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

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 DF508 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 DF508 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 DF508 and R1162X, case 2 DF508 and R1066C and case 3 DF508 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.

Fragile X syndrome (FraX) is caused by the expansion of an unstable CGG repeat located in the Fragile X mental retardation 1 gene (FMR1) gene. Preimplantation genetic diagnosis (PGD) can be proposed to couples at risk of transmitting the disease, that is, when the female carries a premutation or a full mutation. We describe two new single-cell, single-round multiplex PCR for indirect and direct diagnosis of FraX on biopsied embryos. These tests include five unpublished, highly heterozygous simple sequence repeats, and the co-amplification of non-expanded CGG repeats for the direct test. Heterozygosity of the new markers ranged from 69 to 81%. The mean rate of non-informative marker included in the tests was low (26% and 23% for the new indirect and direct tests, respectively). This strategy allows offering a PGD for FraX to 96% of couples requesting it in our centre. A conclusive genotype was obtained in all cells with a rate of cells presenting an allele dropout ranging from 17% for the indirect test to 26% for the direct test. The new indirect test was applied for eight PGD cycles: 32 embryos were analysed, 9 were transferred and 3 healthy babies were born. By multiplexing these highly informative markers, robustness of the diagnosis is improved and the loss of potentially healthy embryos (because they are non-diagnosed or misdiagnosed) is limited. This may increase the chances of success of couples requesting a PGD for FraX, in particular, when premature ovarian insufficiency in premutated women leads to a reduced number of embryos available for analysis.

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN(t)) gene. An oligonucleotide was designed to be specific to the SMN(t) nucleotidic sequence with exonic mismatch G (for SMN(t))-->A (for SMN(c)) at its 3' end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN(t) gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre.

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.

Most of cystic fibrosis (CF) pre-implantation genetic diagnosis (PGD) cases described to date are limited to the detection of DeltaF508. Beside this predominant mutation, over 1000 mutations have been identified, rendering the development of a mutation-based PGD protocol impracticable. This is the reason why we, as well as the others, have developed PGD strategies on the basis of the identification of the pathogenic haplotype instead of the mutation(s). In a previous article, we reported the conditions for the co-amplification of two intragenic polymorphic markers and the F508 locus. Here we describe an improved protocol allowing the additional amplification of two new intragenic markers, intron 1 CA repeat (I1CA) and IVS17bTA. This new protocol should, theoretically, allow us to provide a diagnosis to all couples requiring PGD for CF. Using single lymphoblasts, we have tested four different PCR configurations, including one duplex, two triplexes and one quadruplex PCR. All of them gave results compatible with a clinical application. The number of single lymphoblasts tested in each series varied from 89 to 155. PCR efficiency ranged from 95.4 to 100%. A complete haplotype was achieved for 83.2 to 90.7% of the tested cells, with an allele drop out (ADO) rate comprised between 6.0 and 11.6%. We present here three cases that we performed either with the former test (one case using the triplex PCR combining F508, IVS8CA and IVS17bCA) or with the new one (one case using the triplex combining F508, I1CA and IVS17bTA and one case using a quadruplex test). We obtained two single pregnancies.

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.

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90-98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene.