Abstract
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 ΔF508 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 ΔF508 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 ΔF508 and R1162X, case 2 ΔF508 and R1066C and case 3 ΔF508 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.
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Acknowledgements
We would like to thank Serge Vicaire for his help with the ABI377, the IVF team at the SIHCUS-CMCO and Prof Ingeborg Liebaers, Dr Uwe Strahle, Dr Irwin Davidson and Prof Michel Koenig for their helpful comments on this manuscript. This work was supported by funds from the Association Française contre les Myopathies (AFM), the Association Française de Lutte contre la Mucoviscidose (AFLM-VLM), the Fond pour la recherche Organon (FARO), from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Hôpital Universitaire de Strasbourg.
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Moutou, C., Gardes, N. & Viville, S. Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosis. Eur J Hum Genet 10, 231–238 (2002). https://doi.org/10.1038/sj.ejhg.5200794
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DOI: https://doi.org/10.1038/sj.ejhg.5200794
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