preimplantation genetic diagnosis for single gene disorders: experience with five single gene...

Preimplantation genetic diagnosis for single gene disorders:experience with five single gene disorders

Joyce C. Harper1*, Dagan Wells1, Wirawit Piyamongkol1, Patrick Abou-Sleiman1, Angela Apessos1,Antonis Ioulianos1, Mary Davis2, Alpesh Doshi3, Paul Serhal3, Massimo Ranieri3, Charles Rodeck1 andJoy D. A. Delhanty1

1Department of Obstetrics and Gynaecology, University College London, London, UK2Institute of Neurology, University College London, London, UK3Assisted Conception Unit, University College London, London, UK

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying fivedifferent single gene disorders, during the last 18 months. Diagnoses were performed for myotonicdystrophy (DM), cystic fibrosis (CF) [DF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously,and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2(NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally twoblastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis tocontrol against contamination. For the dominant disorders, where possible, linked polymorphisms providedan additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis dueto allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases,followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping.Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normaldeliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome.In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells.The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection ofextraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminatingDNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which twoblastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplexPCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology toachieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright# 2002 John Wiley & Sons, Ltd.

KEY WORDS: PGD; single gene; PCR

INTRODUCTION

For more than a decade preimplantation geneticdiagnosis (PGD) has been used as an alternative toprenatal diagnosis for a small proportion of patients atrisk of transmitting an inherited disease to theirchildren (Wells and Delhanty, 2001; Delhanty andHarper, 2002). In the majority of centres offering PGDworldwide, embryo biopsy is performed on Day 3,with the removal of one or two cells from the cleavage-stage embryo (ESHRE PGD Consortium SteeringCommittee, 1999, 2000, 2002). Since performing adiagnosis on a single cell is technically difficult, somecentres prefer to make a diagnosis when two cells areavailable (Van de Velde et al., 2000; De Vos and VanSteirteghem, 2001). For the analysis of single genedefects, the polymerase chain reaction (PCR) is used(Wells and Sherlock, 1998). For the analysis of chro-mosomes (Conn et al., 1998; Munne et al., 1998a; VanAssche et al., 1999; Fridstrom et al., 2001) and to

determine embryo sex (Harper et al., 1994; Staessenet al., 1999), interphase fluorescent in situ hybridis-ation (FISH) is the method of choice. More recently,the PGD procedure has been used as a form ofpreimplantation aneuploidy screening (PGD-AS) totry to improve in vitro fertilisation (IVF) pregnancyrates (Munne et al., 1998b; Verlinsky et al., 1998).

PGD and PGD-AS have been hampered by thereports of several misdiagnoses (reviewed in Harperand Delhanty, 2000). In the first series of PGD per-formed in the late 1980s a misdiagnosis of sex occurred.In this case diagnosis had relied on the efficientamplification of a Y chromosome-specific sequencefrom all male embryos (Handyside et al., 1990). Sincethis time, phenomena such as allele dropout (ADO),contamination, chromosomal mosaicism and hybrid-isation failure have revealed PGD to be more techni-cally difficult than was originally imagined.

ADO and contamination are important for PCRdiagnosis (Wells and Sherlock, 1998; Sermon, 2002).ADO is observed when only one of the two alleles ina heterozygous cell is detected after PCR (i.e. ampli-fication failure affecting just one allele) (Ray andHandyside, 1996). In most circumstances this does not

*Correspondence to: J. C. Harper, Department of Obstetrics andGynaecology, University College London, 86–96 Chenies Mews,London WC1E 6HX, UK. E-mail: [email protected]

PRENATAL DIAGNOSIS

Prenat Diagn 2002; 22: 525–533.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 /pd.394

Copyright # 2002 John Wiley & Sons, Ltd. Received: 1 February 2002Accepted: 27 March 2002

pose a major problem for single cell PCR, but it is veryimportant for dominant disorders, where an affectedembryo carries only one copy of the mutant allele. Inthis case if the affected allele does not amplify, theembryo will be diagnosed as normal, when actually itis affected. Contamination is also a major problemfor all PCR diagnoses. Sperm and cumulus cellsembedded in the zona pellucida of the embryo maybecome dislodged during the biopsy procedure andtherefore lead to paternal or maternal contamination,respectively. To overcome this problem, intracyto-plasmic sperm injection (ICSI) is used to achieve fer-tilisation for PCR cycles and the cumulus cells mustbe carefully removed for all PGD cases. It is possiblethat paternal or maternal contamination has causedsome of the misdiagnoses reported (Sermon et al.,1998; Harper and Delhanty, 2000). To overcome theproblem of contamination, either maternal, paternalor from other sources, it has been advisable to per-form multiplex PCR reactions, including ideallylinked (Rechitsky et al., 1998; Dreesen et al., 2000;Piyamongkol et al., 2001a,b) or unlinked polymorphicmarkers, which are informative for the family under-going PGD. For these markers the mother and fathershould have four different alleles, so when examiningthe embryo it should have inherited one allele from thefather and one from the mother. Any other patternwould indicate the presence of additional DNA,suggesting that contamination had occurred. The useof fluorescent PCR is ideal for multiplex PCR from asingle cell.

Here we report on the use of PCR for PGD of fivedifferent single gene disorders: myotonic dystrophy(DM), cystic fibrosis (CF) [DF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and twodisorders for which PGD had not been previouslyattempted, namely neurofibromatosis type 2 (NF2)and Crouzon syndrome.

DM or Steinert’s disease is a progressive autosomaldominant muscular dystrophy. It is caused by anunstable CTG repeat expansion within exon 15 of theDMPK gene on chromosome 19q. Expanded (mutant)CTG repeat sequences are refractory to conventionalPCR, but alleles with a number of repeats within thenormal range can be readily amplified and detected.PGD, with analysis based on the detection of the nor-mal alleles, has been successfully performed (Sermonet al., 1997, 2001) but a misdiagnosis was reported(Sermon et al., 1998) which could have been due tomaternal contamination. Therefore, we have devel-oped two new PGD protocols for DM, which utilisemultiplex fluorescent PCR (Piyamongkol et al., 2001a,b).Ideally a linked polymorphic marker, APOC2, is ampli-fied in addition to the normal DMPK alleles, thusproviding a back-up diagnostic result. We report onPGD for three couples where the females werecarriers using a single-step duplex fluorescent (F)-PCR which will detect the DM triplet repeat withnormal alleles. However, two of the couples were notfully informative at the APOC2 locus and so anunlinked STR marker, D21S1414, was substituted.

This locus provides no direct diagnostic information,but is still useful for contamination detection.

CF, an autosomal recessive disease caused by mu-tation in the CFTR gene, is located on chromosome 7qand was the first single gene defect for which PGD wasoffered (Handyside et al., 1992). The most commonmutation in the Caucasian population is a three basepair deletion known as DF508 and the majority of PGDcycles for CF have been for this mutation (ESHREPGD Consortium Steering Committee, 1999). Herewe report on PGD for this mutation using a single-step triplex F-PCR with primers for the mutation, alinked marker and a contamination marker. A couplealso presented with both partners carrying a mu-tation in exon 4 of CFTR (621+1 G>T). A triplexPCR with direct mutation detection, a linked markerand a contamination marker was used using nestedPCR and SSCP for the mutation detection.

Fragile X syndrome is the most common cause offamilial mental retardation. The most common mu-tation is an expansion of a triplet (CGG)n repeat in the5k untranslated region of the FMR1 gene on Xq27.3.The expansion is refractory to PCR due to preferentialamplification of the smaller allele in heterozygous cellsand the high GC content of the repeat and surround-ing sequences. Detection of the normal and premu-tation alleles has been proposed for use in PGD offragile X syndrome (Black et al., 1995, Sermon et al.,1999, 2001), an approach which has also been usedin the diagnosis of two other triplet repeat disorders,DM (Sermon et al., 1997) and Huntington’s disease(Sermon et al., 1998). We investigated the use of linkedpolymorphic markers flanking the mutation to trackthe normal and premutation carrying maternal chro-mosomes in preimplantation embryos (Apessos et al.,2001). One couple presented where the female wascarrying a large FMR1 premutation for fragile Xsyndrome and the couple were both carriers for CF,DF508. A single-step tetraplex F-PCR was performedemploying amplification of two linked markers flank-ing FMR1, a sequence from the amelogenin gene forsexing, and a DNA fragment encompassing the DF508mutation.

Mutations in the NF2 gene predispose to neuro-fibromatosis type 2 (NF2), a dominantly inheritedcancer predisposition syndrome with an incidence of 1in 33 000–40 000 (Evans et al., 1992). NF2 is charac-terised by the development of histologically benigntumours in the central nervous system. A couple pre-sented where the male carried a splice site mutation inintron 4: 514-2 (A to G) of NF2. The couple neededfertility treatment due to ovulatory problems in thefemale. The molecular diagnosis was performed byduplex F-PCR, followed by analysis of an informativeintragenic single nucleotide polymorphism (SNP) anddetection of the causative mutation using SSCP(Abou-Sleiman et al., 2002a).

Crouzon syndrome is a dominantly inherited cranio-synostosis syndrome that is caused by mutations in thefibroblast growth factor receptor 2 gene (FGFR2) onchromosome 10q (Reardon et al., 1994). It has anestimated birth prevalence of 15–16 per 1 million

J. C. HARPER ET AL.526

Copyright # 2002 John Wiley & Sons, Ltd. Prenat Diagn 2002; 22: 525–533.

births (Cohen and Kreiborg, 1992). The spectrum ofmutations in FGFR2 is relatively limited compared toother genes. To date only 46 mutations have beenidentified, the majority of which are missense, with asmaller number of splice mutations or small insertions/deletions, all of which remain in-frame. For PGD wedeveloped DNA amplification using a multiplex nestedPCR, for the simultaneous amplification of a fragmentof the FGFR2 gene encompassing the mutation and ahighly polymorphic short tandem repeat (STR) locus(unlinked, D21S11). SSCP was used to detect themutation. The genotype of the embryos at the STRwas determined by fragment analysis on an automatedDNA sequencer by fluorescent polyacrylamide gelelectrophoresis (PAGE). Two intragenic SNP hadbeen described in FGFR2 at the time of preparationfor the diagnosis. However, neither was suitable foruse in this case as the parents were uninformative(Abou Sleiman et al., 2002b).

Here we report on 14 PGD cycles for eight patientscarrying five single gene defects, using multiplexF-PCR incorporating polymorphic markers for con-tamination detection.

PATIENTS AND METHODS

All patients had previous genetic counselling beforepresenting to the PGD centre. Two IVF/PGD consul-tations were conducted during which the limitations ofthe PGD procedure were outlined, such as the need forIVF, risk of misdiagnosis, possibility that all embryoscould be affected, PGD pregnancy rates, etc. Blood,DNA or buccal cell samples [obtained by scraping theinside of the cheek with a sterile wooden stick with acotton swab tip and suspending in 3 ml phosphate-buffered saline (PBS) 4% (v/v) bovine serum albumin]were obtained from all couples and affected relativesprior to treatment. This enabled the work-up of aspecific diagnosis for each couple, i.e. analysis oflinked or unlinked markers for contamination detec-tion, development of multiplex PCR using thesemarkers and optimisation of the mutation detection.The necessary investigations required prior to an IVFcycle were conducted, i.e. G test (Ranieri et al., 2001),HyCoSy, dummy embryo transfer, semen analysis, etc.

The time taken to work-up a specific diagnosisvaried from 6 months to over a year. All work-upswere conducted on DNA, single buccal cells from theparents and affected relatives, and single blastomeresdonated from our IVF programme. The results fromthe work-ups were submitted to the Human Fertilis-ation and Embryology Authority (HFEA) for licen-sing purposes and permission was granted for eachdisorder.

The patients underwent routine IVF procedures asoutlined previously (Ranieri et al., 2001). ICSI wasperformed in all cases to reduce the risk of spermcontamination. Oocytes and embryos were cultured inIVF medium (Cook IVF, Brisbane, Australia). Priorto biopsy, all cumulus cells were mechanically removedto reduce the risk of maternal contamination. On Day

3, embryo biopsy was performed on all embryos thatwere considered suitable (depending on number ofcells, grade and the number of embryos available), inCa2+Mg2+-free embryo biopsy medium (Medicult,Surrey, UK), using Research Instrument (Cornwall,UK) micromanipulators. Zona drilling was performedusing acid Tyrode’s solution as described previously(Piyamongkol et al., 2001a). Two blastomeres wereremoved from embryos containing six cells or more byaspiration and washed in PBS. In cases where only asmall number of 6–8-cell embryos are available, weremove a single cell from embryos with less than sixcells. However, in all the cases reported here, embryoswere transferred after a normal result was obtainedfrom two cells. All cells were washed at least threetimes in droplets of PBS/BSA before their transfer in2 ml PBS/BSA to microcentrifuge tubes containing 3 ml125 mg/ml proteinase K and 4r10x4% (w/v) sodiumdodecyl sulphate (SDS). A control blank was pro-cessed for each blastomere using 2 ml of fluid from thefinal PBS wash drop. Cells were lysed by incubation at37uC for 1 h and the proteinase K inactivated byincubation for 15 min at 95uC. After biopsy, embryoswere stored in Cook extended culture medium.

A polymorphic marker was included in each diag-nosis to control against contamination. For thedominant disorders, where possible, linked poly-morphisms provided an additional means of determin-ing the genotype of the embryo hence reducing the riskof misdiagnosis due to ADO. Multiplex PCR was usedin all cases, followed by fragment analysis usingfluorescent DNA sequencers and/or SSCP and silverstaining for genotyping. All diagnoses were performedwithin 24 h and embryo transfers undertaken on Day 4post-insemination. Untransferred embryos were usedto check the original diagnosis.

DM

The method and details of Patients 1 and 2 have beenreported previously (Piyamongkol et al., 2001a). Inbrief, amplification of the expanded allele is notpossible as it is refractory to single cell PCR. Thereforethe diagnosis depends on the identification of thenormal alleles (Brook et al., 1992). In the threepatients, the females carried an expanded allele andso it was important to check for maternal contami-nation. We have previously reported on the use of thelinked marker APOC2, but this marker was onlyinformative for one of our PGD couples (Piyamongkolet al., 2001a,b). Therefore an unlinked STR marker,D21S1414 (which is on chromosome 21), was sub-stituted for contamination detection (Sherlock et al.,1998) for two couples. The multiplex amplified pro-ducts from single cells were each tagged with two dif-ferent fluorochromes using labelled primers. This allowedanalysis to be performed on an automated laserfluorescent sequencer (ALFExpress1) (PharmaciaBiotech, Herts, UK) and also an ABI Prism1310(PE Applied Biosystems, Warrington, UK).

PGD FOR SINGLE GENE DISORDERS 527


CF DF508

One couple presented where both partners werecarrying DF508. A single-step, triplex F-PCR wasemployed which allowed simultaneous amplification ofthe mutation locus together with two polymorphismsused as internal controls. An informative STR(D13S631) and the intron 6-linked polymorphismwere used to detect any contamination and ADO,respectively. Fragment (size) analysis was carried outon the ALFExpress1. The primers used for the mu-tation were as described by Handyside et al. (1992),whereas the primers for the intron 6-linked poly-morphism were modified from the original describedby Chehab et al. (1991).

CF exon 4

One couple presented where both partners carried amutation in exon 4 (621+1 G>T). A single-steptriplex F-PCR was performed for the mutation locusand two polymorphisms (the informative HumTh01STR and the intron 6-linked polymorphism) followedby a second (nested) PCR round for the mutationlocus only. Fragment (size) analysis was performed onthe ALFExpress1 for the two polymorphisms. Themutation was detected by silver-stained SSCP on thesemi-automated GenePhor1 electrophoresis system(Pharmacia Biotech, Herts, UK) using 12.5% polyacryl-amide gels at 15uC. The primers used for the mutationwere designed by Ioulianos et al. (unpublished data),whereas the primers for the intron 6-linked poly-morphism were as described by Chehab et al. (1991).

CF and fragile X

A couple presented where the female carried anexpansion that ranged from the upper permutationto the full mutation (133–300 repeats) and bothpartners were carrying the CF mutation DF508. Asingle-step, tetraplex F-PCR was used with two linkedmarkers flanking to FMR1 [dinucleotide microsatelliterepeat, DXS998 and (CA)n microsatellite polymorph-ism, p39] (Wehnert et al., 1993; Apessos et al., 2001),primers for amelogenin for embryo sexing (Sullivanet al., 1993), and primers for DF508 (Liu et al., 1992).

NF2

The causative mutation was identified as a single basepair substitution affecting a splice site in intron 4 ofthe gene (517-2 A/G). The female partner sufferedfrom polycystic ovary syndrome and had never ovulatedspontaneously; this case has previously been reported(Abou-Sleiman et al., 2002a). Two sets of fluores-cently labelled primers were designed to amplify theregion of the gene harbouring the mutation, and afragment of the 5k untranslated end of the geneencompassing a linked single nucleotide polymorph-ism, G/C substitution at nucleotide 8240. As thelinked polymorphism was found to be informative in

the couple it could be used to infer the presence orabsence of the mutation providing a secondary, inde-pendent means of detecting the mutation, thus reduc-ing the risk of misdiagnosis due to ADO. A duplexF-PCR was developed with simultaneous SSCP usedfor the mutation and SNP.

Crouzon syndrome

The causative mutation was identified in the femalepartner as a de novo G/A single base pair substitutionat codon 568 situated in the alternative coding domainfor the 3k half of the IgIII domain (numbering basedon the sequence published by Zhang et al., 1999). Thepatient was diagnosed with Crouzon syndrome at16 years of age and was mildly affected. This case haspreviously been reported (Abou-Sleiman et al., 2002b).The primers used for the mutation were as describedby Sharma and Litt (1992). The mutation was detectedby silver-stained SSCP on the semi-automated Phast-System1 electrophoresis system using 20% polyacryl-amide gels at 4uC. The patients were also genotyped attwo intragenic single nucleotide polymorphisms bySSCP, a G/A in intron 7 (x133 nucleotides from exon8) on the ALFExpress1 at 15uC using 0.5% MDE gel(Flowgen) and a C/A in intron 8 (+575 nucleotidesfrom exon 8) on the GenePhor1 system at 15uC using12.5% polyacrylamide gels. The patients were unin-formative for the SNP intragenic markers. Thereforethe patients were typed at several STR loci located onchromosomes 13, 18 and 21.

Fluorescently labelled D21S11 locus PCR productswere sized by electrophoresis through 6% denaturingpolyacrylamide gel at 38uC on an automated DNAsequencer (ALFExpress1). Included in each lane weretwo size standards flanking the product, allowing thesizing of fragments up to one base pair apart. TheD21S11 locus was selected for use in the diagnosis asthe patients were found to be fully informative, thusincreasing the probability of detecting contaminantsand particularly maternally derived contamination.The female partner had two alleles, one of 221 bp andthe other of 226 bp. The genotype of the fatherconsisted of a 236 bp allele and a 246 bp allele.

RESULTS

Details of the patients’ ages, previous reproductivehistory, fertility, etc are outlined in Table 1. Only onepatient had fertility problems (NF2). All other patientshad one affected child or had a previous terminationof pregnancy (TOP). The female patients’ ages rangedfrom 27–36 years.

From 14 cycles, embryo transfers were performed in13 cycles resulting in one biochemical pregnancy forCF, one early pregnancy loss for DM and threenormal deliveries; a singleton for Crouzon syndrome,and two pregnancies: a twin and a singleton for DM(Table 2).

In each case the untransferred embryos were used toconfirm the diagnoses performed on the biopsied cells.



The results were concordant in all cases. The inclusionof a polymorphic marker allowed the detection ofextraneous DNA contamination in two cells from onecase. The genotype of the contaminating DNAindicated that it was likely to be maternal in origin.

DM

Multiplex amplification using DM and D21S1414primers on 50 normal heterozygous single buccalcells demonstrated 98% amplification efficiency forboth sets of primers. The DM gene displayed a 4.1%ADO rate, while for D21S1414 it was 14.3%. Thestudies on single buccal cells indicated that the addi-tion of a polymorphic marker to the DM gene analysisas multiplex PCR does not adversely affect the ampli-fication efficiency. Duplex fluorescent analysis for DMand D21S1414 loci was carried out on 23 singleblastomeres giving an amplification efficiency of 95.7%(22/23) for each locus. ADO rates for DM were 4.5%(1/22) while for D21S1414 they were 22.7% (5/22).

A total of four PGD cycles were carried out inthree patients (Figure 1). A total of 53 oocytes werecollected, 39 embryos were biopsied and the diag-nosis gave 12 normal (including one contaminatedwith maternal DNA), 19 affected and eight no result(including one trisomy 21 which could be detected asthe marker was on chromosome 21). A total of nineembryos were transferred in four embryo transferprocedures. The first cycle for Couple 1 did not resultin a pregnancy, but the second resulted in a singletonpregnancy. The first cycle for Couple 2 resulted in atwin pregnancy. The cycle for Couple 3 resulted in afetal sac but no heart beat. Couples 1 and 2 had a CVSthat showed all fetuses to be normal. All babies werefemale and have been successfully delivered.

CF DF508

The triplex PCR protocol for DF508 was tested on 100single heterozygous buccal cells with 96%, 100% and96% amplification efficiency for the mutation locus,the D13S631 STR and the intron 6-linked polymorph-ism, respectively. The ADO rates were 14%, 24%and 10%, respectively. Simplex amplification of each

individual locus produced comparable results thusdemonstrating that the use of the multiplex protocoldid not have a negative effect on the amplificationefficiency. Furthermore, the use of the linked poly-morphism enhanced the reliability of the diagnosticprotocol by significantly reducing the number of cellsfor which a diagnosis was not possible. Prior to clinicalapplication the triplex protocol was also tested on 20(normal) blastomeres from four Day 3 embryos whichwere donated for research. The amplification rateswere similar to those reported above. A single PGDcycle was carried out. Six embryos were biopsied ofwhich two were unaffected, two were carriers, one wasaffected and one gave no result. A total of threeembryos were transferred resulting in a biochemicalpregnancy.

CF exon 4

The two-stage triplex PCR protocol for the exon4 621+1 G>T mutation was also tested on 100 singleheterozygous buccal cells. This produced amplificationefficiencies of 74%, 88% and 91% for the mutationlocus, the HumTh01 STR and the intron 6-linkedpolymorphism, respectively. The ADO rates were 26%,18% and 18%, respectively. Again the introduction ofthe linked polymorphism proved beneficial in maxi-mising the information obtained from the diagnosticresults, despite the slightly low amplification efficien-cies (due to the bad quality of the collected cells). Theprotocol was also tested on 30 (normal) blastomeresfrom six Day 3 embryos, which had been donated forresearch. The amplification rates were 92%, 94% and95%, respectively. One couple underwent two PGDcycles. Sixteen embryos were biopsied in total. Twowere diagnosed as unaffected, five were carriers, threewere affected and five embryos were transferred intotal, in two PGD cycles (two and three embryos,respectively). No pregnancy resulted.

CF and fragile X

For the DXS998 the mother was heterozygous (alleleB 114 bp and C 116 bp) and the father was hemi-zygous for allele A (112 bp). The affected son had

Table 1—Patients’ history

Couple Fertile Female age (years) Affected children (n) Previous PND and TOP (n)

DM 1 Yes 36 1 2DM 2 Yes 32 1 2DM 3 Yes 35 0 2CF: DF508 Yes 29 1 0CF: 621+1 G>T Yes 30 1 0CF and fragile X Yes 36 1 0NF2 No 27 0 0Crouzon syndrome Yes 34 1 RIP 0

The summary for each diagnosis is shown in Table 2.CF, Cystic fibrosis; DM, myotonic dystrophy; NF2, neurofibromatosis type 2; PND, prenatal diagnosis; RIP, rest in peace; TOP, terminationof pregnancy.



inherited the B allele from his mother. For the p39 themother was heterozygous for allele C (152 bp) and G(160 bp) and the father had the C allele. The affectedson had inherited the G allele from his mother. A totalof three PGD cycles were performed and 39 embryosbiopsied. The diagnosis was as follows: three normalfragile X, normal CF; one normal fragile X, normalCF with Turner’s; five normal fragile X, affected CF;12 affected fragile X, normal CF; one affected fragileX, affected CF; and 17 no result. One cycle did notresult in any unaffected embryos for transfer. In theother two PGD cycles, a total of three embryos weretransferred (one and two embryos, respectively) but nopregnancy resulted. Since preparation of this manu-script the couple has undergone another PGD cycle,which was unsuccessful.

NF2

For the work-up, a total of 190 heterozygous singlebuccal cells from the affected male were analysed andamplification of both loci was achieved in 85% of cells,amplification failure at the mutation locus alone wasobserved in 2.6% of cells and at the polymorphismlocus in a further 1.5% of cells. One couple had twoPGD cycles. 18 embryos were biopsied and two werediagnosed as normal, 12 affected and four gave noresult. In the first cycle two normal embryos weretransferred, but no pregnancy resulted. In the secondcycle all the embryos were diagnosed as affected, butsince the couple had fertility problems, they decided totransfer two of the affected embryos, but no pregnancyresulted. No discrepancy was observed between theanalysis of the biopsied cell and the untransferredembryos.

Crouzon syndrome

For the work-up a total of 125 heterozygous singlecells were analysed, 82% of which showed DNAamplification at both loci. Of the cells that amplifiedsuccessfully, 15% showed ADO at the mutation locusand 13% at the polymorphism locus. The protocol wasalso tested on 15 blastomeres disaggregated from twospare Day 3 embryos. The efficiency of amplificationon the blastomeres was similar to that obtained fromthe buccal cells, though data on the ADO rates at themutation locus was unattainable as they were normal(Abou-Sleiman et al., 2002b).

The female was 34 years old and of proven fertility,having conceived an affected girl who subsequentlydied aged 18 months during corrective surgery. Thecouple had two PGD cycles in which 23 embryos werebiopsied. Eight were diagnosed as normal, nine asaffected and six gave no result. Five embryos weretransferred over two embryo transfer procedures andthe second PGD cycle resulted in a pregnancy withtwo sacs, but only one heart beat. The couple did notopt for confirmation by prenatal diagnosis, but afterdelivery the diagnosis was confirmed as a normal male(Abou-Sleiman et al., 2002b).T

able

2—

Summary

of14preim

plantationgenetic

diagnosis(PGD)cycles

foreightcouples

Couple

PGD

cycles

(n)

Oocytes

collected(n)

Biopsied

(n)

Unaffected(n)

Affected(n)

Carrier(n)

No

Diagnosis(n)

Embryos

transferred(n)

Outcome

DM

12

15

10

3a

3–

42

Notpregnant

10

62

3–

12

Delivered

singleton

DM

21

15

14

310

–1

3Delivered

twins

DM

31

13

94

3–

22

Fetalsacbutnoheart

beat

CF

508

16

62

12

13

Biochem

icalpregnancy

CF:621+1G>T

226

91

32

32

Nopregnancy

10

71

03

33

Nopregnancy

CF

andfragileX

317

11

08

–3

0Notransfer

21

13

16

–6

1Nopregnancy

19

15

25

–8

2Nopregnancy

NF2

213

92

4–

32

Nopregnancy

19

90

8–

12b

Nopregnancy

Crouzonsyndrome

210

94

5–

02

Nopregnancy

18

14

44

–6

3Delivered

ForabbreviationsseeTable1.

aOneblastomereresultwasshownto

becontaminated.

bTwoaffectedem

bryostransferred.



Figure

1—

Use

ofSTR

markersforPGD

ofsinglegenedefects.PGD

formyotonicdystrophy(D

M).Lanes

2and4showthePCR

productsover

thetripletrepeatoftheDM

locus.Themother

hasone

norm

alallelewith18repeats

andthefather

hastw

onorm

alalleles,withfourand19repeats.Lanes

1and3show

theuse

ofafullyinform

ativemarker

forthiscouple,whereallfouralleles

havea

differentsize.A

norm

alem

bryowillinheritthenorm

alDM

allelefrom

theirmother,andoneofthenorm

alalleles

from

thefather.TocheckforDNA

contamination,themarker

should

show

one

allelefrom

themother

andonefrom

thefather.Figure

courtesyofWirawitPiyamongkol,University

CollegeLondon(U

CL).Figure

reproduced,withpermission,from

Delhanty

JDA,Harper

JC.

Preim

plantationgenetic

diagnosis.ReprodMed

Rev

(inpress).Copyright#

CambridgeUniversity

Press



CONCLUSIONS

The use of PCR for PGD has been slow to develop, asobtaining a diagnosis from a single cell is a verydifficult technical procedure (Wells and Sherlock,1998; Sermon, 2002) and so the use of linked orunlinked markers is recommended. This is possibleusing multiplex F-PCR on a single cell (Figure 1) butto find informative markers for each PGD couple cantake several months. In this study, the work-ups tookfrom 6 months to over a year. Since the primary aimof PGD is to ensure the transfer of a normal embryo,in our centre we also try to base our diagnosis on theresults of two independent blastomeres from the sameembryo (Apessos et al., 2001; Piyamongkol et al.,2001a; Abou-Sleiman et al., 2002a,b).

From 14 cycles for eight patients, only one cycle didnot result in an embryo transfer as all of the embryosdiagnosed were considered affected. However, in onecycle (NF2) only affected embryos were diagnosed,and since the patient was infertile they chose to havetwo affected embryos transferred. This cycle did notresult in a pregnancy. All five pregnancies that wereobtained were from embryos in which two blastomereswere biopsied for the diagnosis. F-PCR technologyenables a greater sensitivity and two-cell biopsyincreases the accuracy of the diagnosis.

For one patient, a double diagnosis was required asthey were at risk of transmitting CF and fragile X.From three cycles, the majority of embryos were ofgood morphology but from 39 embryos, only threenormal embryos were obtained and no pregnancyresulted. From the ESHRE PGD Consortium SteeringCommittee (2002) data where in the majority of casesonly one disease was diagnosed, from 575 cycles, 7611oocytes were collected, 3454 embryos were analysedand only 1552 embryos were considered transferable(normal or carrier for recessive disorders and normalfor dominant disorders). These data confirm the pre-vious report that a high number of embryos arerequired for successful PGD (Vandervorst et al., 1998)but this is especially true when examining two differentdisorders.

The method we report here for the diagnosis offragile X should be applicable to most patientscarrying fragile X, as long as they were informativefor the markers (Apessos et al., 2001). Dreesen et al.(2000) have reported a method of CF PGD where thesame test can be applied to most patients. They haveused four highly informative linked markers flankingthe CF gene. This can be used for virtually all CFpatients, as long as they are informative for twoflanking markers. Ray et al. (2001) have developed asimilar strategy to cover most of the DM gene. Suchtechniques would allow a more universal test for aparticular disease.

Working up new diagnoses for PGD is very timeconsuming as primers have to be designed to amplifythe mutation and linked or unlinked markers. PCRconditions have to be optimised to reduce the risk ofADO and amplification failure. In our clinic in recentmonths we have had referrals for Treacher Collins

syndrome, brachydactyly type B, pyruvate dehydro-genase complex 1, connexin 26, NF1, Stickler syn-drome, aortic stenosis, brachio-oto-renal syndromeand Wiskott-Aldrich syndrome and been unable tohelp these patients due to limited resources. Indeed,the ESHRE PGD Consortium report lists a very smallnumber of diseases that have been diagnosed by PGD(ESHRE PGD Consortium Steering Committee,2002). Only 14 autosomal dominant, nine autosomalrecessive, eight specific diagnosis for X-linked disease,one mitochondrial disorder and two cases where thepatients carried two disorders were examined, a smallnumber compared to the hundreds of different geneticabnormalities that have been diagnosed by prenataldiagnosis. In our clinic we are concentrating ourefforts on PGD for a handful of disorders, namelyDM, CF, fragile X, polyposis coli, medium chain acylCoA dehydrogenase deficiency (MCAD), thalasse-mias, Li Fraumeni, retinoblastoma, and Huntington’schorea.

For PCR diagnosis in the near future, the mainimprovement will be the increased use of multiplexF-PCR (Harper and Wells, 1999). Whole-genomeamplification has been used for PGD (Ao et al.,1998), but it is not applicable in all situations. The useof micro-arrays on single cells still needs developmentand its use will be limited as in most situations PGDneeds to determine one or two mutations and a markerfor contamination. However, micro-arrays may beuseful for predisposition syndromes where more thanone gene is involved.

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