Cytogenomic Integrative Network Analysis of the Critical ...downloads.hindawi.com/journals/bmri/2018/5436187.pdf · BioMedResearchInternational 0 1000000 2000000 3000000 4000000 Genomic

Research ArticleCytogenomic Integrative Network Analysis of the Critical RegionAssociated with Wolf-Hirschhorn Syndrome

Thiago Correcirca1 Rafaella Mergener1 Juacutelio Ceacutesar Loguercio Leite2 Marcial Francis Galera3

Lilia Maria de AzevedoMoreira4 Joseacute Eduardo Vargas 5 andMariluce Riegel 12

1Post-Graduate Program in Genetics and Molecular Biology Universidade Federal do Rio Grande do Sul (UFRGS)91501-970 Porto Alegre RS Brazil2Medical Genetics Service Hospital de Clınicas de Porto Alegre Rua Ramiro Barcelos 2350 90035-903 Porto Alegre RS Brazil3Department of Pediatrics Universidade Federal do Mato Grosso (UFMT) 78600-000 Cuiaba MT Brazil4Post-Graduate Program in Genetics and Biodiversity Universidade Federal da Bahia Campus Ondina40170-290 Salvador BA Brazil5Institute of Biological Sciences Universidade de Passo Fundo Passo Fundo RS Brazil

Correspondence should be addressed to Mariluce Riegel mriegelhcpaedubr

Received 6 October 2017 Accepted 1 February 2018 Published 12 March 2018

Academic Editor Hesham H Ali

Copyright copy 2018 Thiago Correa et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Deletions in the 4p163 region are associated with Wolf-Hirschhorn syndrome (WHS) a contiguous gene deletion syndromeinvolving variable size deletions In this study we perform a cytogenomic integrative analysis combining classical cytogeneticmethods fluorescence in situ hybridization (FISH) chromosomal microarray analysis (CMA) and systems biology strategies toestablish the cytogenomic profile involving the 4p163 critical region and suggest WHS-related intracellular cell signaling cascadesThe cytogenetic and clinical patient profiles were evaluated We characterized 12 terminal deletions one interstitial deletion tworing chromosomes and one classical translocation 48 CMA allowed delineation of the deletions which ranged from 37 to 256Mbwith breakpoints from 4p163 to 4p1533 Furthermore the smallest region of overlapping (SRO) encompassed seven genes in aterminal region of 330 kb in the 4p163 region suggesting a region of susceptibility to convulsions and microcephaly Thereforemolecular interaction networks and topological analysis were performed to understand these WHS-related symptoms Our resultssuggest that specific cell signaling pathways including dopamine receptor NAD+nucleosidase activity and fibroblast growth factor-activated receptor activity are associated with the diverse pathological WHS phenotypes and their symptoms Additionally weidentified 29 hub-bottlenecks (H-B) nodes with a major role in WHS

1 Introduction

Wolf-Hirschhorn syndrome (WHS OMIM 194190) is awell known genetic condition with estimated prevalence of120000 to 150000 births [1 2] originally described inde-pendently byHirschhorn andCooper [3] andWolf et al [4] inthe 1960sThe core clinical features ofWHSare facial dysmor-phia growth retardation intellectual disability and seizuresMoreover other clinical signs such as microcephaly hypoto-nia congenital heart defects renal abnormalities and skeletalanomalies have also been reported [5] WHS is caused by adeletion in the p163 region (4p163) which has a variable sizethat reflects the spectrum and severity of the disease [5ndash7]

Approximately 55 of individuals with WHS exhibit the4p163 deletion and approximately 40ndash45 are unbalancedtranslocations Complex genomic rearrangements such asring 4 chromosome may also be present in a smaller numberof cases [6 8]

The deleted region required to express the core WHSphenotype has been reported to encompass two adjacentcritical regionsThe first region described as theWHS criticalregion (WHSCR) encompasses a size of 165 kb interval on4p163 [9] Two genes are present in this region the proximalpart of WHSC1 and the negative elongation factor complexmember A (NELFA) WHSC1 is involved in controlling theexpression of several genes and acts as an agent in chromatin

HindawiBioMed Research InternationalVolume 2018 Article ID 5436187 10 pageshttpsdoiorg10115520185436187

2 BioMed Research International

remodeling [10]TheNELFA gene alters the expression of tar-get genes by participating in regulating the elongation of RNApolymerase II transcription [11] The second region is theWHSCR2 which encompasses the leucine zipper-EF-hand-containing transmembrane protein (LETM) gene and the 51015840end of WHSC1 [12 13] LETM1 encodes a protein involvedin mitochondrial metabolism by transporting ions [14 15]Currently the WHSCR is defined at chr4419224ndash2010962position in the reference genome (NCBI BuildGRCh38hg19)[16]

The possibility of using microarrays to characterize chro-mosomal rearrangements has led to several studies aimed atestablishing genotype-phenotype correlations in WHS andmany of them have described the regions of susceptibility toseizures and microcephaly in patients with WHS [1 5 17ndash23] However no consensus has been reached on the exactidentity of the genes and cell signaling pathways involvedin promoting these symptoms Therefore we developed acytogenomic integrative analysis that combines conventionalcytogenetic techniques chromosome microarray analysis(CMA) and systems biology strategies to suggest the mecha-nism underlying the seizures and microcephaly in WHS

2 Materials and Methods

21 Study Design and Sample Selection This was a retro-spective study conducted on 16 samples from patients withclinical suspicions of WHS The patients were followed up atthe public genetic services in collaboration with the BrazilianNetwork of Reference and Information for MicrodeletionSyndrome (RedeBRIM)

22 Cytogenetic Analysis Karyotyping was performed onmetaphase spreads prepared from peripheral blood samplesThe chromosomal analysis was conducted afterGTGbandingat a 550-band resolution and at least 100 cells from eachpatient were analyzed

23 Fluorescence In Situ Hybridization (FISH) The fluores-cence in situ hybridization (FISH) experiments were carriedout using standard techniques with commercially availablelocus-specific probes using a dual-color commercial probefor the WHSCR (Cytocell UK) The probe for the 4p163(red spectrum) contained a sequence that was homologousto the D4S166 locus and covered approximately 223 kb ofthis locus The control probe for the 4q352 region (greenspectrum) contained sequences that were homologous to theCTC-963K6 loci

Hybridizations were analyzed using an epifluorescencemicroscope and the images were captured using a charge-coupled device camera At least 30 cells were analyzedper hybridization We considered a chromosome region asdeleted when the FISH signal from the corresponding probewas absent from one of the homologous chromosomes

24 CMA The deletions were mapped using whole-genomearray-comparative genome hybridization (CGH) using a60mer oligonucleotide-based microarray with a theoretical

resolution of 40 kb (8times 60K Agilent Technologies Inc SantaClara CA USA) The labeling and hybridization were per-formed following the protocols provided byAgilent 2011Thearrays were analyzed using a microarray scanner (G2600D)and the Feature Extraction software (version 951 bothfrom Agilent Technologies) The images were analyzed usingCytogenomics v 20 and 27 with the statistical algorithmADM-2 and a sensitivity threshold of 60

25 Systems Biology Analysis

251 Network Design The Gene Multiple AssociationNetwork Integration Algorithm (GeneMANIA) version3128 (available at httpwwwgenemaniaorg) was usedto analyze the protein-protein interactions (PPI) networksbased on the 343 genes obtained from the GENCODEV24ndashGRCh38hg38-UCSC database In the present studythe association data from GeneMANIA was based on thePPI databases where each interaction between proteinsis experimentally proven [24] or on gene coexpressiondata The interactions based on known protein domainspathways and colocalization were not considered in theanalysis because they could increase the false-positiveratios in PPI networks obtained The outcomes obtainedthrough these search engines were sequentially analyzedusing Cytoscape 341 [25] Nonconnected nodes from thenetworks were not included

(1) Clustering The MCODE tool was used to identify thedensely connected and possibly overlapping regions in theCytoscape network [26] Dense regions corresponded toprotein or compound-protein complexes or their partsThenGene Ontology (GO) enrichment Kyoto Encyclopedia ofGenes and Genomes (KEGG) WikiPathways and Reactomeanalyses were performed using the ClueGOCytoscape plugin[27] Based on the GO predictions a cut-off of a p-correctedvalue le 001 using the false discovery rate (FDR) algorithm(Bonferroni test) was used to describe the mechanisms asdescribed in the discussion section

(2) Centralities Two major parameters of network central-ities (degree and betweenness) were used to identify thehub-bottlenecks (H-B) nodes from the PPI network usingthe Cytoscape plugin CentiScaPe 321 [28] The degree ofcentrality indicates the total number of adjacent nodes thatare connected to a unique node In this study the averagenodal degree of a network was defined as the sum of thedifferent node degree scores divided by the total number ofnodes that composed the entire networks [29] Furthermorewe also analyzed the betweenness which corresponds tothe number of shortest paths between two nodes that passthrough a node of interest [28] The arithmetic average of thebetweenness parameterwas estimated similarly to the averagecentrality degree [29]

26 Ethics Review This study was approved by the ResearchEthics Committee of the Hospital de Clınicas de PortoAlegre (HCPA) with approval number GPPG 10-560 and was

BioMed Research International 3

(a) (b) (c)

(d) (e) (f)

Figure 1 Karyotype results of (a) case 1 showing one normal chromosome 4 (left) and a chromosome 4p (right) (b) case 2 both normalchromosomes 4 (c) case 4 a normal chromosome 4 (left) and a ring chromosome 4 (right) and fluorescence in situ hybridization (FISH)results with locus-specific probes for the Wolf-Hirschhorn syndrome critical region (WHSCR) 4p163 from (d) case 1 (e) case 2 and (f) case4 Absence of red signal on one copy of chromosome 4 indicates deletion of critical region

conducted in accordance with all current institution ethicalrules

3 Results and Discussion

A total of 16 samples from patients whose clinical phe-notypes were indicative of WHS were retrospectively eval-uated (supplementary material S1) Among the patientsseven and nine (4375 and 5625) were women and menrespectively The most frequent clinical findings in ourstudy group were seizures and microcephaly however facialdysmorphia growth retardation and intellectual disabilitywere also observed (Table 1) In addition the cytogeneticand FISH analysis identified 12 classical terminal deletionsone interstitial deletion two ring chromosomes and onetranslocation t(48) (p163p231 Figure 1) Furthermore froma total of 16 samples the CMA was used to further mapeight deletions that occurred caused by terminal or interstitial4p163 rearrangements The deletions ranged in size from37 to 26Mb (Figure 2(a)) and at least seven genes werewithin the smallest region of overlapping (SRO) deletion inthe 4p163 region (Figure 2(b)) At the time of the presentstudy there were no DNA samples available to perform CMAanalysis from the further eight patients

In our study the SRO encompassed the 330 kb terminalregion of the short armof chromosome 4This region extendsfrom 18 to 213Mb in the 4p163 region and it was identifiedas a region of susceptibility to the convulsions and micro-cephaly that are typical of WHS (Figure 2(b)) Comparingthe cytogenomic profile of our samples with that of otherstudies we delineated the proximal and distal breakpoints asthe SRO [1 17] in the 4p163 region (Figure 3) This regionincluded seven candidate genes (LETM1 FGFR3 WHSC1NELFA C4orf48 NAT8L and POLN) These genes couldcontribute to the pathogenic phenotype such as seizures andmicrocephaly and include the LETM1 gene located in ourSRO which contributes to the presence of seizures in ahemizygous deletion [1 12 18 30]

In human cell lines the deletion of LETM1 could alterthe intracellular [Ca2+] levels dysfunctional mitochondrialtransition-pore opening and hyperpolarization [31 32] Fur-thermore thesemitochondrial alterations could contribute tothe emergence of some clinical findings in WHS includingseizures [31 32] All patients in this study presented seizuresymptoms suggesting the putative involvement of this genealthough patients with 4p deletions including LETM1 with-out seizures or patients with seizures and the presence ofLETM1 have been described [1 17 23 33 34] However


Table1Summaryof

cytogeno

micandclinicalfi

ndings

ofeightsam

ples

investigated

usingarray-comparativ

egenom

ehybrid

ization(C

GH)

Case

1Ca

se2

Case

3Ca

se4

Case

5Ca

se6

Case

7Case8

Total

Karyotype

46X

Yt(4

8)

46X

X46

XX

4p-

46X

Xr(4)

46X

X4p

-46

XY4p

-46

XY4p

-46

XY4p

-Molecular

cytogenetic

findings

FISH

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

Dele

tionsiz

e(pb)

3773546

3773546

7175628

8102374

88290

1311073248

2440474

025696727

Genom

icpo

sitionon

chr4

(GRC

h38hg38)

71552ndash3845097

71552ndash3845097

71552ndash7247179

71552ndash8173925

71552ndash8900564

172944

2ndash12802689

68345ndash24473084

68345ndash25765071

Clinica

lfind

ings

Seizures

++

++

++

++

88

Microceph

aly

++

++

++

++

88

Growth

retardation

+NA

NA

+NA

++

+58

Intellectuald

isability

+NA

++

++

+NA

68

Reserved

progno

sisminus

NA

minus+

+minus

+minus

38

Shortu

pper

lip+

NA

++

++

++

78

Smallm

entalregion

+NA

++

++

++

78

Labialdeviations

downw

ard

+NA

++

++

++

78

Hypop

lasticc

olum

ella

+NA

++

++

++

78

Hyperteloris

m+

NA

+minus

++

++

68

Ptosisof

thee

yelid

s+

NA

+minus

++

minusNA

58

Deploym

ento

fhairo

nforehead

ishigh

+NA

+minus

++

++

68

Fine

nose

+NA

++

minusminus

++

58

Cardiovascular

malform

ations

NA

NA

+minus

minusminus

+minus

28

Brainmalform

ations

NA

NA

+NA

minusminus

+minus

28

Cleft

palate

NA

NA

minusminus

NA

NA

+minus

18Re

nalm

alform

ations

NA

NA

minusminus

minusNA

minus+

18Hypospadias

NA

NA

minusNA

NA

NA

minus+

18Feetandsm

allh

ands

+NA

++

++

+minus

68

Narrowfin

gers

+NA

++

++

++

78

Hypotroph

yof

thethenar

+NA

+minus

minusminus

+minus

38

Ham

mer

toe

NA

NA

+minus

minusminus

minusminus

18Ab

norm

alderm

atoglyph

sminus

NA

+NA

NA

NA

NA

NA

18(+)feature

present(minus)feature

absent(NA)n

otavailableFISH

fluo

rescence

insituhybridization


p16

3

q13

1

p12

q13

3q1

32

q12

p13

p14

p15

1p1

52

p15

31p1

532

p15

33p1

61

p16

2

q21

1q2

121

q21

22q2

21

q22

2q2

23

q23

q24

q25

q26

q27

q28

1q2

82

q28

3q3

11

q31

21

q31

22q3

123

q31

3

q32

1q3

22

q32

3q3

3q3

41

q34

2q3

43

q35

1q3

52

p163 p162 p161

WHSCRWHSCR-2

12345

6

p1533

78 25696727 bp

24404740 bp

15000000 bp11300000 bp6000000 bp4500000 bp

11073248 bp8829013 bp

8102374 bp7247179 bp

3773546 bp3773546 bp

(a)

Seizures

Microcephaly

Growth retardation

Facial features

Skeletal malformations

2500000 pb2000000 pb1500000 pb1000000 pb500000 pb

(Simon and Bergemann 2008)


(Endele et al 1999 Zollino et al 2003 Shimizu et al 2014 Hart et al 2014 Bi et al 2016)

(Bayindir et al 2013 Bi et al 2016 Ho et al 2016)

(Shimizu et al 2014 Zollino 2014 Bi et al 2016)

(Engbers et al 2009 Catela et al 2009 Shimizu et al 2014)

(Kerzendorfer et al 2012)

(Buggenhout et al 2004 Maas et al 2008 Nimura et al 2009 Okamoto et al 2013)


(Simon and Bergemann 2008 Misceo et al 2012 Shimizu et al 2014)

tel cen

FGFR3

PIGG

CPLX1 TACC3

LETM1

WHSC1

FGFRL1

CTBP1

NELF-ASLBP

C4ORF48

NAT8L

POLN

ann 2008)

rgemann 2008)

03 Shimizu et al 2

(Kerzendorfer et a

t al 2008 Nimura

2014)

FGFR3

TACC3

LETM1

WHSC

NEL

C4ORF

NAT

PO

SRO

(b)

Figure 2 Cytogenomic profile of chromosome 4 (a) Red horizontal bars show extent of deleted segments on short arm of chromosome 4 ineight samples investigated using array- comparative genome hybridization (CGH) (b) Genes on 4p163p1533 with haploinsufficiency effectsassociated with Wolf-Hirschhorn syndrome (WHS) clinical findings

this may be partially explained by the synergism betweenthe phosphatidylinositol glycan anchor biosynthesis class G(PIGG) complexin 1 (CPLX1) and LETM1 genes frequentlyassociated with WHS convulsions [18]

Furthermore our results showed a 330 kb region alsoassociated with microcephaly The deletion of two genesWHSC1 and NELFA in this region has already been associ-ated with this condition [17 19 35]WHSC1 shows transcrip-tional corepressor activity by expressing a histone methyl-transferase [32 36] which controls the level of histone H3

lysine 36 (H3K36) trimethylation and histone acetylation[10] NELFA encodes a member of the negative elongationfactor involved in regulating the progression of transcriptionby RNA polymerase II [37] and histone mRNA maturationinto mRNAs [11] The haploinsufficiency of NELFA in celllines from patients withWHS is associated with delayed pro-gression from the S- into the M-phase and altered chromatinassembly [35]

The following four additional genes are also located inthe susceptibility region proposed in this study fibroblast


0 1000000 2000000 3000000 4000000Genomic position (bp)

tel cent

Patient 8

Patient 7

Patient 5

Patient 4

Patient 3

Van Buggenhout et al 2004 (5)

Patient 2

Patient I

Maas et al 2008 (9)

Maas et al 2008 (5)


Patient 6

Maas et al 2008 (15)

Engbers et al 2009 (l)


Hannes 2012


WHSCR 2

WHSCR I

Seizure and Microcephaly (SRO)

330000 bp

Figure 3 Smallest region of overlapping (SRO) associated with microcephaly and seizures Bars show deletion sizes and genomic positionon 4p Red horizontal bars indicate seizures and microcephaly phenotype green bars indicate absence of seizures and microcephaly andtwo bars in gray represent critical regions of Wolf-Hirschhorn syndrome (WHS) The smallest region of susceptibility to microcephaly andseizures shown in this study is represented by blue bar covering a 330 kb in size (18 to 213Mb)WHSCRWolf-Hirschhorn syndrome criticalregion

growth factor receptor 3 (FGFR3) N-acetyltransferase 8 like(NAT8L) DNA polymerase Nu (POLN) and chromosome 4open reading frame 48 (C4ORF48I) However these genesare not directly associated with seizures and microcephalybut have a putative involvement in the skeletal developmentand plasticity of the human brain [38ndash40] In additionprevious studies have described the occurrence of seizuresmicrocephaly or both which indicates the contribution ofmultiple deleted genes located adjacent to the SRO delineatedin our study [41]

Thus focusing on the critical 4p163 chromosome regionmapped in our study we explored the molecular inter-action networks and biological pathways involving WHS-associated genes An initial list of 343 genes obtained from theGENCODE V24-GRCh38hg38-UCSC database was usedto construct an interactome network This network wascomposed of 136 nodes and 750 edges (Figure 4(a)) It isimportant to note that for this initial network prediction weconsidered a deletion of 26Mb including our SRO WHSCRand WHSCR2 (Figure 2(a)) Furthermore we performeda cluster analysis that identified two major cluster regionscluster 1 (19 nodes and 56 edges) and cluster 2 (13 nodes

and 56 edges) In these clustered and unclustered nodesmore significant GO categories were identified (Figure 4(a))In the unclustered nodes four discrete molecular functionswere predicted (1) NAD+ nucleosidase activity (119901 valuecorrected = 0002) (2) NAD(P)+ nucleosidase activity (119901value corrected = 75 times 10minus4) These pathways participatein nicotinamide metabolism and calcium signaling therebycontributing to excitability exocytosis motility apoptosisand cell transcriptionmechanisms [42 43] (3) FGFR activity(119901 value corrected = 0006) and (4) FGF binding (119901 valuecorrected = 0004) which is involved in the maintenanceof tissue homeostasis and regulation of metabolic processeswith specific roles such as the regulation of cell migrationproliferation and differentiation [44 45]

The receptors involved in these pathways are encoded byFGFR3 and FGFRL1 which interact with FGFs to activatea downstream signaling cascade The hemizygous deletionsof FGFR3 and FGFRL1 are implicated in the skeletal abnor-malities and facial features typical of WHS [21 46ndash48]Furthermore three GO categories were identified in cluster1 (Figure 4(a)) The dopamine receptor signaling pathway (119901value corrected = 0001) has been implicated in numerous


19 nodes

Cluster 1

Cluster 2

63 edges

Unclustered proteinClustered protein

13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1

Unclustered nodesMaturation of 58S rRNABrush border membrane

Growth factor bindingFibroblast growth factor binding

Bone growthEndochondral bone developmental growth

Fibroblast growth factor-activated receptor activityRough endoplasmic reticulum

MicrovillusMicrovillus membrane

Middle ear morphogenesisResponse to arsenic-containing substance

GTPase inhibitor activityNegative regulation of GTPase activity

Nuclear ubiquitin ligase complexNicotinate and nicotinamide metabolism

Phosphorus-oxygen lyase activityCatecholamine binding

Hydrolase activity hydrolyzing N-glycosyl compoundsNAD+ nucleosidase activity

NAD(P)+ nucleosidase activityDopamine receptor signaling pathway

Positive regulation of B cell proliferationLong term synaptic depression

Positive regulation of vasoconstrictionCiliary membrane

Photoreceptor disc membraneRhodopsin mediated signaling pathway

Regulation of rhodopsin mediated signaling pathwayNegative regulation of epithelial cell apoptotic process

Ubiquitin protein ligase activity involved in ERAD pathwayER-associated ubiquitin-dependent protein catabolic process

Fatty acid degradation

dopamine receptor signaling pathwaynuclear DNA replication

toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)

Figure 4 Graphs representing protein-protein interactions (PPI) network (a) List of 343 genes was obtained from GENCODE V24-GRCh38hg38-UCSC database The data was used to construct networks using Cytoscape software processing (b) Centralities parametersand topological analysis using the CentiScaPe plugin genes in small region overlapping (SRO) in our study are in red


neurological processes including sleep regulation feedingattention cognitive functions olfaction and hormonal reg-ulation [49] Toxin transport (119901 value corrected = 0001) andmolecular functions such as nuclearDNAreplication (119901 valuecorrected = 73 times 10minus4) encompassing the stem-loop bindingprotein (SLBP) andNELFA genes often deleted inWHSwerealso predicted It was not possible to predictGOandpathwayscategories for cluster 2

An alternative strategy to decipher cell signaling path-ways involved in seizures and microcephaly using networksis based on connectivity analysis In this analysis thecentrality properties were evaluated (Figure 4(b)) and 29hub-bottlenecks (H-B) nodes were identified in the initialnetwork The NELFA gene was localized in the SRO definedin this study and the SLBP gene was identified as an H-B in the centrality analysis (Figure 4(b)) There is evidencethat NELFA is necessary for the recruitment of SLBP by itsregulation of histone synthesis during the S-phase [11 50]Therefore the haploinsufficiency of NELFA SLBP or bothcould affect the cell-cycle progression DNA replication andthe chromatin assembly [35] Interestingly NELFA is locatedin the WHSCR and SLBP in WHCR2 suggesting the inde-pendent contribution of both genes in WHS pathogenesis

The WHSC1 protein also located in the SRO affectsthe levels of trimethylated H3K36 (H3K36Me3) which canbe reduced by siRNA-mediated knockdown of the NELF-E component of the NELF complex [10 51] This indicatesanother functional association between WHSC1 and NELFAin controlling gene expression by chromatin remodeling andregulation of the elongation of the transcription respectively[35] The identification of the H-B NELFA SLBP and thehubWHSC1 (Figure 4(b)) using centrality analysis highlightsthe importance of the involvement of these genes in thecore WHS phenotype such as skeletal malformation facialfeatures and microcephaly (Figure 2(b)) [1 17 19 35 52]

The centrality analysis revealed that 90 and 10 of theH-B are located on chromosome 4 and other chromosomesrespectively Considering this evidence we suggest that thediversity of the pathological WHS phenotypes could bedependent on interrelated and close H-Bs located on thesame chromosome To the best of our knowledge this isthe first report of a cytogenomic integrative network analysisusing systems biology to study the critical region associatedwith WHS Our study described the putative cell signalingpathways altered in WHS that contribute to the integrativeunderstanding of the role of contiguous genes in the spec-trum of this syndrome

4 Conclusions

This study combined clinical data and integrative analysiswith conventional cytogenetic techniques CMA and systemsbiology strategies We confined the region of susceptibilityfor microcephaly and seizures to a 330 kb sized regionthat encompassed seven candidate genes (LETM1 FGFR3WHSC1 NELFA C4orf48 NAT8LI and POLN)The networktopological analysis identified 29 H-Bs and the candidategene NELFA was included among these H-Bs In additionsignificant GO categories showed that several cell signaling

pathways are responsible for the seizures and microcephalyin WHS

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the Brazilian Network of Ref-erence and Information in Microdeletion Syndromes (Rede-BRIM) (Grant 4767832016) Thiago Correa was supportedby Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq) Brazil

Supplementary Materials

S1 summary of cytogenomic and clinical findings of 16samples from this study Cytogenetic and clinical profilesfrom the retrospective study conducted on 16 samples frompatients with clinical suspicions of WHS Combining classi-cal cytogenetic methods fluorescence in situ hybridization(FISH) and chromosomal microarray analysis (CMA) wecharacterized 12 terminal deletions one interstitial deletiontwo ring chromosomes and one classical translocation 48CMAallowed delineation of the deletions in 8 samples whichranged from 37 to 256Mb with breakpoints from 4p163 to4p1533 (Supplementary Materials)

References

[1] NM CMaas G Van Buggenhout F Hannes et al ldquoGenotype-phenotype correlation in 21 patients with Wolf-Hirschhornsyndrome using high resolution array comparative genomehybridisation (CGH)rdquo Journal of Medical Genetics vol 45 no2 pp 71ndash80 2008

[2] I W Lurie G I Lazjuk Y I Ussova E B Presman and DB Gurevich ldquoThe Wolf-Hirschhorn syndrome I GeneticsrdquoClinical Genetics vol 17 no 6 pp 375ndash384 1980

[3] K Hirschhorn andH Cooper ldquoApparent deletion of short armsof one chromosome (4 or 5) in a child with defects of midlinefusionrdquoMamm Chrom Nwsl vol 4 no 14 1961

[4] U Wolf H Reinwein R Porsch R Schroter and H BaitschldquoDefizienz an den kurzen Armen eines Chromosomes Nr 4rdquoHuman Genetics vol 1 no 5 pp 397ndash413 1965

[5] M Zollino M Murdolo G Marangi et al ldquoOn thenosology and pathogenesis of WolfndashHirschhorn syndromegenotypendashphenotype correlation analysis of 80 patients andliterature reviewrdquo American Journal of Medical Genetics Part CSeminars in Medical Genetics vol 148 no 4 pp 257ndash269 2008

[6] A Battaglia J C Carey and S T South ldquoWolf-Hirschhornsyndrome A review and updaterdquo American Journal of MedicalGenetics Part C Seminars inMedical Genetics vol 169 no 3 pp216ndash223 2015

[7] A Battaglia T Filippi and J C Carey ldquoUpdate on theclinical features and natural history of Wolf-Hirschhorn (4p-)syndrome Experience with 87 patients and recommendationsfor routine health supervisionrdquo American Journal of MedicalGenetics Part C Seminars in Medical Genetics vol 148 no 4pp 246ndash251 2008


[8] S T South H Whitby A Battaglia J C Carey and AR Brothman ldquoComprehensive analysis of Wolf-Hirschhornsyndrome using array CGH indicates a high prevalence oftranslocationsrdquoEuropean Journal ofHumanGenetics vol 16 no1 pp 45ndash52 2008

[9] T J Wright D O Ricke K Denison et al ldquoA transcript mapof the newly defined 165 kbWolf-Hirschhorn syndrome criticalregionrdquo Human Molecular Genetics vol 6 no 2 pp 317ndash3241997

[10] Y F Lee K NimuraW N Lo K Saga and Y Kaneda ldquoHistoneH3 lysine 36methyltransferaseWhsc1 promotes the associationof Runx2 and p300 in the activation of bone-related genesrdquoPLoS ONE vol 9 no 9 Article ID e106661 2014

[11] T Narita T M C Yung J Yamamoto et al ldquoNELF Interactswith CBC and Participates in 31015840 End Processing of Replication-Dependent Histone mRNAsrdquo Molecular Cell vol 26 no 3 pp349ndash365 2007

[12] M Zollino R Lecce R Fischetto et al ldquoMapping theWolf-Hirschhorn syndrome phenotype outside the currentlyacceptedWHS critical region and defining a new critical regionWHSCR-2rdquoAmerican Journal of Human Genetics vol 72 no 3pp 590ndash597 2003

[13] L Rodrıguez M Zollino S Climent et al ldquoThe new Wolf-Hirschhorn syndrome critical region (WHSCR-2) A descrip-tion of a second caserdquoAmerican Journal ofMedical Genetics vol136 no 2 pp 175ndash178 2005

[14] K S Dimmer F Navoni A Casarin et al ldquoLETM1 deletedin Wolf-Hirschhorn syndrome is required for normal mito-chondrial morphology and cellular viabilityrdquoHumanMolecularGenetics vol 17 no 2 pp 201ndash214 2008

[15] K Nowikovsky T Pozzan R Rizzuto L Scorrano and PBernardi ldquoThe Pathophysiology of LETMrdquo The Journal ofGeneral Physiology vol 139 no 6 pp 445ndash454 2012

[16] S B Cassidy and J E Allanson Management of GeneticSyndromes John Wiley amp Sons Inc Hoboken NJ USA 2005

[17] G Van Buggenhout C Melotte B Dutta et al ldquoMild Wolf-Hirschhorn syndrome Micro-array CGH analysis of atypical4p163 deletions enables refinement of the genotype-phenotypemaprdquo Journal of Medical Genetics vol 41 no 9 pp 691ndash6982004

[18] W Bi S-W Cheung A M Breman and C A Bacino ldquo4p163microdeletions and microduplications detected by chromoso-mal microarray analysis New insights into mechanisms andcritical regionsrdquo American Journal of Medical Genetics Part Avol 170 no 10 pp 2540ndash2550 2016

[19] N Okamoto K Ohmachi S Shimada K Shimojima and TYamamoto ldquo109kb deletion of chromosome 4p163 in a patientwithmild phenotype ofWolf-Hirschhorn syndromerdquoAmericanJournal of Medical Genetics Part A vol 161 no 6 pp 1465ndash14692013

[20] K S Ho S T South A Lortz et al ldquoChromosomal microarraytesting identifies a 4p terminal region associatedwith seizures inWolfndashHirschhorn syndromerdquo Journal of Medical Genetics vol53 no 4 pp 256ndash263 2016

[21] K Shimizu K Wakui T Kosho et al ldquoMicroarray and FISH-based genotype-phenotype analysis of 22 Japanese patientswithWolf-Hirschhorn syndromerdquoAmerican Journal of MedicalGenetics Part A vol 164 no 3 pp 597ndash609 2014

[22] D Concolino E Rossi P Strisciuglio et al ldquoDeletion of a 760kb region at 4p16 determines the prenatal and postnatal growthretardation characteristic of Wolf-Hirschhorn syndromerdquo Jour-nal of Medical Genetics vol 44 no 10 pp 647ndash650 2007

[23] E F Andersen J C Carey D L Earl et al ldquoDeletionsinvolving genes WHSC1 and LETM1 may be necessary but arenot sufficient to cause Wolf-Hirschhorn Syndromerdquo EuropeanJournal of Human Genetics vol 22 no 4 pp 464ndash470 2014

[24] K Zuberi M Franz H Rodriguez et al ldquoGeneMANIA pre-diction server 2013 updaterdquo Nucleic Acids Research vol 41 ppW115ndashW122 2013

[25] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003

[26] G D Bader and C W V Hogue ldquoAn automated methodfor finding molecular complexes in large protein interactionnetworksrdquo BMC Bioinformatics vol 4 no 1 p 2 2003

[27] G Bindea B Mlecnik H Hackl et al ldquoClueGO a Cytoscapeplug-in to decipher functionally grouped gene ontology andpathway annotation networksrdquoBioinformatics vol 25 no 8 pp1091ndash1093 2009

[28] G Scardoni M Petterlini and C Laudanna ldquoAnalyzing biolog-ical network parameters with CentiScaPerdquo Bioinformatics vol25 no 21 pp 2857ndash2859 2009

[29] J E Vargas R Puga J D F Poloni et al ldquoA network flowapproach to predict protein targets and flavonoid backbonesto treat respiratory syncytial virus infectionrdquo BioMed ResearchInternational vol 2015 Article ID 301635 9 pages 2015

[30] S Endele M Fuhry S-J Pak B U Zabel and A WinterpachtldquoLETM1 a novel gene encoding a putative EF-hand Ca2+-binding protein flanks theWolf-Hirschhorn syndrome (WHS)critical region and is deleted in most WHS patientsrdquo Genomicsvol 60 no 2 pp 218ndash225 1999

[31] D S Khurana I Valencia M J Goldenthal and A LegidoldquoMitochondrial dysfunction in epilepsyrdquo Seminars in PediatricNeurology vol 20 no 3 pp 176ndash187 2013

[32] L Hart A Rauch A M Carr J R Vermeesch and MOrsquoDriscoll ldquoLETM1 haploinsufficiency causes mitochondrialdefects in cells from humans with Wolf-Hirschhorn syndromeImplications for dissecting the underlying pathomechanisms inthis conditionrdquo DISEASE MODELS amp MECHANISMS vol 7no 5 pp 535ndash545 2014

[33] D Misceo T Baroslashy J R Helle Oslash Braaten M Fannemeland E Frengen ldquo15Mb deletion of chromosome 4p163 asso-ciated with postnatal growth delay psychomotor impairmentepilepsy impulsive behavior and asynchronous skeletal devel-opmentrdquo Gene vol 507 no 1 pp 85ndash91 2012

[34] B Bayindir E Piazza E Della Mina et al ldquoDravet phenotypein a subject with a der(4)t(48)(p163p233) without the involve-ment of the LETM1 generdquo European Journal ofMedical Geneticsvol 56 no 10 pp 551ndash555 2013

[35] C Kerzendorfer F Hannes R Colnaghi et al ldquoCharacterizingthe functional consequences of haploinsufficiency of NELF-A (WHSC2) and SLBP identifies novel cellular phenotypes inWolf-Hirschhorn syndromerdquo Human Molecular Genetics vol21 no 10 Article ID dds033 pp 2181ndash2193 2012

[36] J Y Kim J K Hae N W Choe et al ldquoMultiple myeloma-related WHSC1MMSET isoform RE-IIBP is a histone methyl-transferase with transcriptional repression activityrdquo Molecularand Cellular Biology vol 28 no 6 pp 2023ndash2034 2008

[37] T Narita Y Yamaguchi K Yano et al ldquoHuman transcriptionelongation factor NELF Identification of novel subunits andreconstitution of the functionally active complexrdquo Molecularand Cellular Biology vol 23 no 6 pp 1863ndash1873 2003


[38] J S Colvin B A Bohne G W Harding D G McEwen and DM Ornitz ldquoSkeletal overgrowth and deafness in mice lackingfibroblast growth factor receptor 3rdquoNature Genetics vol 12 no4 pp 390ndash397 1996

[39] E Wiame D Tyteca N Pierrot et al ldquoMolecular identificationof aspartate N-acetyltransferase and its mutation in hypoacety-laspartiardquo Biochemical Journal vol 425 no 1 pp 127ndash136 2010

[40] S Endele C Nelkenbrecher A Bordlein S Schlickum andA Winterpacht ldquoC4ORF48 a gene from the Wolf-Hirschhornsyndrome critical region encodes a putative neuropeptide andis expressed during neocortex and cerebellar developmentrdquoneurogenetics vol 12 no 2 pp 155ndash163 2011

[41] A D Bergemann F Cole and K Hirschhorn ldquoThe etiology ofWolf-Hirschhorn syndromerdquo Trends in Genetics vol 21 no 3pp 188ndash195 2005

[42] K Nakazawa K Ueda T Honjo K Yoshihara Y Nishizukaand O Hayaishi ldquoNicotinamide adenine dinucleotide glycohy-drolases and poly adenosine diphosphate ribose synthesis in ratliverrdquo Biochemical and Biophysical Research Communicationsvol 32 no 2 pp 143ndash149 1968

[43] D E Clapham ldquoCalcium Signalingrdquo Cell vol 131 no 6 pp1047ndash1058 2007

[44] A A Belov and M Mohammadi ldquoMolecular mechanisms offibroblast growth factor signaling in physiology and pathologyrdquoCold Spring Harbor Perspectives in Biology vol 5 no 6 2013

[45] S Abuharbeid F Czubayko and A Aigner ldquoThe fibroblastgrowth factor-binding protein FGF-BPrdquoThe International Jour-nal of Biochemistry amp Cell Biology vol 38 no 9 pp 1463ndash14682006

[46] H Engbers J J van der Smagt R vanrsquot Slot J R VermeeschR Hochstenbach and M Poot ldquoWolf-Hirschhorn syndromefacial dysmorphic features in a patient with a terminal 4p163deletion telomeric to the WHSCR and WHSCR 2 regionsrdquoEuropean Journal of Human Genetics vol 17 no 1 pp 129ndash1322009

[47] C Catela D Bilbao-Cortes E Slonimsky P Kratsios N Rosen-thal and P Te Welscher ldquoMultiple congenital malformationsof Wolf-Hirschhorn syndrome are recapitulated in Fgfrl1 nullmicerdquoDISEASEMODELSampMECHANISMS vol 2 no 5-6 pp283ndash294 2009

[48] R Simon and A D Bergemann ldquoMouse models of Wolf-Hirschhorn syndromerdquo American Journal of Medical GeneticsPart C Seminars in Medical Genetics vol 148 no 4 pp 275ndash280 2008

[49] J-M Beaulieu S Espinoza and R R Gainetdinov ldquoDopaminereceptorsmdashIUPHAR review 13rdquo British Journal of Pharmacol-ogy vol 172 no 1 pp 1ndash23 2015

[50] L Zheng Z Dominski X-C Yang et al ldquoPhosphorylationof stem-loop binding protein (SLBP) on two threonines trig-gers degradation of SLBP the sole cell cycle-regulated factorrequired for regulation of histone mRNA processing at the endof S phaserdquo Molecular and Cellular Biology vol 23 no 5 pp1590ndash1601 2003

[51] J Sun and R Li ldquoHuman negative elongation factor activatestranscription and regulates alternative transcription initiationrdquoThe Journal of Biological Chemistry vol 285 no 9 pp 6443ndash6452 2010

[52] K Nimura K Ura H Shiratori et al ldquoA histone H3 lysine36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syn-dromerdquo Nature vol 460 no 7252 pp 287ndash291 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


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The Scientific World Journal

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BioinformaticsAdvances in

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BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


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Advances in

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Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


remodeling [10]TheNELFA gene alters the expression of tar-get genes by participating in regulating the elongation of RNApolymerase II transcription [11] The second region is theWHSCR2 which encompasses the leucine zipper-EF-hand-containing transmembrane protein (LETM) gene and the 51015840end of WHSC1 [12 13] LETM1 encodes a protein involvedin mitochondrial metabolism by transporting ions [14 15]Currently the WHSCR is defined at chr4419224ndash2010962position in the reference genome (NCBI BuildGRCh38hg19)[16]

The possibility of using microarrays to characterize chro-mosomal rearrangements has led to several studies aimed atestablishing genotype-phenotype correlations in WHS andmany of them have described the regions of susceptibility toseizures and microcephaly in patients with WHS [1 5 17ndash23] However no consensus has been reached on the exactidentity of the genes and cell signaling pathways involvedin promoting these symptoms Therefore we developed acytogenomic integrative analysis that combines conventionalcytogenetic techniques chromosome microarray analysis(CMA) and systems biology strategies to suggest the mecha-nism underlying the seizures and microcephaly in WHS

2 Materials and Methods

21 Study Design and Sample Selection This was a retro-spective study conducted on 16 samples from patients withclinical suspicions of WHS The patients were followed up atthe public genetic services in collaboration with the BrazilianNetwork of Reference and Information for MicrodeletionSyndrome (RedeBRIM)

22 Cytogenetic Analysis Karyotyping was performed onmetaphase spreads prepared from peripheral blood samplesThe chromosomal analysis was conducted afterGTGbandingat a 550-band resolution and at least 100 cells from eachpatient were analyzed

23 Fluorescence In Situ Hybridization (FISH) The fluores-cence in situ hybridization (FISH) experiments were carriedout using standard techniques with commercially availablelocus-specific probes using a dual-color commercial probefor the WHSCR (Cytocell UK) The probe for the 4p163(red spectrum) contained a sequence that was homologousto the D4S166 locus and covered approximately 223 kb ofthis locus The control probe for the 4q352 region (greenspectrum) contained sequences that were homologous to theCTC-963K6 loci

Hybridizations were analyzed using an epifluorescencemicroscope and the images were captured using a charge-coupled device camera At least 30 cells were analyzedper hybridization We considered a chromosome region asdeleted when the FISH signal from the corresponding probewas absent from one of the homologous chromosomes

24 CMA The deletions were mapped using whole-genomearray-comparative genome hybridization (CGH) using a60mer oligonucleotide-based microarray with a theoretical

resolution of 40 kb (8times 60K Agilent Technologies Inc SantaClara CA USA) The labeling and hybridization were per-formed following the protocols provided byAgilent 2011Thearrays were analyzed using a microarray scanner (G2600D)and the Feature Extraction software (version 951 bothfrom Agilent Technologies) The images were analyzed usingCytogenomics v 20 and 27 with the statistical algorithmADM-2 and a sensitivity threshold of 60

25 Systems Biology Analysis

251 Network Design The Gene Multiple AssociationNetwork Integration Algorithm (GeneMANIA) version3128 (available at httpwwwgenemaniaorg) was usedto analyze the protein-protein interactions (PPI) networksbased on the 343 genes obtained from the GENCODEV24ndashGRCh38hg38-UCSC database In the present studythe association data from GeneMANIA was based on thePPI databases where each interaction between proteinsis experimentally proven [24] or on gene coexpressiondata The interactions based on known protein domainspathways and colocalization were not considered in theanalysis because they could increase the false-positiveratios in PPI networks obtained The outcomes obtainedthrough these search engines were sequentially analyzedusing Cytoscape 341 [25] Nonconnected nodes from thenetworks were not included

(1) Clustering The MCODE tool was used to identify thedensely connected and possibly overlapping regions in theCytoscape network [26] Dense regions corresponded toprotein or compound-protein complexes or their partsThenGene Ontology (GO) enrichment Kyoto Encyclopedia ofGenes and Genomes (KEGG) WikiPathways and Reactomeanalyses were performed using the ClueGOCytoscape plugin[27] Based on the GO predictions a cut-off of a p-correctedvalue le 001 using the false discovery rate (FDR) algorithm(Bonferroni test) was used to describe the mechanisms asdescribed in the discussion section

(2) Centralities Two major parameters of network central-ities (degree and betweenness) were used to identify thehub-bottlenecks (H-B) nodes from the PPI network usingthe Cytoscape plugin CentiScaPe 321 [28] The degree ofcentrality indicates the total number of adjacent nodes thatare connected to a unique node In this study the averagenodal degree of a network was defined as the sum of thedifferent node degree scores divided by the total number ofnodes that composed the entire networks [29] Furthermorewe also analyzed the betweenness which corresponds tothe number of shortest paths between two nodes that passthrough a node of interest [28] The arithmetic average of thebetweenness parameterwas estimated similarly to the averagecentrality degree [29]

26 Ethics Review This study was approved by the ResearchEthics Committee of the Hospital de Clınicas de PortoAlegre (HCPA) with approval number GPPG 10-560 and was


(a) (b) (c)

(d) (e) (f)








Table1Summaryof

cytogeno

micandclinicalfi

ndings

ofeightsam

ples

investigated


egenom

ehybrid

ization(C

GH)

Case

1Ca

se2

Case

3Ca

se4

Case

5Ca

se6

Case

7Case8

Total

Karyotype

46X

Yt(4

8)

46X

X46

XX

4p-

46X

Xr(4)

46X

X4p

-46

XY4p

-46

XY4p

-46

XY4p

-Molecular

cytogenetic

findings

FISH

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

Dele

tionsiz

e(pb)

3773546

3773546

7175628

8102374

88290

1311073248

2440474

025696727

Genom

icpo

sitionon

chr4

(GRC

h38hg38)

71552ndash3845097

71552ndash3845097

71552ndash7247179

71552ndash8173925

71552ndash8900564

172944

2ndash12802689

68345ndash24473084

68345ndash25765071

Clinica

lfind

ings

Seizures

++

++

++

++

88

Microceph

aly

++

++

++

++

88

Growth

retardation

+NA

NA

+NA

++

+58

Intellectuald

isability

+NA

++

++

+NA

68

Reserved

progno

sisminus

NA

minus+

+minus

+minus

38

Shortu

pper

lip+

NA

++

++

++

78

Smallm

entalregion

+NA

++

++

++

78

Labialdeviations

downw

ard

+NA

++

++

++

78

Hypop

lasticc

olum

ella

+NA

++

++

++

78

Hyperteloris

m+

NA

+minus

++

++

68

Ptosisof

thee

yelid

s+

NA

+minus

++

minusNA

58

Deploym

ento

fhairo

nforehead

ishigh

+NA

+minus

++

++

68

Fine

nose

+NA

++

minusminus

++

58

Cardiovascular

malform

ations

NA

NA

+minus

minusminus

+minus

28

Brainmalform

ations

NA

NA

+NA

minusminus

+minus

28

Cleft

palate

NA

NA

minusminus

NA

NA

+minus

18Re

nalm

alform

ations

NA

NA

minusminus

minusNA

minus+

18Hypospadias

NA

NA

minusNA

NA

NA

minus+

18Feetandsm

allh

ands

+NA

++

++

+minus

68

Narrowfin

gers

+NA

++

++

++

78

Hypotroph

yof

thethenar

+NA

+minus

minusminus

+minus

38

Ham

mer

toe

NA

NA

+minus

minusminus

minusminus

18Ab

norm

alderm

atoglyph

sminus

NA

+NA

NA

NA

NA

NA

18(+)feature


absent(NA)n

otavailableFISH

fluo

rescence

insituhybridization


p16

3

q13

1

p12

q13

3q1

32

q12

p13

p14

p15

1p1

52

p15

31p1

532

p15

33p1

61

p16

2

q21

1q2

121

q21

22q2

21

q22

2q2

23

q23

q24

q25

q26

q27

q28

1q2

82

q28

3q3

11

q31

21

q31

22q3

123

q31

3

q32

1q3

22

q32

3q3

3q3

41

q34

2q3

43

q35

1q3

52

p163 p162 p161

WHSCRWHSCR-2

12345

6

p1533

78 25696727 bp

24404740 bp

15000000 bp11300000 bp6000000 bp4500000 bp

11073248 bp8829013 bp

8102374 bp7247179 bp

3773546 bp3773546 bp

(a)

Seizures

Microcephaly

Growth retardation

Facial features


2500000 pb2000000 pb1500000 pb1000000 pb500000 pb











tel cen

FGFR3

PIGG

CPLX1 TACC3

LETM1

WHSC1

FGFRL1

CTBP1

NELF-ASLBP

C4ORF48

NAT8L

POLN

ann 2008)

rgemann 2008)

03 Shimizu et al 2

(Kerzendorfer et a

t al 2008 Nimura

2014)

FGFR3

TACC3

LETM1

WHSC

NEL

C4ORF

NAT

PO

SRO

(b)








tel cent

Patient 8

Patient 7

Patient 5

Patient 4

Patient 3


Patient 2

Patient I

Maas et al 2008 (9)

Maas et al 2008 (5)


Patient 6




Hannes 2012


WHSCR 2

WHSCR I


330000 bp







19 nodes

Cluster 1

Cluster 2

63 edges


13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1



















toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(a) (b) (c)

(d) (e) (f)








Table1Summaryof

cytogeno

micandclinicalfi

ndings

ofeightsam

ples

investigated


egenom

ehybrid

ization(C

GH)

Case

1Ca

se2

Case

3Ca

se4

Case

5Ca

se6

Case

7Case8

Total

Karyotype

46X

Yt(4

8)

46X

X46

XX

4p-

46X

Xr(4)

46X

X4p

-46

XY4p

-46

XY4p

-46

XY4p

-Molecular

cytogenetic

findings

FISH

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

Dele

tionsiz

e(pb)

3773546

3773546

7175628

8102374

88290

1311073248

2440474

025696727

Genom

icpo

sitionon

chr4

(GRC

h38hg38)

71552ndash3845097

71552ndash3845097

71552ndash7247179

71552ndash8173925

71552ndash8900564

172944

2ndash12802689

68345ndash24473084

68345ndash25765071

Clinica

lfind

ings

Seizures

++

++

++

++

88

Microceph

aly

++

++

++

++

88

Growth

retardation

+NA

NA

+NA

++

+58

Intellectuald

isability

+NA

++

++

+NA

68

Reserved

progno

sisminus

NA

minus+

+minus

+minus

38

Shortu

pper

lip+

NA

++

++

++

78

Smallm

entalregion

+NA

++

++

++

78

Labialdeviations

downw

ard

+NA

++

++

++

78

Hypop

lasticc

olum

ella

+NA

++

++

++

78

Hyperteloris

m+

NA

+minus

++

++

68

Ptosisof

thee

yelid

s+

NA

+minus

++

minusNA

58

Deploym

ento

fhairo

nforehead

ishigh

+NA

+minus

++

++

68

Fine

nose

+NA

++

minusminus

++

58

Cardiovascular

malform

ations

NA

NA

+minus

minusminus

+minus

28

Brainmalform

ations

NA

NA

+NA

minusminus

+minus

28

Cleft

palate

NA

NA

minusminus

NA

NA

+minus

18Re

nalm

alform

ations

NA

NA

minusminus

minusNA

minus+

18Hypospadias

NA

NA

minusNA

NA

NA

minus+

18Feetandsm

allh

ands

+NA

++

++

+minus

68

Narrowfin

gers

+NA

++

++

++

78

Hypotroph

yof

thethenar

+NA

+minus

minusminus

+minus

38

Ham

mer

toe

NA

NA

+minus

minusminus

minusminus

18Ab

norm

alderm

atoglyph

sminus

NA

+NA

NA

NA

NA

NA

18(+)feature


absent(NA)n

otavailableFISH

fluo

rescence

insituhybridization


p16

3

q13

1

p12

q13

3q1

32

q12

p13

p14

p15

1p1

52

p15

31p1

532

p15

33p1

61

p16

2

q21

1q2

121

q21

22q2

21

q22

2q2

23

q23

q24

q25

q26

q27

q28

1q2

82

q28

3q3

11

q31

21

q31

22q3

123

q31

3

q32

1q3

22

q32

3q3

3q3

41

q34

2q3

43

q35

1q3

52

p163 p162 p161

WHSCRWHSCR-2

12345

6

p1533

78 25696727 bp

24404740 bp

15000000 bp11300000 bp6000000 bp4500000 bp

11073248 bp8829013 bp

8102374 bp7247179 bp

3773546 bp3773546 bp

(a)

Seizures

Microcephaly

Growth retardation

Facial features


2500000 pb2000000 pb1500000 pb1000000 pb500000 pb











tel cen

FGFR3

PIGG

CPLX1 TACC3

LETM1

WHSC1

FGFRL1

CTBP1

NELF-ASLBP

C4ORF48

NAT8L

POLN

ann 2008)

rgemann 2008)

03 Shimizu et al 2

(Kerzendorfer et a

t al 2008 Nimura

2014)

FGFR3

TACC3

LETM1

WHSC

NEL

C4ORF

NAT

PO

SRO

(b)








tel cent

Patient 8

Patient 7

Patient 5

Patient 4

Patient 3


Patient 2

Patient I

Maas et al 2008 (9)

Maas et al 2008 (5)


Patient 6




Hannes 2012


WHSCR 2

WHSCR I


330000 bp







19 nodes

Cluster 1

Cluster 2

63 edges


13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1



















toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table1Summaryof

cytogeno

micandclinicalfi

ndings

ofeightsam

ples

investigated


egenom

ehybrid

ization(C

GH)

Case

1Ca

se2

Case

3Ca

se4

Case

5Ca

se6

Case

7Case8

Total

Karyotype

46X

Yt(4

8)

46X

X46

XX

4p-

46X

Xr(4)

46X

X4p

-46

XY4p

-46

XY4p

-46

XY4p

-Molecular

cytogenetic

findings

FISH

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

del4p163

Dele

tionsiz

e(pb)

3773546

3773546

7175628

8102374

88290

1311073248

2440474

025696727

Genom

icpo

sitionon

chr4

(GRC

h38hg38)

71552ndash3845097

71552ndash3845097

71552ndash7247179

71552ndash8173925

71552ndash8900564

172944

2ndash12802689

68345ndash24473084

68345ndash25765071

Clinica

lfind

ings

Seizures

++

++

++

++

88

Microceph

aly

++

++

++

++

88

Growth

retardation

+NA

NA

+NA

++

+58

Intellectuald

isability

+NA

++

++

+NA

68

Reserved

progno

sisminus

NA

minus+

+minus

+minus

38

Shortu

pper

lip+

NA

++

++

++

78

Smallm

entalregion

+NA

++

++

++

78

Labialdeviations

downw

ard

+NA

++

++

++

78

Hypop

lasticc

olum

ella

+NA

++

++

++

78

Hyperteloris

m+

NA

+minus

++

++

68

Ptosisof

thee

yelid

s+

NA

+minus

++

minusNA

58

Deploym

ento

fhairo

nforehead

ishigh

+NA

+minus

++

++

68

Fine

nose

+NA

++

minusminus

++

58

Cardiovascular

malform

ations

NA

NA

+minus

minusminus

+minus

28

Brainmalform

ations

NA

NA

+NA

minusminus

+minus

28

Cleft

palate

NA

NA

minusminus

NA

NA

+minus

18Re

nalm

alform

ations

NA

NA

minusminus

minusNA

minus+

18Hypospadias

NA

NA

minusNA

NA

NA

minus+

18Feetandsm

allh

ands

+NA

++

++

+minus

68

Narrowfin

gers

+NA

++

++

++

78

Hypotroph

yof

thethenar

+NA

+minus

minusminus

+minus

38

Ham

mer

toe

NA

NA

+minus

minusminus

minusminus

18Ab

norm

alderm

atoglyph

sminus

NA

+NA

NA

NA

NA

NA

18(+)feature


absent(NA)n

otavailableFISH

fluo

rescence

insituhybridization


p16

3

q13

1

p12

q13

3q1

32

q12

p13

p14

p15

1p1

52

p15

31p1

532

p15

33p1

61

p16

2

q21

1q2

121

q21

22q2

21

q22

2q2

23

q23

q24

q25

q26

q27

q28

1q2

82

q28

3q3

11

q31

21

q31

22q3

123

q31

3

q32

1q3

22

q32

3q3

3q3

41

q34

2q3

43

q35

1q3

52

p163 p162 p161

WHSCRWHSCR-2

12345

6

p1533

78 25696727 bp

24404740 bp

15000000 bp11300000 bp6000000 bp4500000 bp

11073248 bp8829013 bp

8102374 bp7247179 bp

3773546 bp3773546 bp

(a)

Seizures

Microcephaly

Growth retardation

Facial features


2500000 pb2000000 pb1500000 pb1000000 pb500000 pb











tel cen

FGFR3

PIGG

CPLX1 TACC3

LETM1

WHSC1

FGFRL1

CTBP1

NELF-ASLBP

C4ORF48

NAT8L

POLN

ann 2008)

rgemann 2008)

03 Shimizu et al 2

(Kerzendorfer et a

t al 2008 Nimura

2014)

FGFR3

TACC3

LETM1

WHSC

NEL

C4ORF

NAT

PO

SRO

(b)








tel cent

Patient 8

Patient 7

Patient 5

Patient 4

Patient 3


Patient 2

Patient I

Maas et al 2008 (9)

Maas et al 2008 (5)


Patient 6




Hannes 2012


WHSCR 2

WHSCR I


330000 bp







19 nodes

Cluster 1

Cluster 2

63 edges


13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1



















toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



p16

3

q13

1

p12

q13

3q1

32

q12

p13

p14

p15

1p1

52

p15

31p1

532

p15

33p1

61

p16

2

q21

1q2

121

q21

22q2

21

q22

2q2

23

q23

q24

q25

q26

q27

q28

1q2

82

q28

3q3

11

q31

21

q31

22q3

123

q31

3

q32

1q3

22

q32

3q3

3q3

41

q34

2q3

43

q35

1q3

52

p163 p162 p161

WHSCRWHSCR-2

12345

6

p1533

78 25696727 bp

24404740 bp

15000000 bp11300000 bp6000000 bp4500000 bp

11073248 bp8829013 bp

8102374 bp7247179 bp

3773546 bp3773546 bp

(a)

Seizures

Microcephaly

Growth retardation

Facial features


2500000 pb2000000 pb1500000 pb1000000 pb500000 pb











tel cen

FGFR3

PIGG

CPLX1 TACC3

LETM1

WHSC1

FGFRL1

CTBP1

NELF-ASLBP

C4ORF48

NAT8L

POLN

ann 2008)

rgemann 2008)

03 Shimizu et al 2

(Kerzendorfer et a

t al 2008 Nimura

2014)

FGFR3

TACC3

LETM1

WHSC

NEL

C4ORF

NAT

PO

SRO

(b)








tel cent

Patient 8

Patient 7

Patient 5

Patient 4

Patient 3


Patient 2

Patient I

Maas et al 2008 (9)

Maas et al 2008 (5)


Patient 6




Hannes 2012


WHSCR 2

WHSCR I


330000 bp







19 nodes

Cluster 1

Cluster 2

63 edges


13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1



















toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




tel cent

Patient 8

Patient 7

Patient 5

Patient 4

Patient 3


Patient 2

Patient I

Maas et al 2008 (9)

Maas et al 2008 (5)


Patient 6




Hannes 2012


WHSCR 2

WHSCR I


330000 bp







19 nodes

Cluster 1

Cluster 2

63 edges


13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1



















toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



19 nodes

Cluster 1

Cluster 2

63 edges


13 nodes 56 edges

PPI network

A

2

3

5

3

2

2

2

2

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

6

CLuster 1



















toxin transport

0 1 2 3 4 5 6

Genesterm

Genesterm

GO

KEG

GIn

terP

roR

EACT

OM

EW

ikiP

athw

ays

Enric

hmen

t Ana

lysis

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105

(a)

TENC1ADD1

ECI1

PACS2STX18

HTTQDPR MAEA

MAN2B2MRFAP1L1

DHX15

MYL5

RAB28PCGF3

RFC1 SLC30A9

GAK

ACOX3

LRPAP1

RNF4

FAM193A

GRPEL1

KIAA0232

RBPJ

MFSD10

SLBPCTBP1

NOP14GPR125

TACC3

NELFA

WFS1

ACOX3

ADD1

CTBP1

DHX15

ECI1

FAM193A

GAK HTT

KIAA0232

MAEA

MAN2B2

MFSD10

MYL5

NELFA

PCGF3

QDPR

RAB28

RBPJ

RNF4

SLBP SLC30A9

STX18 TENC1

WFS1

FGFR3

NAT8L

LETM1

C4orf48

H-B

H-B

NH-B

WHSC1

0

Betw

eenn

es sc

ore

0 10 20 30 40 50Node degree

12 times 103

10 times 103

80 times 102

60 times 102

40 times 102

20 times 102

(b)







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







4 Conclusions





Acknowledgments




References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


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