Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the detection of the DeltaF508 mutation

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.     

Pregnancy after preimplantation genetic diagnosis for Charcot-Marie- Tooth disease type 1A

Charcot-Marie-Tooth (CMT) disease type 1A is an autosomal dominant peripheral neuropathy characterized by slow progressive distal muscle wasting and weakness, and decreased nerve conduction velocities. Most CMT1A cases (>98%) are caused by a duplication of a 1.5 Mb region on the short arm of chromosome 17 containing the PMP22 gene. A couple with a previous history of CMT followed by termination of pregnancy was referred to our centre for preimplantation genetic diagnosis (PGD). The husband carries the CMT1A duplication which can be detected by polymerase chain reaction (PCR) analysis using polymorphic (CA)n markers localized within the duplication. PCR amplification of genomic DNA of the parents-to-be with one of the two primers labelled with fluorescein, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified fragments allows the distinction between both genotypes. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal allele of the father. PCR with single Epstein-Barr virus-transformed lymphoblasts and blastomeres resulted in 91.4 and 93.5% amplification efficiency respectively, whereas none of the blank controls gave a positive signal. Allele drop-out (ADO) was observed in eight out of 32 lymphoblasts (25%) or in five out of 21 blastomeres (23.8%). However, within this set-up ADO will never lead to transfer of an affected embryo. A first ICSI-PGD cycle did not result in embryo transfer for the patient. A second cycle involved 10 mature oocytes of which eight were fertilized, resulting in five embryos for biopsy. Two unaffected embryos were available for transfer and resulted in a singleton pregnancy. The genotype of the fetus has been confirmed healthy by chorionic villus sampling.      

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.     

PGD for autosomal dominant polycystic kidney disease type 1

Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children.     

Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosis

One major limitation of pre-implantation genetic diagnosis (PGD) practice comes from the need to develop single cell PCR protocols. For a disease such as cystic fibrosis (CF), for which almost 1000 mutations have been identified, the development of a mutation based PGD protocol is impracticable. An elegant way to overcome this problem is to set up an indirect diagnosis using polymorphic markers allowing the identification of the pathogenic haplotype instead of the mutation. We present here a new PGD protocol for CF. Our strategy is based on a multiplex fluorescent PCR co-amplifying the DeltaF508 mutation and two CFTR intragenic polymorphic microsatellites (IVS8CA and IVS17bCA). Such an approach is justified since in 91% of the cases at least one partner of the couple carries the DeltaF508 mutation. The use of intragenic markers reduces the risk of misdiagnosis due to meiotic recombination. In 97% of the single lymphoblasts (151/155) tested a PCR signal was obtained. A complete haplotyping was achieved in 137/151 (91%) lymphoblasts and a 6% rate of allele drop out (ADO) was observed. Three cases were performed. Case one was at risk of transmitting mutations DeltaF508 and R1162X, case 2 DeltaF508 and R1066C and case 3 DeltaF508 and 1341+1A. Considering these three cases and the re-analysis of the affected embryos, we have analysed 62 blastomeres from which we had PCR signal for 58 (94%) and a complete haplotype for 49 (84%). With the degree of polymorphism of the markers used in this work (48 and 39%) and the fact that we co-amplified the F508 locus our test should be suitable for nearly 80% of the couples requesting PGD for CF. This fluorescent multiplex PCR indirect diagnosis provides also a safer test since it allows the confirmation of the diagnosis, the detection of contamination and could give an indication on the ploidy of the embryos tested.     

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy

BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.     

紧密连锁的多态性位点在β地中海贫血植入前遗传学诊断中的应用

目的 探讨与β珠蛋白基因紧密连锁的多态性位点HumTH01在β地中海贫血（β地贫）植入前遗传学诊断（preimplantation genetic diagnosis，PGD）中的作用。方法 对4例已出生重型β地贫患儿的、双方均为β地贫基因携带者的夫妇进行了6个周期的PGD治疗，应用多重巢式PCR同时检测β珠蛋白基因及HumTH01基因，选择健康的胚胎移植入子宫。结果 6个周期共活检44个胚胎，获得44个卵裂球，其中41个卵裂球扩增成功，35个胚胎经PCR分析后获得明确诊断，移植了14个胚胎，获得1例临床妊娠。孕17周时经脐带血穿刺，证实为完全正常胚胎，现已出生一正常女婴。单个卵裂球平均扩增效率为89．7％，等位基因脱扣（allele drop-out，ADO）率为14．4％。HumTH01基因可以帮助检测出ADO及污染的发生。结论 本研究为国内首次报道应用多重巢式PCR同时检测β珠蛋白基因及HumTH01基因对β地贫进行植入前遗传学诊断并成功获得临床妊娠。在PGD中同时检测与β珠蛋白基因紧密连锁的多态性位点可以降低PGD中由于ADO及污染造成的误诊的风险。     

Preimplantation genetic diagnosis of spinocerebellar ataxia 3 by (CAG)(n) repeat detection

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.     

Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples

This study is part of a strategy aimed at using fluorescent polymerase chain reaction (PCR) on informative genetic microsatellite markers as a diagnostic tool in preimplantation genetic diagnosis (PGD) of severe monogenic disease. Two couples, both of whom had previously had children who were compound heterozygote for severe cystic fibrosis mutations, were offered PGD using fluorescent PCR of the highly polymorphic cystic fibrosis transmembrane conductance regulator (CFTR) intragenic microsatellite marker IVS17bTA. Cleavage-stage embryo biopsy followed by PCR resulted in transfer of one unaffected carrier embryo for each couple. This approach eliminates the need for single cell multiplex PCR strategies to detect CF compound heterozygotes. It also provides a control of chromosome 7 ploidy in the blastomeres and a selection against allele dropout by positive detection of each CFTR copy of all genotypes in preimplantation embryos from genetically informative families.     

An evaluation of PGD in clinical genetic services through 3 years application for prevention of beta-thalassaemia major and sickle cell thalassaemia

PGD represents an alternative within prenatal diagnosis services, which avoids terminating affected on-going pregnancies. In Greece, prevention programmes for haemoglobinopathies, including the option of prenatal diagnosis, are well established. Following optimization of a single-cell genotyping strategy (designed to be applicable for the majority of beta-thalassaemia major or sickle thalassaemia genotype interactions) along with close collaboration with an IVF unit, we integrated the option of PGD for at-risk couples with a problematic reproductive history. A total of 59 couples requesting PGD were counselled, of whom 41 initiated 63 PGD cycles. Following standard assisted reproduction treatment for oocyte retrieval, 20 cycles were cancelled (too few oocytes and/or poor quality embryos), but in 43 cycles single blastomeres were biopsied from 3 day embryos and genotyped (total 302). Diagnosis was achieved for 236 embryos, and 100 of 125 unaffected embryos were transferred. Sixteen pregnancies were established, although six were lost within the first trimester. Ten pregnancies underwent second trimester prenatal diagnosis, with nine pregnancies (13 babies: six singletons, two twins and one triplet) confirmed unaffected, although one singleton was a PGD misdiagnosis and terminated. The triplet pregnancy was selectively reduced to twins, and nine pregnancies went to term, with 12 healthy babies born. This report highlights advantages, limitations and approaches towards improvement when incorporating PGD within genetic services for a common recessive disease.     

Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis

Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application.     

Preimplantation genetic diagnosis for neurofibromatosis type 1

PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.     

Preimplantation genetic diagnosis for achondroplasia: genetics and gynaecological limits and difficulties

BACKGROUND: We report the first attempts at preimplantation genetic diagnosis (PGD) and IVF and their accompanying difficulties for achondroplasia (ACH) patients. METHODS: A PGD test was developed using fluorescent single cell PCR on lymphoblasts from patients and controls and from blastomeres from surplus IVF embryos. A specific digestion control based on the use of two fluorochromes was elaborated. Ovarian stimulation and oocyte retrieval were carried out using conventional protocols. RESULTS: We performed 88 single cell tests for which amplification was obtained in 86 (97.7%) single lymphoblasts. Allele drop out (ADO) was observed in two out of 53 (3.7%) heterozygous lymphoblasts. If we combine the results from the blastomere testing from surplus embryos with those from PGD cycles and re-analysis after PGD, we obtained a PCR signal in 84% of cases of which 91% were correctly diagnosed at the G380 locus. A total of six cycles were performed resulting in three embryo transfers. We observed difficulties in ovarian stimulation and oocyte retrieval with affected female patients. No pregnancy was obtained. CONCLUSION: A PGD test for ACH is now available at our centre but our initial practice raises questions on the feasibility of such a test, specially with affected female patients.     

Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders

BACKGROUND: We report on our experience with preimplantation genetic diagnosis (PGD) for single gene disorders (SGDs), from 1999 to 2004, describing strategies and overall clinical outcome of 250 cycles in 174 couples for 23 different genetic conditions. METHODS: PGD cycles included 15 for autosomal dominant, 148 for autosomal recessive and 19 for X-linked SGDs. In addition, 68 cycles of PGD for SGDs were performed in combination with HLA matching. The strategy in each case used an initial multiplex PCR, followed by minisequencing to identify the mutation(s) combined with multiplex PCR for closely linked informative markers to increase accuracy. Linkage analysis, using intragenic and/or extragenic polymorphic microsatellite markers, was performed in cases where the disease-causing mutation(s) was unknown or undetectable. RESULTS: In 250 PGD cycles, a total of 1961 cleavage stage embryos were biopsied. PCR was successful in 3409 out of 3149 (92.4%) biopsied blastomeres and a diagnosis was possible in 1849 (94.3%) embryos. Four hundred and twenty-seven embryos were transferred in 211 cycles, resulting in 71 pregnancies (33.6% per embryo transfer), including 15 biochemical pregnancies, six spontaneous miscarriages, two ectopic pregnancies, which were terminated, and nine pregnancies which are still ongoing. The remaining pregnancies were confirmed to be unaffected and went to term without complications, resulting in the birth of 35 healthy babies. CONCLUSIONS: Minisequencing for mutation detection combined with multiplex fluorescence PCR for linkage analysis is an efficient, accurate and widely applicable strategy for PGD of SGDs. Our experience provides a further demonstration that PGD is an effective clinical tool and a useful option for many couples with a high risk of transmitting a genetic disease.     

Multiple mutation analysis of the cystic fibrosis gene in single cells

PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.     

Preimplantation genetic diagnosis for beta-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci

In order to carry out preimplantation genetic diagnosis (PGD) for beta-thalassaemia, we have applied direct sequencing of single cell PCR products to detect mutations and polymorphic loci within the beta-globin gene. Conventional duplex PCR was used to amplify two regions of the beta-globin gene with an amplification efficiency of 79% for blastomeres. Sequencing data were obtained for 100% of amplified products, with 12% having confirmed allele drop-out (ADO). A double ADO event was observed at least twice, confirming the real risk of such an event during PGD. In one couple, the presence of a polymorphism linked to the female partner's mutation enabled us to eliminate the risk of misdiagnosis due to double ADO without having to amplify both mutations within the same PCR product. We present here the data from eight clinical PGD cycles for three couples resulting in a singleton pregnancy and a twin pregnancy with all babies confirmed to be free from beta-thalassaemia (major).     

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos

Cultured human preimplantation embryos have been used to developmethods which allow preimplantation genetic diagnosis (PGD)analyses by polymerase chain reaction (PCR) and fluorescentin-situ hybridization (FISH) on biopsied blastomeres and trophectodermcells from the same embryo. An experimental design is describedand experiments undertaken, which demonstrate the feasibilityof extending biopsy and PGD procedures currently in use. Wehave shown that dual-stage biopsies are possible, and that thePCR and FISH analyses of the biopsied cell samples are effective.One to two blastomeres were biopsied from an 8- to 10-cell embryoand processed for the simultaneous PCR amplification of a -globinand a cytosine adenine (CA) repeat sequence, or a Y chromosomesequence. FISH procedures were also used to detect the presenceof Y chromosome markers. The biopsied cleavage-stage embryocan be cultured to the blastocyst stage, where the serial biopsyof three to five mural trophectoderm cells provides two furthercell samples. These can be used to repeat and/or undertake additionalPGD analyses. The biopsied blastocyst is either used to confirmearlier diagnoses, or placed in culture for a further 4–24h. Maintenance of a blastocoele cavity, hatching and formationof an outgrowth demonstrates continuing viability followingthe dual-stage biopsy procedures. The PCR DNA amplificationprocedures are effective at the cellular level for both biopsiedblastomeres and mural trophectoderm cells. The FISH techniqueshave shown a definitive Y signal in 50% (one out of two) and100% (two out of two) of the biopsied blastomeres and 72% (twoout of three, four out of five and 7 out of 10) for the trophectodermcell nuclei. Preliminary experiments have demonstrated thatthe FISH preparations can be re-amplified to improve the signal,and dual fluorescent procedures using the X and Y probes areeffective. A retrospective PCR analysis has also been undertakenon preparations of biopsied cells which were previously usedfor PGD analysis by FISH.     

New tools for preimplantation genetic diagnosis of Huntington's disease and their clinical applications

Huntington's disease (HD) is a late-onset neurodegenerative disorder transmitted as an autosomal dominant trait. The causative mutation was characterised in 1993. For HD carriers willing to create a family, prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) based on the mutation identification can be offered. For at-risk persons who do not want to undergo presymptomatic testing (PT), an exclusion test can be proposed. With such a test, only foetuses or embryos that inherit an allele from the unaffected grandparent are considered as unaffected. In cases of PND, if the foetus has one allele of the affected grandparent, termination of pregnancy is proposed. In cases of PGD, only not at-risk embryos are transferred. Since the beginning of our PGD activity, we have had 43 PGD referrals for HD, of which 24 were from patients who know their genetic status and 19 from patients who do not wish to perform PT. We have developed 12 multiplex fluorescent PCR protocols applied at the single-cell level for PGD, some of which target the CAG repeat while others use two different polymorphic microsatellites. We present here these different protocols and their clinical applications, as well as the characterisation and use of a new highly polymorphic intragenic marker. Between May 2001 and December 2003, 39 PGD cycles have been performed for 17 couples, 11 of whom had a known genetic status and six who did not wish to perform PT, resulting in four pregnancies.     

Strategies and outcomes of PGD of familial adenomatous polyposis

Owing to adult onset of hereditary cancer, prenatal diagnosis (PND) raises numerous ethical issues on the acceptability to terminate an affected pregnancy (TOP). PND for these disorders is often considered as unacceptable by couples as well as geneticists and legal or ethical authorities, but preimplantation genetic diagnosis (PGD), even if subject to controversy, seems to be a more acceptable option. Therefore, many couples, who do not want to transmit their cancer to their children, consider PGD as their only reproductive option. This article describes our experience of PGD for familial adenomatous polyposis (FAP). Twelve couples were referred between 2000 and 2005. We developed PGD tests to detect the mutation alone, but we rapidly set up multiplex PCR combining mutation detection and indirect diagnosis. Finally, we set up duplex and triplex indirect diagnoses to be able to offer a PGD, whatever mutation was involved in familial cases. PGD strategies were based on (i) a new double allele-specific PCR approach (D-ARMS) allowing the detection of the wild-type and mutated allele; (ii) PCR fragments sizing and (iii) restriction length polymorphisms. For the 12 referrals, we developed eight tests, and 11 cycles have been performed for four couples, resulting in eight embryo transfers and five pregnancies, with the birth of one healthy boy and two ongoing pregnancies. We are now able to propose PGD to most couples at risk of transmitting FAP to their offspring, whether the mutation is familial or occurred de novo.     

Mutation and haplotype analysis for Duchenne muscular dystrophy by single cell multiple displacement amplification

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder with mutational heterogeneity. The scarcity of DNA from single cells in preimplantation genetic diagnosis (PGD) for DMD limits comprehensive genetic testing. Multiple displacement amplification (MDA) is reported to generate large amounts of template and give the most complete coverage and unbiased amplification to date. Here, we developed mutation and haplotype analysis in conjunction with gender determination on MDA products of single cells providing a generic approach that widens availability of PGD for female carriers with varied mutations. MDA amplified with 98.5% success for single lymphocytes and 94.2% success for single blastomeres, which was evaluated on 60 lymphocytes and 40 blastomeres. A total of six commonly mutant exons, eight short tandem repeat markers within dystrophin gene and amelogenin were incorporated into subsequent singleplex PCR assays. The mean allele dropout rate was 9.0% for single lymphocytes and 25.5% for single blastomeres. None of the blank controls gave a positive signal. Genotyping of each pedigree for three families provided 2-3 fully informative alleles per dystrophin haplotype besides specific mutant exons and amelogenin. We suggest that this approach is reliable to identify non-carrier female embryos other than unaffected male embryos and reduce the risk of misdiagnosis.