochondrial disease experience recurrent or severe infections, most of which are respiratory viral infections, placing them at heightened risk for metabolic decompensation. In general, the toll infection takes on patients with mitochondrial disease is readily appreciated by clinicians: sepsis and pneumonia are the two most common causes of death. Instead of focusing on the bioenergetic costs of infection, as is the current approach, my group addresses the relationship between the immune system and metabolic decompensation. In patients with mitochondrial disease, the immune system is a bit of a paradox: adaptive immune dysfunction leaves patients vulnerable to infection, while robust inflammation from the innate immune system serves as an endogenous toxic insult to end-organs affected by mitochondrial disease. Understanding this tenuous relationship between infection, the immune system and metabolic decompensation in mitochondrial disease is critical for developing new approaches to treatment for this vulnerable population.


Shamima Rahman, FRCP PhD

Presenter: Shamima Rahman, FRCP, PhD

Institution: Great Ormond Street Hospital and UCL Great Ormond Street Institute of Child Health, London, UK

Title: Mitochondrial Phenocopies and Secondary Mitochondrial Dysfunction

Primary mitochondrial diseases have been defined as disorders caused by mutations that lead to oxidative phosphorylation dysfunction or other disturbances of mitochondrial structure and function including perturbed mitochondrial ultrastructure, aberrant synthesis of cofactors and vitamins, or other impaired metabolic processes within the mitochondrion.1 These disorders are characterized by enormous clinical, biochemical and genetic heterogeneity, leading to considerable diagnostic challenges. One of the major difficulties in diagnosing mitochondrial disease is the extremely wide differential diagnosis. Mitochondrial disease presentations may mimic many different diseases and are thus frequently part of the differential diagnosis in a diverse range of clinical scenarios. Many “typical” features of mitochondrial disease, including epilepsy, leukodystrophy, peripheral neuropathy, cardiomyopathy, renal tubulopathy and liver failure, have a host of other possible causes.2 Complex multisystem mitochondrial disorders may be confused with peroxisomal disorders, congenital disorders of glycosylation, lysosomal storage disorders and other syndromic genetic conditions. Furthermore, the central location of the mitochondrion in human metabolism means that secondary mitochondrion dysfunction has been implicated in the pathogenesis of many common disease processes, ranging from Parkinson disease and other neurodegenerative disorders to cancer, diabetes, sepsis and autoimmune diseases. In this presentation I will explore the differential diagnosis of single organ and multisystem mitochondrial disorders – the “phenocopies”. I will then review the rapidly increasing number of genetic disorders with secondary mitochondrial biochemical dysfunction reported since the widescale adoption of exome and genome sequencing, using some examples from the Genomics England 100,000 genomes study.

1. Rahman J, Rahman S. Mitochondrial medicine in the omics era. Lancet. 2018 Jun23;391(10139):2560-2574.

2. Parikh S, Karaa A, Goldstein A, Bertini ES, Chinnery PF, Christodoulou J, Cohen BH, DavisRL, Falk MJ, Fratter C, Horvath R, Koenig MK, Mancuso M, McCormack S, McCormick EM,McFarland R, Nesbitt V, Schiff M, Steele H, Stockler S, Sue C, Tarnopolsky M, Thorburn DR,Vockley J, Rahman S. Diagnosis of 'possible' mitochondrial disease: an existential crisis. J MedGenet. 2019 Mar;56(3):123-130.

Morning Session Leigh Syndrome – Mitochondrial Medicine Society (MMS)

Session

Pluripotent Stem Cell Models for Study of Leigh's Syndrome

Shilpa Iyer, PhD

Presenter: Shilpa Iyer, PhD

Institution: 1Dept of Biological Sciences, Fulbright College of Arts and Sciences, University

of Arkansas, Fayetteville, Arkansas, USA. Email: [email protected]

Title: Pluripotent Stem Cell Models for Study of Leigh's Syndrome

Background: Leigh’s Syndrome (LS), a classic mitochondrial (mt) disease has no current cure

and no adequate cellular model for understanding the rapid fatality associated with the disease.

Fatality results from excess accumulation of mutant mtDNA leading to failure of mt-

bioenergetics. Other symptoms include developmental, neural, cardiac and muscle

impairments. To generate patient and disease specific cell models and to assess the mt-

functional changes associated with LS syndrome, five patient-derived fibroblast cell lines

carrying point mutations in the Complex I (ND3- T10158C; ND5-T12706C) and Complex V

subunits (ATP6- T8993G; T9185C) were used in this study.

Results: Prior to reprogramming, we characterized the patient fibroblast cells to obtain a

metabolic ‘disease signature’ phenotype. A two-photon excited fluorescence microscopy

approach relying entirely on endogenous fluorophores, was used to dynamically quantify

functional metabolic readouts from individual cells. A ratio of FAD/(NADH+FAD) has been

shown to be sensitive to the relative proportion of oxidative phosphorylation to glucose

catabolism. We observed a higher optical redox ratio in cells with Complex V mutations

compared to normal fibroblast cells. This increase was likely due to increased ETC activity or

possible proton leak associated with defective ATP synthase (CV). Using traditional assays,

ETC enzyme activities was measured spectrophotometrically as specific donor–acceptor

oxidoreductase activities in 0.1 M phosphate buffer. We observed an increase in NADH

ferricyanide reductase (NFR) activity in Complex V mutants compared to control fibroblast cells.

NFR activity measures NADH dehydrogenase (Complex I rotenone-insensitive NADH-

reductase). Using the Seahorse extracellular flux analyzer, we conducted extensive analysis to

assess oxidative phosphorylation to glucose catabolism rates in the patient fibroblast and

control cells. Overall, our results indicate that basal oxygen consumption rates and

mitochondrial ATP production was compromised in Leigh’s syndrome cell lines exhibiting

Complex I and Complex V mutations, while real time glycolysis rates was elevated, because the

cells had adapted to a compromised metabolic pathway for their survival. Having established

the metabolic ‘disease signature’ phenotype, we focused our attention to reprogramming these

cells to better understand the developmental impairments seen in Leigh’s syndrome patients.

Using a combined non-viral non-integrating mRNA-miRNA reprogramming strategy, all five

patient fibroblasts were reprogrammed to generate human induced pluripotent stem cells

(hiPSCs). All hiPSCs exhibited hallmarks of a pluripotent cell, based on morphological and

immunocytochemical (hiPSC marker POU5F1, SOX2, TRA-1-60 and SSEA4) analyses. Our

ongoing studies are focused on evaluating mutation burden in fibroblasts and hiPSCs, followed

by comprehensive bioenergetics analyses in the differentiated neurons and muscle cells from

hiPSCs. Results from these studies will now allow us to better understand the pathophysiology

of Leigh’s disease and aid in developing patient specific therapeutic strategies for improving

function in affected cell types.


Session

Leigh Syndrome Registry Data

Mary Kay Koenig, MD

Presenter: Mary Kay Koenig, MD

Institution: University of Texas McGovern Medical School, Houston, Texas

Title: Leigh Syndrome Registry

The International Registry for Leigh Syndrome (TRiaLS) is a patient registry designed to collect information about individuals with Leigh syndrome. The purpose of TRiaLS is two-fold and thus consists of independent two phases. Phase I is designed to ask very basic questions about health history to help determine whether someone is potentially eligible to join a research study. This phase will connect participants with researchers interested in learning more about Leigh syndrome or those conducting clinical trials. Phase I can be completed in less than 15 minutes. Phase II of TRiaLS is more involved. It is designed to provide researchers with detailed medical information about the participants. This phase will help to increase our understanding of Leigh syndrome and can be used to help define the natural history of this condition. Better understanding of the natural history of Leigh syndrome will improve prognostication and treatment as well as assist with clinical trial design. Current status of TRiaLS recruitment will be reviewed.


Session

Leigh Syndrome – the US experience: Data from the NAMDC Registry

Austin Larson, MD

Presenter: Austin Larson1

Authors: Yuelin Long2, John L P Thompson2, Emanuele Barca3, Emily Shelkowitz1, Johan Van Hove1, Amy Goldstein4, Sumit Parikh5, Amel Karaa6, Russ Saneto7, Michio Hirano3

Institutions: 1Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO; 2 Department of Biostatistics, Mailman School of Public Health, Columbia University Medical Center, New York City, NY; 3Department of Neurology, Columbia University Medical Center, New York City, NY; 4Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 5Neurogenetics, Center for Pediatric Neurology, Cleveland Clinic Children's Hospital, Cleveland, OH; 6Department of Genetics, Massachusetts General Hospital, Boston, MA; 7Department of Neurology, University of Washington, Seattle, WA

Title: Leigh Syndrome – the US experience: Data from the NAMDC Registry The North American Mitochondrial Disease Consortium (NAMDC) is a 20 site research network funded by the National Institutes of Health. NAMDC maintains a database that contains extensive clinical data on over 1,500 individuals with primary mitochondrial disease. As of June, 172 patients in the database have a diagnosis of Leigh Syndrome. Of those patients, 33% lack a genetic diagnosis, 16% have a mutation in MT-ATP6, 16% have a mutation in an mtDNA-encoded complex I subunit, 9% have a mutation in a pyruvate dehydrogenase complex gene, 9% have a mutation in SURF1, 6% have a mutation in an nDNA-encoded complex I subunit, 6% have a mutation in an nDNA gene with impact on mitochondrial translation or DNA maintenance, 5% have mutations in other nDNA genes and one individual has a mutation in an mtDNA-encoded tRNA. The most common neurological symptoms for these patients were hypotonia, spasticity and extrapyramidal movement disorders. 56% had developmental regression and 41% had seizures. The most common non-neurological symptoms were growth concerns and gastrointestinal dysmotility. 21% had ophthalmological concerns and 12% had hearing loss. 8% had arrhythmias and 6% had cardiomyopathy. A single patient each had hepatopathy and adrenal insufficiency. No patients had diabetes. The presence of MRI abnormalities of the brainstem had 37% positive predictive value for respiratory insufficiency. The absence of brainstem lesions had 90% negative predictive value for absence of respiratory insufficiency. Future directions for analysis of individuals with Leigh Syndrome in the NAMDC database will include broad molecular genetic testing for those without a clear genetic diagnosis. As the database matures, survival analysis will allow for better data on prognosis of specific subtypes of Leigh Syndrome. Better defining the natural history of Leigh Syndrome will facilitate conduction of clinical trials. Genotype-phenotype correlations will allow for more tailored and evidence-based care of individuals with Leigh Syndrome.


Session

Leigh Syndrome – the United Kingdom (UK) Experience

Shamima Rahman, FRCP PhD

Presenter: Shamima Rahman, FRCP, PhD

Authors: Professor Shamima Rahman and Dr. Helen MacGloin

Institution: Great Ormond Street Hospital and UCL Great Ormond Street Institute of Child Health, London, UK

Title: Leigh Syndrome – the Great Ormond Street Hospital Experience

Background - Leigh syndrome, or subacute necrotising encephalomyelopathy, is the most frequent and most severe clinical presentation of mitochondrial disease in childhood. The condition is genetically heterogeneous, with more than 90 known causative genes, and currently there are no disease-modifying therapies for the vast majority of cases. The prevalence, genetic basis and natural history remain incompletely understood, yet these data are urgently needed to inform the development of novel therapies and to design rational clinical trials using objective outcome measures.

Methods - We performed a retrospective study of children with Leigh syndrome diagnosed in a single UK centre, Great Ormond Street Hospital for Children in London. Leigh syndrome was originally defined neuropathologically, but as brain pathology was not available for the majority of our cases, we used Leigh syndrome spectrum diagnostic criteria recently devised by the ClinGen Gene Curation consortium to select cases. To fulfil these criteria, affected individuals present neurodevelopmental delay and/or regression, with typical neurological and neuroimaging findings and biochemical evidence of mitochondrial dysfunction (elevated lactates in blood and/or cerebrospinal fluid, and/or oxidative phosphorylation (OXPHOS) enzyme deficiencies in a tissue biopsy).

Results - One hundred and thirty-eight affected infants and children were identified (79 males = 57%), presenting between 1982 and 2019. Patients presented from the first day of life to 10 years of age, but more than 70% had presented by the age of 3 years. Thirty percent of cases were from consanguineous families. A definite genetic diagnosis was obtained for 103 cases, and a candidate gene was identified in a research study in another 10 cases, i.e. there was a likely genetic diagnosis in 82% of cases. Seventeen percent had a mtDNA mutation, most commonly in a subunit of complex I or complex V. Nuclear gene mutations included defects of OXPHOS subunits and assembly factors, mitochondrial DNA maintenance, mitochondrial translation, cofactor biosynthesis, the pyruvate dehydrogenase complex and thiamine transport.

Discussion - The extreme genetic heterogeneity observed in our cohort is in keeping with findings from smaller studies in other centres, and emphasises the challenges of collecting sufficiently large cohorts to perform natural history studies and clinical trials in ultra-rare diseases; international multi-centre collaboration is needed. However, the advantages of a single centre study are the consistency of investigations and management. We hope that more detailed studies of this cohort will help to identify biomarkers and outcome measures for Leigh syndrome, and to inform future prospective natural history studies and clinical trials.


Session

Leigh Syndrome – the Australian Experience

David Thorburn, PhD

Presenter: David R. Thorburn, PhD

Authors: Nicole J. Lake1,2,3, Sarah E. Calvo4,5,6, John Christodoulou1,2,7, Vamsi K. Mootha4,5,6, Alison G. Compton1,2,*, David R. Thorburn1,2,7,*

Institution: 1Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia; 2Department of Paediatrics, University of Melbourne, Melbourne, Australia; 3Department of Genetics, Yale School of Medicine, New Haven, USA; 4Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, USA; 5Department of Systems Biology, Harvard Medical School, Boston, USA; 6Broad Institute of Harvard and MIT, Cambridge, USA; 7Victorian Clinical Genetic Services, Royal Children’s Hospital, Melbourne, Australia; *These authors contributed equally

Title: Leigh Syndrome – the Australian Experience

Leigh syndrome (LS) is an infantile-onset neurodegenerative disorder characterised by bilateral symmetrical lesions within the brainstem and basal ganglia, often supported by the finding of elevated plasma or CSF lactate levels. LS is clinically heterogeneous, with variation in age of onset and survival. Onset typically occurs by 2 years of age, with symptoms often presenting during infection or illness after an initial period of normal development. Developmental regression is often episodic and can be accompanied by other neurological symptoms (e.g., ataxia, dystonia, nystagmus, optic atrophy) or cardiac, renal, hepatic or other abnormalities. LS is the most common paediatric presentation of mitochondrial disease, with an estimated birth prevalence of at least 1 in 40,000. Remarkably, this relatively specific phenotype can be caused by mutations in over 80 genes impacting on oxidative phosphorylation (OXPHOS) or affecting pyruvate dehydrogenase and related processes. The causative genes are encoded by both mitochondrial (mt) and nuclear DNA.

Clinical suspicion, imaging and raised lactate levels are the most common prompts for diagnostic investigation of LS. Traditionally, this usually started with muscle and skin biopsies aiming to identify a biochemical diagnosis that could prompt testing of specific candidate genes. No single gene is responsible for more than ~15% of Leigh syndrome so single gene testing has a low diagnostic yield. In order to avoid or minimise invasive biopsies, many centres now offer whole exome sequencing plus mtDNA sequencing while some offer whole genome sequencing sequencing as front line diagnostic methods.

We have acted as the major Australasian referral laboratory for children suspected of mitochondrial disorders for nearly 30 years. We have studied samples from over 200 children suspected of LS and in 1996 suggested criteria to distinguish patients with a stringent diagnosis of LS from patients with “Leigh-like” disease. At that time we performed biochemical and genetic analysis of a cohort of 67 Australasian patients with LS or Leigh-like disease, representing all the known patients at that time with available samples (Rahman et al., 1996, Ann.Neurol. 39:343-51). Recently, we have attempted to determine the potential diagnostic yield of whole exome plus mtDNA sequencing by re-evaluating this clinically well characterised cohort of 67 patients. We received a waiver of consent to study DNA from the 33 patients still lacking a genetic diagnosis by whole exome sequencing supplemented with mtDNA baits. Analysis was

restricted to ~2000 genes encoding known mitochondrial proteins and genetic disorders with overlapping phenotypes.

Careful analysis of sequencing data, supplemented with identification of copy number variants and functional validation, enabled genetic diagnosis where routine filtering yielded no candidates. We have now established the genetic basis in 78% of the total cohort of 67 patients, including 34 of 35 LS patients and 18 of 32 Leigh-like patients. Pathogenic mutations were identified in 18 different genes, with the mode of inheritance being 34% mtDNA, 34% autosomal recessive, 9% X-linked and 22% unknown. Possible genetic diagnoses are under evaluation in 9 of the 15 unsolved patients.

Issues that complicated diagnosis included novel missense variants, synonymous or cryptic splicing defects, copy number variants, finding homozygous protein-truncating variants in two genes associated with LS in a single patient, needing to characterise pathogenicity of variants in novel disease genes and the need for a broader (Mendeliome) gene list in Leigh-like patients. Additionally, two families (only one consanguineous) had two monogenic disorders segregating causing LS in one or more individuals and Leigh-like disease in others.

Based on these data and our experience with the larger cohort we conclude that genomic analysis can potentially identify the genetic basis in over 80% of patients with tightly defined LS and over 50% of Leigh-like patients. It is worth noting that four patients in our cohort had mutations in the SLC19A3 gene, which is a potentially treatable disorder if diagnosed early in the disease course.

A final aspect of the Australian experience that warrants comment is in relation to reproductive options for mtDNA mutations. In about a quarter of families with an mtDNA mutation causing LS or other childhood disorders, the mother has undetectable or a very low amount of the mutation in urine or other appropriate samples. These families can be highly suitable for prenatal diagnosis or pre-implantation genetic diagnosis. This is emphasised by our findings in 22 pregnancies from 15 women expected to have low recurrence risk based on knowledge of the mutation and maternal mutant load (<10% heteroplasmy in urine or other appropriate samples). In each case, only wild-type mtDNA was detected in CVS. The presenter believes that in Australia and internationally our professional community is failing many families at low, but not zero, recurrence risk for mtDNA disease by not making them aware that existing reproductive options could be appropriate for them.

Cytosine methylation in mammalian mitochondrial DNA (mtDNA) is one of the hottest topics in mitochondrial biology and medicine in recent years. It could have profound impact on gene expression, replication and maintenance of mtDNA, and abnormalities in mtDNA methylation might well be a cause of disease with mitochondrial dysfunction. Elucidation of whether mtDNA is methylated or not is thus of pivotal importance. Methylation of mtDNA has been argued for by several recent papers, but after careful reading of them it may be difficult to draw a clear picture of the methylation partly because of conflicting data presented among them. Here, we set out on investigation with a hope of resolving the confusing situation. This study was conducted through collaboration of three research groups who are experts on mtDNA, DNA methylation and mass spectrometry. We carefully prepared and investigated mouse mtDNA using three methods that are based on different principles for detecting 5-methylcytosine; next generation bisulfite-sequencing, a cleavage assay with a methylation-dependent restriction enzyme and mass spectrometric nucleoside analysis. Data from these analyses led us to propose that the 5-

methylcytosine is not present at any specific position(s) in mtDNA and levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in the gene expression or metabolism of mtDNA.


Session

The UMDF Leigh Syndrome Roadmap Project

Bruce Cohen, MD

Presenter: Bruce H. Cohen, MD

Institution: Akron Children’s Hospital, Akron, OH

Title: The UMDF Leigh Syndrome Roadmap Project We are about two decades into the formation of patient advocacy groups that represented the interests of those persons, and their families, touched by mitochondrial disease. As a truly international collaborative, four of the largest patient advocacy groups, The UMDF, Mito Foundation, Mitocon and the Lily Foundation have brought the Leigh Syndrome Roadmap Project (LSRP) group together with the mission focusing on Leigh syndrome. A common independent goal of each PAG has been to build the foundation to support research. As a small organization, with small resources, the initial focus for the UMDF, as an example, was to fund initially one project per year. As a result, the funding went to the single best project. The board of trustees made the decision in the late 1990s to not raise funds for any one mitochondrial syndrome or disease. This allowed the grant funding committee to decide, free of restrictions, as to which project, and then projects, to fund. Over the years, there have been relatively few direct clinical projects that have been funded, in part because the need to fund basic research, and in part because the time was not right and the funds necessary were not available. Twenty years have passed. Each of these organizations has humble beginnings but have formed bonds with their constituents, most importantly the children and adults with mitochondrial diseases. However, each of these organizations also has strong relationships with scientists and clinicians, as well as more recently with the pharmaceutical industry. We have sponsors that support our PAGs, and these PAGs are now very active in both the legislative and regulatory bodies in their respective countries and unions. Over the last twenty years, our collective work on Leigh syndrome has focused on diagnosis, and to a lesser extent, treatment. As clinical trials organized to focus on the treatment of Leigh syndrome it became obvious that there was insufficient solid data in terms of natural history or in terms of disease severity evaluation. This lack of data was a reflection of factors beyond control of investigators. For example, the advent of the genomic evaluation revolution started less than ten years ago. In addition, there were no standards of supportive care, and some clinicians recommend different levels of aggressive care, such as respiratory care, that affect both quality of life and duration of survival. By addressing the broad topics of natural history, outcome measures and preclinical research this group can develop the base for projects to deliver the basis for all future clinical trials. The LSRP will be about achieving results. The members of this project represent the larger group of believers that have a dream that we can take the evaluation and treatment of those with Leigh syndrome quickly to a level beyond what would happen without this effort. In this regard, our community is looking towards our leadership. Funding is initially guaranteed by the PAGs, but with our vision, we hope the Leigh Syndrome Roadmap Project will attract outside funding from competitive grants, investments or philanthropy into the PAGs. The past work will be discussed today, so that we can build the future of treatment for those with Leigh syndrome. The future is ours to define. Our community will make this happen.

Afternoon Session Federal Updates on Research and Clinical Trials for

Mitochondrial Disease

FDA and Rare Disease Drug Approvals

Dragos Roman, MD

Presenter: Dragos Roman, MD

Authors: Dragos Roman, MD, Division of Gastroenterology and Inborn Errors Products

Institution: Food and Drug Administration, Silver Spring, MD, USA*

Title: Facilitating Drug Development in Patients with Mitochondrial Diseases

Rare diseases are defined by the Orphan Drug Act as having a prevalence less than 200,000 in the US. There are over 7000 rare diseases identified to date and advances in molecular diagnosis and genetic research continue to expand the number of phenotypes for which gene specific defects are identified. Overall, up to 30 million patients have rare diseases in the US and there is increasing interest in developing drugs and biologics to treat rare diseases. Drug development in rare diseases faces specific challenges related to the rarity of the condition, the heterogeneity of disease manifestations, the absence of disease-specific endpoints, poorly defined natural histories, a complex genotype-phenotype relationship in a given disease, and geographic dispersion of patients, among others Among rare disease, mitochondrial diseases pose additional unique challenges to drug development because of specific features of mitochondrial biology such as cell- or tissue-level heteroplasmy and the “threshold effect” that needs to be reached before organ-specific disease manifestations become evident. There are 1700 proteins required for mitochondrial function with only 13 encoded by DNA. Currently, drug development in mitochondrial diseases is limited to only a handful ofindications. This presentation will describe some of the regulatory pathways, available programs and recent initiatives from the FDA to facilitate drug development in mitochondrial diseases. * The views expressed in this presentation are those of the speaker, and do not necessarily represent an official FDA position.

Afternoon Abstracts

2:30pm PA-0544 Charles McCall Targeting the PDC/PDK Axis for Inflammatory Shock Syndromes in Inborn Errors of Metabolism

2:45pm PA-0580 Robert K. Naviaux Elevated 1-deoxyceramides in patients with MELAS and NARP—testing a new biomarker of mitochondrial dysfunction

3:00pm PA-0630 Amel Karaa Fatigue, dizziness and nausea: Doc I think I have Mito?

Afternoon Lightning Rounds 2019 PA-0606 Zarazuela

Zolkipli-Cunningham

Quantitative Assessment of Mitochondrial Myopathy

2019 PA-0612 Wolf-Hagen Schunck

Activation of mitochondrial quality control by epoxyeicosanoid-like drugs

2019 PA-0613 Senta Kapnick Understanding the impact of mtDNA copy number regulation on T lymphocyte metabolism and function

2019 PA-0562 Lissa Poincenot Leber’s Hereditary Optic Neuropathy -- Not Just a Young Man's Disease

2019 PA-0649 Andre Mattman A multi-faceted lifestyle intervention for mitochondrial A8344G associated multiple symmetric lipomatosis (MSL): Report of a successful patient initiated novel therapy

2019 PA-0627

Chynna Broxton THERAPEUTIC RESCUE OF DLD-BASED PYRUVATE DEHYDROGENASE DEFICIENCY IN WORM AND ZEBRAFISH ANIMAL MODELS

2019 PA-0635 Olivia Kolenc Characterizing metabolic changes in Leigh’s Syndrome using label-free multiphoton microscopy

2019 PA-0642 Akira Ohtake 5-Aminolevulinic acid and Fe can bring a permanent cure for mitochondrial diseases: Basic experiments and an investigator initiated clinical trial

2019 PA-0644 Min Peng N-Linked Glycosylation of MRS2 Inhibits Mitochondrial Magnesium Import

2019 PA-0647 Bruce Cohen Induced Pluripotent Stem Cells as Models for Studying Mitochondrial Disease



The National Center for Advancing Translational Sciences: (NCATS) Updates on Research

Opportunities and Clinical Trials for Mitochondrial Disease

Anne Pariser, MD

Presenter: Anne Pariser, MD

Institution: Office of Rare Diseases Research, National Center for Advancing Translational Sciences, NIH

Title: The National Center for Advancing Translational Sciences: Updates on Research

Opportunities and Clinical Trials for Mitochondrial Disease

The National Center for Advancing Translational Sciences (NCATS), one of the 27 institutes and centers at the National Institutes of Health (NIH), was established to transform the translational science process so that new treatments and cures for disease can be delivered to patients faster. The Office of Rare Diseases Research (ORDR) within NCATS is dedicated to accelerating the development of treatments to benefit patients with rare diseases. NCATS and ORDR have a number of programs specifically intended to improve the research environment for rare diseases so that no disease and no patient will be left behind, regardless of the number of patients living with a rare disorder. One of ORDR’s programs, the Rare Diseases Clinical Research Network (RDCRN), includes the North American Mitochondrial Disease Consortium (NAMDAC). The RDCRN currently includes 21 different centers of excellence, each of which studies 3 or more related disorders, and which collectively includes more than 200 different rare diseases. The RDCRN provides collaborative awards to further multi-disciplinary rare diseases research, conduct natural history studies and clinical trials, and provide training for young investigators in rare disorders. To date, more than 40,000 patients have participated in clinical studies through the RDCRN, as well as participating in the network through patient advocacy groups affiliated with the research centers. Some of ORDR’s other programs and initiatives include the Genetics and Rare Diseases (GARD) information center, grants for scientific conferences and clinical trial readiness, and “platform” or collective approaches to advancing gene therapies for rare diseases. ORDR has also focused on helping patient groups advance their research agendas through the establishment of patient registries (RaDaR program) and through access to other tools (through the NCATS Toolkit for Patient-focused Therapeutics Development). NCATS additionally supports rare diseases research through programs such as microphysiologic systems, also known as “tissue chips”, and the Therapeutics for Rare and Neglected Diseases (TRND) program that focuses on solving difficult areas of translational research for rare diseases in order to advance the field of rare diseases research. In this presentation, learn about these and other NCATS programs, and how leveraging opportunities available through NCATS can help to advance mitochondrial disorders as well as other related rare diseases.



National Institutes of Health (NIH) Funding for Mitochondrial Disease

Danuta Krotoski, PhD

Presenter: Danuta Krotoski, PhD

Authors: Danuta Krotoski

Institution: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD

Title: National Institutes of Health (NIH) Funding for Mitochondrial Disease Research

The National Institutes of Health (NIH) is the primary Federal agency for conducting and

supporting medical research. NIH funds research through 27 topic specific Institutes and

Centers (ICs) most of which support research to better understand mitochondrial function and

disease. Because of the interest in mitochondrial disorders across the NIH, a Trans-NIH

Mitochondrial Disorders Working Group was established and meets regularly. This presentation

will describe activities of the Working Group, new funding opportunities and recently published

policies and procedures.



DOD Funding for Mitochondrial Diseases

Kathryn Argue, PhD


Friday, June 28, 2019

Morning Session State of Mitochondrial Medicine and The Big Pitch: UMDF

accelerators Research Grant Prize Finals

Welcome: UMDF and the Mitochondrial Medicine Community (Non CME)

The Big Pitch: UMDF accelerators Research

Grant Prize Finals – The accelerators Prize was established in 2019 with an aim to fund promising research from postdoctoral researchers. Come hear our finalists give their ‘fast’

pitch to be awarded the first annual accelerators Research Prize. (Non CME)

Brian Harman and Philip Yeske, PhD

Maybe put the Big Pitch Teaser in here? On Friday morning, our accelerators Prize Finalists (the top 3 Postdoctoral Fellows) will present a 5-minute pitch to a special group of donors known as accelerators: a special group of donors who qualify based upon their level of philanthropic giving to the Foundation. Live Friday and via webcast, accelerators will vote for their favorite proposal. The proposal receiving the most votes will receive the $50,000 accelerators prize.

The winner will be announced live at An Evening of Energy banquet on Friday night.

Rachel GuerraMorgridge Institute for Research

Madison, WI

Accelerator Project:Structural and Functional Characterization of COQ9 in Facilitating Coenzyme Q Biosynthesis and Complex Q Formation

Zachary WilsonUniversity of Utah

Salt Lake City, UT

Accelerator Project:Manipulating Mitochondrial Metabolism Via the Mitochondrial Derived Compartment Pathway

Arwen Gao Ecole Polytechnique Federale de Lausanne (EPFL)

Lausanne, Switzerland

Accelerator Project:Identification of Novel Compounds to Treat Rare Mitochondrial Diseases

2019 Finalists

Become an acceleratorWhen you personally donate $500 or more (cumulatively in a year per household) you unlock your accelerators benefits! No matter how you give – through a special event, to a designated fund or as a tribute to a patient – when you reach the accelerators level you join a group of engaged philanthropists.

There’s still time to become an accelerator and vote for your favorite proposal. See Beth Whitehouse, Director of Development at the UMDF Registration Booth to make your donation today! Donations accepted until 4:00pm EDT on Friday, June 28.

How it works

Morning Session State of Mitochondrial Medicine

Externally-led Patient-Focused Drug Development and Science & Alliance Updates

(Non CME)

Philip Yeske, PhD

Morning Session State of Mitochondrial Medicine –

Medical Community Updates

North American Mitochondrial Disease Consortium (NAMDC) Updates (Non CME)

Speaker Name: Michio Hirano, MD

Mitochondrial Care Network Update (Non CME) Speaker Name: Sumit Parikh, MD

MSeqDR (Non CME)

Speaker Name: Marni Falk, MD

Presenter: Michio Hirano, MD

Authors: Michio Hirano, MD1; Xiomara Q Rosales, MD1; Salvatore DiMauro, MD1; Bruce H Cohen, MD2; Amel Karaa, MD3; Georgirene D. Vladutiu, PhD4; Richard Haas, MB, BChir5; Johan LK Van Hove, MD, PhD6; Fernando Scaglia, MD7,8,9; Sumit Parikh, MD10; Jirair K Bedoyan MD, PhD11 ; Susanne D DeBrosse, MD11; Ralitza H. Gavrilova, MD12; Russell P Saneto, DO, PhD13; Gregory M. Enns MB, ChB14; Peter W Stacpoole, MD, PhD15; Jaya Ganesh, MD16; Austin Larson, MD6; Zarazuela Zolkipli-Cunningham MD17,18; Marni J Falk, MD17; Amy C Goldstein, MD17; Mark Tarnopolsky, MD, PhD19; Andrea Gropman,M.D.20; Isabella Barcelos, MD1; Emanuele Barca, MD, PhD1; Valentina Emmanuele, MD, PhD1; Kristin Engelstad, MS1; Joshua Kriger, BA21; Johnston Grier21; Richard Buchsbaum21; John LP Thompson, PhD21

Institution:

1Department of Neurology, Columbia University Medical Center, New York, NY, USA 2Department of Pediatrics, Northeast Ohio Medical University and Akron Children’s Hospital, Akron, OH, USA 3Genetics Unit, Massachusetts General Hospital, Boston, MA, USA 4Department of Pediatrics, State University of New York at Buffalo, Buffalo, NY, USA 5Departments of Neurosciences and Pediatrics, University of California at San Diego, San Diego, CA, USA 6Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA 7Department of Molecular and Human genetics, Baylor College of Medicine, Houston, TX, USA 8Texas Children`s Hospital, Houston, TX, USA 9Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, New Territories, Hong Kong SAR 10Department of Neurology, Cleveland Clinic, Cleveland, OH, USA 11Departments of Genetics and Genome Sciences and Pediatrics, and Center for Human Genetics, University Hospitals Cleveland Medical Center, Case Western Reserve University, Cleveland, OH, USA 12Departments of Neurology and Clinical Genomics, Mayo Clinic, Rochester, MN, USA 13Department of Neurology, University of Washington, Seattle Children’s Hospital, Seattle, WA, USA 14Department of Pediatrics, Stanford University, Palo Alto, CA, USA 15Department of Medicine, University of Florida at Gainesville, Gainesville, FL, USA 16Genetics and Genomic Sciences at the Icahn School of Medicine at Mount Sinai, New York, NY, USA 17Mitochondrial Medicine Frontier Program, Division of Human Genetics, The Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 18University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 19Department of Neurology, McMasters University, Toronto, ON, Canada 20Department of Neurology, Children’s National Health Network, Washington, D.C., USA 21Department of Biostatistics, Mailman School of Public Health, Columbia University, New York, NY, USA

Title: North American Mitochondrial Disease Consortium A member of the Rare Diseases Clinical Research Network (RDCRN), the North American Mitochondrial Disease Consortium (NAMDC) has established a network of 17 clinical centers in partnership with the United Mitochondrial Disease Foundation (UMDF) with a mission to improve the diagnosis and care, establish the natural history, and support translational research in and investigate treatment of mitochondrial diseases. With support of an NIH American Recovery and Reinvestment Act (ARRA) grant and 7-plus years of a U54 award, NAMDC has already generated a substantial research infrastructure: a powerful Clinical Registry with over 1514 enrolled subjects, a Biorepository with specimen from 304 patients, and a website for education and recruitment of patients, which provide an essential foundation for clinical projects and trials. From this firm base, 19 productive patient-oriented projects have already sprouted: 4 natural history studies (Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE), Pyruvate Dehydrogenase Complex (PDC) deficiency, Alpers syndrome, and Pearson syndrome); 6 surveys; and 9 pilot studies. In addition, a highly successful NAMDC fellowship program has trained six clinician-investigators, all currently active in advanced mitochondrial disease clinical research. A competitive renewal application proposes 5 projects: 1) An expansion of the NAMDC Clinical Registry/Longitudinal Study and Biorepository with detailed analyses of at least six mitochondrial disorders and novel registry-based natural history studies of at least 3 diseases; 2) Application of next-generation sequencing to identify causative mutations in the ~40% of NAMDC Registry subjects whose diseases are genetically undefined in collaboration with the Mitochondrial disease SEQuence Data Resource (MSeqDR); 3) Development and utilization of new mitochondrial functional assays in tissues collected via minimally invasive techniques; 4) Extension of an advanced genetics study of PDC deficiency with the addition of an innovative biomarker newborn screening program and 5) A longitudinal expanded access study of deoxynucleoside therapy for thymidine kinase 2 (TK2) deficiency. In addition, we propose to transform our NAMDC Fellowship into a Career Enhancement program; to initiate new pilot studies; and to transition NAMDC NIH financial support to a broader array of funding sources.

Abstract #: 2019 PA-600

Presenter: Lishuang Shen

Authors: Lishuang Shen1, Elizabeth M. McComick2, Daria Merkurjev1, Colleen C. Muraresku2, Zarazuela Zolkipli-Cunningham2, Marni J. Falk2,3, Xiaowu Gai1,4

Institutions: 1Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 3Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; 4Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Title: MSeqDR updates: A genomic and bioinformatics data hub for the mitochondrial disease community

Background: MSeqDR (https://mseqdr.org) is a centralized genomic bioinformatics Web resource, supported by the United Mitochondrial Disease Foundation, to facilitate research-based diagnoses, genetic analyses, and variant interpretation for researchers and clinicians working in mitochondrial disease. Since its initial release in 2014 and publications in 2016, MSeqDR has been continuously enhanced with additional genomic and phenotypic content, as well as powerful new bioinformatics tools. MSeqDR has now become a major data hub for the mitochondrial disease community, including MitoMap, HmtDB, LeighMap, ClinVar, ClinGen, PhenoTips, and GENOMIT resources, through data sharing, cross-referencing, and active collaboration.

MSeqDR Content Update: The genomic and phenotypic content within MSeqDR has been enhanced by (a) Assembling a large reference dataset for assessing mitochondrial DNA (mtDNA) allele population frequencies. Indeed, MSeqDR generated a novel meta-population mtDNA allele frequency data resource, by compiling close to 90K mtDNA genomes with data from MitoMap, GeneDx, previous publications and other large sequencing initiatives, with substantial amount of data from previously underrepresented populations, such as Chinese and Japanese; (b) Further populating the content of MSeqDR-LSDB, a curated database of pathogenic variants for mitochondrial diseases, genes, and variants by data mining and systematic curation of public genomic resources and literature. MSeqDR-LSDB currents hosts over 12,000 variants from 1607 genes that are potentially associated the mitochondrial diseases, including 265 known mitochondrial disease genes. Among which, the pathogenicity has been assessed for over 8000 variants. Nuclear and mtDNA gene variant deposition by users is also supported in automated fashion with MSeqDR assignment to each variant of a unique identifier, the MSCV accession number, that is recognized as a data repository by ClinVar and journals for data deposition at the time of new variant publication, and provides easily populated data templates to support ClinVar submission; and (c) Compiling, mapping and curating phenotypic terms to capture features most relevant to mitochondrial diseases and harmonized across international registries based on standardized HPO and OMIM terminology ontologies.

MSeqDR Tools Update: We have developed a complementary suite of powerful bioinformatics tools for comprehensively annotating and analyzing mtDNA genome variants, namely MSeqDR-mvTool and Phy-Mer. We have also centralized access to a suite of other tools hosted in or linked to MSeqDR, such as MToolBox from HmtDB, MitoMaster from MitoMap, MitoTIP, and LeighMap. We are further developing a suite of tools, Quick-Mitome, for user-friendly, automated, and rapid Web-based analysis of exome datasets from mitochondrial disease patients. Efforts remain actively underway to allow patient-directed deposition and deidentified analysis of their previously generated exome and genome data from the Mitochondrial Disease Community Registry (MDCR) into MSeqDR for ready analysis in a custom MSeqDR-OpenCGA

annotation and variant analysis resource. MSeqDR also provides users with secure Collaboration Zones that support a range of initiatives, as well as custom tools to support ClinGen gene-disease and variant curation including for a NICHD, NIH U24-funded international initiative to expertly curate Leigh syndrome spectrum diseases in collaboration with the ClinGen consortium.

Conclusion: Overall, MSeqDR now provides extensive sets of unique genomic data, diverse bioinformatics tools that support mtDNA variant detection, annotation, and haplogroup determination, user-friendly exome data analysis, and mitochondrial disease HPO-based clinical feature analyses. In this way, MSeqDR has become recognized world-wide as the genomic and data collaborative hub supporting the complex needs of the mitochondrial disease diagnostic and research community.

Morning Abstracts

11:15am PA-0585 Peter Stacpoole The Changing Landscape of Clinical Trials for Primary Mitochondrial Diseases: 2011 to Present.

11:45am PA-0561 Claudia Pereira Gene replacement reverts myopathy in mouse model with mitochondrial complex I-deficient muscle

12:00pm PA-0517 Stephen Thomas Development of the ProTide Prodrug CERC-913 as a Potential Treatment for Deoxyguanosine Kinase Deficiency

12:15pm PA-0554 Jirair Bedoyan Rationale and feasibility of newborn screening for pyruvate dehydrogenase complex deficiencies

Morning Session Late Breaking Big Data

Patrick Chinnery, MBBS, PhD, FRCP, FMedSci

Presenter: Patrick F. Chinnery, MBBS, PhD, FRCP, FMedSci

Authors: Wei Wei1,2, Ernest Turro3,4,5, Patrick F Chinnery1,2,4 on behalf of the NIHR

BioResource - Rare Diseases+ and the 100,000 Genomes Project - Rare

Diseases Pilot.

Institution: 1Department of Clinical Neurosciences, School of Clinical Medicine, University of

Cambridge, Cambridge Biomedical Campus, Cambridge, UK. 2Medical Research

Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Cambridge,

UK. 3Department of Haematology, University of Cambridge, Cambridge

Biomedical Campus, Cambridge, UK. 4NIHR BioResource, Cambridge University

Hospitals NHS Foundation, Cambridge Biomedical Campus, Cambridge,

UK.5MRC Biostatistics Unit, Cambridge Institute of Public Health, University of

Cambridge, Cambridge, UK.

Title: The inheritance of mitochondrial DNA heteroplasmy is under nuclear genetic

control in humans

Body of Abstract: New mitochondrial DNA (mtDNA) mutations initially affect a small proportion

of the mtDNA molecules in a cell (heteroplasmy), well below the critical threshold required to

cause disease. However, a reduction in the amount of mtDNA per cell during female germ (the

mtDNA bottleneck) leads to the rapid segregation of heteroplasmy, and very different levels

between siblings, with high levels causing mitochondrial diseases. Although primarily governed

by random genetic drift, there is evidence of selection occurring during the inheritance of mtDNA

heteroplasmy in animal models, but it has been difficult to demonstrate this convincingly in

humans. This is important, because it will determine the prevalence of mtDNA disease and

potentially influence clinical recurrence risks within families transmitting mtDNA diseases.

To determine whether there was selection for or against heteroplasmic mtDNA during

transmission in humans we studied 12,975 whole genome sequences, including 1,526 mother-

offspring pairs where 45.1% of the individuals showed heteroplasmy affecting >1% of the

mtDNA molecules. We classified the heteroplasmies as ‘known’ (seen before in 30,506 human

mtDNA sampled from across the globe) or ‘novel’ (not see before). Harnessing genetic

information from both the mtDNA and nuclear genomes, we then determined whether the

nuclear genetic background influenced mtDNA heteroplasmy, and independently validated our

findings in a further 40,325 whole genome sequences.

Novel mtDNA variants were less likely to be inherited than previously seen variants, and the

level of heteroplasmy for known variants tended to increase on transmission. Despite the low

heteroplasmy levels, we saw selection for and against variants in different regions of the

mitochondrial genome. New population-specific heteroplasmies were far more likely to match

the nuclear genetic ancestry of an individual than the mitochondrial genome on which the

mutations occurred.

In conclusion, human mtDNA is shaped by selective forces acting on heteroplasmy within the

female germ line, and the signature of selection can be seen over one generation. The nuclear

genome influences mtDNA heteroplasmy in the population. This will ensure consistency

between these two independent genetic systems, with implications for the origins and treatment

of mitochondrial diseases.

Mitochondrial DNA Deletions in POLG Disease

William Copeland, PhD

Afternoon Session Harnessing the Big Data: Where re e Now?

Presenter: William C. Copeland, PhD

Authors: Scott A. Lujan1, Margaret H. Humble1, Christopher A. Lavender2, Matthey J. Longley1, Adam Burkholder2, Emma L. Blakely3,4, Charlotte L. Alston3,4, Grainne S. Gorman3,4 , Doug M. Turnbull3,4, Robert McFarland3,4, Robert W. Taylor3,4,Thomas A. Kunkel1 and William C. Copeland1

Institution: 1Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA 2 Integrative Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA 3 Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom 4 NHS Highly Specialised Mitochondrial Diagnostic Laboratory, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, NE1 4LP, United Kingdom

Title: Mitochondrial DNA Deletions in POLG Disease

The human mitochondrial genome (mtDNA) encodes 37 genes, including 13 essential subunits of the oxidative phosphorylation system, the indispensable primary metabolic pathway of cellular ATP production. Human cells can have thousands of 16.6 kb circular mtDNA copies, comprising a mix of wild-type and variant sequences (termed heteroplasmy). Focal mitochondrial respiratory deficiency is a pathological hallmark of both mitochondrial disease and aging with acquired mtDNA mutations implicated as symptoms and drivers of both processes. While previous studies suggest little role for substitution mutations in driving the aging process, the clonal expansion of large-scale mtDNA rearrangements are linked to aging, late-onset mitochondrial disorders of mtDNA maintenance and are relevant to competing hypotheses on the general mechanism of mtDNA replication. Here we develop a technique for ultrasensitive deletion detection in a population of circular mtDNA molecules. We use this technology to report deletion maps of skeletal muscle mtDNA from 19 unafflicted individuals between 17 and 93 years of age and from 22 mitochondrial disease patients aged 17 to 80. Mitochondrial disease patients harbor pathogenic variants in POLG, the nuclear gene that encodes the catalytic subunit of DNA Polymerase (Pol ), the mitochondrial replicative polymerase. Age- and disease-correlated patterns among 35 million deletions (~470,000 unique spans) implicate Pol in the formation of mtDNA deletions during both processes. Deletion patterns are consistent with the strand displacement model of mtDNA replication.


Mechanisms of Mitochondrial Dysfunction in Patient Cells and Stem Cell-Derived Models

David Thornburn, PhD

Presenter: David R. Thornburn, PhD

Authors: David R. Thorburn1,2,3, Cameron L. McKnight1,2, Yau Chung Low1,2, David A. Elliott1,2, Andrew G. Elefanty1,2, Richard J. Mills4, James E. Hudson4, Yilin Kang5, Thomas Jackson5, Diana Stojanovski5, Daniella H. Hock5, David A. Stroud5, Luke E. Formosa6, Michael T. Ryan6, Ann E. Frazier1,2

Institution: 1Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia; 2Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia; 3Victorian Clinical Genetic Services, Royal Children’s Hospital, Melbourne, VIC, Australia; 4QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia; 5Department of Biochemistry and Molecular Biology and the Bio21 Molecular Science and Biotechnology Institute, the University of Melbourne, Parkville, VIC, Australia; 6Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

Title: Mechanisms of Mitochondrial Dysfunction in Patient Cells and Stem Cell-Derived Models

Over the last decade, the application of massively parallel sequencing technologies has led to an explosion of our knowledge about the genetic basis of OXPHOS disease, with more than 300 different monogenic OXPHOS disorders now recognised. However, our understanding of the mechanisms linking genotype to phenotype lags well behind. We see many examples where one phenotype can be caused by mutations in many different genes and, conversely, where mutations in one gene can cause many different phenotypes. Human cellular model systems for understanding OXPHOS dysfunction are thus important for validating novel disease genes, for understanding basic biology and for preclinical studies to guide the most appropriate choice of known or novel compounds in clinical trials and in targeted treatment of the diverse range of mitochondrial disorders.

Mitochondrial dysfunction impacts on many intracellular pathways and organelles. Inadequate ATP synthesis is an obvious issue for cells with constant or intermittently high energy demands, particularly neurons and cardiomyocytes. Some organs show marked increases in energy demand during growth and development. Thresholds for ATP requirements and how they respond to changes in demand also vary between different cell types. These features undoubtedly contribute to differential susceptibility of organs to oxidative phosphorylation (OXPHOS) dysfunction, and to why conditions like Leigh syndrome can show substantial pre-symptomatic periods of good health in early life. In addition to ATP demand, OXPHOS defects impact directly on a range of other parameters that influence cellular function. These include abnormalities in mitochondrial membrane potential, metabolite and nucleotide pools, redox balance, production of reactive oxygen species plus mitochondrial quantity and dynamics. Downstream effects of such changes can be mediated by their impact on calcium handling and pathways related to redox signalling, nutrient sensing, mitochondrial biogenesis, other cellular stress responses, cell growth and cell death.

Patient cell lines such as skin fibroblasts have been valuable models in many studies of disease mechanisms and we have used patient skin fibroblasts in the identification of more than 20 novel disease genes. Such studies can show that the cells express a biochemical defect expected for a mutation in the gene of interest, enable better understanding of the role of the

encoded protein and demonstrate rescue of the abnormal biochemical phenotype by lentiviral expression of the wild-type protein. Knockout HEK293T or other human cell lines sometimes have advantages to patient cells, e.g., when fibroblasts do not show a clear biochemical defect, when one lacks the relevant patient cell line or when it is advantageous to have an isogenic control background. This was particularly helpful when we knocked out each of the 30 supernumerary subunits of Complex I and used quantitative proteomics to understand the role of each subunit in the modular assembly of Complex I. More recently, we have also used quantitative proteomics to study OXPHOS complexes, the mito-ribosome and other mitochondrial proteins in patient cell lines with variants of uncertain significance in known and novel disease genes.

An obvious limitation of skin fibroblast cell lines is that they do not represent the primary tissues affected in OXPHOS disorders and thus lack utility in understanding tissue-specific disease mechanisms and in preclinical drug trials. A number of drugs have shown therapeutic promise in pre-clinical studies, but often were studied in only one or two model systems. We have therefore attempted to generate a representative panel of cellular models of OXPHOS disease in human Embryonic Stem Cells (hESCs) as well as induced Pluripotent Stem Cells (iPSCs) from patients. The panel currently comprises 19 different genes, with selection criteria including covering a representative range of mechanisms, preferential selection of the most common defects in children and genes in which the basic mechanisms are unclear. For hESC studies we also prioritised genes where patients are reported to have biallelic Loss-of-Function mutations so that knockout hESC might be expected to be able to differentiate efficiently to relevant cell lineages.

Our initial focus has been on differentiation to cardiomyocytes, where efficient generation of beating cardiomyocytes takes only 2 to 3 weeks. For 2D cultures, cardiomyocytes represent ~70% of the cells. Whole cell label-free quantitative proteomic analyses of pure (FACS-sorted) SURF1-/- cardiomyocytes show a clear defect in assembly of Complex IV with compensatory proliferation of other OXPHOS proteins. Cardiomyocytes derived from hESC with knockouts of genes like SURF1 and AGK show marked abnormalities of calcium handling and in their electrophysiology studied with multi-electrode arrays. Cardiac organoids can be generated in 3D cultures where cells condense around two plastic posts that allow quantification of cardiac contractility parameters in a 96-well screening platform format. These analyses show much potential, albeit one needs to be wary about whether functional deficits measured in stem cell derived models represent deficits of the mature tissue or an impact on normal differentiation.


Integrated Cardiac Mitochondrial Phenome; Half-life of Mitochondrial Proteins, Emphasis on

Cardiac Biology

Peipei Ping, PhD

Presenter: Peipei Ping, PhD

Authors: Ding Wang2,8, Dominic Ng2,8, Howard Choi1,2,4,8, Anders Olav Garlid1,2,8, Edward Lau2,8, Vladimir Guevara-Gonzalez5,8, Wei Wang4,6,7,8, John R. Yates III8,9, and Peipei Ping1,2,3,4,7,8

Institution: 1Cardiovascular Data Science Training Program at UCLA, Depts. of 2Physiology, 3Medicine/Cardiology, 4Bioinformatics, 5Mathematics, and 6Computer Science; 7Scalable Analytics Institute (ScAi); 8HeartBD2K Center of Excellence at UCLA, University of California Los Angeles, CA 90095. 9Department of Molecular Medicine, The Scripps Research Institute (TSRI), La Jolla, CA 92037.

Title: Integrated Cardiac Mitochondrial Proteome and Dynamics, Relevance to Biology and Disease.

Introduction: The mammalian proteome is known to consist of many resident proteins (~1,500 reported) with heterogeneous representation across cell types and tissues.1 A major scientific interest of our team is to understand protein dynamics of the mitochondrial proteome. Our first effort entailed the application of classical 2D methods to measure protein half-life in situ,2 which did not represent protein dynamics in their native environment (e.g., in vivo) and was limited to characterization of just 100+ proteins. Methods: To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (D2O; 2H2O) labeling strategy3 customized to examine individual protein turnover in the mitochondria of both mouse4 and human5-6 in a systematic fashion. Our approach succeeded largely due to two innovative components that enhanced dataset quality and coverage: first, each protein was labeled homogeneously; second, our computational pipeline permits large-scale protein characterization and acquisition of large datasets. Moreover, D2O is inexpensive, lending to a cost-effective method. Briefly, subjects were fed D2O at a minimal level (<5% body water) without physiological impacts and mitochondrial protein dynamics were determined. Samples were prepared for mass spectrometry analysis; a novel multiparameter fitting approach computationally quantified the normalized peak areas of peptide mass isotopomers at initial and steady-state time points, permitting protein half-life determination without plateau-level 2H incorporation. Results and Conclusion: We characterized the turnover rates of 458 proteins in mouse cardiac and mouse hepatic mitochondria and found their median turnover rates of 0.0402 d 1 and 0.163 d 1, respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months ( 60 d). We also determined the proteome dynamics in human plasma; half-life of detected plasma proteins was diverse and spanned over 50-fold in turnover rates from hemoglobin (HBB; half-life: >50 days) to albumin (ALB1; half-life: 22 days) to insulin-like growth factor 2 (IGF2; half-life: <1 day). Many of these proteins serve as effective biomarkers of cardiovascular diseases. Furthermore, we have developed an integrated omics approach, incorporating measurements of transcript abundance, protein abundance, and protein turnover to map the landscape of proteome remodeling in mouse models of cardiac hypertrophy and in human heart failure. Using machine learning supported analytics, we extracted molecular signatures indicative of disease phenotypes; we reported that the integration of transcript abundance, protein abundance, and

protein turnover data leads to 75% gain in discovered disease gene candidates. Moreover, the inclusion of protein turnover measurements allows discovery of post-transcriptional regulations across diverse pathways and implicates distinct disease proteins not found in steady-state transcript and protein abundance data. Our results suggest that multi-omics investigations of proteome dynamics provide important insights into disease pathogenesis in vivo.

References: 1 E Lau, Q Cao, DCM Ng, BJ Bleakley, TU Dincer, BM Bot, D Wang, DA Liem, MPY Lam, J Ge, & P Ping (2016). “A large dataset of protein dynamics in the mammalian heart proteome.” Nat Sci Data. PMID: 26977904. 2 E Lau, D Wang, J Zhang, H Yu, MPY Lam, X Liang, N Zong, TY Kim, & P Ping (2012). “Substrate- and Isoform-Specific Proteome Stability in Normal and Stressed Cardiac Mitochondria.” Circ Res. PMID: 22456183. 3 TY Kim, D Wang, AK Kim, E Lau, AJ Lin, DA Liem, J Zhang, NC Zong, MPY Lam, & P Ping (2012). “Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria.” MCP. PMID: 22915825. 4 E Lau, Q Cao, MPY Lam, J Wang, DCM Ng, BJ Bleakley, JM Lee, DA Liem, D Wang, H Hermjakob & P Ping (2018). “Integrated omics dissection of proteome dynamics during cardiac remodeling.” Nat Comm. PMID: 29317621. 5 D Wang, DA Liem, E Lau, DCM Ng, BJ Bleakley, M Cadeiras, MC Deng, MP Lam, P Ping (2014). “Characterization of human plasma proteome dynamics using deuterium oxide.” Proteomics Clin Appl. PMID: 24946186. 6 MPY Lam, D Wang, E Lau, DA Liem, AK Kim, DCM Ng, X Liang, BJ Bleakley, C Liu, JD Tabaraki, M Cadeiras, Y Wang, MC Deng, P Ping. (2014). “Protein kinetic signatures of the remodeling heart following isoproterenol stimulation.” J Clin Invest. PMID: 24614109.


The Presentation of Mitochondria Disorders in the Newborn: Time for Whole (Mitochondrial and

Nuclear) Genome Sequencing

David Dimmock, MD

Presenter: David Dimmock, MD

Authors: D.P. Dimmock1, L. Salz1 , K. Wigby1,2, L.M. Bird2, M.N. Bainbridge1, S. Batalov1,W. Benson1, J.A. Cakici1, J. Carroll1,2, S. Caylor1, S. Chowdhury1, M.M. Clark1, C.Clarke1, M. Del Campo2, Y. Ding1, K. Ellsworth1, M. Gaughan1, L. Farnaes1,2, A.Hildreth1,2,, M.C. Jones2 , S. Nahas1, E. Sanford1,2, N.M. Sweeney1,2, M. Tokita1,N. Veeraraghavan1, K. Watkins1, T. Wong1, M. Wright1, S.F. Kingsmore1, RCIGMInvestigators

Institution: 1) Rady Children's Institute for Genomic Medicine, San Diego, CA.; 2) University of California, San Diego, San Diego, CA.

Title: The presentation of mitochondria disorders in the newborn: Time for Whole

(mitochondrial and nuclear) Genome Sequencing

INTRODUCTION Genome-wide sequencing has demonstrated significant promise in reducing the time to diagnosis, reducing the costs of care and improving the outcomes of children with suspected genetic disorders. Emerging data have suggested that the key to improved outcomes and reduced costs is the early use of genome-wide sequencing

However the introduction of such powerful genomic tools into clinical practice has led to ongoing conversations about which children are the best candidates for early sequencing and who is best able to make determinations around this.

Classical mitochondrial disease presentations have been extrapolated from adults presenting with subacute illness. Diagnosis of both mitochondrial and nuclear encoded mitochondrial disease continues to present significant problems in children. This is compounded by the nonspecific presentation of neonates and the overlapping clinical presentation with birth asphyxia. However, the first child undergoing rapid genome-wide sequencing in the ICU for an unclear diagnosis was identified as having TWINKLE associated disease

METHODS RCIGM has been involved in several large trials, including the NSIGHT2 study (NCT03211039), in a broad cohort of acutely ill children infants without a clear etiology.

RESULTS In our broadest study, 42% of positive genomic diagnoses were made in newborns who did not demonstrate easily-recognizable and visible malformations or suspected genetic/metabolic diagnosis, representing 10% of such admissions. The most common presenting diagnoses in this cohort were BRUEs, seizures, hypoglycemia and isolated bowel or cardiac defects. Such children did not have genetics consults, are not typically seen by clinical geneticists and have not been enrolled in previous studies. Genomic sequencing had the largest changes in management in such cases. Mitochondrial disorders were significantly over-represented in this cohort.


UMDF Funded Projects and MMS/MRS Cash Award Presentations (NOTE – new presentation

time for this year’s cash awards)

Philip Yeske, PhD


Saturday, June 29, 2019

Morning Session Bench to Bedside in Mitochondrial Disease

MSeqDR Workshop Beech Room

Marni Falk, MD; Lishuang Shen, PhD; and Elizabeth McCormick, CGC


Mitochondrial Disease and Oxygen: Can there be too much of a Good Thing?

Vamsi K. Mootha, MD

Presenter: Vamsi K. Mootha, M.D.

Institution: Harvard Medical School, Boston, MA

Title: Mitochondrial Disease and Oxygen: Can there be too much of a Good Thing?

Mitochondrial dysfunction accompanies a number of conditions – ranging from rare, inborn errors of metabolism to the aging process itself. At present, we have no proven therapies to alleviate mitochondrial dysfunction. In this talk I will present our efforts employing genome-wide mammalian genetic screens to identify pathways that sensitize or buffer cells against mitochondrial toxicity. The results have led to the seemingly paradoxical idea that hypoxia – or low oxygen – can alleviate mitochondrial dysfunction. I will present our latest work in this area, which has mechanistic, evolutionary, and therapeutic implications.


Mitochondrial Transfer – High Throughput Platform

Michael Teitell, MD, PhD

Presenter: Michael Teitell, MD, PhD

Authors: Alexander N. Patananan1, Alexander J. Sercel2, Ting Wu3, Fasih M. Ahsan1, Daniel Braas4, Justin Golovato3, Pei-Yu Chiou5, and Michael A. Teitell1

Institution: 1Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA

2Molecular Biology Interdepartmental Program, University of California, Los Angeles, Los Angeles, CA 90095, USA 3NantWorks, LLC, Culver City, CA 90232, USA 4UCLA Metabolomics Center, University of California, Los Angeles, Los Angeles, CA 90095, USA 5Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA

Title: Mitochondrial Transfer and Fate Transitions

The ability to generate primary cell lines with desired, compatible nDNA-mtDNA sequences is difficult to achieve due to the necessity of controlled mitochondrial transfer. Here, we deploy a massively parallel transfer platform to deliver isolated healthy mitochondria from peripheral blood mononuclear cells into mtDNA-depleted ( 0) primary fibroblasts. The resulting stable isolated mitochondrial replacement (SIMR) fibroblast (SIMR) lines have restored respiration, but retain a 0-like metabolome and transcriptome. Reprogramming of SIMR fibroblasts to pluripotency followed by differentiation to mesenchymal stem cells (MSCs) progressively resets the metabolome and transcriptome profiles to that of reprogrammed and MSC-differentiated control fibroblasts. Despite having non-native mtDNA sequences, the SIMR lines were able to produce adipocytes, osteocytes, and chondrocytes by directed differentiation. Finally, preliminary work has successfully generated SIMR fibroblasts with specific detrimental mtDNA sequences that show impaired respiration and may aid in the modeling of mitochondrial disorders. We show using SIMR fibroblasts that fate transition is necessary to fully reset 0 cell metabolism following mitochondrial transfer, enabling streamlined studies of novel mtDNA-nDNA combinations without co-genome evolution or germline ‘bottleneck’ selection.


Small Molecules Regulating Mitochondrial Translation

Brendan Battersby, PhD

Presenter: Brendan J. Battersby, PhD

Institution: Institute of Biotechnology, University of Helsinki, Helsinki, Finland

Title: Small Molecules Regulating Mitochondrial Translation

Defects in mitochondrial gene expression represent the largest and most diverse group of human mitochondrial disorders. In all cases, these diseases are associated with assembly failures of the oxidative phosphorylation complexes because of impaired protein synthesis on mitochondrial ribosomes. However, the biochemical defect alone cannot account for the exceptional clinical presentations observed in patients. This suggests additional factors underpin the molecular pathogenesis. To uncover these processes, we leveraged the use of small molecule inhibitors of protein synthesis coupled with genetic approaches to reveal the regulatory complexity of mitochondrial gene expression. Our findings establish a new paradigm, demonstrating how nascent chain proteotoxicity arises on mitochondrial ribosomes, which triggers a novel cellular stress response that impinges organelle form and function, and cell fitness. The same stress response manifests in specific classes of human mitochondrial disorders and we show how modulating mitochondrial protein synthesis in these cases can be beneficial, attenuating the proteotoxicity. Therefore, defects to mitochondrial gene expression can generate fundamentally distinct molecular events during protein synthesis, understanding the regulation of these specific processes are critical to the pathogenesis of these genetic disorders.


Vitamin B3/Niacin and Treatment of Mitochondrial Dysfunction

David Livingston PhD

Presenter: David J. Livingston, PhD

Authors: Ana P. Gomes1, Nathan L. Price1, Alvin J.Y. Ling1, Javid J. Moslehi4,5, Magdalene K. Montgomery6, Luis Rajman1, James P. White7, Joao S. Teodoro2,3, Christiane D. Wrann7, Basil P. Hubbard1, Evi M. Mercken8, Carlos M. Palmeira2,3, Rafael de Cabo8, Anabela P. Rolo2,9, Nigel Turner6, Eric L. Bell10, Matthew Baevsky11, Abhirup Das1,6, Michael S. Bonkowski1,11, Karsten J. Koppetsch11, Jonathan N. Kremsky11, Karen Lavery11, James McKearin11, Bruce G. Szczepankiewicz11, David J. Livingston11, and David A. Sinclair 1,6,11

Institution: 1Glenn Labs for the Biological Mechanisms of Aging, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; 2Center for Neurosciences and Cell Biology, 3004-517 Coimbra, Portugal; 3Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, 3004-517 Coimbra, Portugal; 4Department of Medical Oncology, Brigham and Women’s Hospital and Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 5Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; 6Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney NSW 2052, Australia; 7Dana-Farber Cancer Institute, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA 8Laboratory of Experimental Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA; 9Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal; 10Department of Biology, Massachusetts Institute of Technology, Paul F. Glenn Laboratory for the Science of Aging, Cambridge, MA 02139, USA; 11Metro International Biotech, LLC, 100 Barber Ave., Worcester, MA 01606

Title: NAD+ Precursors Restore Mitochondrial Homeostasis and Increase Skeletal Muscle Endurance

During aging there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. An alternate PGC-1 / -independent pathway of nuclear-mitochondrial communication is induced by a decline in nuclear NAD+ and the accumulation of HIF-1 under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1 / -independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible by restoring NAD+ levels. Preclinical studies of skeletal muscle function in rodents have demonstrated that raising NAD+ levels in old mice restores muscle endurance to levels observed in young mice. NAD+ precursors activate sirtuin deacylase SIRT1, which is a key mediator of pro-angiogenic signals secreted by myocytes, thereby increasing capillary density. Future clinical trials with a variety of agents that can raise NAD+ levels, including NAD+ precursor compounds, will demonstrate whether this is an effective mechanism for restoration of mitochondrial ETC function in mitochondrial disease.


Ongoing Clinical Trials Updates (Patients and families have option to attend) - Non CME

10:30am PA-0621 Hilary Vernon TAZPOWER: Trial Results From a Randomized, Double-Blind, Placebo-Controlled, Crossover and Open-Label Extension Trial of Elamipretide in Patients With Barth Syndrome

10:45am PA-0607 Xavier Lloria Long-term (over 24 months) treatment with idebenone may continue to improve visual function response in patients with Leber’s Hereditary Optic Neuropathy (LHON)

11:00am PA-0636 Xavier Lloria Pediatric Leber’s hereditary optic neuropathy (LHON): Real-world efficacy results following long-term idebenone treatment

11:15am PA-0631 Rustum Karanjia Clinical Trial of Elamipretide Topical Ophthalmic Solution for the Treatment of Leber's Hereditary Optic Neuropathy

11:30am PA-0560 Matthew Klein Safety and Efficacy Clinical Trial of EPI-743 in Patients with Mitochondrial Disease and Epilepsy

11:45am PA-0521 Natalie Yivgi Ohana Mitochondrial Augmentation Therapy of Hematopoietic Stem Cells in Mitochondrial Deletion Syndromes: Preliminary Safety and Efficacy Demonstrated in First-in-Human Study

12:00pm PA-0598 Bruce Cohen MMPOWER-2 Open-Label Extension Trial: Results from 12 Months of Elamipretide Therapy in PMM Patients

12:15pm PA-0581 Amel Karaa MMPOWER-3 Trial Update: A Phase 3, Randomized, Double-Blind, Placebo-Controlled Trial of Elamipretide in Patients With Primary Mitochondrial Myopathy

Mitochondrial Medicine 2019: Washington DCAbstracts


Presenter: Stephen Thomas

Authors: Stephen Thomas1, Patrick Crutcher1, Fabrizio Pertusati2, Michaela Serpi2, Elisa Pileggi2,Mark Vanden Avond3, Hui Meng3, Daniel Helbling3, Michael W. Lawlor3, David Dimmock4

Institution: 1 Cerecor Inc, Rockville, MD, 2 Cardiff University, Cardiff, UK, 3 Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI, 4 Rady Children's Hospital, San Diego, CA, USA

Title: Development of the ProTide Prodrug CERC-913 as a Potential Treatment for Deoxyguanosine Kinase Deficiency

Body of Abstract: Loss-of-function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome known as DGUOK deficiency. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. In vitro supplementation of deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK-deficient, patient-derived fibroblasts and myoblasts. CERC-913, a novel ProTide prodrug of deoxyguanosine monophosphate, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC-913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using tissue from DGUOK-deficient rats. DGUOK knockout rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to eight days. CERC-913 increased mtDNA content in DGUOK-deficient hepatocytes up to 2.5-fold after four days of treatment in a dose-dependent fashion, which was significantly more effective than dGMP at the same concentration. Gene expression associated with mtDNA improved after four days of CERC-913 treatment, but disease-associated differences in protein expression were unchanged. Reduction in cell viability noted in DGOUK-deficient cells between four and eight days improved with CERC-913 treatment. The Seahorse Mito Stress Test showed significant differences between untreated wild type and DGUOK-deficient cells on oxygen consumption rate (a key parameter of mitochondrial function), however normalization was not detected after CERC-913treatment. We also evaluated ADMEPK and toxicity profiles of CERC-913 for its potential as a therapeutic for use in patients with DGUOK deficiency. Overall, these early results suggest that primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC-913treatment can improve several disease-associated phenotypes in this model. Furthermore, CERC-913 possesses acceptable drug-like properties for continued evaluation as a therapy for patients with DGUOK deficiency.

Abstract #: 2019-PA-0521

Presenter: Natalie Yivgi-Ohana

Authors: Natalie Yivgi Ohana, PhD1 , Moriya Blumkin, PhD1, Noa Sher, PhD 1, Nira Varda-Bloom, PhD 4, Julia Pansheen 4, Omer Bar Yoseph, MD, PhD 2,3, Noah Gruber, MD 2, Einat Lahav, MD 2, Michal J Besser, PhD 3,5, Jacob Schachter, MD 3,5, Elad Jacoby, MD 2,3,Amos Toren, MD, PhD 2,3 and Yair Anikster, MD, PhD 2,3

Authors: 1 Minovia Therapeutics, Haifa, Israel; 2 The Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel; 3 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Hematology Laboratory, Sheba Medical Center, Ramat Gan,

UMDF Mitochondrial Medicine 2019 Poster Abstracts June 26-29, 2019

Israel; 5 Ella Lemelbaum Institute for Immuno Oncology, Sheba Medical Center, Ramat Gan, Israel

Title: Mitochondrial Augmentation Therapy of Hematopoietic Stem Cells in Mitochondrial Deletion Syndromes: Preliminary Safety and Efficacy Demonstrated in First-in-Human Study

Body of Abstract: Mitochondrial diseases caused by mtDNA deletions or mutations are debilitating and life-threatening, yet to date no effective pharmacologic treatments are available and all treatment is symptomatic. Several decades ago, the capacity of mitochondria to enter living cells and augment endogenous mitochondrial metabolic activity was demonstrated; intercellular transfer of mitochondria has more recently been demonstrated in vitro and in vivo. In preclinical models of mitochondrial and lysosomal disorders, hematopoietic stem and progenitor cells (HSPCs) have been shown capable of carrying and transferring normal organelles into diseased tissues, thereby altering disease phenotype. We demonstrate the enrichment of patient-derived HSPCs with wild-type mitochondria derived from the mother, a process termed mitochondrial augmentation therapy. We report on first-in-man study in four patients with Pearson and Kearn-Sayrne Syndromes (PS and KSS) treated with autologous HSPCs following ex-vivo mitochondrial augmentation.

Mitochondrial augmentation therapy was demonstrated to be safe; anemia, hypocalcemia and alkalosis, related to the CD34+ mobilization prior to administration of augmented CD34+ cells, were rapidly resolved. In patients in which mitochondrial heteroplasmy levels were discernable in peripheral blood, we observed in vivo improved heteroplasmy starting 3-4 months after cellular therapy, which was maintained throughout the follow-up period. Improvement in mitochondrial heteroplasmy and function was in line with clinical findings: following cell therapy, no events of metabolic crisis occurred in any patient, and aerobic ability and muscle strength assessments were superior compared to baseline in all patients. Importantly, functional impairment quality of life, as measured by the International Pediatric Mitochondrial Disease Score (IPMDS) and PEDI questionnaire, was greatly improved after treatment. It is known that there is a high variability in presentation and progression of patients; individual improvements per patient were noted.

Together, these preliminary clinical data suggest that mitochondrial augmentation therapy, in which we enrich HSPCs with mitochondria carrying non-deleted mtDNA sequence, is a potential method to diminish disease progression in patients with mtDNA deletions, and may be extrapolatable to additional indications involving mutations in the mitochondrial DNA. An FDA-cleared phase I/II clinical trial is currently underway in PS patients. In parallel, an allogeneic mitochondrial product is under development, to enable treatment of mtDNA inherited disorders such as Leigh and MELAS syndromes.


Presenter: Atif Towheed

Authors: Atif Towheed1 PhD, Christian L. Hietanen1 DO and Vasudeva G. Kamath1 PhD

Institution: 1Touro College of Osteopathic Medicine, Middletown, New York 10940

Title: Case Study of a Potential Atypical Hypotonia-Cystinuria Syndrome (Cystinuria with Mitochondrial Disease Homozygous 2p16 Deletion Syndrome) and Its Clinical Management

Body of Abstract: Primary mitochondrial disorders present with complex clinical symptoms. These clinical presentations sometimes overlap with nuclear genetic disorders, which make it challenging to diagnose a classical mitochondrial disease. We present a clinical case of mitochondrial dysfunction in a 17-year-old male with a clinical history of lactic acidosis, cystinuria, and urolithiasis. Cystinuria and


urolithiasis were an incidental find during a hospitalization for influenza when the patient was 7 years old. Apart from one of his siblings demonstrating similar phenotype, no other first or second-degree relatives are affected. Past biochemical studies suggest a deficiency in Complex I and Complex IV activity of the mitochondrial electron transport chain (ETC). However, the exact nature of mutation or inheritance (mitochondrial or nuclear) has not been genetically established. Based on his clinical symptoms and biochemical studies of a typical mitochondrial dysfunction, we believe that he may have atypical cystinuria with mitochondrial disease homozygous 2p16 deletion syndrome (hypotonia-cystinuria syndrome).

Several nuclear gene mutations contribute to mitochondrial dysfunction, however, a nuclear gene mutation in an amino acid transporter affecting mitochondrial ETC is intriguing. Based on previous linkage analysis and clinical reports, we hypothesize that this patient has a microdeletion of at least ~100kb on Chromosome 2p16. The region flanked by polymorphic markers D2S119 and D2S2174 onChromosome 2p has two critical genes, SLC3A1, and LRPCC that have previously been linked with mitochondrial disorders. In 2001, Parvari et al., reported microdeletions in Chromosome 2p16 involving type I cystinuria gene (SLC3A1), the protein phosphatase 2Cbeta gene (PP2Cbeta) and an unidentified gene (KIAA0436), implicating them in cystinuria with mitochondrial dysfunction1. Cystinuria type I is due to the loss of SLC3A1, which encodes the heavy chain subunit of the cystine and dibasic amino acid transporter in the renal proximal tubule and small intestine2. However, at the time it was not clearly understood as to how an amino acid channel, SLC3A1 would affect the function of mitochondria. In 2003, using a bioinformatics approach, Mootha et al., reported LRPCC gene to be associated with mitochondrial dysfunction3. In the same report, SLC3A1 was also reported but its relevance was not discussed. Interestingly, both these genes lie proximal to the genetic markers within a distance of 1.9 centiMorgan. The function of LRPCC is not clearly understood but has been proposed to play a role in Complex IV assembly4. We believe that microdeletion in this region of Chromosome 2p leads to deletion of multiple genes involving at least SLC3A1 and LRPCC which would explain concomitant clinical presentation of cystinuria and mitochondrial ETC dysfunction. Additionally, we discuss clinical management (administration of growth hormone) of this complex presentation, which has led to a tremendous improvement in the patient. To further evaluate our hypothesis, we plan to pursue targeted molecular analysis to better understand the gene mutation responsible for the patient’s clinical symptoms.

References

1. Parvari, R., et al. A recessive contiguous gene deletion of chromosome 2p16 associated with cystinuria and a mitochondrial disease. Am J Hum Genet 69, 869-875 (2001).

2. Chabrol, B., et al. Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome. J Med Genet 45, 314-318 (2008).

3. Mootha, V.K., et al. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 115, 629-640 (2003).

4. Alston, C.L., Rocha, M.C., Lax, N.Z., Turnbull, D.M. & Taylor, R.W. The genetics and pathology of mitochondrial disease. J Pathol 241, 236-250 (2017).


Presenter: Alvaro Serrano Russi

Authors: 1Alvaro Serrano Russi MD, 2Jianling Ji, MD FACMG

Affiliations: 1Associate Professor of Clinical Pediatrics; Keck School Of Medicine, Department of Pediatrics; Children's Hospital Los Angeles 2Center for Personalized Medicine; Departmen t of Pathology and Laboratory Medicine; Children's Hospital Los Angeles


Title: Clinical Follow-up of Patient Affected with GFM1 Mitochondrial Elongation Factor Deficiency

Body of Abstract: We present the case of a baby boy born to a 28 year old at thirty nine weeks gestation by cesarean section for breech presentation. His APGAR scores were 8 and 9 with a birth weight of 2795 grams, a birth length of 49.5 cm and head circumference of 33 cm. He was initially well but at 21 hours of life presented with increased work of breathing and blood glucose of 29 mg/dL. He was noted to have low set ears, a sacral dimple and mild growth restriction. Further laboratory showed severe metabolic acidosis with initial blood gas with a pH of 7.1, blood lactate 24 mmol/L, serum bicarbonate 4.4 mmol/L and a base deficit of -22.5. With dextrose 10% at maintenance and 1 mEq/kg/dose of sodium bicarbonate there was gradual correction of lactic acidosis. His GDF15 was 2054 pg/mL (<750).

Mom had no history of illnesses before or during the pregnancy. Maternal serum screening showed increased risk for trisomy 21. NIPT was negative. She had a prior miscarriage. Family history was unremarkable. Paternal ancestry was from Guatemala and El Salvador. His maternal ancestry is from Mexico.

Whole exome sequencing showed a paternally inherited mutation in GFM1 (NM_024996.5) denominated c.748C>T (p.Arg250Trp) and a maternally inherited mutation denominated c.1424delG (p.Arg475Lysfs*78).

To this date, he has not developed cardiomyopathy or liver dysfunction. Carbohydrate deficient transferrin pattern was normal. He was found to have infantile spasms and EEG at 5 months with hypsarrhymia background treated with clobazam and Vigabatrin. Ocular fundus exam was normal at 6 months.

More recently, he presented at 9 months with a viral infection for 3 days with cough congestion and post-tusive emesis. At the ER, his glucose was 51 mg/dL; Lactate of 40 mg/dL and a serum bicarbonate of 24 mEq/L. With intravenous fluids his blood glucose improved to 81 mg/L. Transaminases were within normal limits. He was discharged with no complications.

DISCUSSION

Mitochondrial elongation factor 1 was initially cloned in 2001 as part of researching the process of mitochondrial protein translation. EF-G1 is a GTPase that participates in the elongation of mitochondrial tRNAs and removes the de-acetylated tRNA and replacing with the peptidyl rRNA1. Failure of mitochondrial protein elongation has been associated with disorders of oxidative phosphorylation, with initial patients reported in 2004 presenting with intrauterine growth retardation, congenital microcephaly, profound metabolic acidosis, liver dysfunction and early death2. Two other cases were reported in 2006 presenting with congenital lactic acidosis and dysmorphic features in two siblings of the same family3.

The mutation c.748C>T (p.Arg250Trp) was described in a homozygous state on a patient who died at 2years of age with respiratory insufficiency secondary to pneumonia after presenting with congenital hypotonia followed by seizures at 8 weeks associated with increased lactic acid in body fluids4.

The variant c.1424delG has not been described in the literature. This case serves as a first report of a patient presenting with this combination of alleles.

(1) Hammarsund et al; Identification and characterization of two novel human mitochondrial elongation factor genes, hEFG2 and hEFG1, phylogenetically conserved through evolution; Hum Genet (2001) 109 :542–550

(2) Heister JG, Newbold RF, Trijbels FJ, van den Heuvel LP, Shoubridge EA, et al (2004) Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency.


N Engl J Med 351:2080–2086(3) Shoubridge et al; The molecular basis for tissue specificity of the oxidative phosphorylation

deficiencies in patients with mutations in the mitochondrial translation factor EFG1; Human Molecular Genetics, 2006, Vol. 15, No. 11

(4) Paulien Smits, Hana Antonicka, Peter M van Hasselt, Woranontee Weraarpachai, Wolfram Haller, Marieke Schreurs, Hanka Venselaar, Richard J Rodenburg, Jan A Smeitink and Lambert P van den Heuvel; Mutation in subdomain G’ of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle; European Journal of Human Genetics (2011) 19, 275–279


Presenter: Anne Chiaramello

Authors: Martine Uittenbogaard1, Hao Wang2, Victor Wei Zhang2, Lee-Jun Wong2, Andrea Gropman3, Anne Chiaramello1

Institution: 1George Washington University School of Medicine and Health Sciences, Department of Anatomy and Regenerative Biology, Washington, D.C. 20037, 2Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, TX, 77030, 3Children’s National Medical Center, Division of Neurogenetics and Developmental Pediatrics, Washington, D.C. 20010

Title: The Nuclear Background Modulates the Penetrance and Phenotypic Expression of the Near-Homoplasmic MELAS m.1630 A>G Variant

Body of Abstract: The mitochondrial encephalopathy lactic acidosis and stroke-like episode Syndrome (MELAS, OMIM 540000) is a progressive neurodegenerative disease that results in devastating multi-organ failure as a result of a chronic energy deficit. MELAS patients harbor a mitochondrial variant affecting the oxidative phosphorylation (OXPHOS) pathway responsible for ATP synthesis. Although most MELAS patients harbor the m.3243 A>G variant, the rare pathogenic mitochondrial variant, m.1630 A>G, has recently been reported in one patient with MELAS and the other with mitochondrial neurogastrointestinal encephalopathy (MNGIE). The m.1630 A>G pathogenic variant, which maps in the mitochondrial MT-TV gene, substitutes the residue adenine for guanine in the anticodon-stem of the mt-tRNAVal thereby compromising its secondary structure. Our study focuses on a unique case of a symptomatic proband and her asymptomatic mother, both harboring the m.1630 A>G variant at similar near-homoplasmic levels. Our mitochondrial morphometric analysis reveals a link between defects of the mitochondrial cristae ultrastructure and symptomatic status. Despite harboring near-homoplasmic levels (95%) of the m.1630 A>G variant, the dermal fibroblasts of the asymptomatic mother exhibit a normal OXPHOS metabolism. This stands in contrast with the severely impaired OXPHOS response of the proband’s fibroblasts harboring the mitochondrial variant at near-homoplasmic levels (90%). The proband’s fibroblasts also show a defective metabolic plasticity with a stunted compensatory glycolytic capacity, preventing to switch to glycolysis to offset the severe OXPHOS deficit. Due to the discordant clinical and metabolic phenotype, we performed whole exome sequencing (WES) to investigate whether nuclear variants could alter the penetrance of the m.1630 A>G mitochondrial variant. Our WES analysis reveals the nuclear variant VARS2 as a potential nuclear modifier. The heterozygous nonsense VARS2variant (p.R334.X) is exclusively in the proband’s nuclear genome. It maps in the gene encoding the mitochondrial valyl-tRNA synthetase, which catalyzes the attachment of the valine amino acid to the mutated mt-tRNAVal. This nonsense VARS2 nuclear variant removes two-third of the VARS2 protein, which contains domains necessary for interacting with the mt-tRNAVal. This VARS2 loss-of-function variant results in VARS2 haploinsufficiency and therefore most likely impacts the levels of charged mt-


tRNAVal molecules. Thus, our study provides the first report of an aminoacyl tRNA synthetase VARS2 nuclear variant in the context the m.1630 A>G variant mapping in the corresponding mt-tRNAVal and their potential pathogenic interactions. Our future functional study will elucidate the pathogenic mechanisms by which the nuclear background influences the phenotypic signature of the MELAS m.1630 A>G variant in this rare clinical setting.


Presenter: Martine Uittenbogaard

Authors: Martine Uittenbogaard1, Hao Wang2, Lee-Jun Wong2, Andrea Gropman3, Anne Chiaramello1

Institution: 1George Washington University School of Medicine and Health Sciences, Department of Anatomy and Regenerative Biology, Washington, D.C. 20037, 2Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, TX, 77030, 3Children’s National Medical Center, Division of Neurogenetics and Developmental Pediatrics, Washington, D.C. 20010

Title: Genetic and Metabolic Investigations to Understand the Genotype-Phenotype Correlation in MELAS Patients With The m.3243 A>G or m.14453 G>A variant

Body of Abstract: The mitochondrial encephalopathy lactic acidosis and stroke-like episode syndrome (MELAS, OMIM 540000) is the most common mitochondrial syndromic manifestation of mitochondrial encephalopathy. This childhood-onset progressive neurodegenerative disease remains intractable. Most MELAS patients harbor the m.3243 A>G variant, which maps in the mitochondrial MT-TL1 gene encoding the mt-tRNALeu(UUR). MELAS can be caused by other rare pathogenic mitochondrial variants, such as the m.14453 G>A mapping in the mitochondrial MT-ND6 gene encoding the ND6 subunit of complex I. MELAS variants affect mitochondrial proteins involved in the oxidative phosphorylation (OXPHOS) pathway responsible for ATP synthesis. MELAS patients exhibit a broad and heterogenous clinical spectrum due to the heteroplasmic load of the pathogenic mitochondrial variant, its tissue distribution, the threshold for OXPHOS defects and the nuclear background. To gain insight into the genotype-phenotype correlation, we investigated the metabolic signature in a cohort of 12 patients exhibiting a range of symptomology and heteroplasmy. Patient-derived dermal fibroblasts were used as an ex vivo cellular system to interrogate the functional impact of MELAS variants on energy metabolism and metabolic plasticity. Heteroplasmy was measured by long-range PCR followed by massively parallel sequencing, while the bioenergetic status was determined by live-cell measurement of the oxygen consumption rate, a key functional indicator of the mitochondrial respiration. We found that a decrease in basal respiration and ATP-linked respiration does not necessarily correlate with the heteroplasmic load of the MELAS variant. In contrast, all MELAS patients exhibit a significant deficit in maximum respiration and spare respiratory capacity, regardless of their heteroplasmy, resulting in a diminished capacity to produce extra ATP upon energy demand. We next investigated the metabolic plasticity of MELAS fibroblasts. To assess their ability to undergo energy reprogramming for sustaining an energy crisis by switching to the glycolytic pathway, we performed a Glycolysis Rate assay. It accurately measures the basal glycolysis and the compensatory glycolytic response upon inhibition of mitochondrial ATP production. Most MELAS patients show increased in basal glycolysis, indicating glycolysis as the main pathway for ATP production. However, one MELAS patient with high heteroplasmy has a low basal glycolysis, while his mother with similar heteroplasmy displays a high basal glycolysis. We next investigated their metabolic plasticity. All the MELAS patients, except one, show an inadequate compensatory glycolytic response under conditions simulating an acute ATP crisis. Interestingly, the only MELAS patient with a robust compensatory glycolytic response harbors a high heteroplasmic level of the


m.3243 A>G variant. In conclusion, while heteroplasmy dictates the metabolic phenotype of the MELAS variant, the nuclear background also contributes to the metabolic plasticity of MELAS fibroblasts, a key determinant to curtail the chronic energy crisis associated with MELAS.


Presenter: Siegfried Hekimi

Authors: Siegfried Hekimi

Institution: McGill University, Montreal.

Title: Making Ubiquinone Bioavailable

Body of Abstract: Ubiquinone (UQ) is an obligate electron carrier in the mitochondrial respiratory chain and might also act as a membrane antioxidant. The committed steps of UQ biosynthesis are carried out in mitochondria and appear to be partially inhibited by mitochondrial dysfunction. Patients with mutations in enzymes and regulatory proteins required for UQ biosynthesis exhibit primary UQ deficiency, which presents like mitochondrial disease syndrome (MDS). Conversely, MDS is often accompanied by secondary UQ deficiency. We have constructed a mouse model of UQ deficiency in which UQ biosynthesis can be partially restored, as well as mouse and rat models of stable partial deficiency. We have shown that even near-lethal phenotypes resulting from a 10- to 20-fold reduction in UQ levels can be fully reversed by partial restoration of UQ biosynthesis. However, oral supplementation of UQ only reaches the liver and does not lead to any significant phenotypic rescue. To address this problem, we have constructed a cell line entirely devoid of UQ (lacking the biosynthetic enzymes PDSS2 and MCLK1/COQ7). With these doubly mutant cells we have carried out a drug screen for compounds that can boost UQ uptake. We have screened all FDA-approved drugs and have identified a single compound that is capable of dramatically boosting UQ uptake in both cells and animals. At the meeting, we will describe the origin, structure and mode of action of the compound.


Presenter: Fernando Scaglia

Authors: Kevin E. Glinton1,4, Suzy Boyer4, Elizabeth Mizerik1,4, Alona Birjiniuk2, NatalieVillafranco3,4, Nidhy Varghese3,4, Sarah Elsea1, and Fernando Scaglia1,4,5

Institution: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX77030, 2Department of Pediatrics, Baylor College of Medicine/Texas Children’s Hospital,Houston TX 77030, 3Section of Pediatric Pulmonary Medicine, Baylor College ofMedicine, Houston TX 77030, 4Texas Children’s Hospital, Houston TX 77030, 5BCM-CUHK Center of Medical Genetics, CHUK, Hong Kong SAR

Title: The Utility of a Ketogenic Diet in the Management of Multiple Mitochondrial DysfunctionsSyndrome Type I

Body of Abstract: Background: Pulmonary hypertension is an infrequently observed phenomenon inchildren outside of cases associated with congenital heart disease or lung prematurity. Pulmonaryhypertension (pHTN) is even more rarely described as a feature of metabolic or mitochondrial disordersthough recently, Multiple Mitochondrial Dysfunction Syndromes (MMDS) have been described as a


cause of pHTN associated with pathogenic variants in genes important in Iron-Sulfur Cluster synthesisand assembly. This disorder leads to a constellation of pulmonary hypertension, neurologic symptomsand failure to thrive in affected infants as a consequence of the postulated defects in lipoic acid synthaseand the four lipoic acid dependent enzymes (PDH, α-KGDH, BCKDH and the H-protein of the GCS).Attempts at treating this disease have centered on the use of pulmonary vasodilators though there hasbeen a single report of the use of a ketogenic diet as a means of providing an alternative source ofenergy. Here we report a case of MMDS Type-1 treated with a trial of a ketogenic diet and thiaminesupplementation. In addition, we report findings from her deceased older sibling with same condition.Methods: Patient 1 was a young female diagnosed at the age of 4 months following admission forpulmonary hypertension and signs of right heart failure. Following diagnosis, she was started on empirictherapy with sildenafil/bosentan, thiamine and a ketogenic diet. Patient 2 was the previously affected,deceased older sibling of Patient 1.Results: Trio Whole Exome Sequencing identified bi-allelic pathogenic variants in NFU1, consistent withMMDS-1. The patient’s subsequent biochemical testing in the form of enzyme assay and untargetedmetabolomics, demonstrated features consistent with this diagnosis. The institution of a ketogenic dietdid yield stable ketosis with only rare episodes of hypoglycemia and she remained on the diet for theensuing 3 months. However, the patient did eventually experience an acute decline and passed away atthe age of 7 months. Familial mutation testing on Patient 2 confirmed that he carried this diagnosis aswell. On examination of lung tissue (collected on autopsy), he was found to have features of severepulmonary hypertension and signs of abnormal lung development.Conclusions: Multiple Mitochondrial Dysfunctions Syndrome Type 1 is a rare and particularly devastatingdisease characterized by lactic acidosis, developmental delay/regression and pulmonary hypertension. Aketogenic diet has been explored in one such patient however additional studies into this treatmentmodality are necessary for a better understanding of its utility and effects.


Presenter: Shauna-Kay Rhooms

Authors: Shauna-Kay Rhooms1, Maximino Villanueva1, Edward Owusu-Ansah1,

Institution: 1Columbia University Medical Center, Department of Physiology and Cellular Biophysics, New York, NY 10032

Title: A MitoCarta Screen to Identify Novel Regulators of Complex I Assembly

Body of Abstract: Human mitochondrial diseases are a group of chronic, genetic disorders that resultsfrom failure of the mitochondria to generate enough energy to support cell or organ functions. Studies haveshown that complex I associated deficiencies are the most common enzymatic defects in the oxidativephosphorylation disorders. Appropriately known as the powerhouse of the cell, mitochondria are uniqueorganelles, whose function is to supply energy in the form of adenosine triphosphate (ATP). Comprised ofa series of five protein complexes (Complex I to V), it is essentially the functions of these complexes thatultimately generate the energy used for various cellular functions in the body. Complex I (CI) is the firstand largest enzyme within the series. Human CI is composed of 44 distinct protein subunits assembledthrough a specific process with the assistance of CI assembly factors (CIAFs). Studies have shown thatmany mitochondrial CI diseases do not exhibit any mutations in the 44 known protein subunits or knownCIAFs; which suggests that additional regulators of CI biogenesis are yet to be identified. Like humans,Drosophila generates its energy from the mitochondria; as they require energy to power flight and otherforms of locomotion from mitochondria located in their flight and leg muscles. When genes that regulateany aspect of energy generation in mitochondria are defective in Drosophila, they cause a readilyperceptible locomotory defect that can easily be identified. The MitoCarta list is an inventory of genesencoding proteins that are found in the mitochondrion. Utilizing the Drosophila melanogaster model, wepropose to genetically disrupt all 1158 genes identified as part of the MitoCarta 2.0 list and examine


whether any are novel regulators of CI assembly. Similarities in mechanism of CI assembly in Drosophilaand humans, ease of isolating mitochondria from their flight muscles and numerous tools for geneticanalyses, provide a rationale for uncovering new regulators of mitochondrial CI assembly in Drosophila.We anticipate that analyses of the new regulators will lead to a better understanding of the pathology ofmitochondrial CI diseases, and ultimately uncover novel therapeutic opportunities.


Presenter: Nam Chul Kim

Authors: Yun-Jeong Choe1, Minwoo Baek1, Gerald W. Dorn II2, J. Paul Taylor3, Nam Chul Kim1

Institution: 1University of Minnesota, College of Pharmacy, Duluth, MN 55812, 2Washington University School of Medicine, Center for Pharmacogenomics, St. Louis, MO 63110, 3St.Jude Children’s Research Hospital, Cell and Molecular Biology, Memphis, TN 38105.

Title: The PINK1/Parkin Pathway Mediates Dominant Mitochondrial Toxicity in CHCHD10-induced ALS-FTD

Body of Abstract: Mutations in coiled-coil-helix-coiled-coil-helix-domain containing 10 (CHCHD10) have been identified as a genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. However, the disease-causing mechanism and the nature of mutations have not been fully understood. In this study, we generated a Drosophila model to study the function of CHCHD10 and disease-causing mechanisms. Mutant dCHCHD10S59L expression caused dominant toxicity in Drosophila eyes, motor neurons and muscles as well as HeLa cells while other mutants (R15L, P34S, G58R, and G66V) showed various effects in Drosophila and Hela cells. With the dCHCHD10S59L Drosophila model, we found that CHCHD10S59L is a dominant gain-of-toxicity mutant forming protein aggregates in mitochondria and the PINK1/Parkin pathway is activated by dCHCHD10S59L. The activated PINK1/Parkin pathway actually generates the mutant CHCHD10-dependent toxicity. The genetic reduction of PINK1 and Parkin expression significantly rescued abnormal phenotypes in Drosophila tissues and HeLa cells. In addition to genetic studies, we developed peptide inhibitors for PINK1 kinase. The treatment of the pseudo-substrate inhibitors reversed CHCHD10S59L-induced mitochondrial phenotypes in HeLa cells. With our Drosophila model, we found two downstream substrates of PINK1, mitofusin and mitofillin, are involved in the toxicity-generating process. Recently developed mitofusin2 agonists also restored mitochondrial functions disrupted in HeLa cells, and Drosophila models for dCHCHD10S59L and C9orf72. Our data indicate that chronic activation of the PINK1-mediated pathways by CHCHD10S59L generates dominant toxicity in our model systems and suggest PINK1-mediated pathways as potential therapeutic targets for mutant CHCHD10-induced degenerative diseases and other related diseases.


Presenter: Marissa Cross

Authors: Marissa Cross1, Caroline Trumpff1, Kris Engelstad1, Gabriel Sturm1, Marlon A McGill1,Kalpita Karan1, Xiomara Q. Rosales1, Zachary Anderson2, Joseph Clark2, Sophia Tepler1,Veronica Taleon1, Jennifer Wang1, Jennifer Manly1, Michelle Martinez1, Valerie Medina1,Johanne Fortune1, Grace Liu1, Vincenzo Lauriola1, Dania J Elder1, Todd Ogden1, Michel Thiebaut de Schotten3, Peter Shapiro1, Bruce S McEwen4, Richard P Sloan1, Tor D Wager2, Michio Hirano1, Martin Picard1


Institution: 1 Columbia University Irving Medical Center, New York, NY 10032; 2 University of Colorado, Boulder, CO 80309; 3 Sorbonne Universities and University of Bordeaux, France; 4 The Rockefeller University, New York, NY 10065

Title: The Mitochondrial Stress, Brain Imaging, and Epigenetics Study – MiSBIE

Body of Abstract:

Background Clinical and pre-clinical findings suggest that mitochondrial function influences how metabolic, physiological, neural, and psychological systems operate and interact, but this has not been formally studied in humans. In mouse models of mitochondrial disease, mitochondrial defects cause distinct, multi-systemic stress response signatures involving changes in the hypothalamic-pituitary-adrenal, sympathetic-adrenal-medullary, inflammatory, metabolic, and transcriptional responses (Picard et al. 2015). Here, we take a multi-systemic approach to profile the molecular, genetic, neural, physiological, and psychological changes associated with acute psychological stress exposure among individuals with normal mitochondrial function and in patients with mitochondrial disease.

Methods The Mitochondrial Stress, Brain Imaging, Epigenetics (MiSBIE) study is recruiting participants presenting the m.3243A>G mutation with (n=30) or without MELAS (n=30), a mtDNA deletion (n=30), and healthy controls (n=30). Participants undergo a comprehensive two-day protocol, which includes body composition measurements, medical examination, genetic testing, leukocyte mitochondrial functional profiling (oxygen consumption, enzymatic activities), neuropsychological testing, and evaluations of psychosocial functioning and psychiatric symptoms. A standardized laboratory socio-evaluative stress protocol is used to elicit an integrated whole-body stress response, monitored dynamically via blood-based neuroendocrine and metabolomic analyses, temperature, continuous measures of autonomic and cardiovascular function, neural activity, and functional/structural connectivity within the brain.

Results The two-day protocol is well-tolerated, with a cumulative (n=22) completion rate of 100% to date. The socio-evaluative laboratory stressor elicits robust changes in all monitored parameters, including perceived stress, heart rate, blood pressure, skin conductance, and temperature in participants. The socio-evaluative stress fMRI task also induces robust activation of the expected brain networks (medial prefrontal cortex and striatum). The magnitude of these changes in physiology and neural patterns vary substantially from person to person, and preliminary analyses suggest different response patterns in healthy control, mtDNA mutation, and mtDNA deletion groups, suggesting that mitochondrial function may modulate the stress response.

Discussion These initial preliminary findings demonstrate the feasibility of a controlled psycho-physiological stress paradigm in patients with a range of mitochondrial disorders. Future analyses will test the influence of mitochondrial (dys) function on stress response patterns as a predictor of mitochondrial disease severity and progression.



Authors: Nishitha R Pillai1,2, Noura S AlDhaheri1,2,3, Rajashri Gosh1, Jaehyung Lim2,4, Anuranjita Nayak2,4, Lisa Emrick2,4, Neil Hanchard1,2, Sarah H. Elsea1, Fernando Scaglia1,2,5

Institution: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; 2Texas Children’s Hospital, Houston, TX, USA; 3Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE;


4Department of Neurology, Baylor College of Medicine, Houston Tx, USA; 5Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR

Title: Biallelic variants in COX4I1 Associated with A Novel Phenotype of Developmental Regression, Intellectual Disability and Seizures

Background: Biallelic variants in COX4I1 (OMIM: 123864) have been previously described in a single patient who presented with short stature, poor weight gain, dysmorphic features and features of Fanconi anemia. COX4I1, located at 16q24.1, encodes the subunit IV isoform 1, the principal isoform for COX-IV subunit of cytochrome c oxidase (COX/Complex IV) in humans and other vertebrates. COX plays an important role in oxidative phosphorylation by transferring electrons from cytochrome c to molecular oxygen and contributes to a proton electrochemical gradient across the inner mitochondrial membraneand formation of ATP. Here, we describe a novel COX4I1 variant in two siblings who presented with developmental regression, seizures and pathognomonic changes in brain imaging resembling Leigh syndrome phenotype.

Case reports: First sibling is a 3 year old Iraqi male, born at 37 weeks of gestation to consanguineous parents. After initial normal growth and development, his motor skills regressed at 8 months of age. Brain MRI findings were suggestive of Leigh syndrome. He developed seizures by 2 years of age. A trio WES done as a part of his diagnostic evaluation showed a homozygous novel variant in COX4I1, c.454C>A (p.P152T). In silico analyses suggests that this variant is evolutionarily conserved and constrained. Untargeted metabolomics profile was done on plasma and CSF, which showed elevated lactate along with fumarate suggesting mitochondrial dysfunction.

His older brother who is 11 years of age and born full term had the same phenotype with regression of skills at 11 months of age followed by seizures at one year of life. Upon confirmation of the sibling’s diagnosis through exome sequencing, he had known familial mutation testing for COX4I1, which identified the same variant. A mitochondrial respiratory chain enzyme analysis done on the muscle biopsy specimen showed that complex IV activity was reduced to 16% compared to normal controls, meeting modified Walker criteria further confirming the diagnosis of mitochondrial disease. Chromosome breakage studies done on both siblings were normal.

Conclusions: Ballelic variants in COX4I1 have been described previously to present with short stature, poor weight gain, dysmorphic features and features of Fanconi anemia. Here, we describe siblings with a novel homozygous variant on COX4AI1 presenting with encephalopathy, developmental regression, hypotonia, pathognomonic brain imaging findings resembling Leigh-like syndrome, expanding the known clinical phenotype associated with pathogenic variants COX4I1. We also emphasize the importance of using untargeted metabolomics analysis that could provide functional evidence for pathogenicity of variants in mitochondrial syndromes.


Presenter: Krish Chandrasekaran

Authors: Krish Chandrasekaran, Neda Najimi, Pranith Kumar, Mohammad Salimian, James W. Russell

Institution: Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201 and VA Maryland Health Care System, Baltimore, MD 21201

Title: Nicotinamide Riboside is a Potential Therapy for Diabetic Neuropathy


Body of Abstract: Aim: To test if treatment with a precursor of NAD+, Nicotinamide Riboside (NR), would reverse neuropathy in a mouse model of type 2 diabetes Mellitus (T2DM).

Methods: Adult C57BL6 mice were fed a high fat diet (HFD,:60% calories from fat) for two months until they developed neuropathy. Then, 150 mg/kg or 300 mg/kg NR was mixed with HFD and fed everyday for 2 months. Control animals were fed a control diet (18% calories from fat). Diets had similar protein and carbohydrate contents. At 2 months, blood glucose levels in HFD-fed mice were >200 mg/dL. Neuropathy endpoints were motor sciatic-fibular nerve conduction velocities (MCV), mechanical allodynia (MA), and intraepidermal nerve fiber density (IENFD). Brain NAD+ levels were quantified by liquid chromatography tandem mass spec.

Results: Both MA and MCV improve in HFD mice with NR treatment (P<0.001 mice at 4 months compared to the 2 month time point). There was no change in control diet (CD) animals. HFD animals continued to develop neuropathy over the 4 mo. period. At 4 mo., the IENFD was decreased in the HFD but not the NR group (P<0.001 HFD mice at 4 months vs baseline). NR treatment decreased HFD-induced increase in triglycerides and non-esterified fatty acids, and normalized impaired glucose tolerance test. In HFD mice, there was a decrease in the NAD+ level, in SIRT1 level, and in PGC-1 alpha levels in DRG. NR normalized these measurements.

Conclusion: Oral administration of NR can reverse neuropathy in a model of T2DM.

Supported in part by NIH NIDDK, VA 101RX001030, Diabetes Action Research and Education Foundation.



Authors: Brian J. Shayota1,2, Nhon T. Le N3, Nasim Bekheirnia N1,3,4, Jill A. Rosenfeld2, Amy C. Goldstein5, Michael Moritz6, Dennis W. Bartholomew7, Matthew T. Pastore7, Fan Xia2,8,Christine Eng2,8, Yaping Yang2,8, Dolores J. Lamb3,9, Fernando Scaglia1,2,10, Michael C. Braun1,3,4, Mir Reza Bekheirnia1,2,3,4

Institution: 1Texas Children’s Hospital, Houston, TX, USA, 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 3Baylor College of Medicine, Houston, TX, USA, 4Renal Section, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA, 5Department of Pediatrics and Division of Child Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 6Department of Pediatrics, Division of Nephrology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 7Division of Molecular and Human Genetics, Nationwide Children's Hospital, Columbus, OH, USA, 8Baylor Genetics, Baylor College of Medicine, Houston, TX, USA, 9Department of Urology, Weill Cornell Medicine, New York, NY, USA, 10BCM-CUHKCenter of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR

Title: Expanding the Renal Phenotype of RMND1-related Mitochondrial Disease

Body of Abstract:

The nuclear encoded gene RMND1 (Required for Meiotic Nuclear Division 1 homolog) has recently been linked to RMND1-related mitochondrial disease (RRMD). This autosomal recessive condition characteristically presents with an infantile-onset multisystem disease characterized by severe


hypotonia, global developmental delay, failure to thrive, sensorineural hearing loss, and lactic acidosis. Renal disease, however, appears to be one of the more prominent features of RRMD, affecting patients at significantly higher numbers compared to other mitochondrial diseases. We report the clinical, histological, and molecular findings of four RRMD patients across three academic institutions with a focus on the renal manifestations.

In this study, we identified 4 unrelated cases of RRMD, all of whom developed renal disease characterized as a tubulopathy (3/4), renal tubular acidosis (2/4), interstitial nephritis (1/4), and/or end stage renal disease (ESRD) necessitating renal transplantation (4/4). Molecular testing of the patients revealed the previously reported pathogenic variant c.713A>G (p.N238S) as well as other rare variants including c.485delC (p.P162fs), c.533C>T (p.T178M), and c.1317+1G>C splice variant. Histological evaluation of renal biopsy specimens revealed generalized tubular atrophy and on electron microscopy, abundant mitochondria with pleomorphism and abnormal cristae.

Our experience with RRMD demonstrates a specific pattern of renal disease manifestations and clinical courses. Patients are unlikely to respond to traditional chronic kidney disease (CKD) treatments, making early diagnosis and consideration of renal transplantation paramount to the management of RRMD.



Authors: Brian J. Shayota1,3, Caludia Soler-Alfonso1,3, Mir Reza Bekheirnia1,3, Elizabeth Mizerik1,3,Suzanne W. Boyer1,3, Rui Xiao2, Yaping Yang2, Sarah H. Elsea2, Fernando Scaglia1,3,4

Institution: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 2Department of Molecular and Human Genetics, Medical Genetics Laboratory, Baylor College of Medicine, Houston, TX, USA, 3Texas Children’s Hospital, Houston, TX, USA, 4BCM-CUHK Center of Medical Genetics, Hong Kong SAR

Title: Case Report and Novel Treatment of an Autosomal Recessive Leigh Syndrome Caused by Short-Chain Enoyl-CoA Hydratase Deficiency

Body of Abstract: Short chain enoyl-CoA hydratase (SCEH) deficiency leads to a severe form of autosomal recessive Leigh syndrome with inevitable neurological decline and early mortality. SCEH is most notably involved in valine catabolism, a deficiency of which results in various metabolic alterations, including increased levels of the highly reactive metabolite 2-methacrylyl-CoA. With no proven treatments available to date, it has been speculated that patients may respond to a valine restricted diet and/or N-acetylcysteine supplementation, as suggested by early studies of a very similar inborn error of metabolism, 3-hydroxyisobutyryl-CoA hydrolase deficiency.

We describe a patient with typical Leigh syndrome clinical findings including failure to thrive, profound hypotonia, projectile vomiting, chronic diarrhea, and in the last stages opisthotonic posturing. Diagnostic work-up with Whole Exome Sequencing identified compound heterozygous variants in ECSH1 (c.538A>G p.T180A, c.444G>T p.M148I) and follow-up cultured fibroblast confirmed markedly reduced enzyme activity. Valine-restricted diet was initiated at 6 months of age and N-acetylcysteine supplementation at 9 months with subsequent improvement in growth and slow progress in developmental milestones. However, at 15 months, the patient aspirated during a breakthrough seizure from which he did not recover and died soon after from related complications.

This report highlights some of the challenges that remain in the management and treatment of SCEH deficiency. Primarily, limitations in the availability of a reliable biomarker to assess response to treatment makes it difficult to make a conclusive statement regarding the efficacy of therapeutic approaches. Here


we have demonstrated that in our patient, a valine restricted diet and N-acetylcysteine can be safely administered with the potential for clinical improvement.



Authors: Brian J. Shayota1, Michael L. Kueht2, Rowland Pettit5, Youmna A. Sherif2, Lisa Emrick L3,Michio Hirano M6, Sarah H. Elsea1, Ryan W. Himes4, Fernando Scaglia1

Institution: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 2Department of Surgery, Baylor College of Medicine, Houston, TX, USA, 3Department of Neurology, Baylor College of Medicine, Houston, TX, USA, 4Department of Gastroenterology, Hepatology and Nutrition, Baylor College of Medicine, Houston, TX, USA, 5Baylor College of Medicine, Houston, TX, USA, 6Department of Neurology, Columbia University Medical Center, New York City, NY, USA

Title: Liver Transplantation for the Treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): A Case Report

Body of Abstract:

Mitochondrial Neurogastrointestinal Encephalopathy (MNGIE) (OMIM 603041) is a rare autosomal recessive mitochondrial disorder caused by biallelic variants in TYMP. The loss of thymidine phosphorylase activity results in increased thymidine and deoxyuridine levels in the blood, urine, and CSF, leading to an imbalance in the mitochondrial nucleoside pool and subsequently causing a mitochondrial DNA depletion syndrome. It is characterized by gastrointestinal dysmotility, cachexia, peripheral neuropathy, leukoencephalopathy, ophthalmoplegia, and liver dysfunction. Current therapeutic approaches have focused on reducing plasma thymidine levels with hemodialysis, platelet infusions, and enzyme replacement therapy. However, hematopoietic stem cell transplantation (HSCT) is considered the standard of care, though it carries a substantial risk for morbidity and mortality. Liver transplantation has been suggested as well for the treatment of MNGIE, but only 2 cases have been reported to date, both of which had positive outcomes.

A 23-year-old man with debilitating polyneuropathy, gastric dysmotility, and liver failure was diagnosed with MNGIE based on elevated plasma thymidine levels and a homozygous pathogenic variant in TYMP (c.215-1G>C). Because of his severe liver disease, the risk for HSCT was considered too great and the patient was referred for liver transplantation instead. Following orthotopic liver transplantation, plasma thymidine levels were reduced to normal levels by post-operative day 1. Subsequent measures with untargeted metabolomic studies up to 6 months later remained in the normal range, but revealed a gradual increase in thymidine to four times the initial level. Rising levels of 2-deoxyuridine and pipecolate was detected as well. Post-operatively, the patient had clinically improved strength and sensation, allowing him to walk unassisted again. He also had improved appetite and food tolerance with a return to a more appropriate weight.

This case further supports the use of liver transplantation for the treatment of the clinical manifestations of MNGIE in place of HSCT, particularly for those patients presenting with severe liver disease. However, some of the postoperative metabolomic findings suggest long-term monitoring of certain metabolites may be necessary. Nonetheless, a larger sample size and longer follow-up is necessary to fully understand the appropriate use of this therapeutic intervention.



Presenter: Tal Yardeni

Authors: Tal Yardeni1, Ceylan E. Tanes2, Kyle Bittinger2, Lisa M. Mattei2, Deborah G. Murdock1,Douglas C. Wallace1,3

Institution: 1 Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia; 2Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia;3 Department of Pediatrics, Division of Human Genetics and Metabolism, University of Pennsylvania.

Title: Mitochondrial Influence on the Gut Microbiota Community

Abstract: Changes in the gut microbiota and the mitochondrial genome have both been linked with the development of disease. The question then becomes does the gut microbiome cause the disease or do mitochondrial alterations determine both the microbiome composition and the animal phenotype? Since an individual’s mitochondrial genotype cannot change while his microbiota can, we hypothesize that changes in mitochondrial function control both the animal’s phenotype and gut microbiota composition. To investigate our hypothesis, we examined the gut microbiota of mouse models harboring various mitochondrial DNA and nuclear DNA variants in mitochondrial genes.

These studies revealed that mitochondrial genetic variation directly affects the composition of the gut microbiota community. Moreover, in cross-fostered studies of pups, although the initial microbiota community was that obtained from the nursing mother with different genotype, the microbiota community shifted back toward that characteristic of the pup’s genotype within two months.

Analysis of the physiological effects of the mitochondrial genetic variants suggested that an important influence on the gut microbiota was mitochondrial reactive oxygen species (ROS) production. To confirm that the abundance of ROS could alter the gut microbiota, mice were permitted to age, treated with N-acetyl-cysteine, or genetically combined with the mitochondrially-targeted catalase. All three treatments altered the microbiota from that initially established due to the inherited mitochondrial gene variants.

Thus, the mitochondrial genotype is important in determining both the animal phenotype and the species diversity of the gut microbiome, suggesting that the connection between the gut microbiome and common disease phenotypes might be due to a common underlying mitochondrial genotype.

Abstract #: UMDF 2019 PA-0539

Presenter: Piotr K Kopinski

Authors: Piotr K Kopinski1,2,3, Kevin A Janssen4, Patrick M Schaefer3, Sophie Trefely5,6, Caroline E Perry3, Prasanth Potluri3, Jesus A Tintos-Hernandez3, Larry N Singh3, Kelly R Karch4,Sydney L Campbell5, Mary T Doan6, Helen Jiang6, Itzhak Nissim7, Eiko Nakamaru-Ogiso6,Kathryn E Wellen5, Nathaniel W Snyder6, Benjamin A Garcia4, Douglas C Wallace* 3,8

Institution: 1Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104, USA, 2Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, 3Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Phiadelphia, PA 19104, USA, 4Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, 5Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, 6Drexel University, A.J. Drexel Autism Institute, 3020 Market Street , Philadelphia Pennsylvania 19104 , USA,


7Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA, 8Department of Pediatrics, Division of Human Genetics, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

Title: Regulation of Nuclear Epigenome by Mitochondrial DNA Heteroplasmy

Body of abstract: Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part due to differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A>G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality1. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs2. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially-derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. By contrast, α-ketoglutarate (αKG) levels increase at mid-level heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, while nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause metabolic and histone modifications which explain the transcriptional and phenotypic variability of mitochondrial disease.


Presenter: Matthew C. Dulik

Authors: Matthew C. Dulik1,2, Jorune Balciuniene1, Pushkala Jayaraman1, Hou-Sung Jung1, Juliana Troiani1, Emily Fan1, Morgan Gerace3, Kajia Cao1, Michael Gonzalez1, Chao Wu1, Tolga Ayazseven1, Mahdi Sarmady1,2,4, Robert Wilson1,2,4,5, Doug Wallace2,5, Marni Falk2,3,6

Institution: 1 Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 2 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, 3 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 , 4 Division of Pathology Informatics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, 5 Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 6 Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104.

Title: The CHOP MitoGenome Sequencing and Deletion Clinical Test

Body of Abstract:

Background: Clinical grade interrogation of the mitochondrial genome is essential for patients suspected to have a primary mitochondrial disorder. Mitochondria and their mitogenomes have unique biological features (high copy number, heteroplasmy, NUMTs, etc.) that require special considerations when designing and performing molecular diagnostic testing.

Methods: Here we present the CHOP MitoGenome Sequencing and Deletion clinical test that is validated as a high quality, accurate and sensitive assay. This was achieved by comprehensive


consideration of assay design, equipment performance and bioinformatics tools. Briefly, multiple long range PCRs specifically target full length mitogenomes using high fidelity polymerase followed by independent library preparation and sequencing. The bioinformatics pipeline was extensively validated through the use of positive controls, sample mixtures and manual checking and verification of parameters for all true and false positives variant calls. Finally, signal to noise ratios were examined to assess limits of detection for these assays on our HiSeq 2500 platforms. A separate PCR is performed directly from original blood specimens to compare homoplasmic variants against NGS results to ensure sample integrity is maintained throughout the testing procedure.

Results and Conclusions: Due to the thorough design of this assay, we are able to consistently ensure high quality results for our patients. By using comparisons of data generated independently for the two full length mitogenome libraries, we can simultaneously confirm any identified variants as well as the percentage of heteroplasmy. We can also control for sample swaps during PCR and library preparation. The nature of sequencing mitogenomes with long, detailed haplotypes allows for the identification of contamination by other specimens during DNA extraction, PCR or NGS library preparation. PCR artifacts are readily recognized and potential NUMT interference excluded. Overall, this design results in an extremely sensitive method for accurately identifying mitogenome variants with high confidence including variants at low level heteroplasmy (down to 1%).


Presenter: Yoshitsugu Oikawa

Authors: Yoshitsugu Oikawa1, Rumiko Izumi1, Masashi Koide1, Yoshihiro Hagiwara1, Makoto Kanzaki1, Naoki Suzuki1, Masashi Aoki1 and Takaaki Abe1

Institution: 1 Tohoku University Graduate School of Medicine, Japan

Title: Mitochonic Acid, MA-5 Could be a Candidate Drug for Sporadic Inclusion Body Myositis (sIBM)

Body of Abstract: [Background] Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy after age 50 years and there is no effective treatment. Recent studies reveal that the reduced mitochondrial enzyme activity and mitochondrial dysfunction are involved in the pathogenesis of sIBM. Furthermore, we invented a new candidate drug for mitochondrial disease MA-5and reported (Tohoku J. Exp. Med. 2015, J. Am. Soc. Nephrol. 2016, EBioMedicine. 2017). So, the aim of this study is to clarify the mitochondrial function and effect of MA-5 on sIBM patient’s myoblasts.

[Method] Myoblasts from 3 cases of sIBM patients were collected. The GDF15 level in patient’s serum and the cell culture medium were measured. The cell-protective effect of MA-5 under oxidative stress inducer, BSO and the cellular ATP level was also determined.

Mitochondrial bioenergetic function was determined by flux analyzer. We also evaluated the mitochondria shape by electron microscopy and dynamics by confocal microscopy.

[Result] The GDF15 level in sIBM patient’s serum was elevated in sIBM patient’s serum and mitochondrial bioenergetics was decreased in the sIBM patient’s myoblasts, suggesting the mitochondria dysfunction. The cell death was induced by BSO and MA-5 cancelled it in a dose-dependent manner. The GDF15 level in the culture medium were increased by BSO and that the elevated level decreased by MA-5. In addition, cellular ATP level increased by MA-5. By electron microscopy, damaged cristae shape in the sIBM patient’s myoblast was repaired by MA-5. Furthermore, MA-5 significantly improved the mitochondrial dynamics in sIBM myoblasts.


[Conclusion] Mitochondrial dysfunction was detected in sIBM patient’s myoblasts and MA-5 recover the cell viability, morphology and dynamics, suggesting that MA-5 could be a novel therapeutic drug for the treatment of sIBM. GDF15 was also found to be useful not only for a diagnostic but also as a therapeutic marker or MA-5 treatment.


Presenter: Rachel T. Cox

Authors: Rachel T. Cox1,2, Aditya Sen1, Maithili Saoji1, Kelsey M. Sheard1,2, Sarah A. Thibault-Sennett1,3

Institution: 1Uniformed Services University, Department of Biochemistry and Molecular Biology, 2Uniformed Services University, Graduate Program in Molecular and Cell Biology, Bethesda, MD, 20814, 3present address American Society of Human Genetics, Rockville, MD, 20852.

Title: Modeling mitochondrial disease in Drosophila: how loss of mt-RNase P and the ribonucleoprotein Clu cause decreased mitochondrial function and tissue damage.

Body of Abstract: In order for mitochondria to perform their myriad of functions, the organelle requires 37 products encoded by mitochondrial DNA (mtDNA) and over one thousand encoded in the nucleus. Many nucleus-encoded proteins are also required to transcribe, translate and process mtDNA. Loss from either source leads to mitochondrial disease whose etiology is complex. We use the model organism Drosophila to determine the genes and molecular mechanisms that support mitochondrial function. Drosophila is an excellent model because its body contains well-defined and complex organsystems that are sensitive to mitochondrial perturbations, 75% of human disease genes have a single homolog in flies, and Drosophila mtDNA encodes for the same products as human mtDNA. We study mtDNA transcript processing by mitochondrial RNase P (mt-RNase P). mt-RNase P is a three-protein complex that cleaves the 5’-end of mt-tRNAs which is particularly important for mtDNA as it also cleaves the neighboring mRNA and rRNA from the polycistronic transcript. Patients suffering from mitochondrial disease have been identified with mutations in each of the three proteins. We have shown mt-RNase P is essential in Drosophila and flies lacking each of the three proteins have abnormally processed mtRNA transcripts. We are currently determining how loss of mt-RNase P affects mtRNA junctions in different contexts, determining the tissue specific effects in skeletal muscle and heart that occur from loss of mt-RNase P, and designing Drosophila with human pathogenic mutations in order to better understand the diseases. A second project in the lab involves determining how Clu/Cluh functions in mitochondrial protein import. Drosophila Clu is a ribonucleoprotein that preferentially binds nucleus-encoded mitochondrial mRNAs and the ribosome and appears to be involved in mRNA metabolism. Clu forms dynamic stress-sensitive ribonucleoprotein particles called bliss particles in the cytoplasm. Insulin signaling is necessary and sufficient for particle formation, and lack of vertebrate Cluh was shown to disrupt mitochondrial and cellular metabolism. Clu mutants have decreased mitochondrial protein. In addition, Clu physically and genetically interacts with the PINK1/Parkin mitophagy complex. We hypothesize that Clu could act as a quality control sensor to link mitochondrial protein import with mitophagy. The goal of these lines of research are to understand the basic cellular mechanisms that underlie mitochondrial dysfunction in the diseased state. With this knowledge, it may be possible to better target the root cellular causes of mitochondrial diseases for future therapies.



Presenter: Charles E. McCall

Authors: Peter W. Stacpoole

Institutions: 1Wake Forest University School of Medicine, Winston-Salem, NC, 27101. 2University ofFlorida College of Medicine, Gainesville, FL, 32610

Title: Targeting the PDC/PDK Axis for Inflammatory Shock Syndromes in Inborn Errors ofMetabolism

Background: A recent review identified sepsis and pneumonia among the most common causes ofdeath in children with mitochondrial diseases (Pediatr Neurol 66:82, 2017). Other studies have emphasized the role immunometabolic paralysis in the metabolic decompensation of infected patients with inborn errors of metabolism (Mol Gen Metab 121:283, 2017; Metabolism 81:97, 2018).

We found that pyruvate dehydrogenase kinase (PDK) inactivation of the critical mitochondrial bioenergy homeostat, pyruvate dehydrogenase complex (PDC), creates an imbalance between anabolic stress resistance and catabolic stress tolerance mechanisms mechanistically associated with septic shock anddeath (JCI-I 3: pii99292, 2018). Upregulation of PDK promotes a metabolic crisis in energy conservation that impedes recovery from inflammation-associated shock syndromes. Such perturbation of the PDC/PDK axis disrupts Krebs cycle anabolic activity, in part by increasing conversion of cis-aconitase toitaconate, which inhibits succinate dehydrogenase activity and, ultimately, ATP production. Itaconate also increases expression of the pro-inflammatory protein A20 that promotes TNF-induced apoptosis andactivates transcription and protein expression of antioxidant genes. The prototypic modulator of the PDC/PDK axis is the pyruvate analog and PDK inhibitor dichloroacetate (DCA), which has been used for decades as an investigational drug for the treatment of several congenital and acquired disorders ofmitochondrial fuel metabolism. We recently showed that DCA reactivates PDC, increases anabolic glucose oxidation and reverses shock, restores organ and immune function, and markedly improves survival in septic shock (ibid, 2018). Others report similar findings using DCA to treat animal models ofmyocardial ischemia/reperfusion injury, hemorrhagic shock and traumatic brain injury. Here, we provide new mechanistic evidence of how DCA stimulation of the PDC homeostat restores Krebs cycle function to repress catabolic and restore anabolic energetics.

Methods. We used unbiased metabolomics (Metabolon, Inc., Raleigh, NC) in a published THP-1 monocyte cell culture model of extreme stress that simulates the in vivo biochemical perturbations observed inflammatory shock syndromes in mice. Cell cultures of naïve monocytes received 5 mM DCAor saline (control) for 30 minutes before exposing cells to high dose endotoxin (1 ug/ml) and were observed for up to 24 h. We also investigated DCA’s effects on monocytes tolerized to endotoxin for 24 h and re-stimulated for 3 or 8 h. Metabolon provided bioinformatics and statistical analyses.

Results: DCA stimulation of PDC reversed the endotoxin-induced temporal increases in itaconate, concomitant with increasing levels of the anabolic Krebs cycle oxidative signaling pathways. DCA also reversed itaconate levels in pre-tolerized monocytes. Surprisingly, DCA significantly stimulated branched-chain amino acid catabolism by the branched chain ketoacid dehydrogenase complex, thereby providing acetyl CoA needed for anabolic biosynthetic processes. DCA also increased glutathione synthesis and reduced oxidant stress.

Conclusion: We conclude that DCA stimulation of the PDC homeostat restores Krebs cycle anabolic function during severe stress responses by decreasing cis aconitate metabolism to itaconate. DCA isnow undergoing a pivotal trial in children with PDC deficiency, a cause of cellular energy failure in a population at high risk of succumbing to infection and other acute energy crises. Identifying the


previously unrecognized mitochondria anabolism and catabolism axis supports testing DCA as a safe, novel, rapid-acting and precision-based intervention in inflammatory shock syndromes from sepsis, blood loss, trauma, burns and extreme hypoxemia in patients with inborn errors of metabolism.


Presenter: Amutha Boominathan

Authors: Caitlin Lewis1, Bhavna Dixit1, Carter Hall1, Matthew O’Connor1 and Amutha Boominathan1

Institution: 1SENS Research Foundation, Mountain View, California, 95051, USA

Title: Optimized allotopic expression of mtDNA genes

Body of Abstract: Mutations in mitochondrial DNA (mtDNA) can be inherited or occur de novo at any stage during the life span in humans. Such mutations lead to several debilitating myopathies and current treatment options for such diseases are still in their developing stages. Nuclear expression of mtDNA genes also called “allotopic expression” is one viable approach to treat such disorders. Despite several decades of research in the area, successful allotopic expression of mtDNA genes is still challenging and often controversial.

Here, we applied codon optimization to re-engineer protein coding genes in the human mitochondrial genome and assessed for protein expression. We synthesized 1) codon optimized and 2) minimally codon corrected (recoded) versions for all 13 of the mtDNA coded proteins and tested for transcription and translation. Under transient conditions, all 13 codon optimized constructs exhibited significantly higher protein expression in mitochondria enriched fractions compared to their minimally recoded counterparts. Similarly, steady state mRNA levels for the optimized gene constructs were several fold enriched (~5-200 fold) over recoded constructs in stably selected HEK293 cells. Many of the Complex I protein subunits namely: ND1, ND2, ND3, ND4, ND4L, ND6, and COX2 from Complex IV and ATP8 from Complex V can be robustly expressed following stable selection under these conditions. We assessed the utility of this approach by expressing re-engineered mtDNA gene constructs in cybrid models harboring specific mutations. While some of the allotopic constructs, such as those for ATP8 and ND1 could recapitulate functional rescue, certain others such as ATP6 could not. We hypothesize that codon optimization is a pre-requisite in efficient allotopic expression, however, many of the mtDNA genes will require further engineering in order to achieve appropriate processing and import and restore functional capabilities.



Authors: Fernando Scaglia1,2,3, Allison Tam*1,2,4, Noura S. AlDhaheri*1,2,5, Krupa Mysore6, Mary Elizabeth Tessier6, John Goss2,7, Luis A. Fernandez8, Anthony M. D'Alessandro8, Jessica Scott Schwoerer9, Sarah H. Elsea1, and Gregory M. Rice9.

Institutions: 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston TX, USA; 2 Texas Children’s Hospital, Houston, TX, USA; 3 BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR; 4 Division of Medical Genetics, Department of Pediatrics, University of California San Francisco, California, USA;5 Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE; 6 Department of Gastroenterology and Hepatology, Texas Children’s Hospital, Houston, TX, USA; 7 Division of Abdominal Transplantation,


Baylor College of Medicine, Houston, TX, USA; 8 Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 9 Department of Pediatrics and the Waisman Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA

Title: Liver Transplant: Promising Therapeutic Approach for Patients with Ethylmalonic Encephalopathy

Body of the Abstract:

Background: Ethylmalonic encephalopathy (EE, OMIM #602473) is a rare autosomal recessive disorder caused by biallelic pathogenic variants in ETHE1. It is characterized by neurodevelopmental delay and regression, prominent pyramidal and extrapyramidal signs, recurrent petechiae, chronic diarrhea, and orthostatic acrocyanosis. Laboratory findings include elevated serum levels of lactate and C4-C5acylcarnitines, and elevated urinary excretion of ethylmalonic acid and C4-C6 acylglycines, notably isobutyrylglycine and 2-methylbutyrylglycine. Clinical and laboratory findings are attributed to deficiency of the mitochondrial sulfur dioxygenase resulting in toxic accumulation of hydrogen sulfide metabolites in vascular endothelial cell and mucosal cells of the large intestine leading to the characteristic manifestations in EE including vascular damage in the brain, gastrointestinal tract and peripheral vessels in addition to the observed biochemical abnormalities. Medical management has thus far been directed towards decreasing the accumulation of hydrogen sulfide metabolites using a combination of metronidazole and N-acetylcysteine. More recently, liver transplant has been reported as a new therapeutic option for EE (Dionisi-Vici et al., 2016).Case report: Here we report two patients, including a 13-month old female who presented with clinical features consistent with EE and a newborn male who was suspected to have EE due to the diagnosis of an affected sibling. Both patients had biochemical and molecular confirmation of EE. Both patients underwent liver transplantation at 19 and 13 months of age, respectively. They had clinical and biochemical improvements after liver transplantation. The second case serve as the longest developmental outcome follow-up observed, thus far, following liver transplant for EE. Conclusion: This observation since the original report in 2016 is an additional evidence to validate liver transplant as a promising therapeutic approach for better clinical outcome for what was considered to be a fatal disease until recently.


Presenter: Kirsten E. Hoff

Authors: Kirsten E. Hoff1, Jessica L. Wojtaszek1, Matthew J. Longley1, R. Scott Williams1, William C. Copeland1

Institution: 1Genome Integrity and Structural Biology Laboratory, NIEHS. NIH, DHHS, Research Triangle Park, NC 27709, USA

Title: A Novel Function for the Polymerase Accessory Subunit in Regulating mtDNA copy Number

Body of Abstract: Mitochondria possess a 16.5 kilobase circular DNA genome (mtDNA) containing 37 genes, including 13 that encode components of the electron transport chain, 22 transfer RNAs and 2 ribosomal RNAs. Human mtDNA is copied by the DNA polymerase gamma (Pol ) which is the only known replicative polymerase in mitochondria. Pol is composed from two nuclear encoded subunits: the catalytic subunit encoded by the POLG gene, and a smaller homodimeric accessory subunit encoded by the POLG2 gene. Mutations in POLG or POLG2 adversely affect mtDNA maintenance and cause depletion, deletion and mutation of mtDNA that leads to disease. As the catalytic subunit, POLG harbors the DNA polymerase and exonuclease activities of Pol , whereas POLG2 promotes processivity and enhances


Pol ’s affinity to DNA. POLG2 also exhibits intrinsic binding to double-stranded DNA (dsDNA), although the biological purpose of such binding has remained a long-standing mystery. We examined the DNA binding role of POLG2, and we now report a crystal structure of POLG2 bound to dsDNA. The structure clearly identifies four dsDNA binding loops, two of which are novel and provide critical insight into POLG2 function. We present additional biochemical studies demonstrating POLG2’s ability to bind dsDNA is not essential for stimulation of processive DNA synthesis by Pol . Furthermore, expression of POLG2mutants that disrupt dsDNA binding causes changes to mtDNA copy number and 7S DNA in cultured HEK293 cells. Taken together, our data help to define a biological role for DNA binding by POLG2 which could function to regulate mtDNA levels in vivo.


Presenter: David Bodenstein

Authors: Bodenstein D1, Kim HK1, Brown N1, Navaid B1, Young LT1,2,3, Andreazza AC1,2

Institution: 1Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada, 2Department of Psychiatry, University of Toronto, Toronto, Canada, 3Centre for Addiction and Mental Health, Toronto, Canada.

Title: Role of Mitochondrial DNA Content and Oxidation in Bipolar Disorder Across Brain Regions

Body of Abstract: Bipolar disorder is a complex and chronic mood disorder that severely impacts the quality of life of patients, in addition to family members and partners. However, the underlying pathology remains unknown, though there is accumulating evidence suggesting a role mitochondrial dysfunction. Notably, patients with mitochondrial disease have a higher prevalence of psychiatry conditions. In this study we aim to further characterize mitochondrial dysfunction in bipolar disorder by evaluating mitochondrial dysfunction markers in different brain regions, specifically mitochondrial complex I NDUFS7 levels, mtDNA content, common deletion, and oxidation in the Broadmann area 24, cerebellum, hippocampus, and prefrontal cortex from patients with bipolar disorder, schizophrenia, and non-psychiatric controls. Our results shown significant decreases in NDUFS7 levels in the prefrontal cortex of patients with bipolar disorder, while mtDNA content was significantly increased in the hippocampus of patients with bipolar disorder. Both revealed significant brain region effects, in addition to correlating positively with each other. Furthermore, we demonstrate that there was no change in mtDNA common deletion. Lastly, mtDNA oxidation was significantly decreased in patients with bipolar disorder and schizophrenia in the Broadmann area 24 and cerebellum, respectively. Our findings are consistent with previous studies, which foundsignificant decreases in NDUFS7 protein and mRNA expression in patients with bipolar disorder. Moreover, the significant increases in mtDNA content and decreases in oxidation were not expected and suggest potential compensatory mechanisms within the tissue to maintain mtDNA integrity, thus dysfunction may be arising from irregularities upstream of the mtDNA. Our findings are in line with the brain regions typically studied in bipolar disorder and schizophrenia, confirming the importance of the prefrontal cortex and the cerebellum, respectively, in each disorder.

Abstract#: 2019 PA-0550

Presenter: 1Timothy Etheridge

Authors: 1Timothy Etheridge, 2Bridget Fox, 2Roberta Torregrossa, 2Matthew Whiteman

Institutions: 1Dept. Sport & Health Science and 2University of Exeter Medical School, University of Exeter, St. Luke’s Campus, Magdalen Road, Exeter EX12LU, United Kingdom

Title: Mitochondria-Targeted Sulfide Delivery Molecules Improve Muscle Function in C.ElegansModels of Leigh Syndrome


Body of Abstract: The gas hydrogen sulfide (H2S) has played a pivotal role in human evolution: the first life on earth (primitive bacteria) appeared 2.3 billion years ago in a highly reducing and predominantly H2S gas containing atmosphere, and used H2S as an electron source for metabolic energy. Eukaryotic life then emerged (1.5 billion years ago) and assimilated the prokaryotic bacteria, which evolved into mitochondria, enabling eukaryotic cells to obtain metabolic energy from atmospheric H2S (i.e. sulfur syntropy). Although today’s atmosphere is overwhelmingly O2 rich (80%), human cells have retained mitochondria and have still retained the sulfur syntropy with at least four distinct enzyme systems supplying mitochondria with H2S in times of stress (i.e. as an ‘emergency fuel’). Endogenous H2S stimulates respiration by acting as an electron source in the mitochondrial respiratory chain at sulfide quinone oxidoreductase (SQOR), resulting in complex II and III stimulation, and ATP generation. This suggests molecules able to selectively deliver the gas to mitochondria, could bypass defects in ETC complexes I (and/or pathways leading to it) and potentially ‘normalise’ cellular bioenergetics. To explore this possibility, we have developed a series of novel slow release hydrogen sulfide (H2S) delivery molecules which selectively target H2S to mitochondria (mtH2SD) by several distinct mechanisms (patent awarded 2017). Our first-in-class molecule (AP39) has shown considerable therapeutic efficacy in animal models where mitochondrial dysfunction is secondary to the pathology/disease (e.g. neuroprotection after stroke and cardiac arrest and in Alzheimer’s disease) and increased cerebellar, caudoputamen, cortex and hippocampal levels of H2S indicating blood brain barrier penetration. These studies strongly suggest that mtH2SD could also be effective in disorders causedby mitochondrial defects rather than dysfunction resulting from it. To investigate this, we evaluated the effects of several classes of our mtH2SD in C. elegans as an in vivo model of complex I insufficiency (mutations relevant to Leigh Syndrome), and wild type (N2) worms. We measured muscle function (movement rate/speed) over lifespan as an index of overall mitochondrial health in vivo, and assessed muscle strength and morphology in situ using fluorescent stains to assess mitochondrial structure, and membrane potential. MtH2SD dose-dependently (p<0.0001) increased lifespan in N2 worms and delayed age-associated decline in crawling speed and muscle strength, and muscle mitochondria fragmentation indicating mtH2SD improved overall mitochondrial health. Similarly, in nuo-4 worms deficient in a specific enzymatic domain of complex I (human equivalent of NDUFA10, NADH: ubiquinone oxidoreductase) mtH2SD improved age-associated muscle movement to N2 levels i.e. mtH2SD restored/improved muscle and therefore mitochondrial function in ‘Leigh Syndrome’ worms. Interestingly, in another ETC deficient mutant orthologous to combined oxidative phosphorylation disorder in humans (i.e. generalised ETC failure), AP39 failed to restore survival, movement rate and muscle/mitochondrial health. Thus, specific mitochondrial mutations might be suited to mtH2S-based therapy (i.e. patient-tailored medicine). Collectively, our studies with mtH2SD strongly suggest that modulation of mitochondrial H2S may represent a novel therapeutic opportunity in primary mitochondrial diseases such as Leigh Syndrome, and related disorders.


Presenter: Samantha C. Lewis

Authors: Samantha C. Lewis1, Yonghong Shi2, Lauren Uchiyama1, Marina Besprozvannaya1, Maria Falkenberg2, Jodi Nunnari1

Institution: 1University of California Davis, Department of Molecular and Cellular Biology, Davis, CA 95616, 2University of Gothenburg, Department of Medical Biochemistry and Cell Biology

Title: Twinkle helicase selectively interacts with the inner mitochondrial membrane lipid cardiolipin to scaffold mtDNA replisome recruitment at ER-mitochondria contact sites

Body of Abstract:Mitochondrial DNA synthesis is linked to a small subset of endoplasmic reticulum-mitochondria contact sites, which serve to link successive events of mtDNA replication, ER-mediated mitochondrial constriction, division, and motility thus preferentially distributing nascent mtDNA within human cells. The exact role of this subset endoplasmic reticulum-mitochondria contact site in licensing mtDNA replication is not known.


We find that the helicase TWINKLE assembled into focal structures at ER-mitochondria contact sites in the absence of mtDNA. Genetic perturbation of ER-mito contacts via manipulation of CLIMP63, VAPB, or PTPIP51 protein levels significantly reduced the number of mtDNA-independent TWINKLE foci per cell. We sought to test whether the focal assemblies of TWINKLE directly interact with membrane lipids. In lipid dot blot and liposome sedimentation assays, TWINKLE selectively interacted with the anionic lipids PA, PS and CL in vitro. The recruitment of TWINKLE to liposomes containing the mitochondrial lipid cardiolipin was selectively abrogated by disease mutations in a region that links its primase and helicase domains. In cells, the pharmacological inhibition of cardiolipin biosynthesis significantly reduced both the number of TWINKLE foci at contact sites, and the number of replicating nucleoids per cell, at doses that preserved gross organelle morphology. We propose that ER-mitochondria contacts create cardiolipin-enriched domains of the inner mitochondrial membrane to recruit the TWINKLE helicase, which in turns functions to scaffold the recruitment of remaining mtDNA replisome machinery to initiate mtDNA synthesis on nucleoids destined for distribution by mitochondrial division.


Presenter: Yan Burelle

Authors: Alexanne Cuillerier1, Mathieu Ruiz2, George Cairns1, Jenna Rossi1, Caroline Daneault2,Bertrand Bouchard2, Anik Forest2, Christine Des Rosiers2, Yan Burelle1.

Institutions: 1Faculty of Health Sciences, University of Ottawa, Ottawa, Canada; 2Montreal Heart Institute &Université de Montréal, Montreal, Canada

Title: Coping with respiratory chain deficiency through adaptive optimization of the OXPHOS assembly line: Insights from the hepatic LRPPRC knockout mouse model.

Abstract: LRPPRC is a protein of the PPR family that plays a central role in the stability of mitochondrially encoded mRNA. Mutations of this protein underlies Leigh Syndrome French Canadian (LSFC) variant, a severe disorder mainly characterized by a complex IV (CIV) assembly defect that predominantly affects liver and brain and leads to unpredictable subacute metabolic crises. To better understand the pathophysiology of this disease, we have developed a mouse model harboring a liver-specific Lrpprcknockout (H-Lrpprc-/-) and performed extensive mitochondrial phenotyping. Loss of LRPPRC in the liver causes a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency destabilizes polyadenylated mitochondrial mRNAs, alters mitochondrial ultrastructure, and causes a severe CIV and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency is not limited to OXPHOS, but also includes impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which can be traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. Interestingly, we also show that in LRPPRC deficient mitochondria, residual CIV preferentially assembles in supercomplexes (SC), which allows maintaining nearly normal flux through the respiratory chain despite an 80% loss of CIV activity. This preferential incorporation and stabilization of CIV into SCs is facilitatedby the upregulation of the SC assembly factor SCAFI, and by quantitative as well as qualitative changes in cardiolipins in the SC lipid rafts. Proteomics analysis also reveal several changes that likely take place to compensate the LRPPRC-dependent translation defect, including increased mitochondrial ribosome abundance, upregulation of the mtUPR, and stimulation of the biogenesis program. Altogether, these data illustrate complex and unexpected mechanisms underlying the pathophysiology of genetic OXPHOS disorders and the diversity of adaptive compensatory changes that take place to preserve residual function.



Presenter: Sima T. Tarzami

Authors: Thomas J LaRocca1, Perry Altman

1, Andrew A Jarrah

2, Ron Gordon

1, Edward Wang,

Lahouaria Hadri1, Mark W. Burke

3, Georges E. Haddad

3, Roger J. Hajjar

1and Sima T.

Tarzami3

Institutions: 1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York,

N.Y. 10128 USA, 2Department of Medicine Tufts University School of Medicine, Boston,

M.A. 02111 USA, 3Department of Physiology and Biophysics, College of Medicine,

Howard University, Washington, D.C. 20060 USA

Title: CXCR4 cardiac specific knockout mice develops a progressive cardiomyopathy

Abstract: Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. We previously published that cardiac specific CXCR4 knockout mice show significant hypertrophy and develop cardiac dysfunction in response to chronic catecholamine exposure in an isoproterenol-induced (ISO) heart failure model. We set this study to determine the structural and functional consequences of CXCR4 myocardial knockout in the absence of exogenous stress. Cardiac phenotype and function was examined at 2-, 6- and 12-months of age to determine the regulatory role of CXCR4 in cardiomyopathy. Cardiomyocyte specific-CXCR4 knockout (CXCR4 cKO) mice demonstrate a progressive cardiac dysfunction leading to cardiac failure by 12-months of age. Histological assessments of CXCR4 cKO at 6 months of age revealed significant tissue fibrosis in knockout mice versus wildtype with a subsequent increase in gross heart weights. Furthermore, Quantitative analyses of mitochondrial morphology revealed that CXCR4 cKOs have a significantly higher numbers of mitochondria. There was also a significant difference in the size of mitochondria between the two genotypes as mitochondria were much smaller in the CXCR4 cKO group than in the wild type population. Moreover, CXCR4cKO heart shows dramatic decline in cytochrome c oxidase IV as compared to its littermate control. Thus we believe that a dramatic increase in mitochondria possibly could be the result and it acts as a compensatory mechanism for a reduction in cytochrome c oxidase and ATP synthase in these mice hearts. Our results demonstrated that CXCR4 plays a non-developmental role in regulating cardiac function and that CXCR4 cKO mice develop a progressive cardiomyopathy leading to clinical heart failure most likely due to mitochondria functional abnormalities.


Presenter: Jirair K. Bedoyan

Authors: Jirair K. Bedoyan1,2,3,4, Rosemary Hage5, Ha Kyung Shin6, Kirkland Wilson6, Suzanne D. DeBrosse1,2,3, and Douglas S. Kerr2,4

Institution: Departments of Genetics and Genome Sciences1 and Pediatrics2, Case Western Reserve University (CWRU), Cleveland, OH, 3 Center for Human Genetics, University Hospitals Cleveland Medical Center (UHCMC), Cleveland, OH, 4 Center for Inherited Disorders of Energy Metabolism (CIDEM), UHCMC, Cleveland, OH, 5 Newborn Screening and Radiation Chemistry, Ohio Department of Health Laboratory, Columbus, OH, 6 School of Medicine, CWRU, Cleveland, OH, USA

Title: Rationale and Feasibility of Newborn Screening for Pyruvate Dehydrogenase Complex Deficiencies

Body of Abstract: Pyruvate dehydrogenase complex (PDC) deficiency is a treatable neurometabolic mitochondrial disorder with high morbidity and mortality, and is currently not on the US Recommended Uniform Screening Panel. Prompt and correct diagnosis of this disorder and early initiation of therapy


(e.g., ketogenic diet) is critical for the long-term positive developmental and cognitive outcome of a child affected with primary-specific PDC deficiency, most commonly due to PDHA1 mutations. We present a rationale for newborn screening (NBS) for PDC deficiency using alanine, proline and leucine, and their ratios as screening metrics. This approach makes use of exiting analytical approaches at state NBS laboratories. We have used plasma alanine, proline, and leucine data from affected subjects with pathogenic PDHA1 or PDHB mutations to show that the screening scheme using a combination of Pro:Leu, Ala:Leu, and Ala:Pro ratios are highly sensitive but likely not specific for identifying individuals at high risk for this disorder. This approach is also likely to identify other mitochondrial disorders and other causes of lactic acidosis in newborns. We have evaluated these amino acids and their ratios from dried blood spot specimens of 45,000 de-identified newborns screened through Ohio NBS laboratory to predict the false positive rates given specific cut-offs, known frequency of mitochondrial disorders and the estimated incidence of PDC deficiency. We discuss the sensitivity, specificity, and feasibility of a tier-based NBS approach to identifying newborns with primary-specific PDC deficiency for early intervention. This screening approach lays the foundation for a NBS protocol for identifying newborns at high risk for primary-specific PDC deficiency who might benefit from early known and potential therapeutic intervention(s).


Presenter: Matthew Whiteman

Authors: 1Timothy Etheridge, 2Bridget Fox, 2Roberta Torregrossa, 2Matthew Whiteman

Institutions: 1Dept. Sport & Health Science and 2University of Exeter Medical School, University of Exeter, St. Luke’s Campus, Magdalen Road, Exeter EX12LU, United Kingdom

Title: Mitochondria-Targeted Sulfide Delivery Molecules Reverse Oxidative Damage in Friedreich’s Ataxia Fibroblsts.

Abstract: Friedreich’s Ataxia (spinocerebellar ataxia; FRDA) is a rare autosomal recessive monogenic disease caused by mutations in the Frataxin gene. Clinical manifestations include limb muscle weakness, loss of coordination and heart disorders (atrial fibrillation, tachycardia and hypertrophic cardiomyopathy). Frataxin is a mitochondrial matrix protein required for Fe-S protein assembly in electron transport chain (ETC) constituents need for cellular bioenergetics/ ATP generation. Defective frataxin results in mitochondrial iron accumulation, an overproduction of mitochondrial oxidants (resulting in oxidative stress), impaired ETC activity and ATP synthesis, and mtDNA damage. Effective therapeutic interventions are currently lacking. Several approaches to diminish oxidative stress with antioxidants, including those targeted mitochondria (e.g. Elamipretide, Catena/Raxone, mitoQ etc) or to provide ETC co-factors (e.g. nicotinamide derivatives) have been attempted, but with limited success; possibly because these drugs either combat oxidative stress or impaired ETC activity, but not both. To address this problem, we have developed a series of novel slow release hydrogen sulfide (H2S) delivery molecules which selectively target H2S (MtH2SD) to mitochondria. H2S can bypass defects in ETC complex I and provide electrons to complex II/III via sulfide quinone oxidoreductase (SQOR) to stimulate ETC activity and ATP generation, preserving/restoring cellular bioenergetics in vivo. Our first-in-class molecule (AP39) has shown considerable therapeutic efficacy in animal models of mitochondrial dysfunction (e.g. neuroprotection after stroke and cardiac arrest, and myocardial infarction) and increased cerebellar, caudoputamen, cortex and hippocampal levels of H2S indicating blood brain barrier penetration. Furthermore, AP39 (0.017-0.17 mg/kg) reversed neurological damage and brain atrophy, spatial memory defects and improved mitochondrial bioenergetics in a murine model of Alzheimer’s disease. With the above observations in mind, we evaluated the effects of several classes of our mtH2SD in fibroblasts obtained from FRDA patients and unaffected carriers (Coriell Institute, USA). Mitochondrial and cytoplasmic oxidants were assessed by in situ fluorimetry using MitosoxRed and DCF-DA respectively. ATP levels were measured by luminescence and viability by Trypan Blue. MtH2SD (each 50 nM) significantly lowered mitochondrial and cytoplasmic oxidant production, improved cellular viability and increased ATP levels under basal conditions, and after glutathione-depletion (using BSO; 10 μM). MtH2SD also increased ATP levels after complex I inhibition caused by rotenone (1 μM) treatment. These


preliminary studies suggest that modulation of mitochondrial H2S may represent a novel therapeutic opportunity in FRDA and related ataxias.


Presenter: Barrett Katz

Authors: Katz B,1 Moster M,2 Sadun AA,3 Klopstock T,4 Newman NJ,5 Vignal C,6Carelli V,7 Yu Wai Man P,8 Chevalier C,1 Blouin L,1 Taiel M,1 Sahel JA6,9

Institutions: 1GenSight Biologics, Paris, France. 2Wills Eye Hospital, Departments of Neurology and Ophthalmology, Sidney Kimmel Medical College of Thomas Jefferson University, PHL, PA. 3Doheny Eye Institute and Department of Ophthalmology, UCLA, LA, California. 4Department of Neurology, Friedrich-Baur-Institute, University Hospital of LMU Munich, Germany. 5Departments of Ophthalmology, Neurology and Neurological Surgery, Emory University School of Medicine, ATL, GA. 6Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts and Fondation Ophtalmologique Rothschild, Paris, France. 7IRCCS Institute of Neurological Sciences of Bologna, Bellaria Hospital, Bologna, Italy. 8Moorfields Eye Hospital and UCL Institute of Ophthalmology, London, UK. 9 Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France, and Department of Ophthalmology, University of Pittsburgh School of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.

Title: rAAV2/2-ND4 For the Treatment of Leber Hereditary Optic Neuropathy (LHON): 72-Week Data from the REVERSE Phase III Clinical Trial

Abstract body:

LHON is a mitochondrially inherited disease that causes bilateral central vision loss. A point mutation in the mitochondrial ND4 gene at nucleotide position 11778 accounts for 75% of LHON cases. rAAV2/2-ND4 is a gene therapy enabling allotopic expression and delivery of the wildtype ND4 protein to mitochondria within retinal ganglion cells. The clinical efficacy of rAAV2/2-ND4 (GS010) is currently being assessed in several Phase 3 trials of ND4-LHON subjects.

REVERSE (NCT02652780) is a randomized, multicenter, double-masked, sham-controlled trial of 37 LHON subjects with the G11778A-ND4 mutation. All subjects received a single unilateral intravitreal injection of rAAV2/2-ND4, with the contralateral eye receiving a sham injection. Multiple visual functions and spectral-domain OCT measurements of germane retinal anatomy were monitored for 72 weeks.

At Week 72, an improvement of +15 ETDRS letters was seen in rAAV2/2-ND4 treated eyes; sham-treated eyes also showed improvement in acuity (+12 ETDRS letters). Similarly, contrast sensitivity improved: GS010-treated and sham-treated eyes gained respectively on average +0.21 LogCS and +0.15 LogCS, compared to baseline. The proportion of GS010-treated eyes that achieved a clinically meaningful improvement of 0.3 LogCS or greater (45.9%) was statistically significantly higher than that of sham-treated eyes (24.9%; p=0.0047). A generalized estimating equation model showed drug treated eyes to be significantly more likely to achieve vision of 20/200 or better than sham-treated eyes (p=0.0012). Ganglion cell layer (GCL) volume, papillo-macular bundle thickness and total macular ETDRS thickness were significantly preserved in treated vs. sham eyes, all 3 metrics reaching statistical significance.

Seventy-two weeks after rAAV2/2-ND4 administration, a clinically meaningful improvement in visual functions and sustained preservation of LHON-relevant retinal anatomy were seen in drug-treated eyes


suggesting that the biological targets of this gene therapy were successfully engaged. Week 96 readout of results is expected in mid 2019


Presenter: Anshika Jain

Authors: Anshika Jain1, Nunziata Maio1, Anamika Singh1, Tracey A. Rouault1,

Institution: 1National Institutes of Health, National institute of Child health and Human Development, Bethesda, MD 20892

Title: Identification of a new mutation that causes Multiple Mitochondrial Dysfunctional Syndrome

Body of Abstract: Multiple mitochondrial dysfunctional syndrome (MMDS) is a genetic disorder which results in fatal infantile encephalopathy resulting in seizures and muscle weakening in the infants. This disease is caused by a mutation in NFU1 gene, which appears to prevent NFU1 from transferring an essential cofactor, Fe-S cluster to several mitochondrial enzymes such as succinate dehydrogenase (SDHB) and lipoic acid synthase (LAS), involved in the different stages of respiration. These defects in-turn cause pulmonary hypertension and cardiomyopathy in patients.

An uncharacterized missense mutation c.693A>G (p.Tyr231Cys) in NFU1 has been reported in SNP database. In this work, we characterized the molecular and physiological significance of Y231 mutation and identified the molecular phenotypes that result in MMDS. NFU1 has been previously identified as an Fe-S cluster carrier protein. We characterized the mechanism of its Fe-S cluster acquisition by establishing a direct interaction between NFU1 and Iron-Sulfur cluster scaffold protein (ISCU) via co-immunoprecipitation and yeast-two-hybrid assay. Interestingly, the interaction site on NFU1 was mapped to F230 and Y231 on NFU1 by alanine scanning. Subsequently, to examine the effect of these two residues on the physiological role of NFU1 as an Fe-S cluster carrier, we co-expressed WT-NFU1 or NFU1-FY mutant with ISCU protein in bacteria and purified holo proteins (NFU1/mutant) in vivo anaerobically (to prevent the oxidation of Fe-S cluster). Interestingly, the Fe-S cluster was detected only on the WT protein by UV-visible spectral and ICP-MS elemental analysis. In vitro however, the apo form of both WT and mutant proteins could be successfully reconstituted in the presence of excess Fe and S. Furthermore, we observed a significant decrease in the protein levels of SDHB and LAS (the NFU1 target proteins) conferred by NFU1-FY mutant in the nfu1 knockout background cell lines. These results suggest that the FY motif on NFU1 is important for a biological interaction and therefore, the cluster transfer between ISCU and NFU1, which in turn provides for the downstream proteins. We are currently in the process of conducting a MALDI/MS analysis on the proteins co-purified with NFU1 from HeLa cells to identify new Fe-S proteins targeted by NFU1. Our study not only shows a new motif on NFU1 which causes MMDS but also delineates the mechanism of Fe-S cluster acquisition by NFU1 and its downstream recipient proteins.


Presenter: Matthew Klein

Institution: BioElectron Technology Corporation, Mountain View, CA

Title: Safety and Efficacy Clinical Trial of EPI-743 in Patients with Mitochondrial Disease and Epilepsy

Body of Abstract: Seizures are a common and highly morbid symptom of mitochondrial disease and occur in up to 60% of patients with inherited mitochondrial disease. Saneto et al. recently reported that


over 90% of patients with mitochondrial disease-associated epilepsy have seizures that are refractory to traditional antiepileptic therapies. The refractory nature of seizures that occur in patients with inherited mitochondrial disease may be related to the fact that traditional antiepileptic therapies do not target the energetic pathways that underpin seizure pathology in these patients. Furthermore, traditional antiepileptic therapies have been reported to increase oxidative stress which could have the potential to exacerbate mitochondrial disease pathology. There is increasing evidence that seizure pathology is associated with ferroptosis—a form of programmed cell death that is regulated by the enzymes 15-lipoxygenase and glutathione peroxidase 4 (Gpx4). EPI-743 (Vatiquinone), a novel small molecule therapeutic in development for inherited mitochondrial disease, inactivates 15-lipoxygenase and downregulates the production of oxidized lipid signaling molecules key to the mechanism of ferroptosis. In preclinical studies, EPI-743 has been demonstrated to rescue cells from patients with mitochondrial disease and associated epilepsy by targeting 15-lipoxygenase. In previous clinical studies, vatiquinone therapy has been associated with arrest and prevention of status epilepticus, decrease in seizure frequency and decrease in seizure related morbidity in patients with refractory epilepsy and mitochondrial disease. The purpose of this trial is to study the safety and efficacy of EPI-743 in a larger population of mitochondrial disease patients with associated refractory epilepsy.

This clinical trial is scheduled to be initiated in the second half of 2019 and will be conducted at clinical sites in the United States and Europe. The trial will have a 6-month placebo-controlled phase, followed by a long-term open label extension phase. The purpose of this presentation will be to share details regarding the trial’s scientific rationale, design, inclusion criteria and endpoints.


Presenter: Claudia V. Pereira

Authors: Claudia V. Pereira1#, Susana Peralta1#, Tania Arguello1, Sandra R. Bacman1, Francisca Diaz1 and Carlos T. Moraes1,2*

Institution: 1Department of Neurology, University of Miami Miller School of Medicine, Miami FL, USA.2Department of Cell Biology, University of Miami Miller School of Medicine, Miami FL, USA. #These authors contributed equally to this study.

Title: Gene replacement reverts myopathy in mouse model with mitochondrial complex I-deficient muscle

Body of Abstract: Myopathy can affect both children and adults with mitochondrial diseases which remain with no effective treatment. In addition, the existent animal models that can mimic disease phenotypes are also scarce. To investigate whether gene replacement can be used to treat or cure mitochondrial myopathies, we generated a complex I conditional knockout mouse model lacking NDUFS3, a nuclear-DNA encoded core subunit, in skeletal muscle (smKO). Our model presented a progressive myopathy with onset at 2 months and showed undetectable levels of NDUFS3, at 15 days. The severe and progressive phenotype resulted in shortened life-span, exercise intolerance, increased lactate and mtDNA levels, accompanied by increased mitochondrial proliferation. Next, rAAV9-Ndufs3 was delivered systemically into 15-18 days pre-symptomatic old mice which effectively restored NDUFS3 levels in skeletal muscle. Furthermore, the single viral injection substantially increased smKO mice survival. To further evaluate if gene replacement could also revert the myopathy phenotype in symptomatic mice, a group of 2-month-old mice with clear signs of exercise intolerance was injected with rAAV-Ndufs3. The results showed a remarkable improvement of the biochemical parameters and mitochondrial myopathy, which persisted for at least 4 months after treatment. Our study showed that mitochondrial myopathy can be both prevented and reversed by AAV9-mediated systemic gene replacement, which may indicate that patients with advanced myopathy have a chance of recovery if the molecular defect is corrected.



Presenter: Lissa Poincenot

Authors: Lissa Poincenot1, Alexander Pearson2, Rustum Karanjia2,3,4,5

Institution: 1LHON Project, www.LHON.org, 2The Ottawa Eye Institute, University of Ottawa, Ottawa, Canada, 3Ottawa Hospital Research Institute, Ottawa, Canada, 4Doheny Eye Institute, LosAngeles, CA, USA, 5Department of Ophthalmology, David Geffen School of Medicine atUCLA, Los Angeles, CA, USA

Title: Leber’s Hereditary Optic Neuropathy – Not Just a Young Man's Disease

Body of Abstract: Background: Leber’s hereditary optic neuropathy (LHON) is the most common inherited mitochondrial disease. It is characterized by acute/subacute, painless, profound loss of central and color vision. Over 95% of LHON cases are caused by three mitochondrial DNA (mtDNA) point mutations: m.11778G>A, m.14484T>C, and m.3460G>A. The current literature generally reports that males are four to five times more likely than females to be affected by LHON, with symptom onset reported to be during late teen and young adult life. As a result, LHON is usually described as a “young man’s disease.” However, this may be a self-fulfilling prophecy, where females and members of younger and older age groups are underdiagnosed.

Objective: The goal of this project was to study the epidemiology of LHON using a large international database of people reportedly affected by LHON with knowledge of their genetic mutation.

Methods: 1,517 people affected by LHON with a known pathogenic genetic mutation were included in this study. Self-reported genetic and demographic data was collected (LHON mutation, gender, age of vision loss onset, and country). The data was de-identified and then analyzed.

Results: The data shows that both females and males can have symptom onset at any age. Unlike the traditional 5:1 male:female ratio commonly reported in the literature, we found a 3:1 male:female ratio. Interestingly, below the age of 5 and after the age of 45, the male:female ratio of those becoming affected was approximately 1:1. A dramatic peak in onset of vision loss among males was found at ages 14-26, younger than is usually reported. Female onset occurred more broadly throughout the lifespan, without any comparable dramatic peak of onset age. This study found that 10% of individuals become affected with LHON after the age of 50, twice the 5% that is commonly cited. As per the literature, we found that the m.11778, m.14484 and m.3460 mutations were the most common LHON point mutations in both males and females, with a similar age distribution.

Conclusions/Implications: This is the largest epidemiological study of LHON to date. It shows that women carrying a LHON mutation are at higher risk of losing vision than is generally expected, with 25% of those affected being female. Contrary to the existing literature, LHON is not a disease solely of young men. Rather, LHON is a disease that affects both females and males of all ages. This should prompt physicians to conduct genetic testing for LHON in all patients who meet the clinical criteria, regardless of whether they fit the demographics traditionally associated with the disease. Counseling about LHON should be offered to all maternal bloodline relatives, females and males of all ages, as they are at risk of sudden-onset legal blindness.



Presenter: Magnus J. Hansson1, 2

Authors: Magnus J. Hansson1, 2, Alvar Grönberg1, Johannes Ehinger1, 2, Michael Karlsson1, 2, Eskil Elmér1, 2

Institution: 1NeuroVive Pharmaceutical AB, Lund, Sweden; 2Mitochondrial Medicine, Lund University, Lund, Sweden

Title: The Succinate Prodrug NV354 Demonstrates Positive Effects on Motor Function andMetabolic Blood Parameters in a Model of Rotenone-Induced Complex I Dysfunction

NV354 is a prodrug of succinate in development for treatment of mitochondrial diseases caused by e.g. complex I dysfunction. The objective of the present study was to evaluate therapeutic effects of NV354 in a model of motor dysfunction and metabolic disturbances caused by the complex I inhibitor rotenone (1).

Daily intraperitoneal injections of 2.75 mg/kg rotenone induced weight loss within two days and motor function symptoms, including bradykinesia (slowness of movement) and gait instability within 24 hours. NV354 was administered ad libitum with estimated final received doses ranging from 15 to 72 mg/kg/day for four days (n=6 per group). Behavioral changes, in the form of rearing and postural instability (1), were evaluated in a blinded manner, and arterial and venous blood samples were drawn at end of study after four days.

Treatment with NV354 was associated with overall trends of improvement in health status in all dosing groups. Specifically, NV354 administered ad libitum with a final estimated daily dose of 27 mg/kg significantly improved postural instability and rearing behavior. Mean ± SD values for limb displacement in the postural instability test were 8.0 ± 0.8 cm for normal controls, 12.1 ± 1.7 cm for rotenone-treated animals, and 8.8 ± 1.1 cm for rotenone + NV354 (p<0.05). The number of rears when placed in clear glass cylinders for five minutes were 8.5 ± 2.8, 1.8 ± 2.5, and 7.8 ± 2.3 for controls, rotenone, and rotenone + NV354, respectively (p<0.05). Rotenone administration also caused changes in metabolic blood parameters, including an increase in blood lactate and plasma glucose. Treatment with NV354 reversed rotenone-induced changes in plasma glucose levels, 7.4 ± 0.5, 11.9 ± 3.3, and 7.2 ± 0.6 mM for controls, rotenone, and rotenone + NV354, respectively (p<0.05), and demonstrated a trend to attenuate the lactate increase, 2.6 ± 1.1, 5.9 ± 2.0, and 4.0 ± 2.3 mM for controls, rotenone and rotenone + NV354, respectively (p=0.16).

In conclusion, NV354 demonstrated positive effects on motor function and metabolic blood parameters in the present study, and the results are promising for the therapeutic potential of NV354 in disorders of mitochondrial complex I deficiency.

Reference:

1. Cannon et al. Neurobiol. Dis. 2009 May; 34(2): 279-290


Presenter: Neal D. Mathew

Authors: Neal D. Mathew1, Sujay Guha1, Manuela Lavorato1, Tara L. Gallagher2, Christoph Seiler2,Eiko Nakamaru-Ogiso1 and Marni J. Falk1,3

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2Aquatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104;3Department of Pediatrics, University of Pennsylvania Perelman School of medicine, Philadelphia, PA 19104


Title: Characterization of novel NDUFS2 and NUBPL zebrafish animal models of complex I deficient Leigh syndrome, with validation of therapeutic leads identified in orthologous C. elegans models

Abstract: Background: Complex I (CI) dysfunction impairs neurodevelopment and survival due to pathogenic variants in a range of gene that critically alter cellular metabolism, energy homeostasis, and reactive oxygen species production. Mutations in structural subunits or assembly factors cause a wide range of neuronal and multi-system phenotypes from Leigh syndrome to cerebellar ataxia and exercise intolerance.

Objective: Here, we report phenotypic and metabolic characterization of two novel zebrafish models of complex I disease we generated by CRISPR/Cas9 in a complex I subunit (NDUFS2) and a complex I assembly factor (NUBPL). These models permit cross-species validation of leads identified in high-throughput drug screens underway in C. elegans genetic mutant model of complex I NDUFS2 and NUBPL disease.

Methods: High-throughput analysis of targeted therapeutic leads and an FDA-approved drug library is underway using C. elegans worms that harbor recessive genetic mutations in NUBPL (tm3754) and NDUFS2 (gas-1(fc21)). Primary screen is performed with a high-throughput imaging technique, WormScan (Mathew et al. 2016) that provides an integrated analysis of animal motility, mortality, brood size, and growth. Preliminary hits are then evaluated by automated lifespan and healthspan (activity) analyses at a range of concentrations using a custom robotic analysis system (WormCamp) and on mitochondrial membrane potential and oxidative stress response by Biosorter (COPAS) quantitation. Candidate hits identified in C. elegans screens are validated in CRISPR/Cas9 NUBPL-/- and NDUFS2-/-

knockout zebrafish lines by automated neurobehavioral startle test and gray brain (Leigh syndrome) phenotype.

Results: NUBPL-/- and NDUFS2-/- knockout zebrafish lines have been established by CRISPR/Cas9 within the CHOP Aquatics Core Facility and phenotypically characterized at multiple levels. Specifically, NDUFS2-/- zebrafish have an 80% reduction of complex I ETC activity by 7 days post fertilization (dpf), lack swim bladder development, are hypotonic, and have a 66% reduced swimming activity in dark. NUBPL-/- zebrafish larvae have impaired swim bladder development, and 80% reduced swimming activity in dark. Interestingly, NUBPL-/- also display impaired balance involving circular looping movements upon tail stimulation. This phenotype is consistent with known cerebellar ataxia in NUBPL patients, with additional mechanistic and therapeutic screening studies underway. Glucose at physiologic concentrations (10 mM), which we previously found to rescue lifespan in NDUFS2 (gas-1(fc21) mutant worms, similarly recues swimming activity in the NDUFS2-/- zebrafish, allowing glucose to be used as an effective positive control for high throughput screens underway to identify potent compounds that effectively rescue development, stress resilience, activity, and animal survival.

Conclusion: We have now established matched cross-species C. elegans and zebrafish stable genetic mutant models for both a complex I structural subunit (NDUFS2) and assembly factor (NUBPL), which display unique disease phenotypes at the levels of animal survival, neuromuscular behaviors, swimming activity, and organ-specific physiology. Therapeutic modelling across these evolutionarily conserved model systems will enable prioritization and optimization of a consistent lead therapeutic regimen, including one that may be repurposed from the FDA drug library screen to improve neurodevelopmental function in complex I disease patients with Leigh syndrome.


Presenter: Neal D. Mathew, PhD

Authors: Manuela Lavorato1, Neal D. Mathew1, Elizabeth Herman1, Julian Ostrovsky1, Sujay Guha1, Eiko Nakamaru-Ogiso1, Marni J. Falk1,2

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department ofPediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2Department


of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA19104

Title: Physiological, morphological and behavioral characterization of novel C. elegans and zebrafish models of FBXL4-based mitochondrial respiratory chain disease.

Body of Abstract: FBXL4-related encephalomyopathic mitochondrial DNA depletion syndrome is an autosomal recessive severe, multi-systemic mitochondrial disease with 47 known pathogenic variants reported to date. While FBXL4 protein function is poorly understood, FBXL4 deficiency leads to variable levels of mitochondrial depletion, multiple respiratory chain complex deficiencies, and pronounced lactic acidemia. To better understand FBXL4 functions and therapies, we characterized novel C. elegans and D. rerio models of FBXL4 disease, which share 23% and 65% protein similarity to human FBXL4. Brood size, larval development, body length, neuromuscular activity, and lifespan were characterized in fbxl-1(vc3038) C. elegans strain with a homozygous 707 base pair deletion (ok3741 allele). fbxl-1(vc3038) knockout worms had significant growth delay with reduced median survival (by 13%) and brood size (by 58%) relative to wild-type (N2 Bristol) worms, p<0.0001. Worms’ neuromuscular function was also impaired, with significantly reduced motility (by 60%, body bends/min) and increased, uncoordinated pharyngeal pumping (by 16%), p<0.001. Zebrafish homozygous for a missense mutant FBXL4 allele, fbxl4sa12470,were characterized with microscopy and behavioral assays. No gross morphological defects were seen by stereo microscopy from 0 to 7 days post fertilization (dpf) in any organ of the fbxl4sa12470 larvae, not overt mitochondrial morphology abnormalities in tail muscle by transmission electron microscopy of 7 dpf larvae. However, TEM at 7 dpf did show increased lipid droplets and mildly damaged mitochondria in fbxl4sa12470 liver. Automated video-tracking analysis of fbxl4sa12470 swimming activity (Zebrabox, Viewpoint) also showed decreased swimming activity at 6 dpf relative to wild-type (AB) larvae. Overall, these data demonstrate that FBXL4 deficiency can be effectively modeled in simple translational animals, offering robust translational platforms in which therapies can now be modeled to reverse organ-level and behavioral dysfunction directly relevant to the human disease.


Presenter: Hyunjin Jeong

Authors: Hyunjin Jeong1, Mikaela Dimick1,2, Alysha Sultan1,2, Angela Duong1, Sarah Sohyun Park2,Ben I. Goldstein1,3,4, Ana C. Andreazza1,3,5

Institution: 1Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Canada; 2Centre for Youth Bipolar Disorder, Sunnybrook Health Sciences Centre, Toronto, Canada; 3Department of Human Biology, Faculty of Arts and Science, University of Toronto, Toronto, Canada; 4Department of Psychiatry, Faculty of Medicine, University of Toronto, Toronto, Canada; 5Centre for Addiction and Mental Health, Toronto, Canada

Title: Peripheral Biomarkers of Mitochondrial Dysfunction in Adolescents with Bipolar Disorder

Body of Abstract:Background: Mitochondrial dysfunction has been implicated in the pathophysiology of bipolar disorder (BD). There is a great need to identify biologically relevant peripheral biomarkers to facilitate in the diagnosis and prognosis of BD. Impediment of mitochondrial oxidative phosphorylation results a shift in cellular bioenergetic homeostasis to rely on anaerobic respiration and as a result produces lactate. Lactate level have been shown to be increased in the CNS of patients with BD, compared to healthy controls. Whether peripheral lactate is increased in BD patients remain unknown. Furthermore, there exists a recent surge of investigations looking at circulating cell-free mitochondrial DNA (ccf-mtDNA) as a potential biomarker as they are released from cells under physiological stress, apoptosis, or bioenergetic compromise.


Objectives: To determine whether lactate or ccf-mtDNA differs in serum samples of adolescents with BD and to investigate its relationship with clinical characteristics.

Methods: Serum samples of adolescents with BD (n = 64) and healthy control (n = 44) were analyzed. Serum lactate level was measured using a commercially available colorimetric kit. Serum ccf-mtDNA concentration was measured using real-time quantitative polymerase chain reaction from ccfDNA purified by commercially available spin columns. Clinical demographics (e.g. sex, BMI) and depression and mania scores (KSADS-DEP-P and KSADS-MRS, respectively) were also collected.

Results: There is a statistical trending increase in serum lactate level of patients with BD compared to healthy control (U = 1102.5, p = 0.056) but not ccf-mtDNA (U = 1180.0, p 0.213). A statistically trending correlation between peripheral lactate concentration and ccf-mtDNA concentration was found (ρ = 0.187, p = 0.053). When the correlation was split by diagnosis, the significance was lost in healthy control (ρ = 0.011, p = 0.947) but statistically significant in BD patients (ρ = 0.297, p = 0.017). In the BD cohort, there was a significant correlation between KSADS-DEP-P and ccf-mtDNA (ρ = -0.296, p = 0.032).

Conclusion: Preliminary results indicate that lactate may be suggested as a biomarker for BD and ccf-mtDNA as a biomarker for depression symptomology associated with BD. Interestingly, a strong correlation between ccf-mtDNA and lactate only found in BD may indicate a disease-specific biological difference, however more research is warranted. Whether these biological and clinical relationship is consistent in adult BD patients remains unclear.


Presenter: Arianna Franca Anzmann

Authors: Arianna Franca Anzmann1, Steven M. Claypool2, Hilary J. Vernon1

Institution: 1McKusick-Nathans Institute of Genetic Medicine, 2Department of Physiology, Johns Hopkins University School of Medicine, Johns Hopkins University, Baltimore. MD 21205.

Title: Multi-omics Analysis of a New Cellular Model of Barth Syndrome Uncovers Mechanisms of Mitochondrial Dysfunction

Abstract: Barth syndrome (BTHS) is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia among other features. BTHS is caused by defects in tafazzin (TAZ), a transacylase involved in the final remodeling step of cardiolipin (CL), which results in increased monolysocardiolipin (MLCL) and decreased mature CL on the inner mitochondrial membrane. In order to explore novel areas of cellular dysfunction and identify potential targets for therapeutic intervention we applied a multi-omics discovery approach to a CRISPR-edited TAZ-deficient HEK293 cell line that was developed and phenotypically validated in our laboratory, TAZ∆45. Lipidomics analysis of TAZ∆45 cells showed the characteristic CL abnormalities seen in BTHS including a decrease in remodeled CL, a shift towards unsaturated CL, and an increase in MLCL. Proteomics analysis indicated that compared to wild type (WT), TAZ∆45 cells had differential expression of proteins involved in dynamic responses to mitochondrial stress (i.e. machinery of fission, fusion, and mitophagy) and differential expression of components of the mitochondrial respiratory chain, including decreased expression of several subunits of complex I and complex I assembly factors. Metabolomics analysis revealed decreased NAD+ and increased AMP in TAZ∆45 compared to WT, further highlighting respiratory chain dysfunction as a key feature of TAZ deficiency. Importantly, the most significantly reduced complex I assembly factor in TAZ∆45, NDUFAF1, was also present at lower abundance in multiple BTHS patient-derived lymphoblast cell lines (LCLs) compared to controls. These changes in steady state levels resulted in reduced complex I activity in TAZ∆45 and patient derived LCLs compared to their respective controls. Due to the tissue specific-nature of the clinical symptoms in BTHS (i.e. severe cardiac and skeletal muscle dysfunction, with minimal neurological effects), we aim to determine if complex I dysfunction is more prominent in those tissues with severe clinical effects.



Presenter: Ryan M. Morrow

Authors: Ryan M. Morrow1, Maria Lvova1, Douglas C. Wallace1, William L. Rumsey2

Institution: 1Center for Mitochondrial & Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA; 2GlaxoSmithKline, Stress & Repair DPU, Respiratory TAU, Collegeville, PA.

Title: Mitochondrial DNA Variation and Chronic Obstructive Pulmonary Disease

Body of Abstract: Among smokers a limited number develop the severe pulmonary pathology chronic obstructive pulmonary disease (COPD). We hypothesize that the difference between those that develop COPD and those that do not lies with genetic differences in mitochondrial function resulting from mitochondrial DNA (mtDNA) variation. This hypothesis has been supported by our recent discovery that European mtDNA haplogroup Uk decreases the risk of COPD compared to the most common European haplogroup H.

Leber’s hereditary optic neuropathy (LHON), a mid-life onset blindness caused by mtDNA missense mutations such as ND4 11778G>A, features increased disease penetrance if occurring on the mtDNA haplogroup J background and is exacerbated by smoking. These results are consistent with the hypothesis that predilection to both COPD and LHON in response to smoking stress is modulated by the mtDNA background variation.

Different mtDNAs were introduced into a 143B osteosarcoma cell line to create cybrids representing haplogroups Uk, H, and J with or without ND4 11778A. Baseline mitochondrial function was evaluated in each of the cybrid cell lines by measuring respiration and mtDNA copy number. There was a 25% decrease in state 3 respiration in ND4 11778A on haplogroup H compared to haplogroup H alone. Similarly, state 3 respiration in ND4 11778A on haplogroup J was reduced by 45% compared to haplogroup J alone. The mtDNA genotype was found to affect mtDNA copy number with the highest levels in haplogroup Uk and declining successively through haplogroups H, H11778A, J, and J11778A.Cybrids with ND4 11778A mutation on haplogroup J had an increase in superoxide levels after treatment with the oxidizing agent paraquat, which was not seen in haplogroup H cybrids. In addition, cell viability was decreased in ND4 11778A cybrids on haplogroup J after paraquat treatment. Gene expression analysis is underway to look for differences in mitochondrial biogenesis and antioxidant defense. These results indicate that mtDNA genotype significantly affects mitochondrial function of cells and their sensitivity to oxidative stress.


Presenter: Tamas Kozicz

Authors: T.M. Klein Gunnewiek1, E. J. H. Van Hugte2, M. Frega2, G. Solé Guardia2, K.B. Foreman3,D. Panneman6, K. Linda2, J.M. Keller2, D. Schubert3, D. Cassiman4, E. Morava5, R. Rodenburg6, E. Perales-Clemente7, T.J. Nelson8, N. Nadif Kasri2,3#, T. Kozicz1,5,9#.

Institution: 1Department of Anatomy, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB Nijmegen, the Netherlands; 2Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB Nijmegen, the Netherlands; 3Department of Cognitive Neuroscience, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, 6500 HB Nijmegen, the Netherlands; 4 Department of Hepatology, UZ Leuven, 3000 Leuven, Belgium; 5 Department of Clinical Genomics, Mayo Clinic, 55905 Rochester, MN, USA; 6Radboud Center for Mitochondrial Disorders, Radboudumc, 6500 HB Nijmegen, the Netherlands; 7Department of Laboratory Medicine and Pathology. Mayo Clinic, Rochester, MN 55905, USA; 8Departments of Medicine,


Molecular Pharmacology and Experimental Therapeutics, Division of General Internal Medicine, Division of Pediatric Cardiology. Mayo Clinic Center for Regenerative Medicine, Rochester, MN 55905, USA; 9Department of Biochemistry and Molecular Biology, Mayo Clinic, 55905 Rochester, MN, USA

Title: Brain-on-a-chip as a disease model of impaired neuronal development and network function in individuals with mitochondrial dysfunction

Body of Abstract: Significant patient heterogeneity exists in major depressive disorder (MDD) limiting our understanding of the neurobiology of MDD. Reducing such clinical heterogeneity by selecting a subset of well-characterized subjects from a larger cohort helps our ability to draw meaningful insights about disease-related phenotypes. Mitochondrial dysfunction has been increasingly recognized in the ethology of complex psychiatric disorders, like MDD. More than half of individuals with the condition mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), who carry the pathogenic variant m.3243A>G in the mtDNA, have MDD as a comorbidity.

To understand the underlying molecular pathology, we reprogramed skin fibroblasts from a well-characterized cohort of individuals with MELAS and comorbid MDD into induced pluripotent stem cells (iPS cells) and subsequently to excitatory cortical neurons (iNeurons). iNeurons exhibited isogenic nuclear DNA but different levels of mtDNA heteroplasmy (i.e.: 0% and >80%) in m.3243A>G mutation. We performed a deep in vitro phenotyping of iNeurons to study neural correlates of disease-associated pathology.

High levels of m.3243A>G heteroplasmy (>80%) impaired mitochondrial function, accompanied by reduced complexity of dendritic arbors. Furthermore, we found fewer excitatory synapses and consequently a lower frequency of spontaneous excitatory postsynaptic currents. Neuronal network recordings from micro-electrode arrays showed reduced neuronal network activity and a decrease in synchronous network bursts in iNeurons derived from four distinct MELAS patients with comorbid MDD.

We provide mechanistic insights on how impaired mitochondrial function disrupts neuronal structure and function, neuronal features that are impaired to various degrees in psychiatric disorders as well. Our IPS-derived neuronal model could also serve as a screening platform for identifying novel therapeutic avenues for MDD.


Presenter: Peter J. Oates

Authors: Peter J. Oates1, Jon A. Gangoiti2, James Carr1, Bruce A. Barshop2

Institution: 1Stealth Biotherapeutics, 275 Grove Street, Newton, MA 02466; 2University of California San Diego, San Diego, CA 92093

Title: Baseline Urinary Metabolite Profiles of Primary Mitochondrial Myopathy Subjects in the MMPOWER Study Compared with Literature Healthy Controls

Body of Abstract: INTRODUCTION: MMPOWER was a multi-center, randomized, double-blind, three-dose-ascending, placebo-controlled study of elamipretide (ELAM) for 5 days in 36 subjects with genetically confirmed primary mitochondrial myopathy (PMM). The study demonstrated a dose-dependent (P=0.014) increase in six-minute-walk distance (6MWD) (Karaa et al., Neurology, 90: e1212-e1221, 2018).In the high-dose ELAM group, changes in urinary adipic acid and ornithine correlated with one another and with 6MWD improvement (Muscle & Nerve, 58 (S1): S13, 2018). To further define the metabolic profile of PMM subjects, we report here an analysis of baseline urinary concentrations of selected metabolites in untreated MMPOWER subjects (group PMM) relative to healthy literature controls (group N). METHODS:MMPOWER urine metabolites were quantified by LC-ESI-MS/MS in the UCSD Biochemical Genetics and Metabolomics Laboratory. Reference concentrations of 64 energy-linked metabolites for fasted healthy subjects, 3 years of age to adulthood, were compiled from the Human Metabolome Data Base (HMDB)


and U.S. National Library of Medicine publications. HMDB listings were screened for referenced source, duplicates, and gender differences; multiple data values per publication were averaged. After interquartile range filtering, mean values for each metabolite in group N (average n=7 unique reports/metabolite) and in group PMM (n=33/metabolite) were compared using logarithmic transformation followed by univariate statistical analyses with correction for multiple comparisons at Q=1%. RESULTS: Coefficients of Variation (SD/mean) of 64 metabolites in groups N and PMM averaged 62% and 69%, respectively. Unless otherwise noted, all changes mentioned below were considered statistically significant when P<0.01. Compared to group N, PMM baseline urine concentrations for hexoses, lactate, citrate, aconitate and malate were low (14-48% of N), whereas isocitrate was elevated >2-fold. PMM succinate was also increased ~2-fold, but was variable (P>0.01 vs. N). PMM aspartate was ~8-fold elevated with ~2- to 3-fold rises in glycine, proline, serine, and threonine while ornithine was below normal (42% of N). PMM 3-hydroxybutyrate and adipate were normal, while acylcarnitines were normal or low, e.g., lauroylcarnitine and myristoylcarnitine were 4% and 18% of N, respectively. PMM urate was elevated 36% (P=0.02), while adenine, adenosine, cytidine, hypoxanthine and inosine were 4-26% of N. By contrast, uracil and orotic acid were ~8- and 10-fold above N, respectively. CONCLUSIONS: These data reveal a urinary metabolic baseline profile in PMM subjects suggesting that they exhibit marked energy thriftiness and substantial ATP expenditure to detoxify ammonia via synthesis and excretion of urea, pyrimidines and some NH3-consuming amino acids. Excess flux of ammonia presumably results from enhanced proteolysis consequent to impaired citric acid cycle energy metabolism caused by increased intramitochondrial NADH/NAD+. In addition to robust glycolysis and gluconeogenesis, increased proteolysis and extra-mitochondrial fatty acid oxidation appear to support increased energy production via succinate metabolism through Complex II. These results underpin a previous MMPOWER analysis indicating that increased energy efficiency associated with ELAM treatment in PMM is manifest by a dose-dependent hierarchy of metabolic effects: 1) stabilization or reduction of proteolysis and ATP-requiring ammonia detoxification, 2) reduction of resting fuel consumption and/or increased 6MWD capability.


Presenter: Zarazuela Zolkipli-Cunningham

Authors: Zarazuela Zolkipli-Cunningham1, Elizabeth M. McCormick1, Elle Clendenin1, Colleen C. Muraresku1, Elyse Ryan1, Kathleen Valverde1,2, Patrick F. Chinnery3, Marni J. Falk1,4,Richard H. Haas5,6

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; 2Genetic Counseling Master's Program, Arcadia University, Glenside, PA, 19038, USA;3Department of Clinical Neurosciences and MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK; 4Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; 5Departments of Neurosciences and Pediatrics, University of California San Diego, La Jolla, California, USA; 6Department of Neurosciences, Rady Children's Hospital, San Diego, California, USA.

Title: The significant impact of Mitochondrial Myopathy symptoms Background: Mitochondrial myopathy (MM) refers to genetically confirmed mitochondrial disease that predominantly impairs skeletal muscle, resulting from pathogenic variants in nuclear or mitochondrial (mt) DNA. Exercise intolerance, muscle weakness, fatigue and imbalance are reported as the most commonly experienced and motivating symptoms for clinical trial participation (Zolkipli et al., 2018). The goal of this study was to evaluate the daily impact of these most common MM symptoms, as the patient-focused drug approval process requires understanding of the direct impact of MM symptoms on patient health, functioning and overall well-being.

Methods: The study was performed following CHOP IRB approval (IRB #16-013364). Thirty-four ambulatory and non-ambulatory adult (n=14) and child (n=20) subjects with genetically-confirmed MM


followed in the Children’s Hospital of Philadelphia Mitochondrial Medicine Frontier Program were invited to participate (mean age 28 years, range 5-64 years, 47% male). Parents/caregivers completed the interview for subjects < 18 years. Daily impact of MM symptoms of muscle weakness, exercise intolerance, fatigue, imbalance and neuropathy were individually assessed in symptomatic subjects by semi-structured interview, along with an evaluation of meaningful improvement if relevant symptoms were able to be treated. Interviews were independently completed by two coordinators who recorded subject responses verbatim. A data-driven codebook (NVivo) was developed to conduct qualitative analyses by two independent coders.

Results: The most prevalent symptoms were fatigue (89%), exercise intolerance (88%) and muscle weakness (85%) among the 34 MM subjects. In subjects reporting fatigue and exercise intolerance (n=30) as their top complaints, independent walking, household chores and talking were reported to be most significantly impacted activities. Subjects reporting muscle weakness (n=29, 25% unable to walk unassisted, 30% unable to stand unassisted) as a prevailing symptom revealed functional mobility to be their biggest concern, impacting travel, personal hygiene and grooming, and preparation of meals. Subjects unable to walk with assistance desired the ability to stand. Specific upper extremity weakness impacted upon independent skills such as ability to wash hair (39%), eating meals by mouth (26%) and dressing (17%). In addition, subjects with imbalance (57%) reported concerns regarding personal safety during ambulation, while peripheral neuropathy (43%) limited driving ability. All subjects with symptomatic exercise intolerance reported interest in clinical trial participation. Meaningful improvement in symptoms were reported as incremental gains in their quality of life, including the ability to complete an extra load of laundry, being able to walk and sit unassisted for longer periods, and improved postural stability; or an attenuation in muscle cramps and fatigue.

Conclusion: This is the first study to systematically evaluate the daily impact of MM symptoms in semi-structured interviews. Results indicate the substantial limitations upon activities of daily life imposed by MM, and provide valuable insight into MM patient perception of what gains would constitute meaningful improvement in their lives.


Presenter: Shannon K Kruk BSN, RN

Authors: Shannon K. Kruk BSN, RN a, Susan E. Pacheco MD b, Mary Kay Koenig MD b, Jenna R.E. Bergerson MD, MPH c, Eliza Gordon-Lipkin MD a and Peter J. McGuire MS, MBBCha

Institution: aMetabolism, Infection and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD. bDepartment of Pediatrics, The University of Texas Health Science Center, Houston, TX, cLaboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD.

Title: Vulnerability of Pediatric Patients with Mitochondrial Disease to Vaccine Preventable Diseases

Body of Abstract: Background: Over 80% of patients with mitochondrial disease (MD) experience recurrent or severe infections. The toll infection takes on patients with MD is well recognized by clinicians; up to 50% of cases of infection can be life-threatening or result in significant neurodegenerative sequelae. Previous work by our group has shown that patients with MD can have clinical markers of immunodeficiency including increased rates of infection, memory T-cell defects, and humoral immune defects. The immune phenotype of patients with MD is evolving and is indicative of immune dysfunction.

Methods: Given the recent outbreaks of vaccine preventable diseases (e.g. measles, varicella), and the morbidity and mortality associated with infection in patients with MD, we examined the serum titer status


for most of the childhood vaccines in patients with “probable” or “definite” MD (N = 27) by revised Walkercriteria. Serum titers were determined by clinical testing at the NIH Clinical Center and Mayo Medical Laboratories.

Results: Vaccine uptake in our cohort of patients with MD was ~80% or greater for most childhood vaccines. Patients with MD displayed vulnerability to Hib (33% seronegative) measles (20% seronegative), and varicella (60% seronegative). Up to 56% of our patients with MD were found to be seronegative for 2 or more titers. Three patients treated with IVIG had normal titers for all childhood vaccinations examined.

Conclusions: Infection in patients with MD is associated with high levels of morbidity and mortality. A considerable number of patients with MD are susceptible to vaccine preventable diseases. Monitoring serum titers of patients with MD may be indicated, especially in areas with increased risk of exposure due to decreased rates of herd immunity in the general population from reduced adherence to vaccination recommendations. Understanding the vaccination status of patients with MD is an important part of maintaining the health of this vulnerable population.


Presenter: Gayatri Maria Schur

Authors: Gayatri Maria Schur1, Anna Dedio1, Sofia Miguez2, Sara Nguyen1, Kristin Wade1, Nithya Mitta1, Dah-Jyuu Wang3, James Peterson4, Suraj Serai3, Jie Nguyen3, Elizabeth M. McCormick4, Colleen Muraresku4, Amy C. Goldstein4,5, Rebecca D. Ganetzky4,5, Morgan Gerace4, Zarazuela Zolkipli-Cunningham4, Marni Falk4,5, Chamith Rajapakse2, Shana Erin McCormack1,4,5

Institution: 1Division of Endocrinology and Diabetes, The Children's Hospital of Philadelphia, PA, 19104; 2Department of Radiology, Perelman School of Medicine, University of Pennsylvania, PA 19104; 3Department of Radiology, The Children's Hospital of Philadelphia, PA, 19104; 4Mitochondrial Medicine Frontier Program, Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104; 5Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania 1Children’s Hospital of Philadelphia, PA, 19104;

Title: MRI Muscle-bone Phenotyping in Pediatric Mitochondrial Disease

Body of Abstract: Background: Impaired skeletal muscle oxidative phosphorylation (OXPHOS) capacity is a feature of primary mitochondrial disease and a wide range of other chronic conditions. Magnetic resonance imaging (MRI) may serve as a safe, non-invasive, reproducible method of quantitatively assessing muscle OXPHOS capacity in children for research and clinical purposes, including longitudinal tracking of the effects of interventions. Although our group has demonstrated the utility of creatine chemical exchange saturation transfer (CrCEST) MRI in adults1, its utility in children has not been tested.

Methods: Participants included individuals with genetic disorders affecting mitochondria (ages 7 to 25 years) and healthy controls with a similar distribution of age, sex, BMI, ancestry, and Tanner stage. OXPHOS capacity of each calf muscle was assessed using CrCEST MRI at 3T. Images are taken at baseline and following standardized plantar flexion exercise. Ectopic fat distribution in muscle is assessed by 8-point Dixon imaging and bone strength using macro- and micro-structure with digital topological analysis.

Results: Interim analysis was performed of 6 pediatric participants, (4 affected, 2 control). Affected individuals were all female, median age 13 years (IQI,12.8-15.4), and median BMI Z-score -0.05 (IQI, -1.87-1.84). Genetic diagnoses for the affected group included primary mitochondrial disorders (m.3243A,


AFG3L2, MPV17) and Trisomy 21. Controls were a 9 year old male (BMI Z-score 1.77) and a 24 year old female (BMI 18.8 kg/m2). Five out of 6 completed MRI scans were included in preliminary muscle CrCEST analysis (one requires additional analysis due to field inhomogeneities). The median recovery time constant (τCr) in controls for the medial gastrocnemius, lateral gastrocnemius, and soleus were 129 (IQI, 95-162), 223 (IQI, 196-250), and 208 (IQI, 148-267) seconds; median for the same muscle groups in affected individuals were 165 (IQI, 99-174), 236 (IQI, 139-334), and 194 (IQI, 118-268) seconds, respectively. Prolonged τCr in 2 of 3 muscle groups in the affected group suggests reduced OXPHOS capacity. Four out of 6 scans were included in bone strength assessment (due to scan availability). In controls, median trabecular and cortical bone thicknesses were 0.207mm (IQI, 0.203-0.211) and 2.32mm (IQI, 2.26-2.37), respectively, and for affected individuals, 0.201mm (IQI, 0.201-0.202) and 1.86mm (IQI, 1.75-1.96). Prolonged τCr in the affected group suggests reduced OXPHOS capacity relative to controls. Greater trabecular and cortical bone thickness in controls suggests greater bone strength relative to affected individuals, but age- and sex-adjusted comparisons will be needed.

Conclusions: CrCEST imaging prior and after controlled exercise can be used to noninvasively quantify muscle mitochondrial function in children with mitochondrial disorders. As we previously observed in adults, preliminary analysis suggested children also have the greatest difference detectable in the lateral gastrocnemius muscle. Bone structure phenotyping using MRI is also feasible in children with mitochondrial disorders. We continue to evaluate both affected and control participants across the range of ages and stages of pubertal development, with the goal of validating a clinically useful MRI-based test to quantify tissue-specific mitochondrial function in a range of pediatric diseases.

This study is funded by the Children’s Hospital of Philadelphia Mitochondrial Medicine Frontier Program.

Reference: 1DeBrosse et al., (2016) JCI Insight. (PMID: 27812541)


Presenter: Ruzzenente Benedetta

Authors: Ruzzenente B1, Assouline Z2, Barcia G2, Rio M2, Boddaert N3, Munnich A1,2, Rötig A1, Metodiev MD1

Institution: 1 INSERM UMR1163, Université Paris Descartes - Sorbonne Paris Cité, Institut Imagine, Paris, France ; 2 Departments of Pediatrics, Neurology and Genetics, Hôpital Necker-Enfants-Malades, Paris, France; 3 Department of pediatric radiology, INSERM 1000 and INSERM UMR1136, Hôpital Necker-Enfants-Malades AP-HP, Université Paris Descartes -Sorbonne Paris Cité, Institut Imagine, Paris, France.

Title: Inhibition of Mitochondrial Translation in Fibroblasts From a Patient Expressing the KARS p.(Pro228Leu) Variant and Presenting with Sensorineural Deafness, Developmental Delay, and Lactic Acidosis.

Body of Abstract: Aminoacyl-tRNA synthetases are ubiquitous enzymes, which universally charge tRNAs with their cognate amino acids for use in cytosolic or organellar translation. In humans, mutations in mitochondrial tRNA synthetases have been linked to different tissue-specific pathologies. Mutations in the KARS gene, which encodes both the cytosolic and mitochondrial isoform of lysyl-tRNA synthetase, cause predominantly neurological diseases that often involve deafness, but have also been linked to cardiomyopathy, developmental delay, and lactic acidosis. Using whole exome sequencing, we identified two compound heterozygous mutations, NM_001130089.1:c.683C>T p.(Pro228Leu) and NM_001130089.1:c.1438del p.(Leu480TrpfsX3), in a patient presenting with sensorineural deafness, developmental delay, hypotonia, and lactic acidosis. Nonsense-mediated mRNA decay eliminated the truncated mRNA transcript, rendering the patient hemizygous for the missense mutation. The c.683C>T


mutation was previously described, but its pathogenicity remained unexamined. Molecular characterization of patient fibroblasts revealed a multiple oxidative phosphorylation deficiency due to impaired mitochondrial translation, but no evidence of inhibition of cytosolic translation. Reintroduction of wild-type mitochondrial KARS, but not the cytosolic isoform, rescued this phenotype confirming the disease-causing nature of p.(Pro228Leu) exchange and demonstrating the mitochondrial etiology of the disease. We propose that mitochondrial translation deficiency is the probable disease culprit in this and possibly other patients with mutations in KARS.


Presenter: Metodi D. Metodiev

Authors: Juliette Pulman1, Benedetta Ruzzenente1, Lucas Bianchi1, Marlène Rio2, Nathalie Boddaert3, Arnold Munnich1,2, Agnès Rötig1 and Metodi D. Metodiev1,

Institution: 1INSERM UMR1163, Université Paris Descartes—Sorbonne Paris Cité, Institut Imagine, 75015 Paris, France; 2Departments of Pediatrics, Neurology and Genetics, Hôpital Necker—Enfants Malades, 75015 Paris, France and 3Department of Pediatric Radiology, INSERM 1000 and INSERM UMR1163, Hôpital Necker—Enfants Malades AP-HP, Université Paris Descartes—Sorbonne Paris Cité, Institut Imagine, 75015 Paris, France

Title: Mutations in the MRPS28 Gene Encoding the Small Mitoribosomal Subunit Protein bS1m in a Patient with Intrauterine Growth Retardation, Craniofacial Dysmorphism and Multisystemic Involvement

Body of Abstract: Mitochondria contain a dedicated translation system, which is responsible for the intramitochondrial synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides essential for the biogenesis of oxidative phosphorylation (OXPHOS) complexes I and III–V. Mutations in nuclear genes encoding factors involved in mitochondrial translation result in isolated or multiple OXPHOS deficiencies and mitochondrial disease. Here, we report the identification of disease-causing variants in the MRPS28 gene, encoding the small mitoribosomal subunit (mtSSU) protein bS1m in a patient with intrauterine growth retardation, craniofacial dysmorphism and developmental delay. Whole exome sequencing helped identify a seemingly homozygous missense variant NM 014018.2:c.356A>G, p.(Lys119Arg) which affected a highly conserved lysine residue. The variant was present in the mother in a heterozygous state, but not in the father who likely carried a large deletion spanning exon 2 and parts of introns 1 and 2 that could account for the apparent homozygosity of the patient. Polymerase chain reaction (PCR) amplification and Sanger sequencing of MRPS28 cDNA from patient fibroblasts revealed the presence of a truncated MRPS28 transcript, which lacked exon 2. Molecular and biochemical characterization of patient fibroblasts revealed a decrease in the abundance of the bS1m protein, decreased abundance of assembled mtSSU and inhibited mitochondrial translation. Consequently, OXPHOS biogenesis and cellular respiration were compromised in these cells. Expression of wild-type MRPS28 restored mitoribosomal assembly, mitochondrial translation and OXPHOS biogenesis, thereby demonstrating the deleterious nature of the identified MRPS28 variants. Thus, MRPS28 joins the increasing number of nuclear genes encoding mitoribosomal structural proteins linked to mitochondrial disease.


Presenter: James T. Peterson

Authors: James T. Peterson1, Claire Newman1, Elizabeth M. McCormick1, Rebecca Ganetzky1,2,Amy Goldstein1,2, Zarazuela Zolkipli-Cunningham1,2, Colleen Muraresku1, Donna Divito1,Sara Nguyen1,3, Kristin Wade3, Marni J. Falk1,2, Shana E. McCormack1,2,3


Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, The Children's Hospital of Philadelphia (CHOP), Philadelphia, PA 19104; 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;3Division of Endocrinology and Diabetes, Children's Hospital of Philadelphia, Philadelphia, PA 19104

Title: Energy Intake Quantitative Assessment in Patients with Primary Mitochondrial Disease

Abstract: Background: Little evidence exists regarding the merits of different approaches to nutrition in mitochondrial disease (MD), partly due to the lack of convenient, validated tools to quantify energy intake in affected individuals. Objective: We evaluated the extent to which an existing age-stratified (pediatric and adult) food frequency questionnaire (FFQ) produces valid nutrient intake estimates in individuals with MD. Results were compared with those obtained in the same participants using the more time-consuming “gold-standard” reference of three 24-hour diet recalls (3DR). Methods: Participants included genetically-confirmed primary MD patients age ≥8y who consume some food by mouth. 3DRs were conducted by the CHOP/Penn Center for Human Phenomic Science (CHPS) Bionutrition cores via telephone. Concordance between energy intake (mean kcal/day), macronutrient and micronutrient intake from 3DR and FFQ were calculated using Lin’s Concordance Correlation Coefficient (CCC). We tested the association between quality of life (QOL) scores (NMDAS/NMPDS) and 3DR outcomes. The statistical plan was formulated with assistance from CHOP/CHPS Biostatistics core, and analysis done with Stata v15.1. Results: Interim analyses are reported (n=21 with complete data, of N=58 participants enrolled to date). 71% of participants were adults, and 52% were female. By 3DR, adults (18y+; mean BMI 25.0 kg/m2, ±5.7 kg/m2 SD) consumed a mean of 89% ± 27% of their Recommended Daily Allowance (RDA) for total daily kcal, and received 48% ± 13% of kcal from carbohydrate, 33% ± 8% from fat, and 18% ± 6% from protein. Pediatric participants (mean BMI 53 ± 35%ile) consumed a mean of 100% (± 54%) of RDA for total kcal, and received 50% ± 17% of kcal from carbohydrates, 35% ± 14% from fat, and 15% ± 5% from protein. CCC between FFQ and 3DR for kcal/day data was 0.55 (95%CI: 0.25, 0.75; p=0.001). Secondary endpoint CCC’s (95%CI; p-value): Fat: 0.69 (0.41, 0.85; p<0.001) %Kcal from fat: 0.64 (0.37,0.81; p<0.001), Protein: 0.27 (0.01, 0.50; p=0.05), %Kcal from protein: 0.61 (0.28, 0.81; p=0.001), Carbohydrates: 0.54 (0.23, 0.75; p=0.001), %Kcal from carbohydrates: 0.72 (0.44, 0.87; p<0.001), Calcium: 0.43 (0.18, 0.63; p=0.001), Vitamin B1: 0.26 (0.04, 0.46; p=0.021), Vitamin B2:0.34 (0.05, 0.58 p=0.024). In qualitative analyses, participants reported that nutrition was important for maintaining activity. Barriers to receiving appropriate nutrition included financial restrictions, limited appetite, and dietary preferences. Conclusions: Although on average, the diet consumed by individuals with MD resembles population references, there is wide variability. Our preliminary data suggest the FFQ can be used to assess nutrition in individuals with MD who eat by mouth, as the CCC exceeded 0.4, which is generally considered acceptable. We identified the highest concordance between the FFQ and 3DR for % of energy intake from each macronutrient, lower concordance for absolute energy and macronutrient intake, and lowest for micronutrients. Our project will find the most appropriate uses for the more convenient FFQ tools in MD. Our overarching goal is to identify nutritional patterns that most closely associate with highest physical and mental function, and use these insights to develop and test individualized dietary interventions to optimize QOL in MD.


Presenter: Robert K. Naviaux

Authors: Kefeng Li1,2, Lin Wang1,2, Jane C. Naviaux1,5, Jonathan Monk1,2, Robert K. Naviaux1-4


Institution: The Mitochondrial and Metabolic Disease Center1, Departments of Medicine2, Pediatrics3,Pathology4, and Neurosciences5. University of California, San Diego School of Medicine, 214 Dickinson St, Bldg CTF, Rm C102, San Diego, CA 92103. Email: [email protected]

Title: Elevated 1-deoxyceramides in Patients with MELAS and NARP—Testing a New Biomarker of Mitochondrial Dysfunction

Abstract: Background. Serine palmitoyl-CoA transferase (SPT) is the rate-limiting enzyme in sphingolipid synthesis. When alanine is elevated and serine is decreased, which can occur in many inherited forms of mitochondrial disease, SPT can use alanine to make 1-deoxysphinganine. 1-deoxysphinganine is then used to make a series of 1-deoxyceramides by ceramide synthase. 1-deoxyceramides are powerful mitochondrial oxphos inhibitors and pro-apoptotic signaling molecules.

Methods. Samples of EDTA-anticoagulated whole blood stored for 7-21 years at -80˚C were analyzed. Samples from 33 patients with MELAS (A3243G; ages 0.3-51 years; median heteroplasmy = 40%, IQR = 26-55%), 12 with NARP (7 T8993G and 5 T8993C; ages 0.8-46 years; median heteroplasmy = 68%, IQR = 52-85%), and 48 age-, sex-, and storage time-matched disease controls were analyzed. Twelve different 1-deoxyceramide species were quantified by stable isotope dilution, reverse phase HPLC, and scheduled multiple reaction monitoring (sMRM) by electrospray ionization tandem mass spectrometry (LC-MS/MS).

Results. Two very long chain 1-deoxyceramide species, m18:1/22:0 (Control = 118 ± 8 (SEM) nM; MELAS = 218 ± 27 nM; p < 0.0001; NARP = 178 ± 25 nM; p < 0.005) and m18:1/24:0 (Control = 438 ± 32 nM; MELAS = 718 ± 87 nM; p < 0.001; NARP = 641 ± 83 nM; p < 0.01), were significantly increased in patients with MELAS and NARP regardless of age or heteroplasmy. A significant correlation with heteroplasmy was found by multiple linear regression analysis (r2 = 0.54; p < 0.004). Overall, 10 of 12 measured 1-deoxyceramides were increased in MELAS and at least 2 of 12 were increased in NARP.

Limitations: The results reflect whole blood concentrations. Plasma was not available at the time of this study. The effect of the long storage times is unknown.

Conclusions. Quantitative 1-deoxysphingolipid analysis shows promise as a minimally invasive diagnostic test for mitochondrial dysfunction in MELAS and NARP. 1-deoxyceramides correlate with heteroplasmy and may act as a feedback regulator of mitochondrial quality control by eliminating cells with the greatest mitochondrial dysfunction. The utility of 1-deoxyceramide analysis as a general biomarker of mitochondrial dysfunction, and the correlation with other markers like lactate, FGF21, and GDF15, is unknown.


Presenter: Amel Karaa

Author: Amel Karaa, on behalf of the MMPOWER-3 Investigators

Institution: Genetics Unit, Massachusetts General Hospital, Boston, MA 02114

Title: MMPOWER-3 Trial Update: A Phase 3, Randomized, Double-Blind, Placebo-Controlled Trial of Elamipretide in Patients With Primary Mitochondrial Myopathy

Body of Abstract: INTRODUCTION: Primary mitochondrial myopathy (PMMs) are genetically-defined disorders of the mitochondrial respiratory chain affecting predominantly, but not exclusively, skeletal muscle. Muscle Involvement may present as fatigue, exercise intolerance, and muscle weakness. These symptoms adversely affect physical function and exercise capacity, as well as quality-of-life (QoL). To date, there are no approved treatments for patients with PMM. Elamipretide, an investigational therapy being evaluated in patients with PMM, localizes to the inner mitochondrial membrane where it associates with


cardiolipin (CL), to restore cristae architecture resulting in improved ATP generation and reduced oxidative stress.

OBJECTIVES: MMPOWER-3 is an ongoing, phase-3 randomized, double-blind, placebo-controlled trial evaluating the efficacy, safety, and tolerability of elamipretide in patients with molecularly confirmed PMM. In Part 1, patients are administered daily subcutaneous (SC) injections of 40 mg elamipretide orplacebo for 24 weeks. In Part 2, eligible patients may be enrolled in a 144-week open-label treatment extension. METHODS: Here we present baseline characteristics of the subjects that have been evaluated, to date, for participation in the trial. We also provide a status on subject progress as related to trial milestones and time points. Updates to this information will be provided. RESULTS: To date, among the 224 subjects who have been screened, 147 have been randomized totreatment (average age, 44.2 years). Based on available data entries, the study population consists of37% males and 63% females. Among the 147 patients, 124 (84%) have self-identified as being white. Inaddition, genetic test results showed that the majority (111/147 [75%]) of patients have mutations inmitochondrial DNA (mtDNA), while the rest were represented by nuclear DNA defects (nDNA). To date, 93% (136/147) of these patients have had a Week-4 Visit, 71% (105/147) have had a Week-12 Visit; 49% (72/147) have completed Part 1. Of the patients who have completed Part 1 of the MMPOWER-3 trial and eligible to participate in the 144-week open-label extension portion (Part 2), only three individuals elected not to participate. CONCLUSIONS: Subjects continue to enroll into the MMPOWER-3 trial. The baseline characteristics ofthe subjects enrolled to date in MMPOWER-3 are consistent with what has been observed in the early phase elamipretide clinical development program. Full enrollment is expected to be completed soon which will enable additional detailed analyses to be presented at the meeting.


Presenter: Liam Coyne

Authors: Liam Coyne1*, Xiaowen Wang1*, Frank Middleton2, and Xin Jie Chen1,2

Institution: 1Department of Biochemistry and Molecular Biology and 2Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, NY 13210. *Equal contributors

Title: Protein Misfolding on the Inner Mitochondrial Membrane is Toxic and has Pathological Consequences

Body of Abstract: Numerous devastating neurodegenerative diseases are caused by mutations in inner mitochondrial membrane (IMM) quality control proteases. These proteases maintain proteostasis by degrading misfolded IMM proteins, suggesting the accumulation of misfolded membrane proteins may underlie disease. However, whether IMM protein misfolding is sufficient to cause disease is unclear due to the numerous additional processes IMM proteases participate in. To overcome this, we developed a system in which we express a single misfolded IMM protein, the ADP/ATP exchanger adenine nucleotide translocase 1 (Ant1)1—3. Dominant missense mutations in Ant1 are associated with progressive external ophthalmoplegia, muscle weakness, dementia, and neuropsychiatric conditions. We previously showed that equivalent mutations in Ant1’s yeast ortholog, AAC2, induce its misfolding and dominantly kills yeast cells independent of nucleotide transport. Strikingly, we report here that combining two pathogenic mutations into the same protein results in a substantial enhancement of toxicity, despite drastically reduced protein levels compared with the single mutant counterparts. Thus, severely misfolded protein in the IMM is a dominant and potent cell killer. To uncover the pathophysiological potential of IMM protein misfolding, we generated a mouse model expressing the equivalent double mutant Ant1 allele. In early litters, several heterozygous knock-in (KI/+) mice developed severe neurodegeneration, paralysis, and death around 11 months old. This suggests that IMM protein misfolding can cause neurodegeneration. Non-paralytic KI/+ mice replicate some of the dominant features of Ant1-induced disease. Like in yeast, levels of the double mutant Ant1 are severely reduced in transfected human cells, and barely detectable


in heterozygous or homozygous KI mice, consistent with its degradation due to misfolding. In summary, we established a unique mouse model to study the consequences of IMM protein misfolding, which revealed that minute levels of misfolded IMM protein are sufficient to cause disease. Contrary to popular belief, our data also indicate that dominant Ant1 diseases are unlikely to be caused by nucleotide imbalance. We speculate that targeting IMM proteostasis is a potential strategy for the treatment of dominant Ant1 diseases, as well as IMM protease-induced neurodegeneration.

This work is supported by grants from the National Institute on Aging (NIH) to X.J.C. and an F30 predoctoral fellowship to L.P.C.

References:

1. Wang, X., Zuo, X., Kucejova, B. & Chen, X. J., 2008, Nat Cell Biol. 10, 1090-7 2. Wang, X. & Chen, X. J., 2015, Nature. 524, 481-4 3. Liu, Y., Wang, X., Coyne, L. P., Yang, Y., Qi, Y., Middleton, F. A. & Chen, X. J., 2019, Mol Biol

Cell, mbcE19010046


Presenter: Xiaobai Patrinostro

Authors: Xiaobai Patrinostro1, Neil Otto1, Travis Cordie1, Jarryd Campbell2, Stephen Ekker2, David Largaespada1, Beau Webber1, Branden Moriarity1

Institution: 1B-MoGen Biotechnologies, Minneapolis MN 55413, 2Mayo Clinic, Rochester, MN 55905

Title: Generation of de-novo site specific mitochondrial gene deletions in mammalian cells

Abstract: Mitochondria are double membrane bound organelles that are found in all eukaryotic cells and carry a specialized circular 16.5kb genome. Inherited or spontaneous mutation of genes encoded within the mitochondrial genome (mtDNA) can result in devastating human genetic diseases. These diseases display a wide array of medical phenotypes from subtle muscular weakness to extreme neuromuscular difficulties such as loss of balance and coordination, seizures, stroke, dementia and death. Furthermore, it is estimated that 1 in 10,000 children are diagnosed with a mitochondrial disease each year. Despite the importance of the mtDNA in maintaining normal cellular function, as well as its role in promoting mitochondrial dysfunction when mutated, no method exists to alter the mtDNA in a site-specific manner. To address this deficiency in the field, our team has developed a novel method to introduce targeted deletions within the mtDNA utilizing MitoTALENs (Mito-TAL). Our strategy employs two paired site-directed mito-TAL nickases to ‘seed’ targeted deletions within individual mitochondrion genomes, which are subsequently enriched via heteroplasmic shift using mito-TAL nucleases that selectively deplete remaining WT mitochondrial genomes. We have efficiently induced mtDNA deletions in zebrafish embryos, and both human and mouse cell lines. In mouse NIH3T3 fibroblasts, we deleted a mtDNA region corresponding to same mtDNA deletion seen in the human Kearns-Sayre Syndrome (KSS). This deletion resulted in decreased mitochondria basal respiration, ATP production and maximal respiration. Thus, our technology represents the first reliable method to introduce site specific deletions in the mitochondrial genome, resulting in functional phenotypes in mammalian cells. We are currently expanding the innovations in mammalian cells by engineering targeted and precise mtDNA deletions systemically in mice. We will also develop inducible systems for expressing the mitoTALENs, which will enable us to create conditional loss of function of mitochondrial genes in mice. The inducible mouse model system could be highly useful in settings in which the mitochondrial deletion would cause a selective disadvantage, such as during early mouse development. The production of these novel mouse models will be valuable for both academic and pharmaceutical companies to test novel regenerative stem cell technologies.



Presenter: Peter W. Stacpoole

Authors: Delia Khayat 1, Tracie L. Kurtz 1 and Peter W. Stacpoole 1,2.

Institution: Departments of Medicine 1 and Biochemistry and Molecular Biology 2, College of Medicine, University of Florida, Gainesville, FL.

Title: The Changing Landscape of Clinical Trials for Primary Mitochondrial Diseases: 2011 to Present

Abstract: In 2011 we reviewed the status of interventional clinical trials for primary mitochondrial diseases (PMDs), noting the intellectual chasm between nonclinical research on mitochondrial biology generally and the paucity (and failure) of randomized controlled trials (RCTs) for PMDs (Mitochondrion 11:679, 2011). At that time there were no therapies for these disorders that had been approved by the U.S. Food and Drug Administration or by any similar regulatory agency in a foreign country. We now report how this landscape has changed during the ensuing 8 years.

Methods. We reviewed published data from PubMed, clinicaltrials.gov and the clinical trials registries of the WHO International Clinical Trials Registry Platform, the European Union and the Japanese National Institute of Public Health from December, 2011 through March 26, 2019, for information on the following search terms: mitochondria; mitochondrial diseases; clinical trials (mainly open label, nonrandomized); RCTs (mainly specifying a particular disease phenotype, e.g., mitochondrial myopathy, rather than a genetically discrete group) and RCTs in primary (aka genetic) mitochondrial diseases. We also reviewed the evolving literature on several related topics, including funding opportunities and challenges for rare disease RCTs; advances in developing clinical and biochemical endpoints used in clinical trials; regulatory flexibility in evaluating data from RCTs; attitudinal changes in the lay and professional communities regarding RCTs for primary mitochondrial diseases; repurposing approved therapies; potential molecular-oriented therapies; and a widening of the conceptual and therapeutic windows in the evaluation and treatment of rare and common acquired mitochondrial disorders. Results. The figure summarizes the differences in publication citations, according to specific categories.

Amajor Red: citations in original study using only PubMed (2011); light blue: citations using PubMed through March 26, 2019; dark

blue: citations using all citations listed above through March 26, 2019.


inflection point differentiates the number of Clinical Trials identified between the time periods. There is a 2.7-fold increase in RCTs for PMDs between 2011 and 2019, using only the PubMed search engine, and a 4.3-fold increase in RCTs for PMDs between 2011 and 2019, when including international registries. Funding for such trials remains challenging, especially for those that are investigator-initiated and reliant mainly or solely on peer-reviewed grant support. Many academic-based investigators may be naïve to the regulatory complexities and costs of conducting FDA-compliant trials that could lead to product approval. Nevertheless, consilience between the lay and professional mitochondrial disease communities to engage in RCTs has greatly increased, as has progress in developing new disease and treatment biomarkers and potential molecular therapies. Finally, the continued advancement of general knowledge of mitochondrial biology has increased appreciation for the fundamental role these remarkable organelles play in the etiopathology of many other rare and common illnesses. Such knowledge is leading to repurposing established drugs for PMDs and emphasizes the therapeutic potential of mitochondrially-targeted small molecules for an increasing spectrum of human diseases. Supported by FDA grant R01 FD005407 and STTR grant 4R42HD0889804 from NICHD.


Presenter: Ibrahim George-Sankoh

Authors: Ibrahim George-Sankoh1, 3,, Laura Macmullen1 , Deanne Taylor2, 3, Batsal Devkota3,Rebecca Ganetzky1, 2, Marni J Falk1, 2.

Institutions: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia PA; 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 3Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA.

Title: “MMFP Tableau: Enabling Precision Mitochondrial Medicine through Novel Integration, Visualization, and Complex Analytics of Clinical and Research Data”

Abstract: Precision Mitochondrial Medicine will require novel informatics approaches that support intuitive mining of traditional clinical evaluations integrated with objective outcome assessments at both individual patient and cohort levels. To this end, we have developed and launched a novel data management approach that readily collates, integrates, validates, and allows direct clinician visualization and complex analytic capabilities of all clinical and research data generated within The Children’s Hospital of Philadelphia (CHOP) Mitochondrial Medicine Frontier Program (MMFP).

Within health systems, data is stored in a range of siloed domains in diverse formats and for a wide variety of purposes. We sought to develop a robust data integration tool that efficiently extracts and unifies updated medical, genomic, clinical and research data collected in all potential domains within a single database server. A key concern was maintaining data integrity without duplication or loss, regular streaming updates, selective accessibility to identified vs deidentified data, and connectivity between the electronic medical record (EPIC) and research databases (REDCap, Excel, OnCore, etc).

Our data integration solution adopts a data warehouse built in Alteryx. Alteryx serves as a data staging warehouse that pulls from all desired data sources to enable sophisticated analytics for supervised and unsupervised models, allowing novel algorithms to be developed by our in-house data integration bioinformatics team that support custom analyses of high dimensionality data. These integrated data are then directly exported to a commercial resource, Tableau, which is hosted in-house in a virtual machine (VM) readily accessible via the Web with password protection by clinicians, scientists, and researchers. Tableau supports data visualization in intuitive reports and charts, with user manipulation to rapidly gain desired insights and analytics within the Tableau environment. Launched within the CHOP MMFP in 2019, this unique data integration resource now enables efficient and rapid individual patient or cohort analyses of individuals readily grouped by specific genes, mutations, laboratory values, geographic factors, age, medications, HPO-based phenotypes, procedures and assessments, and outcome


measures including survey results, among others. Indeed, any data field present in EPIC can be readily visualized in MMFP Tableau.

Our ultimate goal is to enable prioritization of precision medical care and personalized clinical trial outcome measures that leverage direct clinician mining. This will be aided by machine learning approaches to predict mitochondrial diagnosis, prognoses, biomarkers, and therapeutic response based on complex arrays of molecular, biochemical and clinical outcomes. This solution is also readily sharable to potentially expand the CHOP MMFP database with a broader national and international network of institutional partners working to provide precision mitochondrial medicine.


Presenter: Laura E.D. MacMullen

Authors: Laura E.D. MacMullen1, Amy C. Goldstein1, Zarazuela Zolkipli-Cunningham1, Rebecca D. Ganetzky1,2, Ibrahim George-Sankoh3, Deanne M. Taylor3, Batsal Devkota3, Marni J. Falk1,2

Institution: 1Children’s Hospital of Philadelphia, Mitochondrial Medicine Frontier Program, Philadelphia, PA 19104, 2Department of Pediatrics, University of Pennsylvania and Perelman School of Medicine, Philadelphia, PA 19104; 3Children’s Hospital of Philadelphia, Department of Biomedical and Health Informatics, Philadelphia, PA 19104

Title: Bridging the Clinical-Research Gap: Using Data Integration to Achieve Precision Mitochondrial Medicine through Regular Incorporation of Patient-Reported Outcome Measures in Clinical Care

Abstract: Mitochondrial disease is highly heterogeneous in genetic etiology and clinical presentation. We now recognize patients present on average with 16 different symptoms1, with highly different clinical presentations both between and within families. A blanket approach to clinical care is insufficient to manage the nuances of these complex disorders. Providing precision mitochondrial medicine requires tracking patient-specific outcomes and individually targeted therapies, a challenge that can be approached by effectively harnessing existing clinical data generated through the course of care and objectively tracking patient progress in the domains that are self-reported to matter most, including fatigue and quality of life1.Here, we report our center’s experience launching an integrated data management system that blends data from multiple different sources using high-powered predictive, spatial and statistical analytics combined with dynamic, interactive data visualization and reporting that is readily accessible to clinicians and researchers. This system allows us to regularly collect and assess definite primary mitochondrial disease patient and parent-reported data within the domains of fatigue, function, and quality of life. REDCap survey software is used to program and administer electronic surveys, which are age-dependent to account for varying developmental stages and quantitatively track over time individual patient changes in domains directly relevant to their well-being and health. This enables real-time objective assessment of the impact of disease progression or exacerbations, various clinical interventions including new or altered medications, surgical interventions or gastrostomy tube placement, and physical therapy response. To date, quality of life data have been collected on 30 mitochondrial disease patients over a one month period using the PedsQL Generic Core Scales2, with an overall completion rate of 73%. Fatigue data have been collected on 46 mitochondrial disease patients over a four month period using the Modified Fatigue Impact Scale3. Function scores were obtained using the Karnofsky4/Lansky5 Performance Status scales on 45 patients over a four month period. Survey frequency is scheduled at 3 month intervals for infants, 6 month intervals for children, 12 month intervals for adults, or at custom intervals, as needed, to track the impact of clinical interventions. Additional data are collected in an ongoing fashion as patients present for clinical care.


Real-time integration of self-reported data into our newly developed clinical-research data integration and management system, MMFP Tableau, allows for rapid visualization and charting of outcomes over time, at both patient-specific and cohort levels. Data is mapped and interpreted in contextual relation to interventions and clinical data tracked in the electronic medical record (EMR). This unique data management system allows for data-driven knowledge acquisition that supports rational approaches to treatment, evaluation and tracking of clinical outcomes, as well as rapid recruitment and targeted cohort identification for clinical trials. We continue to develop and improve upon our patient survey instruments and data integration system design to optimize performance, functionality, efficiency and usability, all in an effort to leverage data-driven insights to bring the best possible precision care and treatment opportunities to our mitochondrial disease patients.

1Zolkipli-Cunningham Z, Xiao R, Stoddart A, McCormick EM, Holberts A, Burrill N, et al. Mitochondrial disease patient motivations and barriers to participate in clinical trials. PLoS ONE. 2018; 13(5): e0197513. 2Varni JW, Seid M, Kurtin PD. PedsQL 4.0: Reliability and validity of the pediatric quality of life inventory Version 4.0 generic core scales in healthy and patient populations. Med Care. 2001;38(8):800–812.3Larson RD. Psychometric properties of the modified fatigue impact scale. Int J MS Care. 2013:15(1):15-204Schag CC, Heinrich RL, Ganz PA. Karnofsky performance status revisited: reliability, validity, and guidelines. J Clin Oncol. 1984 Mar;2(3):187-93.5Lansky SB, List MA, Lansky LL, Ritter-Sterr C, Miller DR. The measurement of performance in childhood cancer patients. Cancer. 1987 Oct 1;60(7):1651-6.


Presenter: Elizabeth M. McCormick

Authors: Elizabeth M. McCormick1, Marie T. Lott2, Matthew C. Dulik3,4, Lishuang Shen5, Marcella Attimonelli6, Ornella Vitale6, Amel Karaa7, Renkui Bai8, Daniel E. Pineda-Alvarez9, Larry N. Singh2, Christine M. Stanley10,11, Stacey Wong9, Anshu Bhardwaj12, Rong Mao13,14, Neal Sondheimer15, Vincent Procaccio16, Douglas C. Wallace2,3, Xiaowu Gai5,17, Marni J. Falk1,3

Institutions: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; 2Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; 3Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; 4Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; 5Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA; 6Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy; 7Genetics Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; 8GeneDx, Gaithersburg, MD 20877, USA; 9Invitae, San Francisco, CA 94103, USA; 10Variantyx, Inc, Framingham, MA 01701, USA; 11QNA Diagnostics, Cambridge, MA 02139, USA; 12CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh – 160036, India; 13ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT 84108, USA; 14Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA; 15Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada; 16Biochemistry and Genetics Department, MitoVasc Institute, UMR CNRS 6015– INSERM U1083, CHU Angers, 49933 Angers, France; 17Keck School of Medicine, University of Southern California, Los Angeles, CA 90033,USA.


Title: Standards and guidelines for mitochondrial DNA variant interpretation

Abstract: Background: Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given certain features of the mtDNA genome, including variant heteroplasmy, threshold effect, absence of splicing, presence of haplogroups, and maternal inheritance. Currently there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting.

Methods and results: An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium and formed a working group seeking Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics (ACMG) and Association of Molecular Pathology (AMP) standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications and additional guidance for mtDNA variant classification.

Conclusion: The 2015 ACMG/AMP variant interpretation guidelines were critically reviewed and specified for application in mtDNA variant assessment. These specifications allow for consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, mtDNA genomic databases and computational algorithms, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA.


Presenter: Divakar S. Mithal

Authors: Sarah A. Reilly,1,2, Lauren E. Hitchins2, Divakar S. Mithal1,3

Institution: 1Department of Pediatrics, Northwestern University, Chicago, IL, 2Section of Genetics, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, 60611, 3Section of Neurology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, 60611

Title: A text-based search of electronic medical records accurately identifies patients with mitochondrial disease and enables analysis of clinical and genetic data

Body of Abstract:Introduction: Mitochondrial diseases are a heterogenous group of genetic disorders with highly variable symptomatology. Retrospective reviews provide insight regarding the natural history, clinical characteristics and genetic etiology of these disorders. Given the more recent advances in diagnostics for mitochondrial disorders, retrospective identification of patients with confirmed mitochondrial disease is appealing but remains challenging. We designed a text-based search method to identify patients with mitochondrial disease accurately and at a high frequency.

Methods: Electronic medical records (EMR) were searched using a text-based algorithm through a Lurie Children’s internal review board approved study. A list of search terms referencing mitochondrial metabolism was derived manually. All patients within the Lurie Children’s EMR system from 1986 to 2018 were queried with the list of search terms. Resulting charts were reviewed by clinicians individually to confirm or reject the underlying diagnosis as a primary mitochondrial disorder. The group of patients that appeared to have mitochondrial disease was then stratified by the degree of certainty for diagnosis using clinical and testing information available in the patient charts. The EMR were queried subsequently for a variety of clinical parameters that were analyzed for each subgroup.

Results: Of the 548 patient charts that resulted from the initial text-based search, 113 (20.6%) had high suspicion for mitochondrial disease once reviewed by a clinician. Chart review yielded a “positive” group with high likelihood of mitochondrial disease and a “negative” control population with alternative


diagnoses. The “negative” control population consisted of 216 charts (39%) that were identified by broad search terms referencing metabolism. The “positive” group consisted of 332 charts (61%) that were identified with more targeted terms. On clinician review, 32% of the charts from the “positive” group were identified as having high suspicion for mitochondrial disease, whereas only 3% of the charts from the “negative” group were identified as such. Within the clinician-suspected mitochondrial patient group, 64% had a confirmed genetic diagnosis and 25% had either diagnostic enzyme testing in the context of a convincing clinical picture or highly suspicious but non-confirmatory genetic testing, rendering them “likely” mitochondrial. Patients labeled as “likely” mitochondrial often harbored at least one variant of unclear significance, resulting in possible novel variants for over 10% of mitochondrial disease patients identified through this algorithm.

Conclusions: The text-based search algorithm in this work demonstrates the feasibility of mining historical EMR data to identify patients with mitochondrial disease. Subgroup analysis of identified patients provided novel clinical insight for mitochondrial patients. Once patients are identified, evaluation of specific genetic etiologies expands the knowledge base concerning mitochondrial disease.


Presenter: Elizabeth M. McCormick

Authors: Elizabeth M. McCormick1, Kierstin Keller2, Lishuang Shen3, Danuta Krotoski4, Xiaowu Gai3,5, Marni J. Falk1,6, Zarazuela Zolkipli-Cunningham1,6, and Shamima Rahman7

Institutions: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Center for Mitochondrial and Epigenomic Medicine, Department of Pathology, Children's Hospital of Philadelphia, Philadelphia, PA, USA; 3Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA; 4IDDB/NICHD, National Institutes of Health, Bethesda, MD, USA; 5Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 6University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; 7Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, London, United Kingdom.

Title: Standardization of Leigh syndrome spectrum definition and application within the ClinGen Gene Clinical Validity Curation Framework.

Abstract: Background: Leigh syndrome (LS) is a progressive neurodegenerative disorder first described in 1951 as a neuropathological diagnosis of bilateral necrotic lesions of the brainstem and basal ganglia. Over time, the definition of this clinical syndrome has evolved to incorporate various neurodevelopmental and metabolic findings that have been seen in those with LS. Additionally, the term “Leigh-like syndrome” (LLS) has been utilized to describe individuals with some but not all features of LS. There is significant overlap in these two clinical entities and the terms have been applied inconsistently despite stringent LS diagnostic criteria. As part of the NICHD U24 ClinGen Mitochondrial Disease Gene Curation Expert Panel, we sought to again refine the definition of this spectrum of neurodegenerative diseases to improve consistency in how the terms are applied as well as used in gene-disease association curation.

Methods: We proposed to a global mitochondrial disease expert panel the term Leigh syndrome spectrum (LSS) that would incorporate LS and LLS into a single clinical entity. We then systematically reviewed historic and current literature to ensure this updated definition would be relevant for both prior reported and newly diagnosed individuals, as imaging and laboratory techniques have significantly changed since the first description of LS in 1951. Finally, we sought to apply this new definition to animal models of LS and LLS to determine how these models could be consistently assessed for the presence of LSS.


Results: We propose the term LSS to refer to the collection of individually rare genetic diseases characterized by either typical neuropathologic findings of Leigh syndrome; OR (in the absence of neuropathology) neuroimaging findings of bilateral, typically symmetric T2-weighted hyperintensities on MRI or hypodensities on CT scan in brainstem, basal ganglia, thalamus, cerebellum, and/or spinal cord (minimum findings being isolated basal ganglia or isolated brainstem changes); ANDneurodevelopmental delay and/or stepwise neurodevelopmental regression (basal ganglia and brainstem signs); AND one or more of 1) elevated lactate in plasma or CSF, AND/OR 2) MRS lactate peak, AND/OR 3) evidence of OXPHOS enzyme activity deficiency (<30%) in affected tissue (muscle or liver) assessed by spectrophotometric assay.

Within the ClinGen Gene Clinical Validity Curation Framework, the approach to scoring affected individuals reported in the literature was weighted, where emphasis was placed on neuropathologic findings, compared to clinical and biochemical findings. This methodical approach was also utilized in animal model curation, where similar criteria and weighting were applied to reported neuropathological features, MRI findings, biochemical evidence of mitochondrial dysfunction, and/or neurocognitive or developmental deficits. In addition, we devised a standardized curation approach to research-level data for biochemical function, protein interaction, expression, and cell lines.

Conclusion: We propose a refined and encompassing term, LSS, to further specify manifestations of LS and including individuals previously referred to as having LLS. This updated definition and systematic scoring system will allow for increased consistency in phenotype review and curation of case- and research-level human genetic variants and animal models, leading to more accurate assertions of gene-disease associations for Leigh syndrome spectrum disorders.


Presenter: Russell L. D’Souza

Authors: Russell L. D’Souza1, Peter J. McGuire1

Institution: 1Metabolism, Infection, and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD.

Title: Generation of Cerebral Organoids to Understand Neurodegeneration in Mitochondrial Disorders

Abstract: Mitochondria are ubiquitous organelles that generate ATP through the process of oxidative phosphorylation (OxPhos) which is required for various cell processes. Certain organs of the body such as the brain require a higher energy demand and have a greater number of mitochondria in them to fulfill these energy requirements. Therefore, these organs tend to be greatly affected in the event of a mitochondrial disease (MD). Patients with MD are a central cause of neurodegeneration that is characterized by behavioral, motor, and cognitive impairment. Although, the use of cell culture and mouse models has helped us understand the pathophysiology of the brain in MD, we still lack a model system that closely resembles the human brain. To circumvent this problem, our lab has made an effort to generate human cerebral organoids from healthy and two patient derived iPS cells.

P1: 13 yo male that shows a multisystem disease which includes musculoskeletal and neurologic decline with illness. OxPhos enzyme activities using mitochondria derived from LCLs show reduced Complex III activities and lower respiration rates. Neurological events of the brain indicate a higher occurrence of stroke like episodes. Whole genome sequencing revealed a 5Kb deletion from exon 1 of the MPPB gene. This gene is a mitochondrial processing peptidase that cleaves the target sequence of newly imported proteins in the mitochondria. Reported literature suggests that defects in the MPPB gene affects the biogenesis of


FeS proteins. Our biochemical assays reveal the unsuccessful incorporation of the RIP protein into Complex III. Mitochondria isolated from cerebral organoids did not affect the expression levels of BCS1L or the RIP protein. Further investigation of the effects of the MPPB gene is ongoing in the laboratory.

P2: 4 yo male with a classic case of Leigh’s Disease. This patient has a stop codon mutation in the exon 4 of the Surf1 gene. We have derived iPS cells from the patient’s PBMCS using gene editing methods. Generation of cerebral and mid-brain organoids is ongoing in the laboratory.


Presenter: Patrick F. Chinnery

Authors: Wei Wei1,2, Ernest Turro3,4,5, Patrick F Chinnery1,2,4 on behalf of the NIHR BioResource - Rare Diseases+ and the 100,000 Genomes Project - Rare Diseases Pilot.

Institution: 1Department of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK. 2Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Cambridge, UK. 3Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK. 4NIHR BioResource, Cambridge University Hospitals NHS Foundation, Cambridge Biomedical Campus, Cambridge, UK.5MRC Biostatistics Unit, Cambridge Institute of Public Health, University of Cambridge, Cambridge, UK.

Title: The Inheritance of Mitochondrial DNA Heteroplasmy is Under Nuclear Genetic Control in Humans

Body of Abstract: New mitochondrial DNA (mtDNA) mutations initially affect a small proportion of the mtDNA molecules in a cell (heteroplasmy), well below the critical threshold required to cause disease. However, a reduction in the amount of mtDNA per cell during female germ (the mtDNA bottleneck) leads to the rapid segregation of heteroplasmy, and very different levels between siblings, with high levels causing mitochondrial diseases. Although primarily governed by random genetic drift, there is evidence of selection occurring during the inheritance of mtDNA heteroplasmy in animal models, but it has been difficult to demonstrate this convincingly in humans. This is important, because it will determine the prevalence of mtDNA disease and potentially influence clinical recurrence risks within families transmitting mtDNA diseases.

To determine whether there was selection for or against heteroplasmic mtDNA during transmission in humans we studied 12,975 whole genome sequences, including 1,526 mother-offspring pairs where 45.1% of the individuals showed heteroplasmy affecting >1% of the mtDNA molecules. We classified the heteroplasmies as ‘known’ (seen before in 30,506 human mtDNA sampled from across the globe) or ‘novel’ (not see before). Harnessing genetic information from both the mtDNA and nuclear genomes, we then determined whether the nuclear genetic background influenced mtDNA heteroplasmy, and independently validated our findings in a further 40,325 whole genome sequences.

Novel mtDNA variants were less likely to be inherited than previously seen variants, and the level of heteroplasmy for known variants tended to increase on transmission. Despite the low heteroplasmy levels, we saw selection for and against variants in different regions of the mitochondrial genome. New population-specific heteroplasmies were far more likely to match the nuclear genetic ancestry of an individual than the mitochondrial genome on which the mutations occurred.

In conclusion, human mtDNA is shaped by selective forces acting on heteroplasmy within the female germ line, and the signature of selection can be seen over one generation. The nuclear genome influences mtDNA heteroplasmy in the population. This will ensure consistency between these two independent genetic systems, with implications for the origins and treatment of mitochondrial diseases.



Authors: Xilma Ortiz-Gonzalez1,2, Jesus A Tintos-Hernandez2 and Douglas C Wallace2

Institution: 1Division of Neurology, 2Center for Mitochondrial and Epigenomic Medicine, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania

Title: Examining Mitochondrial Quality Control in TBCK-encephaloneuronopathy Using Human iPSC Derived Neurons

Body of Abstract: Mitochondrial dysfunction is well known to contribute to neurodegenerative disease. TBCK- encephaloneuronopathy is a novel pediatric neurodegenerative syndrome where mitochondrial and lysosomal dysfunction appear to converge. Our cohort of patients with homozygous null TBCK mutations present with progressive central and peripheral neurodegenerative features. Intriguingly, they also exhibit mitochondrial dysfunction, including mtDNA (mitochondrial DNA) depletion. We found that patient derived exhibit upregulation of mitophagy and mtDNA depletion. To determine if abnormal mitochondrial quality control is present in neurons, we derived IPSC from patients and assayed autophagic flux, mitochondrial copy number and mitophagy. Our data suggest that indeed autophagic flux is increased in TBCK-null iPSC-neurons and that the mtDNA depletion phenotype observed in patients is recapitulated in this model. Therefore, using this is a disease relevant model to screen for rescue strategies to target mitochondrial dysfunction in neurons in order to develop better therapeutic interventions in the future.


Presenter: Amy Goldstein

Author: Amy Goldstein, on behalf of the MMPOWER-3 Investigators

Institution: Children’s Hospital of Philadelphia, Philadelphia, PA. 19104

Title: Natural History of Disease Progression in Patients with Primary Mitochondrial Myopathy

Body of Abstract:INTRODUCTION: Primary mitochondrial myopathies (PMMs) are genetic disorders that negatively impact the efficiency of the mitochondrial respiratory chain and the production of adenosine triphosphate (ATP). As a result, patients with PMM experience fatigue, exercise intolerance, and muscle weakness, which adversely affect physical function and quality-of-life (QoL). Despite our understanding of the pathophysiology of PMM, the characterization of disease progression in this patient population remains limited. Data from subjects who were screened and enrolled into the late-stage elamipretide development program (i.e. RePOWER Registry and MMPOWER-3 interventional trial) may serve to augment our understanding of the natural progression of disease in patients with PMM.

OBJECTIVES: RePOWER was a global, prospective, non-interventional study that enrolled ambulatory subjects between 16 years and 80 years of age with signs and/or symptoms of PMM. RePOWER sought to obtain demographic, genetic/phenotypic, and functional assessment data in patients with PMM. RePOWER also served to identify potential participants for recruitment into MMPOWER-3, a 24-week, double-blind, randomized, placebo-controlled study to investigate the efficacy and safety of elamipretide in patients with PMM. METHODS: Disease progression has been evaluated by analyzing the data from the non-interventional time that elapsed between enrollment visit in RePOWER and the screening visit for the interventional study, MMPOWER-3. Only those subjects who both enrolled in RePOWER and were screened for MMPOWER-3 (R-M3) are included in the analysis. Evaluations, including the 6-Minute Walk Test (6MWT), Total Fatigue on the Primary Mitochondrial Myopathy Symptom Assessment 4 point scale (PMMSA), and the Neuro-QoL Fatigue Short Form, needed to be performed at both clinic visits (enrollment into RePOWER and screening for MMPOWER-3).


RESULTS: RePOWER evaluated 414 subjects with an average age of 43.6 yrs, the majority of whom were white (92%) and female (60%) and primarily classified as having mtDNA mutations (77%). Asscreening for enrollment into the MMPOWER-3 trial continues, the R-M3 population will continue toexpand. CONCLUSIONS: The R-M3 population demonstrated signs of disease progression, on preliminary assessment of functional and subject-reported assessment scores, over the evaluable time frame.


Presenter: Lawrence I. Grossman

Authors: Siddhesh Aras1, Neeraja Purandare1, Douglas C. Wallace2, and Lawrence I. Grossman1

Institution: 1Wayne State University, Center for Molecular Medicine and Genetics, Detroit, MI 48201.2University of Pennsylvania, Center for Mitochondrial and Epigenomic Medicine, Philadelphia, PA 19104.

Title: MNRR1, a Therapeutic Target in a MELAS Model for Mitochondrial Disease

Body of Abstract: We asked whether manipulating gene expression could reverse the pathophysiology of a cellular model of mitochondrial disease. We used as a model 143B cybrid cells containing 72.8% MELAS (mitochondrial encephalomyopathy, lactic-acidosis, and stroke-like episodes) mitochondria, containing the mtDNA A3243G mutation in tRNALeu(UUR). Compared to the parental cell line with 100% wild-type mtDNA, these heteroplasmic cells displayed reduced oxygen consumption, reduced ATP level, slower growth, and a higher level of ROS. To modulate gene expression, we utilized expression of the MNRR1 (CHCHD2) gene. MNRR1 protein is a bi-organellar regulator of mitochondrial function. In the mitochondria, MNRR1 functions by (a) increasing OxPhos, and (b) Inhibiting apoptosis. In the nucleus, MNRR1 is a transcription factor for a plethora of stress-responsive genes. When expressed in the cybrid MELAS cells, MNRR1 (a) induces mitochondrial biogenesis, and (b) increases cellular oxygen consumption and ATP production, and (c) reduces ROS levels. At the same time, MNRR1 expressing MELAS cells display an increase in the cellular markers of UPRmt and autophagy that, at least in part, contributes to the MNRR1-mediated rescue of cellular physiology. By using MNRR1 mutants that favor either its mitochondrial or its nuclear function, we were able to show that stimulating its nuclear function is sufficient to rescue cellular physiology. We discuss these results in terms of several modalities for using MNRR1 as a mechanistic target for the design of specific therapeutics in patients with MELAS and possibly other mitochondrial diseases.


Presenter: Melissa A. Walker M.D., Ph.D.

Authors: Melissa A. Walker1,3, Amel Karaa2,3

Institution: 1Massachusetts General Hospital, Department of Neurology, Boston, MA 02114, 2Massachusetts General Hospital, Department of Pediatrics, Boston, MA 02114, 3Harvard Medical School, Boston, MA 02114

Title: Standardization of Acute Metabolic Stroke Care at a Large Academic Center

Body of Abstract: In contrast to well-established protocols for ischemic stroke, standard of care protocols for metabolic stroke have yet to be established. L-arginine infusion for acute metabolic stroke in Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes (MELAS) is a broadly accepted but still emerging therapy. We hypothesize that the creation and implementation of a standardized protocol for l-arginine infusion in acute metabolic stroke in MELAS will increase treatment rates, decrease hospitalization time, decrease cost, decrease patient morbidity, and increase establishment long-term care with expert mitochondrial providers at our large, adult and pediatric tertiary care center.


To improve the acute metabolic stroke care provided at Massachusetts General Hospital (MGH) and partner institutions, we have undertaken the following steps: (1) We have therefore analyzed data from our anonymized patient database (the Partners Patient Data Registry) to understand the demographics of MELAS patients presenting with stroke to MGH as well as the percentage and demographics of the subset treated with l-arginine infusion. (2) We have interviewed a multidisciplinary group (mitochondrial physicians, general neurology attending physicians, general neurology resident physicians, neurologic nurses, nurse managers, and neurologic pharmacists, and MELAS patients) to identify problematic aspects of and/or barriers to acute stroke care in MELAS. (3) We have developed a template for acute metabolic stroke care in MELAS compatible with the EPIC hyperspace system to ultimately be implemented at MGH and partner institutions and made available for broader sharing and application.

We aim to prospectively analyze the impact of this protocol for continued improvement in patient care. Ongoing analysis and quality improvement will be essential to create and increase the availability of the highest quality mitochondrial medical care.


Presenter: Bruce H Cohen

Authors: Bruce H Cohen 1, Amy Goldstein,2 Richard Haas,3 Jerry Vockley,4 Amel Karaa5

Institution: 1Akron Children’s Hospital, Akron, OH. 44308, 2Children’s Hospital of Philadelphia, Philadelphia, PA. 19104, 3UC San Diego School of Medicine, San Diego, CA. 92093, 4Children’s Hospital of Pittsburgh, Pittsburgh, PA. 15224, 5 Massachusetts General Hospital, Boston, MA. 02114

Title: MMPOWER-2 Open-Label Extension Trial: Results from 12 Months of Elamipretide Therapy in PMM Patients

Abstract: INTRODUCTION: Patients with primary mitochondrial myopathy (PMM) have genetic disorders of the mitochondrial respiratory chain affecting predominantly, but not exclusively, skeletal muscle. Cardinal symptoms include fatigue, exercise intolerance, and muscle weakness. These symptoms negatively affect physical functioning, exercise capacity, and quality-of-life (QoL). It is estimated that there are approximately 36,000 adult patients in the USA who have been coded as having a primary mitochondrial disease based on a USA commercial insurance claim database analysis. Currently, there are no FDA approved therapies for the treatment of these mitochondrial diseases. Elamipretide, an investigational agent, is currently being evaluated in a large clinical development program referred to as MMPOWER asa potential therapy for the treatment of PMM.

OBJECTIVES: The objective of this analysis is to present assessment data from the 12-month visit for patients continuously enrolled and receiving elamipretide therapy in the MMPOWER-2 OLE trial.

METHODS: Patients enrolled in MMPOWER-2 OLE (N=28) who had a 12-Month visit were included in the MMPOWER-2 OLE analysis cohort (n=24). These subject have received elamipretide 40 mg SC daily for 12 months. Demographic data as well as longitudinal trends in functional assessments and patient-reported outcome questionnaires were evaluated. As there is no control arm in the MMPOWER-2OLE, a “natural history control” dataset will be included for comparative purposes of available assessments.

RESULTS: Of patients with 12-month data, there were 3 males and 21 females (mean age 50.3 years). Distance walked in the 6MWT was maintained throughout the 12- month observation period (median values, baseline=402 meters and end of observation=397m) for the patients treated daily with


elamipretide. At the end of the 12-month treatment period, patients showed continued improvement intheir self-reported fatigue assessment scores. The PMMSA Total Fatigue score was reduced (-2.21 points) reflective of an improvement in fatigue rating. Results from the Neuro-QoL Fatigue Short Form showed a similar result related to fatigue with a change in the T-Score of -6.6). Improvements were also observed in each of the analyses performed on the EuroQoL-5 Dimension Individual Domain questionnaire. Changes in individual domain scores are indicative of a beneficial effect as reflected bythe Problem-to-No Problem transition analysis, where “Problem” is defined as a domain score of 2-5 and“No Problem” as a domain score of 1. The safety profile of elamipretide is consistent with results obtained from previous PMM trials. As observed in the blinded MMPOWER-2 study, the most commonly reported AEs were injection site reactions.

CONCLUSIONS: Patients receiving continued therapy with elamipretide demonstrated a favorable treatment effect over the duration of the additional 12-month observation period.



Authors: Melissa A. Walker1,2, Sam Nicholas Russo3,4, Mary Kay Koenig3,4, Amel Karaa2,5

Institution: 1Massachusetts General Hospital, Department of Neurology, Boston, MA 02114, 2Harvard Medical School, Boston, MA 02114, 3University of Texas Health Science Center at Houston, Department of Pediatrics, 4McGovern Medical School, 5Massachusetts General Hospital, Department of Pediatrics, Boston, MA 02114

Title: Leigh Syndrome as a Phenotype Of Homoplasmic m.8344A>G Mutation

Body of Abstract: Correlation of phenotype with mutation heteroplasmy remains an outstanding question in the field of mitochondrial disease with few, if any, clear thresholds corresponding to a given phenotype. The m.8344A>G mutation is most commonly associated with the myoclonus epilepsy and ragged red fiber syndrome (MERRF) at varying levels of heteroplasmy. However, a handful of cases (from the United States, Australia, China, Japan, United Kingdom) have been previously reported in which individuals homoplasmic or nearly homoplasmic for this mutation in blood have presented with a picture of bulbar palsy, respiratory insufficiency, and progressive neurologic decline to a comatose state, almost uniformly following a respiratory illness. MRI brain in all affected individuals revealed symmetric T2 hyperintense lesions of subcortical grey matter structures, consistent with Leigh Syndrome.

Here, we review this literature and present two, unrelated cases with clinical, biochemical, and neuro-imaging findings with the additional reporting of spinal lesions (not previously published in such cases). Both cases are pediatric female patients of Hispanic heritage. While one individual had an early course of failure to thrive, hearing loss, myopathy, myoclonus, and epilepsy consistent with a classic MERRF phenotype and prior imaging without Leigh Syndrome lesions, the second patient (who had a prior history of failure to thrive and short stature) presented initially with bulbar findings and subsequently was additionally diagnosed with sensory ataxia. Initial imaging revealed Leigh-like lesions. Her course was relatively attenuated compared to prior cases. She did not immediately require mechanical ventilation upon developing bulbar symptoms and did demonstrate some interval improvement on repeat neuro-imaging prior to further disease progression.

Together, ours and previously published cases support a homoplasmic or near homoplasmic 8344A>G phenotype of Leigh Syndrome lesions on neuro-imaging with clinical features of bulbar palsy, ventilator dependence, and progressive decline in arousal leading to comatose state. The reporting of this phenotype in individuals of diverse ancestry argues against a haplotype specific phenomenon. Further research will be required to understand the mechanism by which homoplasmic and near homoplasmic m.8344A>G mutations produce Leigh Syndrome and to understand this phenotype as a common final pathway among the diverse genetic lesions that result in Leigh Syndrome.



Presenter: Lishuang Shen

Authors: Lishuang Shen1, Elizabeth M. McComick2, Daria Merkurjev1, Colleen C. Muraresku2,Zarazuela Zolkipli-Cunningham2, Marni J. Falk2,3, Xiaowu Gai1,4

Institutions: 1Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 3Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; 4Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Title: MSeqDR Updates: A Genomic and Bioinformatics Data Hub for the Mitochondrial Disease Community

Abstract: Background: MSeqDR (https://mseqdr.org) is a centralized genomic bioinformatics Web resource, supported by the United Mitochondrial Disease Foundation, to facilitate research-based diagnoses, genetic analyses, and variant interpretation for researchers and clinicians working in mitochondrial disease. Since its initial release in 2014 and publications in 2016, MSeqDR has been continuously enhanced with additional genomic and phenotypic content, as well as powerful new bioinformatics tools. MSeqDR has now become a major data hub for the mitochondrial disease community, including MitoMap, HmtDB, LeighMap, ClinVar, ClinGen, PhenoTips, and GENOMIT resources, through data sharing, cross-referencing, and active collaboration. MSeqDR Content Update: The genomic and phenotypic content within MSeqDR has been enhanced by (a) Assembling a large reference dataset for assessing mitochondrial DNA (mtDNA) allele population frequencies. Indeed, MSeqDR generated a novel meta-population mtDNA allele frequency data resource, by compiling close to 90K mtDNA genomes with data from MitoMap, GeneDx, previous publications and other large sequencing initiatives, with substantial amount of data from previously underrepresented populations, such as Chinese and Japanese; (b) Further populating the content of MSeqDR-LSDB, a curated database of pathogenic variants for mitochondrial diseases, genes, and variants by data mining and systematic curation of public genomic resources and literature. MSeqDR-LSDB currents hosts over 12,000 variants from 1607 genes that are potentially associated the mitochondrial diseases, including 265 known mitochondrial disease genes. Among which, the pathogenicity has been assessed for over 8000 variants. Nuclear and mtDNA gene variant deposition by users is also supported in automated fashion with MSeqDR assignment to each variant of a unique identifier, the MSCV accession number, that is recognized as a data repository by ClinVar and journals for data deposition at the time of new variant publication, and provides easily populated data templates to support ClinVar submission; and (c) Compiling, mapping and curating phenotypic terms to capture features most relevant to mitochondrial diseases and harmonized across international registries basedon standardized HPO and OMIM terminology ontologies.

MSeqDR Tools Update: We have developed a complementary suite of powerful bioinformatics tools for comprehensively annotating and analyzing mtDNA genome variants, namely MSeqDR-mvTool and Phy-Mer. We have also centralized access to a suite of other tools hosted in or linked to MSeqDR, such as MToolBox from HmtDB, MitoMaster from MitoMap, MitoTIP, and LeighMap. We are further developing a suite of tools, Quick-Mitome, for user-friendly, automated, and rapid Web-based analysis of exome datasets from mitochondrial disease patients. Efforts remain actively underway to allow patient-directed deposition and deidentified analysis of their previously generated exome and genome data from the Mitochondrial Disease Community Registry (MDCR) into MSeqDR for ready analysis in a custom MSeqDR-OpenCGA annotation and variant analysis resource. MSeqDR also provides users with secure Collaboration Zones that support a range of initiatives, as well as custom tools to support ClinGen gene-disease and variant curation including for a NICHD, NIH U24-funded international initiative to expertly curate Leigh syndrome spectrum diseases in collaboration with the ClinGen consortium.


Conclusion: Overall, MSeqDR now provides extensive sets of unique genomic data, diverse bioinformatics tools that support mtDNA variant detection, annotation, and haplogroup determination, user-friendly exome data analysis, and mitochondrial disease HPO-based clinical feature analyses. In this way, MSeqDR has become recognized world-wide as the genomic and data collaborative hub supporting the complex needs of the mitochondrial disease diagnostic and research community.


Presenter: Johan L.K. Van Hove

Authors: Marisa W. Friederich1, Abdallah F. Elias2, Austin A. Larson1, Aaron Landry3, Logan Ellwood-Diegel1, David Mirsky4, David Dimmock5, Jaclyn Haven2, Hua Jiang1, Kenneth N. MacLean1, Maike Friederich1, Ruma Bannerjee2, Johan L.K. Van Hove1

Institutions: 1. Section of Clinical Genetics and Metabolism, Department of Pediatrics, University of Colorado, Aurora, Colorado, USA; 2. Department of Medical Genetics, Shodair Children’s Hospital, Helena, Montana, USA; 3. Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA; 4. Department of Radiology, University of Colorado, and Children’s Hospital Colorado, Aurora, Colorado, USA; 3. Rady Children’s Institute for Genomic Medicine, Rady Children’s Hospital, San Diego, California, USA

Title: Mutations in SQOR Encoding Sulfide:Quinone Oxidoreductase Are a Potentially Treatable Cause of Leigh Disease

Abstract: Objective: Hydrogen sulfide is a gaseous transmitter formed mainly from cysteine, and catabolized by sulfide:quinone oxidoreductase (SQOR gene). Toxic exposure to hydrogen sulfide causes inhibition of complex IV. We describe a first patient with mutations in SQOR.

Methods: Mutations were identified by exome sequencing and confirmed by Sanger sequencing. SQOR enzyme activity was measured spectrophotometrically and protein levels evaluated by western blotting. Results: Following a brief illness, a four-year-old girl presented comatose with lactic acidosis, a seizure, and multiorgan failure. After stabilization with supportive care, she remained comatose, hypotonic, had episodic neuro-storming episodes, with hypoalbuminemia and elevated lactate, and died shortly after. Brain MRI showed increased T2 signal intensity in basal ganglia, cerebral peduncles, dentate nuclei, and diffusion restriction in medial temporal lobe, posterior thalami, mammillary bodies and posterior hippocampi. An elevated lactate peak was seen on MRS. Her eight-year-old sister, with a recent history of migraines, subsequently presented with a rapidly evolving comatose episode with lactic acidosis. On brain MRI, she had diffusion restriction in the basal ganglia, and left cerebral cortex and to a lesser extent the right. MRS showed elevated lactate. She died after three days. In patient 1, muscle and liver tissue had isolated decreased complex IV activity, but normal blue native PAGE with in-gel activity staining, normal complex IV subunit protein levels, and normal complex I assembly. Exome sequencing revealed a homozygous c.637G>A, p.E213K mutation in SQOR. The SQOR protein was strongly reduced, and SQOR enzyme activity was 6% of controls, whereas sulfide generating enzyme levels were unchanged. Expression in E. coli resulted in a very low yield. The Glu-213 is remote from the active site, but its change to lysine disrupts hydrogen bonding of neighboring residues Arg-217 and Arg-222. Conclusion: The mutation introduced loss of the SQOR protein and enzyme activity. The resulting hydrogen sulfide caused complex IV inhibition compatible with its known toxicity, resulting in energy failure. This represents a new cause of Leigh disease, which is likely treatable with avoidance of catabolism, dietary precursor restriction and parenteral hydroxocobalamin, similar to treatment of cyanide intoxication.



Presenter: Newell Belnap

Authors: Newell Belnap1,2, Abby Moskowitz1, Isabelle Schrauwen1,2, Cherae Bilagody1,2, Lorida Llaci12, Richa Pandey1,2, Kelsey Chain1, Raj Gupta1, C4RCD Research Group1, 2, Vinodh Narayanan1,2, Sampathkumar Rangasamy1,2

Institutions: 1) Neurogenomics Division, Translational Genomics Research Institute (TGen), Phoenix, AZ; 2) Center for Rare Childhood Disorders (C4RCD), Translational Genomics Research Institute (TGen), Phoenix AZ

Title: Investigation of a Recurrent Splice Site Mutation in Mitochondrial Methionyl-tRNA Formyltransferase (MTFMT) Using Cellular and a Novel Mouse Model

Abstract: Mutations of the nuclear encoded mitochondrial protein MTFMT causes combined oxidative phosphorylation deficiency and Leigh syndrome. At the Center for Rare Childhood Disorders (C4RCD), we have identified a recurrent homozygous nonsynonymous mutation in MTFMT c. 626C>T, in three children from two unrelated families. This mutation generates a splicing suppressor in the coding region of exon 4, resulting in exon skipping and a truncated protein (p. R181SfsX5). This mutation is estimated to account for about 0.1% of all alleles in patients with combined OXPHOS deficiency.

In Family 1, an affected boy had a neurological presentation with delayed development, nystagmus, optic atrophy, elevated CSF lactate, and an abnormal MRI with areas of frontal, pericallosal demyelination and basal ganglia necrosis. His affected older sister had delayed cognitive development, Wolff-Parkinson-White (WPW) syndrome, normal brain MRI, and elevated CSF lactate. In Family 2, the affected girl had progressive dystonia, brain MRI that showed bilateral striatal FLAIR hyperintensities, and elevated CSF lactate. RNA sequencing from blood samples in the homozygous patients, identified a dramatic reduction in read counts of exon 4 confirming skipping of this exon. Through differential gene expression analysis, we found that glutaminase, a vital enzyme that helps in the conversion of glutamine to glutamate is highly expressed in homozygous patients. This result suggests the activation of a compensatory metabolic pathway in homozygous patient due to the primary oxidative phosphorylation deficiency.

Examination of mitochondrial function in patient derived fibroblast cultures identified increased generation of reactive oxygen species due to compromised complex 1 activity when compared to the heterozygous patients and the unaffected healthy controls. Further, anti-sense oligonucleotide (morpholino) treatment of patient-derived fibroblasts counteracted the skipping of exon 4 induced by the point mutation and increased the level of MTFMT mRNA containing exon 4. This result suggests the possibility of using such antisense oligonucleotides as a treatment modality to restore inclusion of exon 4 in these patients. In collaboration with Jackson Labs we have created heterozygous MTFMT-Mut-Hum (mutant MTFMT with c. 626C>T variant human exon 4) and breeding of these heterozygous resulted in successful generation of homozygous MTFMT-Mut-Hum animals. Pre-clinical studies of these oligonucleotides in our animal model will validate it as a potential therapy for this recurrent mutation in the MTFMT gene.


Presenter: Ailian Du

Authors: Ailian Du1, Xiya Shen, Gailing Liu1, Yuanyuan Li1, Qing Lv1

Institutions: 1Tongren Hospital, Shanghai Jiaotong University School of Medicine, Department of Neurology, Shanghai, 200050, PR China


Title: Mitochondrial Encephalopathy Presented with Cyclic Vomiting and Dementia Syndrome in a Chinese Family

Abstract: Objective To studied the clinical and genetical characteristic of a Chinese family presenting with cyclic vomiting and dementia syndrome. Methods The proband (II-5) was detailed evaluated for clinical and pathological properties in muscle biopsy. Mitochondrial DNA sequencing was performed on the proband and next generation sequencing (NGS) for nuclear DNA mutations were performed on 2 patients and one normal sibling. Results Three male affected members (II-2, II-4, II-5) of the family presented with combination of cyclic vomiting, epilepsy, encephalopathy, renal failure, dementia, and diabetes. One female member presented with dementia and urinary incontinence. And one female sibling keeps normal at the age of 81. Muscle biopsy from the proband showed typical red ragged fibers and paracrystalline inclusions. MtDNA sequencing was normal in blood of the proband. NGS sequencing results showed that several gene mutations in the proband and the affected sister are highly related to mitochondrial function, including PUS1, OTOA, AGK, SHANK2, and GTPBP10. Conclusion Cyclic vomiting and dementia syndrome in this Chinese family may be caused by a nuclear gene mutation which need further investigation.


Presenter: Colleen C. Muraresku

Authors: Nicole M. Engelhardt1,2, Colleen C. Muraresku1, Kathleen D. Valverde2, Katherine L Nathanson3,4, Marni J. Falk1,5

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104; 2Arcadia University Genetic Counseling Glenside, Pennsylvania, PA 19104; 3Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104; 4Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104; 5Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Title: Cancer Occurrence in Primary Mitochondrial Disease Patients and Their Families

Abstract: Introduction: Mitochondria play a central role in apoptosis, reactive oxygen species (ROS) production and scavenging, intermediary metabolism, and energy production. Abnormal functioning of these mitochondrial processes is a known mechanism of neoplasm formation. While mitochondrial dysfunction is the hallmark of primary mitochondrial disease (MD), the occurrence of cancer among individuals with primary MD has not been well studied. We postulate that primary MD may be protective from cancer development, as individuals with primary MD develop multi-systemic involvement of nearly every organ, but in our clinical experience do not develop cancer as a common clinical manifestation regardless of genetic subtype or patient age.

Methods: A brief survey was sent to 99 adults or parents of children with molecularly confirmed primary MD followed in the Mitochondrial Medicine Frontier Program at the Children’s Hospital of Philadelphia, one survey per family (CHOP IRB #08-6177). The survey was conducted online using Research Electronic Data Capture (REDCap). Participants responded to questions about their personal and family histories of cancer. Results were compared to general population cancer occurrence.

Preliminary Results: Interim analysis was performed of 17/99 individuals surveyed who responded during the initial two week response period (a response rate of 16.7%), with this study ongoing. Individuals affected with MD ranged from 3 – 45 years old. Thirteen of the respondents had a nuclear gene cause for the primary MD in the family (76%) and 4 had a mitochondrial DNA cause (24%). Ethnic background was noted to be 73% Caucasian, 6.7% African American, and 6.7% Asian. Families identified as being 13.3% Ashkenazi Jewish and 13.3% self-reported more than one race. Across the 17 families, information was reported for 114 individuals. Among these 114 individuals,12 non-MD individuals in 9


unrelated families had experienced cancer (10.5%), which includes 8 grandparents affected with cancer (16.7% of all respondents). No individuals affected with MD had a personal history of cancer. Two asymptomatic carriers of low-level mtDNA mutations in 2 separate families at the time of the survey reported having colon or thyroid cancer. Additional results and analyses are pending.

Conclusions: Preliminary data with interim analysis of ongoing cancer history analysis is suggestive that MD affected patients do not commonly experience cancer, with no instances reported in affected individuals across 17 unrelated families. Additional analysis of the full data set is necessary to determine if MD affected status may be protective of cancer, or if low-level mutation carriers without mitochondrial disease have an increased cancer risk for specific cancers.


Presenter: Nikol Volfová

Authors: Nikol Volfová1, Lukáš Alán2, Tereza Daňhelovská1, Marie Rodinová1, Jana Sládková1, JanaKřížová1, Hana Hansíková1, Jiří Zeman1, Markéta Tesařová1

Institution: 1Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, Prague, Czech Republic, 12800, 2Czech Academy of Sciences, Institute of Physiology, Czech Republic, 14220.

Title: Mutations in the DNM1L Gene (Drp1) and Their Impact on the Mitochondria, Peroxisomes and Mitochondrial Energetic Metabolism

Body of Abstract: The DNM1L gene encodes the dynamin-1-like protein (Drp1), which is part of the GTPase group of hydrolase enzymes and is important in the mitochondrial and peroxisomal fission process. Drp1 has three domains: a GTPase domain, a middle domain and a GTPase efector domain. Several mutations in the GTPase domain and the middle domain have been described previously. In human fibroblasts, we studied consequences of two heterozygous DNM1L mutations c.176C>T (p.Thr59Ile) in the Drp1 GTPase domain, essential for GTP hydrolysis, and c.1084G>A (p.Gly362Ser), in the Drp1 middle domain necessary for its oligomerization. We confirmed mutation-specific effect of the mutations on the mitochondrial ultrastructure and mitochondrial and peroxisomal fission process. Furthermore, we have observed differences in bioenergetic metabolism in transfected cells lines. The respiration and ATP production were decreased in cells with the GTPase domain mutation and, on the contrary in cells with the middle domain mutation, production of ATP and respiration were slightly increased, compared to control fibroblasts. Our data confirm a dominant-negative effect of these mutations in the DNM1L gene on the function of Drp1. Supported by research projects AZV 17-30965A, RVO-VFN64165/2012, SVV 260367.


Presenter: Zarazuela Zolkipli-Cunningham

Authors: Zarazuela Zolkipli-Cunningham1, Jean Flickinger2, Allan Glanzman2, Colleen C. Muraresku1, Elizabeth McCormick1, Simone Udeh1, Brianna Soreth1, Laura MacMullen1,George Ibrahim-Sankoh1, Rui Xiao3,4, Marni J. Falk1,4.

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics; 2Center for Rehabilitation, Children’s Hospital of Philadelphia; 3Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania, Philadelphia, PA 19104, USA; 4Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.

Title: Quantitative Assessment of Mitochondrial Myopathy


Background: ‘Mitochondrial myopathy’ (MM) refers to genetically-confirmed mitochondrial disease that predominantly impairs skeletal muscle function. Increasing pursuit of MM treatment trials has created a clear need for outcome measures that reliably capture the severity of MM patient-prioritized symptoms, disease progression, and response to interventions. Currently, no validated, objective outcome measures capture the multi-dimensional domains of MM, including muscle weakness, fatigue, and imbalance (Zolkipli et al. 2018). The goal of this study was to establish clinically-meaningful quantitative measures of MM.

Methods: An infrastructure of physical therapists, bioinformatician, and clinical coordinators was assembled within The Children’s Hospital of Philadelphia (CHOP) Mitochondrial Medicine Frontier Program (MMFP), which follows a clinical cohort of 195 genetically-confirmed individuals with mitochondrial disease in existing clinical research protocols (IRB #08-6177, PI Falk; IRB #16-013364, PI Zolkipli). Physical therapy (PT) evaluations are routinely included in MMFP clinical assessments. An array of objective measures are used to quantify strength by hand-held dynamometry (Mentiplay et al., 2015); functional dexterity (FDT, Sartorio et al., 2013) and 9-Hole Peg Test (9HPT); balance (McKay et al., 2016); and endurance by 30 seconds sit-to-stand (30SSTS) and 6-minute walk test (6MWT) to accurately capture the multi-dimensional domains of MM. Results are normalized to published normative data (McKay et al., 2016) and displayed as Z-scores, where ≤ -2 Standard Deviation (SD) is abnormal. 6MWT per minute distances are analyzed by a linear mixed-effects model.

Results: We have completed detailed PT evaluations in 120 subjects to date, including 45 subjects with genetically confirmed MM. Mean age was 23.1 years (range 7-66 years), and 53% were male. In genetically-confirmed MM subjects (n=45), dynamometry strength measurements in adult and child MM subjects revealed the greatest weakness in elbow flexion, wrist extension, and handgrip in the upper extremities, and hip flexion and ankle dorsiflexion in the lower extremities (≤ -2 SD), as well as knee flexion only in child MM subjects. These results demonstrate there exists both proximal and distal muscle weakness in MM, where only 2 subjects had co-existing peripheral neuropathy. Muscle weakness was symmetric in all MM subjects. The FDT (-4.5 SD) and 9HPT (-5.3 SD) revealed significantly impaired hand dexterity in MM, and standing tandem stance with eyes open (-2.6 SD) and closed (-2.7 SD) revealed impaired balance in MM. MM subjects also had increased fatigue on 30SSTS (-2.2 SD). 6MWT minute distance analysis revealed a significant decline in distance walked by 1.2 meters per minute (p-value=0.00082) consistent with poor endurance, while non-MM individuals with fatigue (n=12) did not show a significant decline in distance walked (p-value=0.9). Real-time integration of data into our newly developed clinical-research data integration and management system, MMFP Tableau, enabled rapid visualization of these objective clinical outcomes over time and facilitated ready completion of robust statistical analyses.

Conclusion: We have established an array of quantitative PT assessments that accurately reflect MM multi-dimensional and patient-important domains of muscle weakness, impaired dexterity, fatigue, and imbalance. Development of a composite measure to display the collective results as one overall integrated measure of MM is underway, which will hold utility as a clinically-meaningful objective outcome measure for future MM trials.


Presenter: Xavier LLòria

Authors: Xavier Llòria,1 Magda Silva,1 Guenther Rudolph,2 Felice Lob,2 Bettina von Livonius,2 Claudia Catarino,3 Thomas Klopstock3

Institution: 1Santhera Pharmaceuticals Ltd, Pratteln, Switzerland; 2University Hospital of the Ludwig-Maximilians-Universität, Munich, Germany. 3Friedrich-Baur-Institute, Munich, Germany.

Title: Long-term (over 24 months) Treatment with Idebenone may Continue to Improve Visual Function Response in Patients with Leber’s Hereditary Optic Neuropathy (LHON)


Body of abstract:Background: LHON causes a progressive, profound, usually irreversible, bilateral loss of visual acuity (VA). Three primary mitochondrial DNA (mtDNA) mutations cause over 90% of cases. The only approved treatment for LHON is idebenone (150 mg tablets) at a dose of 900 mg/day. Here we present VA outcome data from an international Expanded Access Program (EAP) of idebenone in a sub-group of patients treated for a minimum of 24 months.

Methods: Patients with confirmed mtDNA mutations were treated with idebenone under Named Patient Regulations, and followed in routine clinical practice. VA and safety data were collected at each visit. Along-term (LT) efficacy cohort was defined as patients carrying a primary mutation, having initiated treatment within 1 year from onset of VA loss (OVL) and a treatment duration of at least 24 months. Efficacy was assessed as evidence of clinically relevant recovery (CRR) from nadir (VA improvement from off-chart to reading 5 letters on the ETDRS chart, or improvement of 10 letters), at 8 different time points: baseline (BL), 6 months, 9 months, 12 months, 15 months, 18 months, 24 months and 30 months.

Results: 111 patients, 40 of whom fulfilled the LT treatment criteria, participated in the EAP. The patients (82.5% male) had a median age of 22.1 (6.9 – 65.8), a median time since OVL of 4.0 (0.3 – 11.5) months and were treated for a median of 35.7 (24.5 – 59.5) months. G11778A was the prevalent mutation (55.0%), followed by G3460A (22.5%) and T14484C (22.5%). 26/40 (65%) of patients experienced a CRR, of which 42.3% occurred within 6 months and 57.7% within 12 months. The last patient experienced an initial CRR at 26.5 months. The magnitude of VA improvement from nadir (median ETDRS letters) at initial CRR was 14 letters, which improved to 31 letters at last visit. Best median VA (expressed as logMAR) were 1.20, 1.34, 1.15 at BL, 9 months and 30 months, respectively. Safety signals observed in the LT treatment cohort were consistent with the overall EAP population.

Conclusions: A steady improvement in VA can be observed in patients who maintain idebenone treatment, with 65% of patients experiencing an initial CRR by 26.5 months after treatment initiation. Some patients experienced a transient deterioration during the first 9 months after treatment initiation. However, this should not be considered as treatment failure, as these results indicate improvements can occur up to 30 months of treatment.


Presenter: Xavier Llòria

Authors: Thomas Klopstock1, Xavier Llòria2, Magda Silva2, Claudia Catarino1, Felice Lob3, Bettina von Livonius3, Günther Rudolph3

Institution: 1Friedrich-Baur Institute, University Hospital of the Ludwig-Maximilians University, Munich, Germany; 2Santhera Pharmaceuticals Ltd, Pratteln, Switzerland; 3Department of Ophthalmology, University Hospital of the Ludwig-Maximilians University, Munich, Germany

Title: Responder analysis of chronic Leber’s hereditary optic neuropathy (LHON) patients to idebenone in a placebo controlled, randomized clinical trial (RHODOS)

Body of Abstract: Introduction: LHON is a mitochondrial genetic disorder causing severe, bilateral central vision loss in both eyes. Here we report on the response outcome to idebenone in a double-blind, randomized, placebo controlled clinical (RHODOS) which evaluated the efficacy and safety of idebenone over 24 weeks.

Methods: RHODOS studied patients from 14-64 years of age with a confirmed primary mutation and vision loss due to LHON within 5 years. Post-hoc response analysis evaluated efficacy as a clinically relevant recovery (CRR) at week 24 (last visit; defined as improvement from off-chart to reading 5 letters


on the ETDRS chart, or improvement of 10 letters) from baseline (BL), in the subpopulation of patients > 1 year since symptoms onset at start of treatment.

Results: At BL, 53/82 patients (idebenone: 34; placebo: 19) presented with chronic LHON. 35.3% of idebenone-treated vs. 10.5% of placebo-treated patients achieved CRR from BL (p=0.0596). By eyes: 23.5% idebenone-treated vs. 5.3% of placebo-treated eyes achieved CRR (p=0.0163). Magnitude of recovery at CRR (lines): idebenone, 2-10; placebo, 2-5. Time to CRR from BL (months): idebenone, mean 2.8 (range 0.9-6.2); placebo, mean 3.4 (range 1.1-5.7).

Conclusions: Idebenone treatment induced CRR in a significantly larger proportion of patients/eyes than placebo in chronic LHON.


Presenter: Sujay Guha1

Authors: Sujay Guha1, Chigoziri Konkwo1, Seinn Wai1, Matthew A. Churgin2, Anthony D Fouad2, Rui Xiao3, Eiko Nakamaru-Ogiso1, Christopher Fang-Yen2, Marni J. Falk1, 4

Institution: 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2 School of Engineering and Applied Sciences, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104; 3 Department of Statistics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104. 4 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.

Title: Automated High throughput screening of disease and drug effects on survival, activity, and mitochondrial stress in Caenorhabditis elegans animal models of primary mitochondrial disease

Abstract: Simple model animals of mitochondrial disease, such as the 959-cell nematode C. elegans,offer a valuable means by which to understand the complex impact of different mitochondrial diseases and candidate therapies in a living system. Animal survival and activity, in particular, convey particularly meaningful and relevant integrated physiologic outcomes by which to assess the impact of mitochondrial disease and guide therapeutic optimization. However, traditional manual microscopic assessments of animal survival, activity, and mitochondrial stress have been labor-intensive and tedious, limiting throughput and cost efficiency.

Here, we report our experience using a novel automated WormCamp robotic method we constructed to monitor lifespan and healthspan (activity) of C. elegans (worm) populations. Based on the initial prototype of single worm semi-automated lifespan analyses in 240 well plate format in the WorMotel analysis system (Churgin M et al, eLife, 2017), the newer robotic system permits fully automated analysis of 90 different 24-well plate format with populations of approximately 50 worms seeded per well. Worms are added to plates containing test therapies at the L4 larval stage, and then imaged by a high-resolution camera moved between plates with a robotic gantry for 5 minutes each twice daily for their entire adult lives (mean lifespan 2 weeks), both before and after intense blue light exposure to stimulate worm activity. Pixel changes between pre-post light images are used to determine worm relative activity and viability. Automated data analysis is performed using a custom MATLAB code.

We also report a novel screen to efficiently quantify the C. elegans mitochondrial unfolded protein response (UPRmt), which is similar to that of mammalian systems. UPRmt in C. elegans, is assessed using a GFP reporter for hsp-6. For example, feeding RNA interference-based knockdown of K09A9.5 (gas-1, NDUFS2 orthologue) in the hsp-6p::gfp reporter worm strain strongly induces UPRmt, which is rapidly quantified by Union Biometrica Biosorter based on fluorescence intensity in living worms. Preliminary studies


demonstrate that treatments of either glucose (10mM) or N-acetylcysteine (2.5 mM) significantly reduce this mitochondrial stress response.

Therapeutic screening of all known mitochondrial disease therapies, and broader drug libraries, is now readily achievable and underway to determine effective therapies, concentrations, and combinations that restores animal survival, activity, and mitochondrial stress to healthy levels in diverse RC disease models. Therapeutic leads have been identified, which will be further discussed, to be used as positive controls in high-throughput drug screening necessary to identify the most potent therapeutic leads that improve overall survival and function to take forward to clinical trials in mitochondrial disease patients with distinct genetic disorders.


Presenter: Wolf-Hagen Schunck

Authors: Wolf-Hagen Schunck1,2, Victor Samokhvalov3,Tim Wesser2, Anne Konkel2, Robert Fischer2,John Seubert3

Institutions: 1Max Delbrueck Centre for Molecular Medicine, Berlin, Germany, 2OMEICOS Therapeutics GmbH, Berlin, Germany; 3Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Canada

Title: Activation of Mitochondrial Quality Control by Epoxyeicosanoid-Like Drugs

Abstract: Epoxyeicosanoids comprise a family of bioactive lipid mediators generated by cytochrome P450 enzymes from long-chain polyunsaturated fatty acids (PUFAs). Epoxyeicosanoids exert cell and organ protective effects under diverse stress conditions that cause mitochondrial dysfunction. Prominent examples include ischemia/reperfusion-induced damage of the heart, brain and kidney. Studies in cardiomyocytes revealed that epoxyeicosanoids limit stress-induced mitochondrial dysfunction by (i) preserving the function of existing mitochondria, (ii) increasing autophagy potentially resulting in the removal of damaged mitochondria, and (iii) enhancing the biogenesis of intact mitochondria.

OMEICOS Therapeutics developed a small molecule (OMT28) that mimics the structure and biological activities of omega-3 PUFA-derived epoxyeicosanoids. Preclinical studies indicate therapeutic efficiencies of orally administered OMT28 in atrial fibrillation (AF) and age-related macular degeneration (wet AMD). OMT28 successfully passed Clinical Phase I and is currently investigated in a prospective randomized phase II trial in AF patients.

OMT-28 is effective in the nanomolar range and improves Ca2+-handling, mitochondrial function and cell viability in cardiomyocytes under stress conditions, such as hypoxia-reoxygenation (HR). OMT28 attenuates HR-induced decreases in mitochondrial respiratory function as well as of cellular NAD/NADH- and ADP/ATP ratios. Moreover, OMT28 prevented the HR-induced decline in the ratio of mitochondrial (COX-1) vs. nuclear (SDH-A) gene expression, indicating stimulation of mitochondrial biogenesis. The mode of action includes activation of PI3K/Akt/eNOS/PKG signaling as well as of sirtuin-1 and PPARα.

We hypothesize and would like to discuss that epoxyeicosanoid-mediated activation of mitochondrial quality control may have a therapeutic potential not only in secondary mitochondrial dysfunction, but also in primary mitochondrial diseases, where enhanced mitochondrial quality control might result in shifting heteroplasmy and decreasing pathogenic mtDNA mutation load.



Presenter: Senta M Kapnick

Authors: Senta M Kapnick, Rylee Genner, Peter McGuire

Institution: National Human Genome Research Institute, National Institutes of Health, Bethesda, MD,20892

Title: Understanding the Impact of Mtdna Copy Number Regulation on T Lymphocyte Metabolism and Function

Body of Abstract: Mitochondria contain multiple copies of circular, double-stranded, maternally-inherited DNA (mtDNA) approximately 16kB in length that encode for 37 genes: 2 rRNAs, 22 tRNAs, and 13 proteins all important for energy production via the respiratory chain. Loss of mtDNA copy number or integrity in tissues is associated with mitochondrial depletion syndromes, a group of clinically heterogenous disorders caused by mutations in genes important for mitochondrial maintenance. Mitochondrial transcription factor A (TFAM) is an essential protein that binds mtDNA regulating packaging, replication, initiation of mitochondrial transcription, and ultimately mtDNA copy number and mitochondrial biogenesis. Total disruption of TFAM by gene targeting in mice results in embryonic lethality, while heterozygous mice exhibit reduced mtDNA copy number and respiratory chain deficiency in cardiac tissue. Although TFAM has been recently shown to also play a role in neurodegeneration, its contribution to mitochondrial maintenance in other tissues with high energetic demands remains poorly understand. To mount effective immune responses, T cells must migrate between tissues, clonally expand, and secrete effector molecules; highly energetic processes now known to be dependent on changes in mitochondrial-mediated metabolism. To better understand the role of mtDNA copy number regulation in T lymphocyte function, we disrupted TFAM expression in T cells using a loxP-flanked Tfamallele in combination with a Cre-recombinase transgene under the control of the CD4 promoter. TFAM-deficient T cells undergo normal thymic development and exhibit expected total numbers in the periphery. While T cell receptor-mediated activation of naïve CD8+ T cells results in reduced proliferative capacity, stimulated CD8+ T cells display increased expression of activation markers and enhanced cytotoxic function, on a per cell basis. Although mtDNA content is significantly reduced in TFAM-deficient T cells, MitoTracker staining suggests increased mitochondrial mass. Studies are currently underway investigating bioenergetics and expanded immune responses in TFAM-deficient T cells. By understanding how loss of TFAM alters T cell metabolism, we expect to contribute to our knowledge ofthe link between mtDNA copy number regulation and immune cell function. This work has the potential to aid in the development of improved strategies for evaluating and treating immune dysfunction in patientswith mitochondrial disease associated with mtDNA depletion.


Presenter: Xavier Llòria

Authors: Xavier Llòria1, Magda Silva1, Claudia Catarino2, Felice Lob3, Bettina von Livonius3,Günther Rudolph3, Thomas Klopstock2

Institution: 1Santhera Pharmaceuticals Ltd, Pratteln, Switzerland; 2Friedrich-Baur Institute, University Hospital of the Ludwig-Maximilians University, Munich, Germany; 3Department of Ophthalmology, University Hospital of the Ludwig-Maximilians University, Munich, Germany

Title: Evaluating Long-Term Real-World Data (RWD) from Patients with Leber’s Hereditary Optic Neuropathy (LHON) in Light of the Recommendations of the International Consensus Statement


Body of Abstract: Introduction: LHON is a rare mitochondrial genetic disorder that results in severe, bilateral central vision loss in both eyes. Idebenone has been shown to be efficacious in treating LHON. The International Consensus recommends 1) a minimum treatment duration of 12 months before evaluating efficacy and 2) maintaining treatment for 12 months after best corrected visual acuity (BCVA) improvement has stabilized.

Methods: A retrospective evaluation of patients treated with idebenone (900 mg/day) was conducted to assess the efficacy of idebenone in real-world clinical practice. Efficacy was evaluated as clinically relevant recovery (CRR; BCVA improvement from off-chart to reading 5 letters, or 10 letters on-chart improvement [ETDRS chart]), time to initial response and response magnitude over time. Inclusion criteria: <12 months since most recent eye onset; provided post-baseline data.

Results: 47% of patients (41/87) achieved CRR at the last observation (mean 23.8 months from baseline). Of the 41 responders, 66%, 83% and 90% responded by 12, 18 and 24 months of therapy. The average magnitude of response increased from 0.4 at time of first CRR to 0.7 logMAR at last visit.

Conclusion: RWD supports the Consensus. Some patients may not respond until 24 months of continuous therapy. Early discontinuation may prevent the full magnitude of response


Presenter: Bryn D. Webb

Authors: Bryn D. Webb1, Ananya Swaroop1, Mingma Sherpa1, Carmen Argmann1, Eric E. Schadt1,Sander M. Houten1

Institution: 1Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Title: In Vivo Studies of Mitochondrial Methionyl-tRNA Synthetase (MARS2) Deficiency

Body of Abstract:Objective: To further characterize the pathophysiology of MARS2 deficiency using a newly generated knock-in mouse model.

Background: We previously identified by WES a novel mitochondrial disorder caused by recessive single nucleotide variants in mitochondrial methionyl amino-acyl tRNA synthetase (MARS2), which presents with clinical symptoms of developmental delay, poor growth, and sensorineural hearing loss. The MARS2compound heterozygous mutations identified in the index family, c.550C>T;p.Gln184* and c.424C>T;p.Arg142Trp, led to: decreased MARS2 protein levels in patient lymphoblasts; decreased Complex I and IV enzyme activities in patient fibroblasts; and reduced protein levels of NDUFB8 and COXII, representing Complex I and IV respectively, in patient fibroblasts and lymphoblasts. Overexpression of wild-type MARS2 in patient fibroblasts rescued complex I and IV subunit deficiencies (Webb et al., 2015).

Methods: A knock-in mouse model was generated by CRISPR/Cas9 technologies on a C57BL/6 background. The Mars2 c.403C>T;p.Arg135Trp variant corresponding to the MARS2 p.Arg142Trp change was generated. Mice were bred to homozygosity, and littermates were sacrificed and tissues and organs harvested at different time points. Immunoblotting with a mitochondrial oxidative phosphorylation antibody cocktail was completed to assess for mitochondrial dysfunction. RNA-seq was completed with RNA generated from wild-type and homozygous mutant mice liver and gastrocnemius samples. Differentially expressed gene (DEG) signatures were identified using an in-house pipeline, which utilizes RNAStar, FeatureCounts, and Limma/Voom.


Results: Homozygous mutant mice are viable and survive to adulthood, but do not exhibit gross hypotonia or motor delay. However, immunoblotting studies revealed a biochemical phenotype of reduced protein levels of NDUFB8 in liver and kidney samples from homozygous mutant mice. RNA-seq analysis revealed 465 DEGs in liver and 99 DEGs in gastrocnemius (p-value <0.05). Gene ontology pathway analysis highlighted involvement of innate-immune response, regulation of glucose import, and phosphatidylinositol-3-kinase signaling pathways. We noted considerable overlap of our DEG lists with a previously identified innate antiviral immune response signature identified in Tfam+/- mice, which serve as a model for mitochondrial depletion.

Conclusion: Homozygous MARS2 Arg135Trp mice are a viable model to further study MARS2 deficiency. Additionally, study of this model highlights the connections between mitochondria and the innate immune system.


Presenter: Megan Barksdale

Author: Megan Barksdale1, Sheila Clever1, Rebecca Ganetzky1,2

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, 19104 USA, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 USA

Title: ATP Profiling of Diverse Known and Novel MT-ATP6 Variants, Using Transmitochondrial Cybrids

Abstract: Background: Mitochondrial Complex V (CV) is the bioenergetic pump in the mitochondria that directly generates chemical energy in the form of ATP. CV is comprised of 17 subunits that function together to release the mitochondrial membrane potential generated by the electron transport system, causing spinning of the catalytic domain and ultimately ATP synthesis. The vast majority of reported CV deficiency is due to mutations in subunit a, which is encoded on the mtDNA by MT-ATP6. MT-ATP6 has a high degree of genetic variability. These variants are challenging to evaluate due to the limited availability of CLIA-approved biochemical testing to evaluate the function of mitochondrial complex V (CV). Previous reports on the biochemical effects of pathogenic variants in MT-ATP6 show high variability in effects. We hypothesized that the biochemical variability was related to diverse samples in terms of tissue type, genetic defect and methodology. Therefore, we sought to systematically analyze ATP levels in transmitochondrial cybrids with a range of variants in MT-ATP6.

Methods: ATP production use evaluated using a luciferase/luciferin-based assay (CellTiter-Glo, Promega) in transmitochondrial cybrid lines with homoplasmic pathogenic MT-ATP6 mutations (n=2, m.8993T>G, m.8993T>C) and novel homoplasmic MT-ATP6 variants (n=2, m.8612T>C, m.9041A>G). When available, maternal cell lines that were homoplasmic wild type were used as control. ATP was measured at baseline and after 24 hours of treatment with Oligomycin, Rotenone, and 2-Deoxy-D-glucose (2DG) at a range of concentrations. Results were normalized to protein concentration was determined.

Results: At baseline control cell lines produce significantly more ATP than all MT-ATP6 variant cell lines tested. This effect is genotype-specific, and is most profound in the m.8993T>G line, while very subtle in the m.8993T>C line. Variant cell lines are more sensitive to the CV inhibitor Oligomycin, while they show no increased sensitivity to the CI inhibitor rotenone. The overall pattern of ATP production is consistent across lines with MT-ATP6 variants.

Conclusion: This research has shown that at baseline cells with MT-ATP6 mutations produce less ATP than control lines and that cells and have increased oligomycin sensitivity. In addition to these results, this reaction to Oligomycin allows us to create profiles for different variants. These results suggest that


ATP measurements at baseline and oligomycin treatment is beneficial to classify variants in MT-ATP6.These results suggest that the m.8612T>C and m.9041A>G variants in MT-ATP6 are pathogenic.


Presenter: Amel Karaa, MD

Institution: Massachusetts General Hospital, Boston, MA

Title: Telemedicine in the Mito Clinic: The MGH experience.

Body of Abstract: Telemedicine is a good health care option for rare diseases enhancing access to disease-specific health care experts and covering patients in underserved geographical areas. These two aspects have been found to be major gaps in mitochondrial care by the Mitochondrial Medicine Society (MMS) and the Mitochondrial Care Network (MCN). The Mito Clinic at the Massachusetts General Hospital (MGH) has been utilizing telemedicine since 2013. Here we review our experience in the last 2 years (09/2016 to 01/2019). Amongst 81 virtual visits (VV) conducted in the Mito clinic, 70 pertained to confirmed primary mitochondrial disease (PMD) patients or patients with suspected PMD who were being seen for follow up. All visits were conducted with patients in their home (94%) with some exceptions. Half the patients were alone at the visit (51.5%) whereas the other half (47.5%) were accompanied by a family member (a parent, spouse, sibling or a child). The average age of patients participating in these VVs was 39.7 years (min 9-max 72) and most were females (68.5%). The main reason for the VV included: discussion of test results (40%), genetic counseling to obtain further diagnostic testing (20%), and specific symptom assessment/urgent care (7%). Regular, periodic scheduled follow-up and active “check-in” by provider to monitor sick patients represented 24% and 4% of the VV respectively. Conversion of scheduled face-to-face visits to VVs was performed in 7% of the cases during snow storm to accommodate patients and prevent hazardous travel. One or several interventions resulted from these VV including ordering more follow-up tests for specific medical issues (41.5% of cases), referrals to specific specialists (23%), new prescription or medication dose adjustment(19%), and other changes in management (nutrition, exercise, health counseling in general and supportive care). In 20% of the cases, no changes were made to the patient’s management following a VV. It is interesting to note that only 7 of our patients are wheelchair bound and have limited mobility which does not seem to be a major factor in patients choosing this method for follow up, moreover, patients satisfaction scores with VV remain very high. These VV saved our patients 22533.3 miles of physical travel and an average of $45.5/patients in travel cost and parking fees. VV also decreased the cost of PMD office visit from ~ $68-234/visit (depending on the level of complexity) to 0-$25 (when paying out of pocket for the VV). Telemedicine holds promise to improve access and value in mitochondrial care at MGH, we are further clarifying the appropriate use, expansion, and clinical outcomes in our Mito Clinic while remaining mindful of the challenges related to clinical limitations, legal and social barriers, as well as reimbursement issues for a subset of our patients.



Authors: A. Karaa, A. Larson, J. Gannon, JB. Le Pichon, K. Mann, S. Parikh

On behalf of the MCN

Institutions: Massachusetts General Hospital, Children's Colorado Hospital, Children's Mercy Hospital, MitoAction, The Cleveland Clinic

Title: Are we Ready for Telehealth in Mitochondrial Medicine?


Body of Abstract: Previous work by the Mitochondrial Medicine Society (MMS) and the Mitochondrial Care Network (MCN) revealed disparities in mitochondrial care delivery with most experts and sites being located on the coasts and big US cities while a large swath of mid-country states remain without providers. An immediate solution to this problem is to utilize telehealth to allow increased geographical coverage and access to mitochondrial disease experts. An MCN telehealth task force was created to assess site resources, interest and potential virtual medicine expansion. A 32-question electronic survey was developed and answered by the MCN directors. Results show that most MCN sites (66.7%) already have an established telehealth program at variable degrees of maturity (43% of sites have a fully launched or expanding program). Multiple telehealth options are available across sites including virtual consultations with patients/families directly in the home setting (33.5%) or supported by another local medical facility with or without a health care professional present (66.5%). Telehealth visits are mainly used in the outpatient setting (38%) to discuss test results, urgent care, patient monitoring and to provide second opinions for those who can see new patients (9% of sites). Although most MCN directors were enthusiastic about using Telehealth resources and were interested in participating in a mitochondrial disease telehealth network (76% of respondents), several concerns were raised. Multiple state licenses are required to see patients residing in different states then the consulting providers. This is even required for follow-up patient visits in 28.5% of the MCN sites. Physician-liability insurance might prevent access to new patients across state lines and the patient cost of telehealth remains high with the majority of sites billing patients directly. MCN sites with telehealth capabilities use different video conferencing software primarily provided and managed by their own institutions.

Telehealth is an innovative, potentially new avenue to expand patients’ access to mitochondrial disease experts and care in areas without specialized resources or MCN site availability. State regulations and operational costs are the major rate-limiting factors to such an expansion. The MCN is investigating whether an independent telehealth consult services may be worth developing and implementing within the US.


Presenter: Eiko Nakamaru-Ogiso

Authors: Eiko Nakamaru-Ogiso1, Jianping Kong1, Jeremy N. Leipzig2, Kelly Getz3, Theodore Schurr4, Richard Aplenc3,5, and Marni J.Falk1.5

Institution: 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, 2Center for Biomedical Informatics, 3Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 4 Laboratory of Molecular Anthropology, Department of Anthropology, University of Pennsylvania, Philadelphia, PA 19104; 5Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Title: Mitochondria Haplogroup Modulates Cardiac Toxicity from Anthracycline Chemotherapy inAcute Myeloid Leukemia Patients

Abstract: Background: Anthracycline chemotherapy is a major treatment strategy for many forms of cancer, including acute myeloid leukemia (AML). While anthracyclines significantly increase overall cancer survival rates, their cardiac toxicity is a dose-limiting side effect. Further, cardiovascular morbidity and mortality are significantly increased after anthracycline exposure in long-term cancer survivors. The cardiotoxic mechanisms of anthracyclines remain unclear, but are postulated to involve DNA damage, increased reactive oxygen species production, and mitochondrial dysfunction. Current pediatric AML therapy requires high anthracycline doses with an increased risk of short and long term cardiac toxicity. Interestingly, clinical studies have suggested that ethnic background plays a role in the frequency and severity of anthracycline cardiotoxicity among AML patients.


Objective: To determine whether mitochondrial haplogroup conferred altered sensitivity cardiotoxicity from the anthracycline doxorubicin (DOX), mitochondrial genome-wide association study (GWAS) was performed on AML patients, with validation of DOX sensitivity in lymphoblastoid cells derived from the most sensitive and insensitive mitochondrial haplogroups.

Methods: Clinical oncology group (COG) AAML1031 trial enrolled ~1100 patients, with cardiac function values collected from ~90% of patients. 200 patients were selected for mitochondrial DNA (mtDNA) genome sequencing, including those who had shown either the largest drop or no drop in cardiac function after DOX treatment. Cardiotoxicity was measured on cardiac echocardiogram as clinically asymptomatic left ventricular systolic dysfunction (LVSD) with a fall in left ventricular (LV) ejection fraction. NCI clinical toxicity criteria were used to code LVSD grading from 0 (no toxicity) to 5 (death). Based on the relationship analysis performed between mtDNA haplogroup and LVSD clinical toxicity grades, two healthy lymphoblastoid cell lines, GM19328C (GM, Kenya, female, L0a’b) and HG02048 (HG, Vietnamese female, M5) were purchased from Coriell Institute, which respectively represent LVSD low (African) and LVSD high (South Asian) populations. Cells were compared following DOX treatment on outcomes including viability and mitochondrial physiology (FACS analyses, high resolution respirometry, and electron transport chain enzyme activity analyses by spectrophotometry).

Results: Clinical data combined with mitochondrial genome analysis demonstrated that mtDNA haplogroups M, R, and X (which exist in Hispanics/South Asian population) have increased LVSD high outcomes, and are more likely to get DOX cardiotoxicity than mtDNA haplogroups K, L0, A, and J (which exist in non-Hispanics/Africans population). To functionally validate and further evaluate the mechanism underlying this association, we sought to determine whether any intrinsic differences in DOX sensitivity exist in healthy cells based solely on divergent mitochondrial haplogroups. We found that while HG cells (LVSD high) showed high sensitivities toward DOX, with reduced survival by 30% at 0.1 μM and by 50% at 0.5 μM after 24 hours, GM cells (LVSD low) were virtually unaffected. Parallel results were seen on mitochondrial physiology analyses, where significantly increased mitochondrial superoxide levels by ~30% after DOX exposure (0.1 μM for 24 hours) occurred only in HG cells. After low dose (0.1 μM DOX) treatment for 24 hours, maximum respiratory capacity and uncoupled electron transfer activities were significantly decreased in HG cells but increased in GM cells by ~10-15%. This stimulating effect in GM cells is consistent with the increased complex II activity by ~20%.

Conclusions: The increased risk of cardiac toxicity following anthracycline chemotherapy appears to be modulated by mitochondrial DNA haplogroups. Cellular validation studies demonstrate that haplogroup M5 (HG, Vietenamese) lymphoblastoid cells are indeed uniquely sensitive to DOX, with dose-dependent reductions in viability and respiratory capacity, and increased mitochondrial oxidative stress. These data are suggestive that DOX dosing in AML patients might benefit from a pharmacogenetic approach, where mitochondrial haplogroup analysis may enable dose modulation to reduce the risk of cytotoxicity.


Presenter: Richard Haas, MD

Author: Richard Haas,1 Amy Goldstein,2 Jerry Vockley,3 Bruce H Cohen4 Amel Karaa5

Institution: 1UC San Diego School of Medicine, San Diego, CA, 2Children’s Hospital of Philadelphia, Philadelphia, PA, 3Children’s Hospital of Pittsburgh, Pittsburgh, PA, 4Akron Children’s Hospital, Akron, OH, 5Massachusetts General Hospital, Boston, MA

Title: MMPOWER-2 Open-Label Extension (OLE) Trial: Six-Month Treatment Effect on Patients with Baseline 6MWT between 100-450 Meters

Abstract: INTRODUCTION: Primary mitochondrial myopathy (PMM) is a group of genetic disorders of the mitochondrial respiratory chain affecting predominantly, but not exclusively, skeletal muscle. Cardinal symptoms include fatigue, exercise intolerance, and muscle weakness. These symptoms negatively affect physical functioning, exercise capacity, and quality-of-life (QoL). An analysis of a USA commercial insurance claim database places estimates at approximately 36,000 adult patients in the USA who have


been evaluated for having a primary mitochondrial disease. To date, there are no FDA approved treatments for affected patients. Elamipretide, an investigational therapy being evaluated in patients with PMM, localizes to the inner mitochondrial membrane where it associates with cardiolipin (CL), to restore cristae architecture resulting in improved ATP generation and reduced oxidative stress. OBJECTIVES: A post-hoc subgroup analysis was conducted to evaluate the impact of 6 months ofcontinuous elamipretide therapy in patients whose 6-Minute Walk Test (6MWT) results were between 100-450 meters at pre-treatment baseline (elamipretide naïve) before entering into the MMPOWER program. This analysis simulates the design of MMPOWER-3 (Phase 3 elamipretide trial) patient population. METHODS: Patients enrolled in MMPOWER-2 OLE (N=28) who met the elamipretide-naïve 6MWT range (100-450 meters) and had a 6-Month MMPOWER-2 OLE visit were included in the analysis cohort (n=21). These subjects have received elamipretide 40 mg SC daily from entry into the MMPOWER-2 OLE (baseline) through 6 months (evaluation endpoint). We analyzed the change in the 6MWT, Primary Mitochondrial Myopathy Symptom Assessment (PMMSA) Total Fatigue score, Neuro-QoL Short Form Fatigue, EQ-5D-5L, and overall safety via reported adverse events (AE). RESULTS: The analysis cohort of patients (2 male and 19 female) self-reported being white (100%) andhad an average age 49 years (range 19-66 years). Distance walked in the 6MWT was maintained throughout the 6- month observation period for the patients treated daily with elamipretide. Importantly, patients continued to show improvements compared with their baseline scores in their self-reported symptom questionnaire scores. The PMMSA-Total Fatigue scores demonstrated a reduction infatigue/improvement (-1.95 points), the Neuro-QoL Short Form Fatigue demonstrated agreement with these results as demonstrated by reduction in T-Score of -5.81. All of the individual domains in the EQ-5D-5L assessment improved with continued elamipretide therapy. Patients also reported changes inscores indicative of a beneficial effect as evidenced by the transition from having some degree of“Problem” (domain score of 2-5) to having “No Problem” (domain score of 1). The safety profile ofelamipretide was shown to be consistent with results from previous trials, with the most commonly reported AEs being injection site reactions. CONCLUSIONS: Patients receiving continued therapy with elamipretide demonstrated a favorable treatment effect over the duration of the assessed 6-month period. Safety analysis results were consistent with those observed in other clinical studies of elamipretide administered to patients with PMM.


Presenter: Hilary Vernon

Authors: Hilary Vernon, MD, PhD1; William Reid Thompson III, MD2; Anthony Aiudi, PharmD3; John J Jones, PharmD3; Jim Carr, PharmD3, Brittany DeCroes Hornby, PT, DPT, PCS4

Institution: 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine and Kennedy Krieger Institute; Director, Barth Syndrome Clinic at Kennedy Krieger Institute, Baltimore, MD, 21287 2Division of Pediatric Cardiology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287 3Stealth BioTherapeutics, Newton, MA, 02467 4Department of Physical Therapy, Kennedy Krieger Institute, Baltimore, MD, 21205

Title: TAZPOWER: Trial Results From a Randomized, Double-Blind, Placebo-Controlled, Crossover and Open-Label Extension Trial of Elamipretide in Patients With Barth Syndrome

Body of abstract: Background: Barth syndrome (BTHS) is a rare, X-linked infantile-onset debilitating disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delays and neutropenia. BTHS is caused by defects in the TAZ gene that encodes tafazzin, a transacylase responsible for the


remodeling and maturation of the mitochondrial phospholipid cardiolipin (CL). CL is critical to normal mitochondrial structure and function (i.e. ATP generation). In patients with BTHS, the ratio of structurally immature monolyso-cardiolipin (MLCL) to structurally mature tetralineoyl-cardiolipin (L4-CL) is increased. An increased MLCL:L4-CL ratio has diagnostic utility and has been correlated with greater clinical severity.

TAZPOWER is the first prospectively designed clinical trial to evaluate the use of elamipretide in patients with BTHS.

Objectives: Determine the clinical efficacy, safety, and tolerability of elamipretide in patients with genetically confirmed BTHS.

Methods: Part 1 (N=12) was a 28-week crossover design with 1:1 randomization of patients to elamipretide 40 mg subcutaneously (SC) daily, administered for 12 weeks, followed by a washout period of 4 weeks, and then matching placebo for 12 weeks, or vice versa. Efficacy was measured through functional assessments (6-Minute Walk Test [6MWT], muscle strength via handheld dynamometry [HHD], 5-times-sit-to-stand [5XSST], and SWAY Balance score), patient-/clinician-/caregiver-reported questionnaires (including the BarTH Syndrome Symptom Assessment [BTHS-SA], PROMIS Short Form Fatigue, and Global Impression scales)), and biomarkers, including the MLCL:L4-CL ratio. Safety was evaluated through reported adverse events (AEs) and laboratory assessments. Subgroup analyses were performed to examine the impact of baseline disease characteristics on treatment effects. Part 2 is an open-label, long-term assessment (≤168 weeks) of elamipretide in endpoints that are similar to those evaluated in Part 1.

Results: Elamipretide provided clinically meaningful improvements in individual subject functional performances and patient-/clinician-/caregiver-reported reported symptom questionnaires. In particular, a treatment difference was observed for the Clinical Global Impression (CGI) of Change (p=0.0042). In Part 2 OLE, improvement has been observed relative to Part 1 Baseline across all primary and secondary endpoints. At the Part 2 Week 12 Visit, subjects (N=10) improved across all measured endpoints by an average of 60.5 meters (N=10) on the 6MWT, -1.6 on the BTHS-SA, -0.4 on the Patient Global Impression (PGI) of Symptoms, -0.2 on the CGI of Symptoms, -6.0 on the PROMIS Short Form Fatigue, 3.4 on the SWAY Balance score, 37.9 on the HHD, and -0.46 on the 5XSST. Similar trends were seen in the cohorts of patients that have completed Part 2 Week 24 and Part 2 Week 36 Visits. Treatment with elamipretide was well tolerated. Injection site reactions were the most commonly reported AEs.

Conclusions: Elamipretide was well tolerated and clinical benefit was demonstrated in TAZPOWER, compared to published literature. More rapid benefits with elamipretide may be realized in patients with BTHS who have a lower MLCL:L4-CL ratio, however, patients with BTHS with higher MLCL:L4-CL ratios may realize the therapeutic benefits of treatment with elamipretide with continued therapy.



Authors: Melissa A. Walker1,2,3, Vamsi K. Mootha2,3,4,5

Institution: 1Massachusetts General Hospital, Department of Neurology, Boston, MA 02114, 2Harvard Medical School, Boston, MA 02115, 3Massachusetts General Hospital, Department of Molecular Biology, Boston, MA, 02114, 4Department of Systems Biology, Harvard Medical School, Boston, MA 02115, 5Howard Hughes Institute, 6Broad Institute, Cambridge, MA, 02142

Title: Are Leigh Syndrome Lesions Reversible?


Body of Abstract: Leigh syndrome (LS) is the most common pediatric manifestation of mitochondrial respiratory chain disease, originally defined by autopsy findings of bilateral, symmetric brainstem or basal ganglia gray matter lesions characterized by gliosis, vacuolation, capillary proliferation, and relative sparing of neurons. Since Denis Leigh’s original descriptions, the advent of neuro-imaging has largely subsumed the role of histology in the diagnosis of LS; leading to increased number of diagnoses (including cases with milder clinical phenotypes) presenting with the characteristic T2 hyperintense, bilateral, symmetric lesions of the brainstem and/or basal ganglia. Intriguingly, serial application of magnetic resonance (MR) imaging has occasionally revealed cases in which some or all LS lesions no longer demonstrate T2 hyperintensity, suggestive of lesion resolution or reversal. Recent work in a LS mouse model has shown such imaging findings with corresponding resolution of inflammatory markers and other histologic abnormalities in animals treated with chronic hypoxia, raising the possibility that the disease may be reversible. Here, we present two unpublished cases of LS patient with resolution or amelioration of some or all lesions on MRI, review similar published cases, and discuss the implications of these findings in the context of emerging preclinical mouse studies.



Authors: Hilary Vernon, MD, PhD1; William Reid Thompson III, MD2; Anthony Aiudi, PharmD3; John J Jones, PharmD3; Jim Carr, PharmD3, Meagan Elliott,3 Brittany DeCroes Hornby, PT, DPT, PCS4

Institution: 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine and Kennedy Krieger Institute; Director, Barth Syndrome Clinic at Kennedy Krieger Institute, Baltimore, MD, 21287 2Division of Pediatric Cardiology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287 3Stealth BioTherapeutics, Newton, MA, 02467 4Department of Physical Therapy, Kennedy Krieger Institute, Baltimore, MD, 21205

Title: TAZPOWER: Study Design of A Randomized, Double-Blind, Placebo-Controlled Crossover and Extension Trial of Elamipretide in Subjects with Barth Syndrome and Baseline Characteristics.

Body of abstract: Background: Barth syndrome (BTHS) is a rare, X-linked infantile-onset debilitating disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delays and neutropenia. BTHS is caused by defects in the TAZ gene that encodes tafazzin, a transacylase responsible for the remodeling and maturation of the mitochondrial phospholipid cardiolipin (CL). CL is critical to normal mitochondrial structure and function (i.e. ATP generation). In patients with BTHS, the ratio of structurally immature monolyso-cardiolipin (MLCL) to structurally mature tetralineoyl-cardiolipin (L4-CL) is increased. An increased MLCL:L4-CL ratio has diagnostic utility and has been correlated with greater clinical severity. TAZPOWER is the first prospective clinical trial designed to evaluate the use of elamipretide in patients with BTHS. Objectives: The trial was designed to evaluate the efficacy of elamipretide through functional and, patient-/clinician-/caregiver-reported outcome assessments, as well as, safety/tolerability, including AEs and laboratory tests. Subgroup analyses examined impact of age, 6MWT, and MLCL:L4-CL ratio on endpoints. Methods: Part 1 (N=12) was a 28-week crossover design with 1:1 randomization of patients to elamipretide 40 mg subcutaneously (SC) daily, administered for 12 weeks, followed by a washout period of 4 weeks, and then matching placebo for 12 weeks, or vice versa. Efficacy was measured through functional assessments (6-Minute Walk Test [6MWT], muscle strength via handheld dynamometry


[HHD], 5-times-sit-to-stand [5XSST], and SWAY Balance score), patient-/clinician-/caregiver-reported questionnaires (including the BarTH Syndrome Symptom Assessment [BTHS-SA], PROMIS Short Form Fatigue, and Global Impression scales)), and biomarkers, including the MLCL:L4-CL ratio. Safety was evaluated through reported adverse events (AEs) and laboratory assessments. Inclusion criteria consisted of genetic confirmation of BTHS, male age ≥12 years, and impaired ambulation. Additionally, subjects were required to have stable cardiac and hematologic comorbidities and could not have had a history of heart transplant. Subgroup analyses were performed to examine the impact of baseline disease characteristics on treatment effects. Part 2 is an open-label, long-term assessment (≤168 weeks) of elamipretide in endpoints that are similar to those evaluated in Part 1 Results: Twelve males were enrolled in this study; the baseline characteristics consisted of an average age of 19.5 years, weight of 50.8 kg, height of 167.3 cm, and body mass index of 17.6 kg/m2. At enrollment, these subjects had mild to moderate impairment based on functional tests ECGs and echocardiograms were within normal limits. The most common baseline comorbid medical diagnoses included cardiomyopathy (66.6%), neutropenia (58.3%), and hypotonia (50%). For the purposes of subgroup analyses, the median age was 16.5 years, median 6MWT distance was 458.5 meters, and median MLCL:L4-CL ratio was 17.3. Conclusions: TAZPOWER was designed to evaluate the clinical impact of elamipretide in targeting an underlying defect of BTHS (mitochondrial dysfunction and energy production). Results from TAZPOWER Parts 1 and 2 are the subjects of an accompanying presentation.



Authors: Hilary Vernon, MD, PhD1, Brittany DeCroes Hornby, PT, DPT, PCS2; William Reid Thompson, III, MD3; Anthony Aiudi, PharmD4; John Jeffrey Jones, PharmD4, Jim Carr, PharmD4.

Institution: 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine and at the Kennedy Krieger Institute; Director, Barth Syndrome Clinic at Kennedy Krieger Institute, Baltimore, MD, 21287, 2Department of Physical Therapy, Kennedy Krieger Institute, Baltimore, MD, 21205, 3Division of Pediatric Cardiology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287, 4Stealth BioTherapeutics, Newton, MA, 02467

Title: TAZPOWER: Biomarker Cardiolipin Ratios and Clinical Symptom Severity From a Randomized, Double-Blind, Placebo-Controlled, Crossover and Open-Label Extension Trial of Elamipretide in Barth Syndrome

Body of abstract:Background: Barth syndrome (BTHS) is a rare, X-linked infantile-onset debilitating disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delays and neutropenia. BTHS is caused by defects in the TAZ gene that encodes tafazzin, a transacylase responsible for the remodeling and maturation of the mitochondrial phospholipid cardiolipin (CL). CL is critical to normal mitochondrial structure and function (i.e. ATP generation). In BTHS, the ratio of structurally immature monolyso-cardiolipin (MLCL) to structurally mature tetralineoyl-cardiolipin (L4-CL) is increased (MLCL:L4-CL ratio) which have diagnostic utility and correlate with greater clinical severity. In preclinical studies, elamipretide stabilized L4-CL, reduce oxidative stress, improve ATP generation, and increase TAZ gene expression. The efficacy and safety of elamipretide are being studied in TAZPOWER, the first prospective clinical trial to evaluate elamipretide in BTHS patients.

Objectives: Determine the effect of elamipretide therapy on MLCL:L4-CL ratios and efficacy assessments in genetically-confirmed BTHS patients.

Methods: Part 1 (N=12) was a 28-week crossover design with 1:1 randomization to elamipretide 40 mg subcutaneously (SC) daily, administered for 12 weeks, followed by a 4-week washout period, and then


matching placebo for 12 weeks, or vice versa. Efficacy was measured through functional assessments (6-Minute Walk Test [6MWT], muscle strength via handheld dynamometry [HHD], 5-times-sit-to-stand [5XSST], and SWAY Balance score), patient-/clinician-/caregiver-reported questionnaires (including the BarTH Syndrome Symptom Assessment [BTHS-SA], PROMIS Short Form Fatigue, and Global Impression scales), and biomarkers, including the MLCL:L4-CL ratio. Safety was evaluated through reported adverse events (AEs) and laboratory assessments. Subgroup analyses were performed to examine the impact of baseline disease characteristics on treatment effects. Part 2 is an open-label, long-term assessment (≤168 weeks) of elamipretide in endpoints that are similar to those in Part 1.

Results: In Part 1, at the end of Treatment Period 2, irrespective of randomization sequence, a mean reduction in MLCL:L4-CL ratio of 4.7 from Baseline was observed. Additionally, results demonstrated an inverse correlation between baseline MLCL:L4-CL ratio and 6MWT improvement. Currently, for all 10 subjects continuing to Part 2, the MLCL:L4-CL ratio decreased from Part 1 Baseline. A mean improvement of -7.4 (paired t-test; p=0.03) from Baseline was observed for the 10 subjects at the Part 2, Week-12 Visit, the only visit all subjects completed to date. Using a last observation carried forward method (due to variable duration of participation in Part 2 and the nature of future visits being missing completely at random [MCAR]), a mean improvement of -9.4 (paired t-test; p=0.02) from Baseline was observed at the last available visit (N=10). Additionally, 23 of the 26 (88.5%) MLCL:L4-CL Part 2 ratios were lower than Baseline values. Part 2 OLE 6MWT data and patient-/clinician-/caregiver-reported outcomes show improvement with continued elamipretide therapy. TheMLCL:L4-CL ratio data, along with the improvements in these outcomes, suggest a physiologic basis to improvements in efficacy assessments in Part 2 OLE.

Conclusions: Part 2 OLE functional and patient-/clinician-/caregiver-reported outcomes show improvement with continued elamipretide therapy. The MLCL:L4-CL ratio data along, with the outcomes improvements, suggest a physiologic basis to the effects of continued elamipretide therapy in Part 2 OLE.


Presenter: Cameron L. McKnight

Authors: Cameron L. McKnight1, David A. Elliott1, Andrew G. Elefanty1, Richard J. Mills2, James E. Hudson2, David A. Stroud3, Michael T. Ryan4, David R. Thorburn1,5, Ann E. Frazier1

Institution: 1Murdoch Children’s Research Institute, Royal Children’s Hospital, and Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia. 2QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia, 3Department of Biochemistry and Molecular Biology, University of Melbourne/Bio21, Melbourne, VIC, Australia, 4Department of Biochemistry and Molecular Biology, University of Monash, Melbourne, VIC, Australia, 5Victorian Clinical Genetic Services, Royal Children’s Hospital, Melbourne, Australia

Title: Using Human Pluripotent Stem Cell Models of Mitochondrial Disease to Identify Candidate Drug Treatments

Abstract: Objective: Affecting approximately 1 in 5000 live births, mitochondrial diseases are both clinically and genetically heterogeneous often presenting with highly tissue specific phenotypes. Despite a number of agents showing therapeutic promise, there are currently no clinically validated treatment options available. With nearly 300 genetic causes of oxidative phosphorylation (OXPHOS) disorders, proving therapeutic efficacy remains challenging since homogeneous groups of patients are difficult to identify. This project aims to differentiate human pluripotent stem cell (hPSC) models of mitochondrial disease to a cardiomyocyte cell fate in order to facilitate preclinical treatment studies and investigation of the underlying cellular mechanisms of disease in a clinically relevant cell type.

Methods: Using CRISPR/Cas9 gene editing we generated knockout models of nuclear-encoded OXPHOS genes in human embryonic stem cells (hESC) allowing us to study tissue-specific effects via differentiation.


In general, we focused on generating cell lines for common disease genes where patients have presented with biallelic Loss-of-Function mutations in 15 different genes, including SURF1, that cover a range of primary and secondary roles in OXPHOS biogenesis. However, for COA6, a complete knockout was not viable in hESC culture, so the p.W59C patient mutation was generated in a hESC line with a heterozygous 80bp COA6 deletion. These strategies allow us to model clinically relevant null mutations for multiple genes on a single isogenic background.

Results: Using these techniques two SURF1-/- and COA6del80/p.W59C hESC clones were generated for each gene and all display OXPHOS complex IV (CIV) deficiency, as in patient samples. Both SURF1-/- clones form beating cardiomyocytes and maintain decreased CIV expression post-differentiation. Additionally, these cells show abnormal calcium handling and a significant decrease in contraction force in a cardiac organoid system. SURF1 is a CIV assembly factor and label-free quantitative proteomic analysis of contractile cardiomyocytes shows downregulation of CIV subunit proteins while most other mitochondrial proteins – including other CIV assembly factors – trend toward upregulation.

Although COA6 is also a CIV assembly factor, the COA6del80/p.W59C clones fail to differentiate to cardiomyocytes under standard conditions. However, adding drugs targeting specific mitochondrial pathways in the differentiation medium has shown some success in getting the cells past their block in differentiation. To ensure we are comparing differentiated cells of equivalent maturity we have also generated inducible correction lines for both COA6 and SURF1, allowing us to express the wildtype protein throughout the differentiation. These correction lines also support associated phenotypes being a direct result of the intended mutation and not an off-target effect.

Conclusion: Data from the SURF1-/- model are promising for other stem cell models of mitochondrial disorders already generated within our group. The proteomic data demonstrates compensatory mitochondrial proliferation in response to decreased complex IV assembly. Despite some mitochondrial disease stem cell models exhibiting blocks in differentiation, preliminary drug screens using the COA6lines have identified promising pathways to target in downstream screens on mature cells. Currently, these hESC models are being used for further investigation of disease-specific treatment options.


Presenter: Chynna Broxton

Authors: Chynna N Broxton1, Corey Burrough1, Prabhjot Kaur1, Manuela Lavorato1, Sujay Guha1,Tara Gallagher2, Christoph Seiler2, Eiko Nakamaru-Ogiso1, Marni J Falk1,3

Institutions: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2Aquatics Core Facility, The Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA 19104; 3Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104

Title: Therapeutic Rescue of Dld-Based Pyruvate Dehydrogenase Deficiency in Worm and Zebrafish Animal Models

Abstract: BACKGROUND: Dihydrolipoamide dehydrogenase (DLD) is a key mitochondrial enzyme that serves as the E3 subunit of three mitochondrial matrix dehydrogenases involved in glucose metabolism (PDH), the tricarboxylic acid (TCA) cycle (AKGDH), and amino acid catabolism (BCAADH). Autosomal recessive DLD disease causes a rare form of PDH deficiency that manifests as Leigh syndrome with neurodevelopmental disabilities, lactic acidosis, liver dysfunction, and/or failure to thrive. Full


understanding of the biochemical mechanisms driving these complex phenotypes remains largely unknown, and current therapies are non-specific and generally ineffective.

GOAL: Here, we report development and characterization of two novel genetic animal models of DLD disease in zebrafish (D. rerio, vertebrate) generated by CRISPR/Cas9 knockout of DLDH, and in nematodes (C. elegans, invertebrate) generated by RNA interference (RNAi) to knockdown DLD-1expression. Both models displayed survival and multi-system phenotypes, permitting objective modeling of disease mechanisms and novel therapeutic leads.

METHODS: DLD depletion in C. elegans was achieved by feeding wild-type (N2 Bristol) worms E.coli expressing a dsRNA targeting DLD-1 messenger RNA. Brood size, survival, activity, growth (length), DLD-1 expression, and mitochondrial unfolded protein response (UPRmt) were quantified in the resulting DLD-1-/- animals. High throughput semi-automated analyses of animal growth (length) and mitochondrial stress response (UPRmt) were also measured in day 1 adult worm populations using the COPAS Biosorter (Union Biometrica). DLDH-/- zebrafish mutants were generated using CRISPR/Cas9 gene editing by microinjection at the 1-cell embryo stage. Animal viability, organ development, swimming activity, and DLDH expression, were evaluated in DLDH-/- relative to AB (wild-type) zebrafish. Liver physiology was studied by Oil-Red-O staining and transmission electron microscopy (TEM). Therapeutic impact on DLD-1-/- C. elegans UPRmt induction and growth (length), as well as DLDH-/- zebrafish survival, liver morphology, and swiming behavior were tested for 5 therapies, including a PDH activator (dichloroacetate, DCA), PDH cofactors (thiamine, riboflavin, lipoic acid), and a lipid lowering drug that functions as a signaling modifier to inhibit mTORC1 and activate AMPK and PPAR (probucol).

RESULTS: C. elegans: RNAi resulted reduced DLD-1 expression by 90% in young adult worms, brood size by 90%, and adult growth (length) by 23% on adult day 3. Mutants had an ~ 8-fold mean increase in UPRmt as assessed by HSP6::GFP fluorescence, which was significantly rescued by treatment from early development with DCA (56% reduction) or thiamine (18% reduction) relative to untreated DLD-1-/- worms. The PDH cofactors riboflavin and thiamine significantly increased worm growth (length) by 33% and 16%, respectively. Zebrafish: DLDH-/- zebrafish had grossly enlarged livers by 5 days post fertilization (dpf). DLDH-/- liver TEM showed pronounced mitochondrial degeneration with increased lipid droplets, confirmed by Oil-Red-O staining. Reduced swimming activity was seen at 7 dpf by automated behavioral analysis (Zebrabox), with 100% animal mortality occurring by 11 dpf. Remarkably, pre-treatment of dldhmutant zebrafish larvae from 2 dpf with lipoic acid extended animal survival by 3 days and rescued their liver phenotype, with a 58% decrease in liver size compared to untreated DLDH-/- larvae. Further, the metabolic modifier, probucol, also reduced liver disease by 49% compared to untreated mutant fish. Combined treatment analyses remain underway.

CONCLUSION: We have generated the first viable DLD disease translational models in any species. Specifically, DLD deficiency in both C. elegans and zebrafish replicates the classical hallmarks of mitochondrial and liver dysfunction typical of human DLD disease. Therapeutic modeling has provided objective evidence for benefit of several current standard of care treatments used in human DLD disease patients. High throughput drug screens are underway to evaluate novel therapies that may rescue the diverse phenotypes of DLD disease model animals, and prioritize therapeutics leads to test in human clinical treatment trials of DLD subjects.


Presenter: Rebecca D. Ganetzky

Authors: Rebecca D. Ganetzky1,2, Colleen Muraresku1, Marni J. Falk1,2

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, 19104 USA; 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 USA.

Title: Dysmorphology is a Common Manifestation of Pediatric Primary Mitochondrial Disease


Abstract: Background: Congenital anomalies occur in up to 3% of all births, and are a common manifestation of inherited genetic conditions that disrupt development. Dysmorphic facial features have been noted in a few individual primary mitochondrial diseases, especially pyruvate dehydrogenase complex deficiency. However, the prevalence of major or minor dysmorphic features has not been assessed in mitochondrial disease as a whole. We sought to systematically characterize differences in facial morphology, as well as other structural differences, in a large, diverse cohort of patients with primary mitochondrial disease.

Methods: A retrospective chart review was conducted on a cohort of patients with molecularly confirmed mitochondrial disease in the Mitochondrial Medicine Frontier Program at the Children’s Hospital of Philadelphia (n=207). Patients without a documented examination by a clinical geneticist were excluded (n=21). A second subgroup of patients with severe primary lactic acidosis (persistent lactate >10 mM with normal hemodynamics) without a definite genetic diagnosis were also included (n=5). Physical exam data was collected, including any anthropometric data measured (head circumference, inner canthal distance, outer canthal distance, interpupillary distance, ear length, internipple distance, chest circumference, total hand length, palm length and/or foot length) along with any documented dysmorphic features. Percentiles for anthropometric data were calculated using the Handbook of Physical Measurements (3rd edition). Photographs, when available, were also examined to ensure consistency with documented examinations. Statistical analyses were performed in R studio.

Results: On whole mitochondrial disease cohort analysis, no significant variation from the median values was seen for any of the anthropometric data. However, dysmorphic features were significantly more common in patients with pediatric-onset primary mitochondrial disease (n=70) as compared to adults. Minor anomalies were present in the adult cohort at a frequency similar to that expected in general population. However, within the pediatric subgroup, 50% (n=35) were noted to have at least one dysmorphic feature. The most common abnormalities were nasal differences, especially anteverted nares (n=9), lip variation, including thin upper lip (n=6) and/or a prominent flat philtrum (n=6), and ear anomalies, particularly low-set ears (n=6). Major structural anomalies were seen in a small cohort of patients (n=7), including 3 patients who had agenesis or dysgenesis of the corpus callosum. In addition, a laryngeal cleft was seen in a patient with FBXL4 deficiency; polysplenia and an accessory lung lobe were seen in a patient with ECHS1 deficiency (as previously reported); soft cleft palate, congenital diaphragmatic hernia, horseshoe kidney and ambiguous genitalia in a patient with LONP1 deficiency; and foreshortened long bones in two patients (with mutations in MRPS22 and PDHA1). The presence of major structural anomalies correlated with early disease-onset before age 1 year (p=0.005, Chi-square test). Further subgroup analysis is ongoing to identify gene-specific recurrent patterns of morphologic differences.

Conclusion: Overall, major and minor congenital anomalies are a common finding in children with primary mitochondrial disease, where structural differences correlate with age of onset. Clinically significant structural differences were seen in nearly 1/3 of mitochondrial disease cases with onset before one year of age, highlighting the importance of screening for congenital anomalies in this population. The presence of specific dysmorphic features, including anteverted nares, prominent flat philtrum, and thin upper lip may be particularly recurrent in mitochondrial disease. Ongoing subgroup analysis will help identify dysmorphic features that may assist with clinical diagnosis or management in mitochondrial disease.


Presenter: Isabella Peixoto de Barcelos

Authors: Isabella Peixoto de Barcelos1, Cesar Augusto Alves2, Amy Goldstein1,3, Colleen Muraresku1, Can Ficicioglu3,4, Marc Yudkoff3,4, Giulio Zuccoli2 and Rebecca Ganetzky1, 3, 4.


Institution: 1- Mitochondrial Medicine Frontier Program at Children’s Hospital of Philadelphia, PA. 2- Neuroradiology Division in the Radiology Department at Children’s Hospital of Philadelphia, PA. 3- Department of Pediatrics of Perelman School of Medicine at the University of Pennsylvania. 4- Section of Biochemical Genetics, Division of Human Genetics of the Children’s Hospital of Philadelphia, PA.

Title: A Recurrent Point Mutation on PDHA1 Causes Pyruvate Dehydrogenase Complex Deficiency (PDCD) without Lactate Elevation.

Abstract: Pyruvate dehydrogenase complex deficiency (PDCD) is a primary mitochondrial disease due a defect in the pyruvate dehydrogenase complex, responsible for the conversion of pyruvate to acetyl-CoA. Deficiency leads to the accumulation of lactate, a hallmark of this disease. It is clinically characterized by neurologic symptoms, such as seizures, developmental delay, hypotonia and muscle weakness starting in the first years of life. This condition can be associates with dysgenesis of the corpus callosum and Leigh-like brain findings. Here we report three cases, with genetic confirmation of a known pathogenic PDHA1mutation (c.491A>G, p.N164S) with normal levels of plasma alanine and plasma and/or CSF lactate, an unexpected finding for this primary mitochondrial disorder.

Patient 1. Female, 23-years-old, Ashkenazi Jewish, presented with stroke-like episodes when six-years-old, followed by several clinical transient focal neurologic deficits (ptosis, focal weakness, ophthalmoplegia) and mild motor (left foot dystonia) and cognitive impairment. Seizures started around 17 years old with a good response to ketogenic diet. Brain MRI demonstrated several lesions in the brainstem, and the cortex was also involved with a stroke-like pattern. Pre-treatment lactate and alanine in the CSF were normal on multiple occasions. She had one elevated alanine during her first presentation. Laboratory results demonstrated that the inactivated PDC activity in fibroblasts was 34% of control.

Patient 2. Male, four-years-old, Dominican, presenting with previous developmental delay, failure to thrive, hypotonia, and spontaneous central respiratory failure at three-years-old. He had recurrent episodes of central respiratory failure, accompanied by ophthalmoplegia and ptosis. EEG was normal during these episodes. He was stabilized on the ketogenic diet with no further episodes for a year. MRI and spectroscopy studies were performed showing diffuse abnormal T2 signal involvement of the brainstem and no lactate peaks. Lactate and alanine in blood were normal during crisis and multiple times thereafter. Laboratory results demonstrate inactivated PDC activity in fibroblasts was 10%.

Patient 3. Male, eight-years-old, Somali, presenting with seizures starting at 2 days of life, and after that, lethargy, emesis, and acute respiratory distress as a Leigh-like syndrome around ten-months-old. The patient had two subsequent spontaneous episodes of respiratory failure, ultimately, requiring tracheostomy placement. After that, the patient developed hypotonia and chronic encephalopathy. Lactate and alanine in plasma were normal. Laboratory results demonstrate inactivated PDC activity in fibroblasts was 21%. The description of his image reported basal ganglia and sub insular infarcts.

Here we present three patients with the same recurrent point mutation in PDHA1, who each presented with deep brain abnormalities on brain MRI, but with persistently normal or near-normal levels of lactate and alanine in the blood, CSF and brain even during episodes of acute neurologic decompensation. Importantly both affected males also had the distinct feature of episodic spontaneous respiratory failure, which ultimately did respond to ketogenic diet in one case. These patients have a shared and unique phenotype of PDCD and highlight the importance of considering PDCD on the differential even when lactate levels are normal or near-normal.



Authors: A. Karaa, EL. Kelly, M. Guyette


Institutions: Massachusetts General Hospital, Boston, MA

Title: Fatigue, dizziness and nausea: Doc I think I have Mito?

Body of Abstract: Small fiber polyneuropathy (SFPN) arises when small myelinated or unmyelinated fibers become damaged through several processes including mitochondrial dysfunction. SFPN can thus occur as part of many diseases leading to sensory, autonomic and enteric dysfunction and result in one or several multisystemic symptoms often leading to referrals for “rule out mitochondrial disease”. We set to better understand the characteristics of symptoms in diseases where SFPN plays a major pathophysiology such as Fabry disease (FD, 23 patients) and connective tissue diseases (CD, 12 patients with Ehlers Danlos Syndrome). We compared those symptoms to patients with primary mitochondrial disease (PMD, 20 patients with the following mtDNA mutations: m.10158T>C (1), m.3243A>G (7), mtDNA deletion (4), and m.8344A>G (4), as well as mutations in POLG (3), and mRRM2B (1) genes) and healthy individuals (H, 7). The goal was to catalogue the specific symptoms experienced by each group and to identify any similarities or differences in SFPN manifestations.

All 62 individuals enrolled answered a SFPN-specific questionnaire (46 questions) addressing the different clinical aspects of SFPN symptoms (sensory, circulatory, gastrointestinal (GI), pelvic, generalized and miscellaneous manifestations) and underwent a thorough neurological exam. Participants were primarily Caucasian females (n female (F)/male (M)) in early adulthood (median age in years): FD (13F/10M; 42.6), PMD (12F/8M, 43.6), CD (11F/1M, 38.6) and H (5F/2M, 39.5). Overall, symptoms of SFPN were statistically (p<0.001) more severe in the CD group compared to FD (LSM 1.44 +/- 0.22 STD) and PMD (LSM 1.52 +/- 0.22 STD). Scores in all 3 groups were much higher than in healthy volunteer: (LSM 3.26 +/- 0.29) in CD, (1.82 +/- 0.27) in FD and (1.74 +/- 0.27) in PMD compared to control. CD patients had earlier symptoms onset (21.8 year) compared to FD (22.7 year) and PMD (29.6 year) and were more likely to be diagnosed with fibromyalgia, chronic fatigue and Lyme disease. Fatigue was the most bothersome symptom in all 3 disease groups but felt worse in CD patients (p<0.05). FD and PMD had similar complaints and symptoms intensity, but CD patients complain of much more intense sensory and GI problems compared to PMD patients (p<0.001). Specifically, CD patients experience more paresthesia, dizziness and postural symptoms, nausea, vomiting and abdominal pains than PMD patients (one-way anova with Bonferroni correction p<0.001). The presence and severity of the SFPN symptoms assessed by the questionnaire did not always correlate with obvious clinical exam signs.

SFPN symptoms cause a significant disease burden in patients with FD, PMD and CD but although FD and PMD are very similar in the type and intensity of symptoms experienced which are clearly much more prominent than in healthy control, these symptoms are statistically more diffuse, intense and disabling in the CD group. Adult patients who primarily complaints of fatigue, autonomic circulatory symptoms and GI dysmotility should be considered for CD diagnosis and are less likely to have PMD.


Presenter: Rustum Karanjia

Authors: Rustum Karanjia1,2; Alfredo A. Sadun2

Institutions: 1. Ophthalmology, University of Ottawa, Ottawa, ON, Canada. 2. Ophthalmology, Doheny Eye Centers & David Geffen School of Medicine UCLA, Los Angeles, CA, United States.

Title: Clinical Trial of Elamipretide Topical Ophthalmic Solution for the Treatment of Leber's Hereditary Optic Neuropathy

Body of Abstract Introduction


Leber’s Hereditary Optic Neuropathy is a mitochondrial disease characterized by sudden and profound vision loss in both eyes. Elamipretide has been demonstrated to colocalize with cardiolipin in the inner membrane of mitochondria, where it is thought to improve mitochondrial bioenergetics. The purpose of this study was to look at the safety, tolerability and potential efficacy of topical Elamipretide in patients affected with LHON.

Materials and Methods

Twelve patients affected with LHON were included in this study. Patients between ages 18 and 50 with decreased vision, for at least 1 year and no more than 10 years, and a genetically confirmed diagnosis of 11778 LHON were eligible for this trial. The primary outcome measure was the assessment of adverse events from the administration of topical Elamipretide 1%. Secondary outcome measures looked at changes in best corrected visual acuity (BCVA), color vision, visual field mean deviation and electrophysiological outcomes. For the first 52 weeks of the study patients were randomized to one of two arms, Elamipretide in both eyes or Elamipretide in one eye and vehicle in the other eye. An open label extension (OLE) where both eyes are being treated with Elamipretide was offered to the patients upon conclusion of the masked portion of the trail.

Results

There were no serious adverse events, relatable to treatment in the masked portion of the study. These findings held for the OLE, where all patients opted to continue treatment. There was an improvement in BCVA of more than 1 line in 25% of treated eyes at 52 weeks. This improvement continued in the OLE to include 42% of treated eyes by week 84 (p = 0.01). There was also a corresponding improvement in the visual field of 5 dB by week 84 of the OLE. This was reflected in a significant improvement in quality of life assessment (VFQ 25). There was no significant improvement in the other secondary outcome measures by week 52. Data collection and analysis is ongoing.

Discussion

Elamipretide topical solution is safe for ocular administration with no serious adverse events reported. Initial analysis of the secondary outcome measured showed significant improvement in visual acuity and visual fields. The clinical meaningfulness of these changes remains to be established.


Presenter: Kelly M. Scheulin

Authors: Kelly M. Scheulin1-3, Franklin D. West1-3 and Shilpa Iyer4

Institutions: 1Regenerative Bioscience Center, 2Biomedical and Health Science Institute Neuroscience Program, 3Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602; 4Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701

Title: mRNA Reprogramming Of Patient-Specific Mitochondrial Disease Fibroblasts Into Human Induced Pluripotent Stem Cells

Abstract: Mitochondrial diseases present in a wide range of phenotypes in patients caused by mutations in mitochondrial DNA (mtDNA) resulting in mitochondria dysfunction. The mutations are associated with mitochondrial encephalopathy with lactate acidosis and stroke-like episodes (MELAS), Leigh syndrome (LS), epilepsy, and myopathy. Advancements in Food and Drug Administration approved therapeutics have been hindered due to limited knowledge of disease progression and lack of an adequate model to study key disease mechanisms. To further identify aberrations of key cellular pathways that are potential therapeutic targets, human induced pluripotent stem cells (hiPSCs) can be


generated from somatic cells of mitochondrial disease patients. Patient-specific hiPSCs can be differentiated into unique cell populations such as cardiomyocytes and neurons and be utilized to study basic disease mechanisms or for high-throughput/high content drug screening. In this study, we determined the ability of 7 mitochondrial disease patient fibroblast cell lines carrying Complex 1 (SBG3-T10158C; SBG-4-T12706C) and Complex V (SBG1& SBG2- T8993G;SBG3- T9185C) and two mt-tRNA mutations [ SBG6-GA3243G (tRNALeu(UUR)) and SBG7-G14739A (tRNAGlu(GAA))] and a control BJ fibroblast line to successfully reprogram into hiPSCs utilizing non-viral, non-integrating mRNA reprogramming technology. Patient fibroblasts were reprogrammed in a complete xeno-free culture environment using non-modified RNAs (StemRNA™-NM Reprogramming Kit, Stemgent®) to generate hiPSCs. Putative hiPSCs from all 7 reprogrammed lines showed morphology changes indicative of an iPSC-state including colony formation, high nucleus to cytoplasm ratio and large prominent nucleoli. Cells from all 7 hiPSC lines showed positive expression for the iPSC markers POU5F1, SOX2, SSEA4, TRA-1-60, and TRA-1-81. The mitochondrial mutation burden is heterogenous making it critically important to ensure that mtDNA burden is maintained after reprogramming. Next-generation sequencing will demonstrate if the mtDNA burden is consistent between fibroblasts and derived iPSCs. These results demonstrate that patient-specific disease fibroblasts can be successfully reprogrammed into hiPSCs with pluripotent morphology and marker expression using a non-viral, non-integrating mRNA reprogramming approach for understanding and treating mitochondrial diseases.


Presenter: Suraiya Haroon, PhD

Authors: Suraiya Haroon1, Heeyong Yoon1, Bruce Osei-Frimpong1, Adele Donahue 2, Christoph Seiler2, Eiko Nakamaru-Ogiso1, Marni J. Falk1,3

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department ofPediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2Aquatics Core Facility, Childrens’ Hospital of Philadelphia, Philadelphia, PA 19104; 3Department ofPediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA19104

Title: Cysteamine Bitratrate and N-acetylcysteine Rescue Brain Death and Neuromuscular Activity in a Novel SURF1-knockout Zebrafish Animal Model of Leigh Syndrome

Abstract: BACKGROUND: SURF1 is an assembly factor required for complex IV formation. Without SURF1, the assembly of complex IV is greatly impaired, which disrupts bioenergetic capacity. Indeed, autosomal recessive pathogenic variants in SURF1 are one of the more common etiologies of Leigh syndrome, with basal ganglia strokes, seizures, failure to thrive, lactic academia, and complex IV deficiency. Unfortunately, no FDA approved therapies or cures currently exist for SURF1-based Leigh syndrome. GOAL: We sought to develop a vertebrate animal model of SURF1 disease in zebrafish in order to improve the understanding of disease pathophysiology and identify promising therapeutic leads.

METHODS: CRISPR/Cas9 was used to generate multiple genetic SURF1 mutant models. One of the models resulting from targeting the site homologous to the human R192 amino acid (aa) have an early stop codon at aa210, where the full length protein is 310 amino acids. The second model resulting from the same targetting method has a complex insertion/deletion mutation that changes a donor site for splicing at exon 6 and exon 7 junction. The third model resulted from targeting aaL105 has an early stop codon at aa129. All models were outcrossed and bred to maintain a homozygous mutant line. Larval fish in the first week of life were studied to characterize SURF1 RNA and protein effects, COX deficiency by immunoblot, electron transport chain enzyme activities by spectrophotometry and assembly by blue native gel analyses, and ATP levels. Gross phenotypes evaluated included swimming activity (Zebrabox) at baseline and in the presence of pharmacologic inhibitors of respiratory chain function, as well as brain death and neuromuscular functions, and heart rate in response to acute respiratory chain


inhibition at 6-7 days post fertilization (dpf). Pharmacologic therapies were evaluated by pretreating fish from 5 dpf.

RESULTS: The genetic changes in SURF1 was confirmed in two of the CRISPR/Cas9 generated by cDNA sequencing and severe complex IV (COX) deficiency was confirmed by immunoblot analysis. None of the mutant strains displayed gross morphologic, early survival, or fertility defects. However, SURF1-/- larvae demonstrated increased sensitivity to low doses of the complex IV inhibitor, sodium azide, manifesting as gray brain (brain death), impaired neuromuscular behaviors (startle and touch response), and slowed heartrate. Multiple drugs at varying concentrations were screened to determine if these abnormalities could be prevented. Significant neuroprevention was identified with two cysteine donor molecules, namely cysteamine bitartrate and N-acetylcysteine; glutathione redox analyses are under way to better understand the therapeutic mechanisms of these therapies.

To translate this to a high throughput assay, we identified lower azide concentrations that impaired swimming activity as a readout of neuromuscular function, without inducing brain death. In addition, one of the models was found to have reduced swimming activities in dark even without azide stress, with drug screens now underway to identify therapeutic leads that improve swimming activity in SURF1-/- larvae. Interestingly, ETC enzyme activities completed in the first SURF1-/- deletion zebrafish linedemonstrated severely (90%) reduced CIV activity at baseline in 7 dpf larvae, which increased to >95% CIV deficiency upon azide stress doses that induce brain death, demonstrating severe stress sensitivity to loss of residual CIV activity in SURF1 disease.

CONCLUSION: We have developed a series of viable SURF1-/- zebrafish lines with severe complex IV deficiency, reduced swimming activity, and increased stress sensitivity upon mild complex IV inhibition with sodium azide that manifests as brain death, similarly as seen in humans with Leigh syndrome. Pre-treating SURF1-/- fish with cysteamine bitartrate or N-acetylcysteine prevented the brain death and neuromuscular dysfunctions observed after sodium azide treatment. These data support the pursuit of clinical trial with this class of therapies in patients with SURF1-based Leigh syndrome.


Presenter: Suraiya Haroon, PhD

Authors: Suraiya Haroon1, Ortal Nakash1, Sujay Guha1, Eiko Nakamaru-Ogiso1, Marni J. Falk1,2

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department ofPediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA19104

Title: Therapeutic Rescue of Neuromuscular Defects in a C. elegans Animal Model of POLG Disease

Abstract: BACKGROUND: POLG is the nuclear-encoded sole polymerase that replicates the mitochondrial genome (mtDNA), with a polymerase domain that adds nucleotides and an exonuclease domain that performs proofreading to correct errors during mtDNA replication. POLG mutations result in mtDNA depletion or an increase in mtDNA mutations, which over time disrupt bioenergetic capacity. In humans, autosomal dominant and recessive variants in POLG result in a variety of diseases involving neurologic dysfunction and myopathy. Unfortunately, no FDA approved therapies or cures currently exist for any POLG disease.

OBJETIVE: We sought to identify novel therapeutic leads for POLG disease in a recently characterized POLG disease C. elegans model polg-1(srh1) that harbors an error-prone allele (Haroon S et al, 2018).

METHOD: The polg-1(srh1) worms exhibit hallmark features of human POLG disease, with 56% reduced mtDNA content, 70% increased mtDNA mutation frequency, 90% decrease in mitochondrial respiratory reserve capacity, neuromuscular dysfunction, and 19% decreased median lifespan. Their chemotaxis is abnormal, representing a neuromuscular defect that can be screened to identify novel


therapeutic leads to improve neuromuscular activity in human patients with POLG disease. Worms are grown on NGM plates with each drug under analysis from birth to day 5 of adulthood. At adult day 5, worms are placed on the NGM plates 5 cm from a chemoattractant (isoamyl alcohol) for 1 hour, at which time animal distance from attractant is measured and averaged by treatment group. Each drug condition was tested using 25-30 worms and statistical analysis is performed of mean distance relative to untreated polg-1(srh1) animals by Student’s t-test.

RESULTS: We have completed preliminary analysis of 5 drugs that rescue lifespan in complex I disease gas-1(fc21) worms at 2-3 different concentrations in polg-1(srh1). Treatment with rapamycin (2.5 μM, 5 μM, 10 μM), probucol (2.5 mM, 5 mM and 10 mM), N-acetylcysteine (1.3 mM, 5 mM), nicotinic acid (0.63 mM, 1.3 mM and 2.5 mM), and glucose (10 mM) individually did not rescue neuromuscular function of polg-1(srh1) animals. However, treatment with either N-acetylcysteine at 2.5 mM, or glucose at 1mM or 5mM concentration, significantly improved their neuromuscular function by ~30%. Interestingly, treatment with an FDA-approved vasodilating drug not previously considered for treating mitochondrial disease restored the neuromuscular function of the polg-1(srh1) by 64% at 200μM and by 95% at 100μM. Additional drug analyses of candidate therapies and FDA approved drug libraries, together with mechanistic studies of therapeutic leads, remain underway.

CONCLUSION: Quantifying neuromuscular function in polg-1 mutant worms has identified 3 therapeutic leads at specific concentrations for POLG disease. Mechanistic investigations will assist in the rational prioritization of optimal therapies to bring forward for clinical trials to improve neuromuscular function in this severe and currently untreatable mitochondrial disease.


Presenter: Olivia Kolenc

Authors: Olivia Kolenc1, Ajibola B. Bakare2, Isaac Vargas Lopez1, Joshua Stabach2, Kyle P. Quinn1,Shilpa Iyer2

Institution: 1University of Arkansas, Biomedical Engineering, College of Engineering, Fayetteville, AR 72701, 2University of Arkansas, Biological Sciences, J. William Fulbright College of Arts & Sciences, Fayetteville, AR 72701

Title: Characterizing Metabolic Changes in Leigh’s Syndrome Using Label-free MultiphotonMicroscopy

Body of Abstract: Understanding the metabolic consequences of mutations affecting key protein complexes in the electron transport chain (ETC) is critical to identifying effective therapeutic interventions for Leigh’s Syndrome (LS). However, there is a lack of non-invasive quantitative functional biomarkers to diagnose and evaluate mitochondrial disorders. The objective of this study is to evaluate the sensitivity of label-free multiphoton microscopy to functional changes in patient-derived cells with different protein complex mutations.

Five LS and LS-like patient-derived fibroblast cell lines carrying mutations in either Complex I or Complex V of the ETC, as well as a normal control fibroblast line, were cultured to evaluate functional metabolic differences. Using label-free multiphoton microscopy, random fields were imaged with a 20x objective (1.0 NA) (n=4 dishes/ cell line) and NADH and FAD autofluorescence intensities were collected at 755 nm and 855 nm, respectively. NADH and FAD intensities were normalized to concentrations of fluorescein as in previous studies. An optical redox ratio of FAD/(NADH+FAD) was computed from each image field.

A higher optical redox ratio was associated with both the Complex V mutations (p=0.0006) and the Complex I mutations (p=0.008) in comparison to normal fibroblasts (Figure 1). While the cell lines carrying Complex V mutations had a higher redox ratio overall, the redox ratio was also sensitive to differences in specific cell lines. These findings, along with additional metabolic characterization, could contextualize cellular efforts in compensating for ETC defects.


This work demonstrates the potential of non-invasive, label-free imaging for characterizing bioenergetics in Leigh’s Syndrome patient-derived cells. Our findings suggest that through multiple imaging outcomes of mitochondrial structure and function, we can discriminate some effects of different mitochondrial DNA mutations. Future work will explore applications for this imaging approach in drug delivery platforms.

Figure 1. (a) Representative redox ratio maps of fibroblasts derived from Leigh’s Syndrome patients with Complex I and Complex V mutations in comparison to normal cells. (b) Average redox ratios revealed significant differences between diseased and normal cells, and indicated altered metabolic activity in Complex I and Complex V mutated cells.

Abstract #: 2019 PA-0636 Presenter: Xavier Llòria

Authors: Magda Silva1, Xavier Llòria1, Claudia Catarino2, Felice Lob3, Bettina von Livonius3,Thomas Klopstock2

Institution: 1Santhera Pharmaceuticals Ltd, Pratteln, Switzerland; 2Friedrich-Baur Institute, University Hospital of the Ludwig-Maximilians University, Munich, Germany; 3Department of Ophthalmology, University Hospital of the Ludwig-Maximilians University, Munich, Germany

Title: Pediatric Leber’s Hereditary Optic Neuropathy (LHON): Real-world Efficacy Results Following Long-Term Idebenone Treatment

Body of Abstract: Introduction: LHON is a rare mitochondrial genetic disorder that results in severe, bilateral central vision loss. The Expanded Access Program (EAP), a named patient program under local regulations, provides insights into the potential of idebenone in pediatric (<12 years old) patients in a real-world setting. Methods: A retrospective medical chart analysis of visual acuity (VA), expressed as logMAR, from enrolled patients was performed. VA efficacy was determined as a clinically relevant recovery (CRR), defined as an improvement from off-chart to reading one line on the ETDRS chart, or an on-chart improvement of two lines. Results: Five patients were below 12 years of age at baseline (BL), with a time since LHON onset of 1.7 months to 5 years. The median best VA at BL was 0.94 logMAR (range 0.16 – 1.20). Following a median treatment duration of 33.6 months (range 6.8 – 40), median best VA at last visit (LV) was 0.08 logMAR (-0.18 – 1.36). Three patients achieved CRR from nadir (and BL) in both eyes, with a magnitude of recovery from 2 – 9 lines at first observation of CRR, which increased to 4 – 12 lines by LV. Conclusions: Idebenone was safe and efficacious in pediatric patients, which is consistent to results seen in the adult LHON population.

(b)



Presenter: Enrico Bertini

Authors: Enrico Bertini1, Daniela Verrigni1, Alessandra Torraco1, Michela Di Nottia1, Giulia Trani1,

Fiorella Piemonte1, Rosalba Carrozzo1

Affiliations: 1Unit of Neuromuscular and Neurodegenerative Disorder, and Laboratory of Molecular Medicine, Bambino Gesu’ Children’s Research Hospital, P.sa S Onofrio, 4 001645, Rome, Italy

Title: A Single Center Experience Addressing the Genetic Epidemiology of Leigh Syndrome

Abstract Body: In the interval of 20 years, from 1997 to 2017 we have diagnosed 250 conditions of genetically proven mitochondrial disorders. Out of this cumulative number, 53 were phenotypically diagnosed as Leigh syndrome.

All patients were diagnosed by histopathology, biochemistry (spectrophotometric assay and when necessary, validation by WB and Blue Native Gel Electrophoresis-BNGE) and molecular genetics. Since 2013 we started using the NGS technology with a targeted “MitoExome”, then we followed using the clinical exome (TruSight One Expanded Sequencing Panel, Illumina, which covers 6700 gene diseases), that in our experience scans quite well the mtDNA genes as the nuclear DNA disease genes of the mitochondrial machinery.Overall we found 40% of patients (21/53) with a Leigh syndrome caused by a defects in the mtDNA genes, and particularly the most frequent involved genes/mutations were 13513G>A in MTND5 (6/21) and 8993T>G in MTATP6 (6/21) or 9176T>G in MTATP6 (3/21). Overall, these 3 mutations accounted for 70 % (15/21) of the mtDNA genes/mutations in our series.Mutations in the nDNA were found in 32/53 (60%) patients assessed at neuroimaging as Leigh syndrome. Three genes were mostly involved by heterogeneous biallelic mutations, SURF1 (7/32, 22%), SUCLA2(6/32, 18%) and SUCLG1 (6/32, 18%) followed by mutations in PDHA1 (4/32, 12%). In SUCLA2 we detected a founder effect mutation c.850C>T [p.Arg284Cys] / c.850C>T [p.Arg284Cys] in three unrelated families originating from southern Italy. We detected as well in one family an index patients with compound heterozygous mutations in NDUFAF6 (c.532G>C [p.Ala178Pro] / c.420+784C>T); the intonic mutation c.420+784C>T was also found in additional 2 families originating from southern Italy in heterozygosity followed by another centre (Besta Institute, Milano) and not reported so far. Finally in our series of nDNA mitochondrial defects, subunits and assembling factors of Complex 1 were quite frequent (6/32) as a whole but were observed only once in the following genes: NDUFA12, NDUFAF4, NDUFS1, NDUFA10, NDUFA1, NDUFAF6. Additional rare mutated genes in our series that were observed only once in the mtDNA were: MTND1, MTND3, MTND6, and in the nDNA was SERCA1.All this information has been included in the Italian Registry of mitochondrial disorders leaded by the Italian advocacy group MITOCON.


Presenters: Austin Larson and Brian Shayota

Authors: Kimberly Kripps,a, * MD,, Warapan Nakayuenyongsukb,*, MD, Brian J. Shayotaf,* ,MD, Johan Van Hovea, MD, William Berquistc, MD, Natalia Gomez-Ospinac, MD, Carlos Esquivelc, MD, Waldo Concepcionc, MD, Jacinda B. Sampson, MD, PhD, David Cristind,MD, Whitney Jacksond, MD, Samuel Gilliland, MDe, Michael L. Kuehtg, MD, R Pettit, Youmna A. Sherifh, MD, Lisa Emrickf, MD, Michio Hiranoj, MD, Sarah H. Elseaf, PhD, Ryan Himesi, MD, Fernando Scagliaf,k,l*, MD, Gregory M. Ennsc,*, MD, Austin Larsona,*,MD

Institutions: aChildren’s Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Center, Aurora, Colorado bUniversity of Nebraska Medical Center,


Omaha, Nebraska; cDepartment of Pediatrics, Stanford University School of Medicine, Stanford, CA; dDivision of Gastroenterology and Hepatology, University of Colorado Anschutz Medical Center, Aurora, Colorado; eDepartment of Anesthesia, University of Colorado Anschutz Medical Center, Aurora, Colorado; fDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; gDepartment of Surgery, Baylor College of Medicine, Houston, TX; hDepartment of Neurology, Baylor College of Medicine, Houston, TX; iDepartment of Gastroenterology, Hepatology and Nutrition, Baylor College of Medicine, Houston, TX; jDepartment of Neurology, Columbia University Medical Center, New York City, NY; kTexas Children’s Hospital, lJoint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR

* denotes equal effort among these authors

Title: Liver Transplantation in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)

Body of Abstract: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the TYMP gene, resulting in thymidine phosphorylase (TP) deficiency and elevated thymidine and deoxyuridine, impairing mitochondrial DNA replication. Symptoms include progressive and fatal gastrointestinal dysmotility, neuropathy, myopathy, ophthalmoparesis and ptosis. Hematopoietic stem cell transplantation (HSCT) restores sufficient TP enzyme activity to reduce thymidine and deoxyuridine to normal levels but is associated with a high mortality rate in MNGIE patients. TP is also highly expressed in liver and orthotopic liver transplant (OLT) has been published for two adult patients with MNGIE resulting in normalization of thymidine and short-term positive clinical outcomes. We report one pediatric patient and three additional adult patients that underwent OLT for MNGIE.

Patient 1 is a male with progressive liver failure at 14 months of age. Sequencing showed TYMP(c.516+2 T>A). Plasma thymidine level was 25.61 μmol/L (normal <0.25 μmol/L). OLT was performed at 26 months. Follow-up thymidine levels dropped to near-normal levels. Exams 3 years post-OLT showed no evidence of ophthalmoplegia or myopathy. Areflexia persists. MRI at 3.5 years old showed unchanged T2 hyperintensity of the white matter from pre-OLT. Profound sensorineural hearing loss was identified after transplant and cochlear implants placed. No pre-transplant hearing evaluation was done, so onset of hearing loss is unknown.

Patient 2 had new neurological symptoms at 35 years and MRI showed leukodystrophy. Electromyography showed neuropathy. Ophthalmology exam showed ophthalmoplegia and ptosis, which had been present since adolescence. She had no intestinal symptoms. Exome sequencing showed biallelic variants in TYMP: c.866 A>C (p.E289A) and c.653 T>G (p.I218S). Pre-OLT thymidine level was 6.63 μmol/L. The patient elected to undergo OLT instead of HSCT. OLT from a living unrelated donor was performed at 37 years of age. Thymidine dropped to 1.01 μmol/L. She has had stable neurological symptoms for one year after OLT and eats a regular diet.

Patient 3 presented with abdominal pain, fatigue, weakness, paresthesias and was TPN-dependent. TYMP sequencing showed homozygous c.214+1G>T. Plasma thymidine level was 3.095 μmol/L (normal <0.7 μmol/L). Brain MRI showed diffuse leukoencephalopathy. OLT was done at age 21 years. Post-OLT thymidine level was normal. 6 weeks post-OLT, she was able to tolerate half her nutrition via gastro-jejunal tube feeding. BMI at diagnosis was 12.74 kg/m2, and at 10 months post-transplantation was 12.5 kg/m2. Neurological symptoms were stable post-OLT.

Patient 4 had fatigue, weakness and paresthesias at age 14 years. At 17, he was found to have cirrhosis. At 22 years, he had weight loss and dysmotility and was found to have thymidine level of 17 μmol/L (normal <0.7 μmol/L) and homozygous TYMP c.215-1G>C. OLT was done at age 23 years. Thymidine was persistently normal post-OLT. Gastrointestinal symptoms improved with subsequent weight gain over 6 months after OLT. He had improvement in strength, stability, and proprioception but continued to have decreased muscle mass, ophthalmoplegia, areflexia and paresthesia.


This case series supports initial efficacy of OLT in patients with MNGIE and hepatopathy or other contraindications to HSCT, though longer-term follow up is needed to compare clinical outcomes to HSCT.


Presenter: Ethan Perlstein, PhD

Authors: Ethan Perlstein, Nina DiPrimio, Sangeetha Iyer, Jessica Lao, Joshua Mast, Tamy Portillo Rodriguez, Feba Sam, Hillary Tsang, Kausalya Murthy, Gabriela Colmenares, Aras Rezvanian, Madeleine Prangley, Zach Parton, Chris Spiewak

Institution: Perlara PBC, 2625 Alcatraz Ave #435, Berkeley, CA 94705

Title: Precision Drug Repurposing for Inborn Errors of Metabolism

Body of Abstract: Conventional drug discovery involves human cell-based phenotypic screens or human protein target-based in vitro (or in silico) screens followed by validation in mice. While model organisms routinely identify and elucidate fundamental principles and mechanisms in biology, simple animals remain largely absent from or under-utilized by academic and biopharma drug discovery efforts, especially for ultra-rare inborn errors of metabolism. Mitochondrial diseases are well-suited to invertebrate models because causal genes, underlying biochemical defects and pathophysiology are evolutionarily conserved. Here we describe results from precision drug repurposing screens using yeast, worm and fly patient avatars of congenital disorders of glycosylation, PMM2-CDG and NGLY1 deficiency, as well as preliminary screening results using yeast models of Leigh syndrome, including mutants deficient in Complex I, IV or V. Combining insights from yeast cells, worms and patient fibroblasts led us to repurpose a Japanese drug approved in the 1990s for PMM2-CDG under a compassionate use individual IND at Mayo Clinic. Yeast and worm patient avatars of inborn errors of metabolism combine with patient cell and tissue models to create a precision drug repurposing and drug discovery engine for metabolic diseases at a fraction of the cost ($25,000 to $250,000) and time (2 to 6 months) compared to traditional approaches.


Presenter: Sampath Rangasamy

Authors: Sampath Rangasamy1, 2, Lorida Llaci1 2, Gabrielle Mills1, Newell Belnap1, 2, Richa Pandey1,

2, Cherae Bilagody1, 2, Raj Gupta1, Kelsey Chain1, C4RCD Research Group1, 2, V Narayanan1, 2

Institutions: 1 Neurogenomics Division, Translational Genomics Research Institute (TGen), Phoenix, AZ; 2 Center for Rare Childhood Disorders (C4RCD), Translational Genomics Research Institute (TGen), Phoenix AZ

Title: Mutations in Mammalian Dynamin 1 (DNM1) cause Epileptic Encephalopathy with Mitochondrial Dysfunction

Abstract: The epileptic encephalopathies (EE) (MIM 308350) are severe childhood brain disorders characterized by severe epilepsy syndromes, which differ by age of onset and seizure type. Whole exome sequencing (WES) has led to the identification of several causal genes in individuals with epileptic encephalopathies (EE), and the number of genes associated with EE is steadily increasing. We at the Center for Rare Childhood Disorders (C4RCD), and others have identified de novo missense mutations in the Dynamin 1 gene (DNM1, OMIM: 602377) in patients with early infantile EE (infantile spasms or Lennox-Gastaut syndrome (OMIM: 616346). Early onset epileptic encephalopathy, intractable seizures, intellectual disability, developmental delay, and hypotonia are the common neurological deficit described in patients with DNM1. Dynamin 1 (DNM1), with a molecular mass of ~100 kd, functions in GTPase mediated endocytosis, synaptic transmission, and activation of signaling mediated by PKC. DNM1 is highly


expressed in neurons, but not widely expressed in other tissues. We describe the phenotypic and genetic spectrum in a cohort of patients (n=5) with early infantile epileptic encephalopathy caused by the dynamin 1 mutation. Patient-derived DNM1 mutant fibroblast cell studies have demonstrated impaired clathrin mediated endocytosis (CME). Unexpectedly, mitochondrial dysfunction (complex I or IV deficiency) was the primary finding in two of the patients in our cohort. Functional assessment of mitochondria through Seahorse XF Analyzer (Agilent, CA) indicated a significant decrease in the ATP generation and mitochondrial spare capacity in DNM1 mutant patient cells compared to control cells. Morphological analysis of patient derived fibroblast cells revealed mitochondrial elongation possibly due to alterations in fission dynamics. Together, our results demonstrate mitochondrial dysfunction in the DNM1 epileptic encephalopathy. Dynamin superfamily members play a critical role in mitochondrial fission and fusion. Nevertheless, mammalian Dynamin 1 has not shown to involved in mitochondrial function, and our results suggest a potential relationship between mitochondria and dynamin 1 protein.


Presenter: Akira Ohtake

Authors: Akira Ohtake1,2, Masaru Shimura3, Minako Ogawa-Tominaga3, Naoko Nozawa4, Takuya Ishii4, Kiwamu Takahashi4, Yasushi Okazaki5, Kei Murayma3

Institution: 1Department of Pediatrics, Faculty of Medicine, Saitama Medical University, Saitama, Japan; 2Center for Intractable Diseases, Saitama Medical University Hospital, Saitama, Japan; 3Department of Metabolism, Chiba Children’s Hospital, Chiba, Japan; 4Division of Pharmaceutical Research, SBI Pharmaceuticals Co., Ltd., Tokyo, Japan; 5Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Tokyo, Japan

Title: 5-Aminolevulinic acid and Fe can bring a permanent cure for mitochondrial diseases:Basic experiments and an investigator initiated clinical trial

Body of Abstract:Background: Many drugs are now in development for mitochondrial diseases (MD), but there is no drug for permanent cure. 5-aminolevlinic acid (5-ALA) is a heme precursor, and heme proteins play an important role at the catalytic site of subunits of mitochondrial respiratory chain enzyme complexes II, III and IV. Methods: Using patients’ skin fibroblasts with culture media supplemented with four different concentrations of 5-ALA/sodium ferrous citrate (SFC): 0/0μM, 50/25μM, 100/50μM, 200/100μM, first the oxygen consumption rates (OCR) were analyzed by microscale oxygraphy using the XF96 extracellular flux analyzer. Next the amounts of ATP production were measured by the luminescence assay. Results: According to increasing of the 5-ALA/SFC concentration, the OCR in the fibroblasts of all patients are improved. The amounts of ATP production in patients’ fibroblasts were increased by 5-ALA/SFC administration. Conclusion: These results provide strong evidences for the efficacy of 5-ALA and SFC treatment in patients with MD. Multicenter, randomized withdrawal, double-blind, placebo-controlled, phase III confirmatory clinical trial are now ongoing in Japan. I’d like to introduce the brief summary of this clinical trial, too.


Presenter: Min Peng, PhD

Authors: Min Peng1, Yi Cheng1, Jianping Kong1, Neal Mathew1, Miao He2, Yair Argon2, Marni J. Falk1,3, Eiko Nakamaru-Ogiso1

Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department ofPediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; 2Department


of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia and Universityof Pennsylvania Perelman School of Medicine; 3Department of Pediatrics, University ofPennsylvania Perelman School of Medicine, Philadelphia, PA 19104

Title: N-Linked Glycosylation of MRS2 Inhibits Mitochondrial Magnesium Import

Abstract: BACKGROUND: N-linked glycosylation of cytosolic proteins has important roles in biological processes, including in cell-to-cell recognition, growth, differentiation, and programmed cell death. While O-linked glycosylation of mitochondria-localized proteins is now well-recognized, the extent and functional significance of N-linked glycosylation of mitochondrial proteins remains uncertain. GOAL: We sought to discern the extent and functional consequences of mitochondrial protein N-glycosylation. Here, we utilize a variety of methodologies to evaluate the N-glycosylation status and functional significance of N-glycosylation on the nuclear-encoded inner mitochondrial membrane protein necessary for mitochondrial magnesium uptake, MRS2.

METHODS: A variety of methodologies were used to evaluate and confirm the N-glycosylation status of MRS2, including a variety of lectin binding assays, PNGase F digestion, SDS-PAGE and immunoblotting of whole cell and tissue fractions as well as isolated mitochondria and inner mitochondrial membrane fractions from mouse liver and human embryonic kidney (HEK293) cells transfected with human MRS2 (both wild-type and mutated at the canonical N-glycosylation site). Mitochondrial matrix magnesium levels were assayed by spectrofluorometry of Fura-2 fluorescence.

RESULTS: Our studies using classical methods directly demonstrate that the mitochondrial magnesium transporter (MRS2) is N-glycosylated, validating prior mass spectrometry analyses and consistent with in silico predictions given it has a canonical N-glycosylation site. MRS2-DYKDDDDK tagged protein was then isolated with anti-flag beads from HEK293 cells that had been transfected with human MRS2-DYKDDDDK tagged cDNA and digested with PNGase F to remove glycans, revealing an additional (lower molecular weight) MRS2 band. A similar gel shift was observed following PNGase F digestion of mouse liver mitochondria protein. Finally, lectin Con A bound to MRS2 isolated from mouse liver mitochondria and overexpressed MRS2 protein in HEK293 cells transfected with MRS2-DYKDDDK tag. These data collectively provide strong evidence to support that the MRS2 protein is N-linked glycosylated.

To study the functional significance of MRS2 N-linked glycosylation on mitochondrial inner membrane magnesium transport, cultured HEK293 cells were treated with an N-linked glycosylation inhibitor (tunicamycin), α-glucosidase inhibitor (castanospermine), or glucose inhibitor (2-deoxyglucose). Isolated mitochondria from the inhibitor treated or control HEK293 cells were then studied to quantify matrix mitochondrial magnesium level by spectrofluorometry of the fluorescent dye Fura-2 that binds free magnesium. Results demonstrated that MRS2 deglycosylation increased mitochondrial magnesium uptake. Validation studies are actively underway to confirm loss of MRS2 N-linked glycosylation, and corresponding reduction in mitochondrial magnesium import, occurs when a point mutation is introduced at the canonical N-glycan attached site in an asparagine of the MRS2 protein.

CONCLUSION: We demonstrate that MRS2, a nuclear-encoded inner mitochondrial membrane protein necessary for mitochondrial magnesium import, is N-glycosylated in both mouse and human cells. Functional validation revealed this unique modification plays a modulatory role in magnesium channel import function, where N-glycosylation has an inhibitory effect on mitochondrial matrix magnesium uptake.


Presenter: Isabella Peixoto de Barcelos

Authors: Isabella Peixoto de Barcelos1, Cesar Augusto Alves2, James Peterson1, Rebecca Ganetzky1, 3, 4, Elizabeth McCormick1, Colleen Muraresku1, Zarazuela Zolkipli-Cunningham1, 3, Marni Falk1, 3 and Amy Goldstein1, 3.


Institution: 1Mitochondrial Medicine Frontier Program, Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104; 2Neuroradiology Department at Children’s Hospital of Philadelphia, PA. 3Department of Pediatrics of Perelman School of Medicine at the University of Pennsylvania. 4Section of Biochemical Genetics, Division of Human Genetics of the Children’s Hospital of Philadelphia, PA.

Title: Clinical and Neuroradiological Findings in Patients with Single Large-Scale Mitochondrial DNA (mtDNA) Deletion Syndromes.

Abstract: Single Large-Scale mtDNA Deletion Syndromes (SLSMDS) are a subgroup of primary mitochondrial disorders classically divided into three overlapping clinical phenotypes which include Pearson-marrow syndrome, chronic progressive external ophthalmoplegia (CPEO), and Kearns-Sayre syndrome (KSS), in addition to those who do not fit a defined phenotype.

We report ten patients (7 males; 3 females) ranging from 2 - to 20 years old from a cohort of 25 patients with a molecularly confirmed diagnosis of SLSMDS. Demographics and clinical features are summarized in Table 1.

The ten patients were previously classified as 6 with classic KSS, one with CPEO-plus spectrum, 2 with Pearson-marrow syndrome, and 1 Pearson-marrow survivor who transitioned to KSS. The mtDNA deletions seen in these patients ranged in size from 3.9 to 5kb and were detected at various heteroplasmy levels on blood.

In reviewing neuroimaging from these patients, neuroradiologic patterns stratified the cohort into two main different subgroups. Subgroup 1, comprised of seven patients, had classic findings described in KSS, including brainstem and globus pallidus involvement, with areas of restricted diffusion, and a variable component of white matter lesions with predominant U-fiber involvement. Subgroup 2 had three patients with diffuse involvement of the white matter, without selective U fibers distribution, or other a gradient of distribution of the lesions. Moreover, differently, from what was observed in subgroup 1, these patients had an absence of involvement of the basal ganglia and brainstem. Advances in genetic testing are increasing the diagnosis of SLSMDS in patients who do not fit into one of the “classic” phenotypes.

Furthermore, by refining the radiologic phenotypes, clinicians may recognize SLMDS radiographically and correlate with “non-classic” clinical presentations. Increasing the index of suspicion of SLSMDS is essential to help narrow the diagnostic testing, identify the diagnosis more rapidly, and address medically actionable issues seen in this disease spectrum, including the potential need for a prophylactic pacemaker for cardiac arrhythmia and screening of endocrinopathies such as diabetes mellitus and hypoparathyroidism.


Presenter: Bruce H. Cohen

Authors: Bruce H. Cohen1*, Liya Yin2, Yeong-Renn Chen2, William M. Chilian2, and Patrick T. Kang2

Institutions: Department of Integrative Medical Sciences, Northeast Ohio Medical University2 andDepartment of Pediatrics and Rebecca D. Considine Research Institute, Akron Children’s Hospital1*

Title: Induced Pluripotent Stem Cells as Models for Studying Mitochondrial Disease

Abstract: Introduction: Evidence-based decisions are lacking because of too many unknowns in each patient. To gain insight into this problem, we propose to obtain blood samples from patients, reprogram the blood cells into induced pluripotent stem cells (iPSCs) for rapid scalable expansion, and to differentiate these stem cells into cardiomyocytes (CMs), a cell-type that is critically dependent on mitochondrial function for survival, as the disease model.


Methods: Blood samples from 30 de-identified enrolled patients (20 patients with mitochondrial disease and 10 healthy donors) were collected at Akron Children’s Hospital, and 3 of these blood samples (1 healthy control, 1 Leigh Syndrome, and 1 MELAS patient) were used in this study. The peripheral blood mononuclear cells (PBMCs) were successfully isolated from these samples, and the 4 essential reprogramming factors, Oct-4, Sox-2, Klf-4 and c-Myc, were then transduced into these cells via Sendai virus infection to create human iPS cells. After reprogramming, cells were initially co-cultured with inactivated mouse embryonic fibroblasts (iMEFs) as feeder cells and selected colonies of iPS cells were eventually transferred to feeder-free matrigel culturing system. Essential 8 medium was changed daily. The confluent iPS cells were then differentiated into cardiomyocytes following monolayer culture method using combinations of growth factors including activin A, BMP-4 and bFGF. Bioenergetics flux of iPS-CMs was measured by the Seahorse Bioscience XF Analyzer.

Results: Successful reprogramming of the three specimens was achieved, with the cardiac mass created by the Control-iPS cells and LS-iPS cells demonstrated beating phenomena (vigorous and non-vigorous respectively). OXPHOS studies suggest that Control-iPS relied on OXPHOS while the MELAS-iPS relied more on glycolysis.

Summary: Three iPS cell lines derived from blood samples of healthy donor, Leigh Syndrome and MELAS patients were established. As the disease modeling platform, both the iPS and iPS-CM cells derived from mitochondrial disease patients exhibited compromised mitochondrial respiration and elevated glycolysis. This model may serve as a method of rapid screening of pharmacological agents that can be used to improve a specific patient’s disease state.


Presenter: Andre Mattman

Authors: Elizabeth Nadeau1, Michelle Mezei2, Marc Cresswell3, Isabelle Dupuis3, Emily Allen2,Andre Mattman2

Institution: 1Patient co-investigator, 2Adult Metabolic Diseases Clinic, Vancouver General Hospital, University of British Columbia, Vancouver, BC 3 Dept of Radiology, St Paul’s Hospital, Vancouver, BC

Title: A multi-faceted lifestyle intervention for mitochondrial A8344G associated multiple symmetric lipomatosis (MSL): Report of a successful patient initiated novel therapy

Body of abstract: An adult female patient carrying the mitochondrial DNA A8344G mutation had multiple symmetric lipomatosis (MSL) as the primary disease manifesation, as did two of her siblings. Relatedly, her daughter developed severe cadiomyocyte lipid deposition culminating in fatal infantile histiocytoid cardiomyopathy. Other family members had typical neurologic manifestations of the mitochondrial disease syndrome (myoclonic epilepsy with ragged red fibres) without MSL. The patient required major lipoma reduction surgery after a rapid rate of lipoma progression starting in the 6th decade of life. Following a difficult recovery from the surgical procedure, the patient independently researched an alternative therapy for her disease. The lifestyle intervention was multi-faceted (dietary, physical activity, meditation) and progressive over the course of two years. It was informed by her personal review of the medical literature in addition to consultation with multiple individuals including family members, professors, social media user groups, and her metabolic disease health care professionals. A carbohydrate reduced (5 – 10% of calories) modified ketogenic diet was a major part of the treatment regimen owing to note of its incidental success in MSL management when her brother started similar dietary therapy for management of glioblastgoma multiforme. Intermittent fasting was also implemented and progressed over two years. Exercise regimens and return to work protocols werefurther incorporated. The outcome of her multi-facted intervention was extremely positive in all of the targeted regards: weight loss (85 - 60 kg), lipoma size stabilization and then reduction by 50% in transverse diameter as measured by serial MRI, improvement in physical activity and strength,


improvement in laboratory markers of insulin resistance (fasting glucose 107 mg/dL to 93 mg/dL), and improved subjective sense of well being accompanied by a return to full time work. Two years into the progression of her self-designed therapy, she continues with her dramatic improvements in subjective and objective measures of health. In summary, a potential non-surgical therapy for mitochondrial disease associated MSL appears feasible over the short term. The success of the lifestyle intervention in MSL therapy is unprecedented and, importantly, fully patient intiated. The success of this novel therapy provides potential insight into the mechanism of MSL exacerbation: suggesting insulin resistance or other lifestyle modifiable factors as mediators of disease progression.


Presenter: Sanna Matilainen M.D.

Authors: Sanna Matilainen1*, Philipp Gut2,3*, Jesse G. Meyer4*, Pieti Pällijeff1, Christopher Carroll5,Christopher Jackson1, Shamita Mahzabin1, Pirjo Isohanni1, Berge Minassian6, Elsebet Ostergaard7, Jason Locasale8, Birgit Schilling4, Anu Suomalainen1 & Eric Verdin2,4*Equal contribution

Institutions: 1 Research Program of Stem Cells and Metabolism, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; 2 Gladstone Institutes and University of California, San Francisco; 3 Nestlé Institute of Health Sciences, EPFL Innovation Park, Lausanne, Switzerland; 4 Buck Institute for Research on Aging, Novato, CA, USA; 5 Genetics Research Centre, Molecular and Clinical Sciences, St. George’s, University of London, London, UK; 6 Program in Genetics and Genome Biology, The Hospital for Sick Children, Institute of Medical Science, University of Toronto, Toronto, Division of Neurology, Department of Pediatrics, University of Texas Southwestern, Dallas, USA; 7 Department of Clinical Genetics, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark; 8 Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Duke Molecular Physiology Institute, Duke Cancer Institute, Durham, North Carolina, USA;

Title: Protein Hypersuccinylation Occurs in Succinyl-CoA Ligase Deficiency, and Leads to Disruption of Metabolic Networks Including Glycolysis

Abstract: Succinyl-CoA ligase (SCL) deficiency due to mutations in the genes SUCLA2 and SUCLG1causes a mitochondrial encephalomyopathy. We find that cell lines from patients with SCL deficiency accumulate succinyl-CoA, leading to global protein hypersuccinylation, a post-translational modification that has not previously been linked to mtDNA depletion syndromes. Hypersuccinylation is specific to SCL deficiency, and is consistently present in non-proliferating cell lines and tissues of SUCLA2 and SUCLG1patients. Reduction of mitochondrial DNA, a hallmark of the disorder, is observed in induced pluripotent stem cell-derived neurons, but not in replicating cells, suggesting specificity to tissue and cell identity.

Label-free proteomic profiling in fibroblasts and myotubes from patients identifies nearly 1000 succinylated lysine residues among many important metabolic pathways, including nearly all glycolytic enzymes. Metabolomics analysis proposes glycolysis to be functionally affected in SUCLA2 patients, in particular at the level of the final step of glycolysis through impairment of the irreversible reaction of phosphoenol-pyruvate (PEP) to pyruvate. Many of these pathologically-succinylated proteins are regulated by the desuccinylase SIRT5, suggesting that a subset of the “succinylome” is susceptible to succinylation independent whether the cause is loss of sirtuin function or increased substrate availability. These data proposes a role for succinylation in metabolic regulation, post-translationally modifying metabolic proteins in response to changes in metabolic flux, hypersuccinylation contributing to the pathophysiology of SCL deficiency.



Presenter: Joel Meyer

Authors: Jessica H. Hartman1, Joel N. Meyer1

Institutions: 1Duke University, Durham, NC

Title: Swim exercise in Caenorhabditis elegans protects dopaminergic neurons from age- and rotenone-induced degeneration

Abstract:Physical exercise exerts positive impacts on cognitive function, maintenance of skeletal muscle, and protection from age-related diseases. However, molecular mechanisms underlying those protections are not well understood. Furthermore, it is unknown what effects regular exercise training may have on other health-modifying factors such as environmental toxicant exposures. In this study, we used C. elegans to study the impact of long-term exercise training on dopaminergic neuronal health at baseline and after exposure to the pesticide rotenone. For exercise experiments, beginning at adult day 1, animals were transferred to unseeded agar plates without (control) or with liquid (causing worms to swim) for 90 minutes twice daily. This regimen was carried out for six days, and dopaminergic neuronal health was tested following exercise on adult day 6 and adult day 10. Exercise caused a reduction in age-related morphological changes to the dendrites of dopaminergic CEP neurons that was apparent by adult day 6 (p<0.05) and was much more dramatic by day 10 (p<0.0001). This was accompanied by a 20% greater (p<0.01) basal slowing rate in exercised animals (locomotion on food versus off food), a behavioral readout of dopaminergic neuron function. Furthermore, exercise was protective against dopaminergic neurodegeneration from rotenone exposures initiated on day 6. We also performed whole-animal RNA-seq analysis to determine pathways involved in exercise conditioning. Enriched pathways included lipid biosynthesis and metabolism, nucleoside metabolism, chemosensation, cellular signaling, and cellular respiration. In ongoing and future studies, we will interrogate pathways changed by exercise to determine which genes are involved in regulating exercise-induced neuroprotection. Together, these data will provide critical insights into the mechanisms underlying exercise benefits.


Documents

Mitochondrial Medicine 2019: Washington DC€¦ · Mitochondrial Medicine 2019: Washington DC 6FLHQWLÀF DQG &OLQLFDO 0HHWLQJV June 26-29, 2019 Hilton Alexandria Mark Center Alexandria,