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Preimplantation Genetic Diagnosis for Monogenic

Disorders and Chromosomal Rearrangements – The German

Perspective

Koehler U, Schoen U, Mayer V, Holinski-Feder E

J. Reproduktionsmed. Endokrinol 2013; 10 (Sonderheft

1), 38-44

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38 J Reproduktionsmed Endokrinol 2013; 10 (Special Issue 1)

PGD – The German Perspective

Preimplantation Genetic Diagnosis for MonogenicDisorders and Chromosomal Rearrangements –

The German PerspectiveU. Koehler, U. Schoen, V. Mayer, E. Holinski-Feder

Since its dawn in the late 1980s, preimplantation genetic diagnosis (PGD, or Präimplantationsdiagnostik, PID) has evolved into a well-established tech-nique, which can be offered to couples at risk of transmitting a mutation or a chromosomal aberration to their offspring. Polar bodies as well as day 3blastomeres and day 5 blastocysts (trophectoderm) can be employed for the detection of a specific gene mutation or unbalanced karyotypes. For the latter,array comparative genomic hybridisation (array CGH) has replaced fluorescence in situ hybridisation (FISH) approaches. Furthermore, as blastocysts seemto exhibit less mosaicism compared to blastomeres, current PGD protocols focus on the analysis of blastocysts, however polar body testing is still appliedfor maternally derived conditions. In November 2011, the German embryo protection law (ESchG) has been supplemented by §3a, which defines theconditions for the legal implementation of PGD (PräimpG) in Germany. J Reproduktionsmed Endokrinol 2013; 10 (Special Issue 1): 38–44.

Key words: reimplantation genetic diagnosis, PGD, polar body diagnosis, blastocyst, trophectoderm biopsy, monogenic disease,reciprocal translocation, Robertsonian translocation, mosaicism, polymerase chain reaction (PCR), array CGH

Received: May 22, 2012; accepted after revision: June 28, 2012From the MGZ – Medizinisch Genetisches Zentrum, München, GermanyCorrespondence: Udo Koehler, PhD, MGZ-Medizinisch Genetisches Zentrum, Bayerstraße 3–5, D-80335 München; e-mail: koehler@mgz-muenchen.de

Introduction

Preimplantation genetic diagnosis is thegenetic analysis of polar bodies (PB),blastomeres or blastocysts before trans-ferring the embryo into the uterus withinan in-vitro-fertilisation (IVF) cycle andintracytoplasmic sperm injection (ICSI).Different PGD approaches are applieddepending on legal regulations and tech-nical preferences throughout reproduc-tive and genetic centers worldwide (seealso http://www.drze.de/in-focus/preim-plantation-genetic-diagnosis/modules/legal-regulation-of-pgd). In the majorityof countries, PGD is applied primarily topolar bodies and totipotent blastomeres(day 3 cleavage stage embryos), how-ever, a switch to pluripotent trophecto-derm (TE) samples from blastocysts(day 5) can be observed, as these cellsare considered to yield more reliable re-sults. The legal situation in Germany isambiguous even though a “Präimplanta-tionsdiagnostikgesetz” (PräimpG) [1]was published in November 2011 as asupplement of the Embryo ProtectionAct (ESchG) [2]. In contrast to othercountries, preimplantation genetic diag-nosis of totipotent day 3 blastomeres re-mains prohibited in Germany, whereasgenetic testing of pluripotent trophecto-derm samples after biopsy of day 5 blas-tocysts will be in accordance with thePräimpG. However, guidelines regulat-ing the conditions under which a PGD

can be performed in licensed centers af-ter genetic and social counselling and apositive vote from a clinical ethics com-mittee, is still lacking as of May 2012.

A prerequisite for PGD is an intracyto-plasmic sperm injection within the set-ting of an assisted reproductive technol-ogy (ART). Sir Robert Edwards, a pio-neer in the field, settled the foundationfor ART in the 1960s and 1970s. PGDthen was first performed in 1989, whenHandyside et al. [3] successfully deter-mined the sex of an embryo in couples atrisk for recessive X-linked diseases; thiswas followed by single cell diagnosis forthe monogenic disease cystic fibrosis[4]. First pregnancies after PGD werereported by Handyside et al. in 1990 [5].Since then, PGD has evolved rapidly andis applied for more than 200 single genedisorders and chromosomal abnormali-ties [6]. Furthermore, applying improvedmolecular genetic techniques, PGD pro-tocols for every gene causing a mono-genic inherited disease are imaginable.

Indications for PGD

PGD is in accordance with German law,when (1.) a genetic condition in the fe-male or the male partner can lead to asevere disease in the offspring or (2.)when a severe genetic impairment of theembryo which leads to a miscarriage canbe avoided. The underlying genetic aber-

ration can be a single gene mutation or achromosome rearrangement (transloca-tion, inversion). Furthermore, employ-ing array CGH, embryos carrying denovo chromosome aneuploidies can bedetected, thus preventing pregnancieswhich would lead to a miscarriage.

Sample Options

Maternally derived gene mutations orchromosomal translocations can be ana-lysed through preimplantation geneticdiagnosis of polar bodies [7], blasto-meres [8], or blastocysts [9], whereaspaternally derived genetic conditionscan only be detected in blastomeres orblastocysts. Polar bodies I and II (PB I,PB II) are haploid cells, which can beaspirated from the oocyte. Blastomeresare diploid totipotent embryonic cells(day 3 cleavage stage embryo). Blasto-cysts are day 5–6 embryos after the innercell mass (ICM, embryonic cells) hasseparated from the trophectoderm (tro-phoblast cells).

Polar Body Biopsy

Polar bodies accumulate during meiosisas by-products of the mature oocyte.Polar body I is present before fertilisa-tion as a product of meiosis I, whereaspolar body II forms after fertilisation(meiosis II). PB I reflects the geneticconfiguration of the oocyte in a mirror

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PGD – The German Perspective

J Reproduktionsmed Endokrinol 2013; 10 (Special Issue 1) 39

image fashion, whereas the genome ofPB II is identical to the oocyte, providedthat no genetic recombination occurredduring the first meiotic division. Thus,analyzing the polar bodies can detectmutations and chromosomal aberrationspresent in the oocyte [7]. The disadvan-tage of polar body analysis is that it pro-vides an insight only into the maternalgenome, which in the case of a recessivedisease can lead to the rejection ofheterozygously mutated oocytes whichmay otherwise have developed intohealthy embryos. Additionally, no pro-spective rating of the blastocyst develop-ment can be made at the time of polarbody diagnosis.

Blastomere Biopsy

Blastomeres are embryonic cells ofcleavage stage embryos. A biopsy at daythree after fertilisation removes one (ortwo cells) from the totipotent 8 cell em-bryo. The biopsy of two blastomeres inthe early days of PGD revealed an inap-propriate decrease in viability of the em-bryo, so that current approaches employthe biopsy of a single blastomere. Thesesamples still are the preferred ones forPGD in the majority of genetic centers,even though they exhibit a higher rate ofchromosomal instability compared toblastocysts [10, 11]. Therefore, a changein PGD protocols to a trophectodermbiopsy of blastocysts is about to occur.

Blastocyst Biopsy

At day 5 after fertilisation, the blastocysthas differentiated into the embryoblast(inner cell mass, ICM) and the tropho-blast (trophectoderm, TE). Trophoblastcells are no longer totipotent, but pluri-potent. After implantation into theuterus, the trophoblast cells constitutethe chorionic villi and the placenta,whereas the fetus evolves from the em-bryoblast. Trophoblast cells are geneti-cally identical to the genome of theembryoblast and thus represent an idealsample with which to test the embryonicgenome without affecting the embryoniccells themselves. Schoolcraft et al. couldshow that analysis of day 5 blastocysts isas reliable as analysis of day 3 cleavagestage embryos [12]. Compared to day 3blastomeres, blastocysts tend to presentmore homogeneous karyotypes and aless mosaicism. Although PGD follow-ing blastocyst biopsy most probably re-

veals less aneuploidies due to the self-selection processes in the embryoniccells [13], it has yet to be shown in alarger cohort of samples that mosaicismin trophoblast cells and the embryoblastdoes not lead to misdiagnoses; Johnsonet al. showed a high concordance ofPGD results between the trophectodermand the inner cell mass [14]. As a conse-quence, trophectoderm cells seem to bethe ideal sample for PGD. However,blastocyst biopsy is more challengingthan blastomere biopsy, and only somecenters have changed their protocols sofar (Fig. 1).

Diagnostic Approaches

Different technical approaches are em-ployed in PGD: PCR based techniques

[15, 16], fluorescence in situ hybridisa-tion (FISH) [17, 18], and comparativegenomic hybridisation techniques (CGH)[19–21]. Within the PGD procedure,microarray based comparative genomic

Figure 1. Trophectoderm biopsy. Courtesy of Kinder-wunschzentrum Regensburg, Professor Dr. med. BerndSeifert and Bernd Paulmann.

Table 1. Selection of single gene disorders for which PGD protocols for polar bodiesand day 5 blastocyts are established in Germany

Disease Gene

Adenylosuccinase deficiency ADSLAndermann syndrome SLC12A6Aromatic L-amino acid decarboxylase deficiency DDCCeroid lipofuscinosis CLN3Charcot-Marie-Tooth disease, X-linked (CMTX1) CX32 (GJB1)Cystic fibrosis CFTRDesbuquois syndrome CANT1Familial adenomatous polyposis 1 APCFragile X syndrome FMR1Freeman Sheldon syndrome MYH3Gorlin Goltz syndrome PTCH1Haemophilia A F8Huntington disease HTTHydrocephalus, X-linked L1CAMHypophosphatasia, perinatal lethal ALPLJoubert syndrome INPP5EKrabbe disease GALCLeigh syndrome SURF1Marfan syndrome FBN1Mitochondriopathy, Lactat acidosis BOLA3Mucopolysaccharidosis type IIIC GNPTABMucopolysaccharidosis type IIIA SGSHMuscular dystrophy type Duchenne DMDMyotonic dystrophy DMPKNeurofibromatosis type 1 NF1Nonketotic hyperglycinemia GLDCNorrie syndrome NDPOrnithine transcarbamylase deficiency OTCPeters-Plus syndrome B3GALTLRetinoblastoma RB1Simson-Golabi-Behmel syndrome GPC3Spinal muscular atrophy SMN1Spinocerebellar ataxia 1 SCA1Surfactant deficiency SFTPBTuberous sklerosis, type 1 and 2 TSC1/TSC2Van der Woude syndrome IRF6Walker Warburg syndrome POMT1Zellweger syndrome PEX1, PEX6

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PGD – The German Perspective

hybridisation (array CGH) is replacingFISH and the time-consuming conven-tional or metaphase comparative ge-nomic hybridisation [22–25]. Mutationanalyses as well as array CGH protocolsare optimized to meet the narrow timeslot of a maximum of 24 hours betweenthe trophectoderm biopsy at day 5 andthe embryo transfer in the same cycle onday 6.

Monogenic Diseases

A mendelian gene disorder (monogenicdisease) originates from a mutation of asingle gene. In the case of an X-chromo-somal recessive disorder, as a rule, boysare more severely affected than girls;female carriers of the mutation may behealthy or only moderately affected. Inautosomal dominant disorders the recur-rence risk is 50% independent of gender.In the case of an autosomal recessivelyinherited disorder, the recurrence riskis 25%, again independent of gender.The setup of a PGD for a monogenic dis-order is a very individual procedure, asthe distinct mutation as well as geneticmarkers need to be tested in this setting.A protocol of an established PGD pro-cedure is therefore of limited use forcouples having different mutations evenin the same gene. The setup requires ap-proximately 3–6 months and the incor-poration of genetic information from theparents and, wherever applicable, theindex patient and, in some cases, addi-tional family members. All new assayshave to be verified in a single cell testsystem (e.g. single lymphocyte cells,buccal cells). The major challenges ofmutation analysis within a PGD setupare the limited amount of DNA, allelicdropout (ADO), contamination, and theshort time slot between biopsy and re-porting the result. If detection of the mu-tation by sequencing or fragment lengthanalysis is not applicable, a selection ofinformative microsatellite markers en-closing the mutated part of the gene canbe employed for an indirect diagnosis[26]. The segregation analysis (haplo-typing) of family members then enablesthe detection of the affected oocytes orembryos without detecting the mutationitself. The polymorphic markers are alsoused to exclude contamination and todiminish the effects of allelic dropoutwhich could lead to misdiagnosis if oneallele is preferentially amplified in thePCR (Tab. 1).

Example 1: PGD for

Haemophilia A

Haemophilia A (OMIM *306700) is asevere X-linked recessive disorder whichaffects blood coagulation. The diseaseis caused by mutations in the gene forcoagulation factor VIII (F8, OMIM*300841), resulting in low or no activityof this protein. One of the most commonmutations in severe haemophilia A is alarge genomic inversion in intron 22 ofF8 which leads to a reduced protein ac-tivity of less than 1%. This inversion canonly be detected by an indirect mutationanalysis employing amplification of

polymorphic microsatellite marker se-quences within and close to the putativemutation of the F8 gene, which deter-mines the status of the cell. Figure 2 il-lustrates the result of fragment lengthanalysis, Figure 3 shows the correspond-ing pedigree with transmission of af-fected and unaffected alleles.

Example 2: PGD for Spinal

Muscular Atrophy (SMA)

Spinal muscular atrophy type 1 (SMA1),an autosomal recessive disorder, iscaused by the degeneration of spinalcord neurons. The incidence of SMA1 is

Figure 2. Polar body diagnosis for haemophilia A: fragment length analysis of amplified polymorphic markers ofthe carrier mother and of PB I and PB II. Arrows indicate polymorphic marker alleles linked to the mutation. Thedetection of the wild type haplotype in PB I and the haplotype linked to the mutation in PB II indicates the presenceof the mutation in the oocyte.

Figure 3. Polar body diagnosis of haemophilia A: The pedigree shows PCR product sizes (in bp) of five polymorphicmarkers linked to the F8 gene. Red PCR products are linked to the mutation, green and blue ones are linked to thewild type. As an example, the result of the fragment length analysis is shown for an affected oocyte indicated bythe result of PB I (blue) and PB II (red).

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J Reproduktionsmed Endokrinol 2013; 10 (Special Issue 1) 41

about 1 in 10000 newborns. About 95%of SMA1 patients show a homozygousdeletion of exon 7 within the survivalmotor neuron 1 gene (SMN1, OMIM*600354). SMN2 (OMIM *601627) is ahighly homologous gene copy to SMN1differing in only a very few nucleotides.The loss of SMN1 activity is mainlycaused by deletions in SMN1 or by geneconversion from SMN1 to SMN2. Onlyone nucleotide discriminates SMN1from SMN2 in exon 7. Dreesen et al.were the first to describe a PGD for SMAin 1998 [27]. Figures 4–6 illustrate theresults of fragment length and sequenceanalysis of exon 7 in two trophectodermbiopsies (TE1, TE2). The carrier parentsdisplay both nucleotides (C and T) inexon 7, whereas only the T-nucleotidewas detected in the affected son, indicat-ing a homozygous deletion of the SMN1gene.

Chromosomal Rearrange-

ments and Aneuploidies

Approximately 1 in 500 individuals car-ries a balanced reciprocal translocation.In this type of translocation, terminal seg-ments of any size from different chro-mosomes are exchanged. By definition,no loss (deletion) or gain (duplication) ofchromosomal material occurs in thesebalanced chromosomal alterations. ARobertsonian translocation, which occursin approximately 1 in 1000 individuals isthe exchange of whole arms of the acro-centic chromosomes 13, 14, 15, 21 and22. Carriers of these translocations arehealthy, however, offspring of either aRobertsonian or a reciprocal transloca-tion carrier may exhibit an unbalancedchromosomal constitution. Affected, un-balanced embryos can be identified with-in a PGD setting by performing an arrayCGH, which also is capable to detect an-euploidies of all other chromosomes.

Fluorescence in situ Hybridisa-tion (FISH)FISH employs fluorescently labelledDNA fragments to detect homologouschromosomal regions. Selected probesdesigned for the detection of chromo-somal translocations verify the presenceor absence of an unbalanced karyotypein polar bodies or embryos [28]. FISHhas limited value for aneuploidy testing,as only a maximum of 12 different chro-mosome probes can be administered in asingle PGD setup.

Array Comparative GenomicHybridisation (array CGH)Comparative genomic hybridisation en-ables the detection of unbalanced chro-mosomal rearrangements and aneuploi-dies of all 24 chromosomes with a singleapproach. Wells et al. demonstrated thefeasibility of conventional or metaphaseCGH for single cells and thus proved thereliability of CGH for PGD [19, 20]. Ar-ray CGH, which employs a microarray

instead of metaphase chromosomes is awell-established technique in postnatalas well as in prenatal genetic diagnosticsfor the detection of unbalanced copynumber variations (CNVs). In PGD, ar-ray CGH has the unquestionable benefitof not only detecting imbalances causedby a chromosome rearrangement, butalso revealing aneuploidies of all chro-mosomes simultaneously [22–25]. Ar-ray CGH protocols for bacterial artificial

Figure 4. PGD for Spinal Muscular Atrophy, SMA. Fragment length analysis for multiplex-PCR products of twopolymorphic markers generated from DNA of two trophectoderm biopsies (TE1, TE2). Arrows indicate the alleleslinked to the mutation. The TE1 sample shows no alleles linked to the mutation, therefore the corresponding em-bryo is unaffected. TE2 shows one allele linked to the mutation and one allele linked to the wild type, respectively.This embryo is a heterozygous carrier of the mutation.

Figure 5. PGD for Spinal Muscular Atrophy, SMA. DNA sequence analysis for amplification products of exon 7 inmother, father, affected son and two trophectoderm biopsies (TE1, TE2). The mother and father are heterozygousfor the nucleotide (C or T) in exon 7 corresponding to the presence of gene copies of SMN1 and SMN2. The sonshows only the T-nucleotide of the SMN2 gene and thus carries a homozygous deletion in SMN1. Both TE samplesshow both nucleotides at this base position, indicating that the blastocysts are either wild type or heterozygouscarriers of the deletion.

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chromosome (BAC) arrays have beenmodified in such way as to enable theanalysis within the challenging timeframe of 12–24 hours. BlueGnome Ltd.developed the 24sure technology whichis comprised of a whole genome amplifi-cation system (WGA, SurePlex), DNAfluorescent labelling kits, BAC arraysand evaluation software (BlueFuseMulti). After amplification of the sampleDNA, it is fluorescently labelled and

hybridised together with a differentiallylabelled reference DNA on a 24sure ar-ray, which consists of several thousandBAC clones of human DNA. This arrayformat is optimized for PGD in such waythat it only reflects the very robust re-gions of the human genome; genomicvariants of unknown significance areabsent from the array, thus avoidingquestionable interpretations of the re-sults. Figure 7 illustrates the unbalanced

array CGH result of a trophectodermsample (TE1); the male partner is carrierof a balanced reciprocal translocation(karyotype 46,XY,t(7;12)(q32;q24.1));Figure 8 illustrates the array CGH resultof another trophectoderm sample (TE2)of the same cycle presenting balancedfor the translocation, but aneuploid forchromosome 18 (trisomy 18); Figure 9illustrates the array CGH result of a bal-anced and euploid trophectoderm sample(TE3). Array CGH is a highly reliableprocedure that detects chromosomal im-balances in as few as 12 hours, thusavoiding cryopreservation (vitrification)of the embryos.

Treff et al. applied a different type of ar-ray based upon single nucleotide poly-morphism (SNP) probes (262K SNP ar-ray, Affimetrix) [23]. These arrays alsoaccomplish the diagnosis in a very shortperiod of time. Handyside et al. [28]combined closely spaced informativeSNP loci to detect not only copy numbervariations but also single gene mutationsby haplotyping the genome of the em-bryo in one single approach (Karyo-mapping).

Aneuploidy Testing

Compared to the targeted investigationof embryos from translocation or inver-sion carriers, the term preimplantationgenetic screening (PGS) describes thetesting of chromosomal aneuploidiesoocytes or embryos of women with re-current miscarriages, repeated IVF fail-ure or advanced maternal age. Data foraneuploidy testing mainly revealedthrough FISH testing of only a limitednumber of chromosomes showed no oruncertain benefit [29, 30], whereas arrayCGH revealed a striking improvement inpregnancy rates. Yang et. al. [31] pub-lished results from a pilot study, whichrevealed a pregnancy rate of 69,1% aftertransfer of a single embryo employingarray CGH of day 5 blastocysts com-pared to a rate of 41,7% in a controlgroup. However, more randomized con-trolled trials have to prove the benefit ofthe aneuploidy testing before it can beoffered couples without a familial risk inthe context of an assisted reproductivetechnology. Aneuploidy testing of blas-tocysts may then become a powerful toolto select a single euploid embryo fortransfer, which may lead to an improvedpregnancy rate.

Figure 6. PGD for Spinal Muscular Atrophy, SMA. Pedigree illustrating allele sizes of two microsatellite markerslinked to SMN1 and SMN2. Blue and green alleles are linked to the mutation, the orange and red ones to the wild type.

Figure 8. Array CGH profile of an aneuploid (trisomy 18) of another trophectoderm sample (TE2) of the same cycle.

Figure 7. Array CGH profile of an unbalanced rearrangement of chromosomes 7 and 12 after PGD of a trophecto-derm sample (TE1).

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J Reproduktionsmed Endokrinol 2013; 10 (Special Issue 1) 43

Conclusion

Since the first preimplantation geneticdiagnosis in the late 1980s and early1990s, PGD has evolved into a well-established technique for the analysis ofboth single gene mutations, chromo-some rearrangements and aneuploidies[32, 33]. Array CGH has replaced FISHand its limited informative value [34].It can be expected that the pregnancyrate after comprehensive chromosomescreening will improve remarkably andthat a transfer of a single embryo is as-pired to avoid twin pregnancies [31, 35].Scott et al. [36] were the first to reportthe delivery of a chromosomally normalchild from an oocyte with reciprocalaneuploid polar bodies but an euploidblastocyst, which calls polar body diag-nosis into question. Improved PGD pro-tocols including PCR based detection ofchromosomal imbalances [37], karyo-mapping [28, 38] and the approach ofBrezina et al. [39] who employed a SNP-based array CGH for the simultaneousdetection of aneuploidies and singlegene disorders are very promising anddocument the high standard of geneticanalyses. However, further randomizedclinical trials have to be evaluated in or-der to strengthen support for the benefitsof PGD [40]. Best practice guidelines forpreimplantation genetic diagnosis aresupported though the ESHRE PGD con-sortium [41–44]. Despite the undoubtfulpositive value of PGD, it is worth men-tioning that PGD may lead to a higheramount of ART cycles without transfer-rable embryos, because no unaffected orwild type embryos is left after PGD. Thelegal regulation varies greatly in coun-tries offering PGD - a fact that is unlikelyto change in the near future. For in-stance, social sexing or the selection of aso-called savior sibling (HLA matching)

is routinely performed within a PGD set-ting in some countries. Hence, legal andethical issues have to be considered veryclosely [45]. As of May 2012 the legalsituation in Germany is still ambiguous;a rapid regulation of the law is requiredin order to serve couples with these verypromising techniques.

Acknowlegement

We thank the kïz – Kinderwunsch imZentrum, München, the KITZ – Kinder-wunschTherapie im Zentrum, Regens-burg and the Kinderwunsch CentrumMünchen for their collaboration.

Conflict of Interest

No potential conflict of interest to thisarticle was reported.

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