Single cell pcr amplification of microsatellites flanking the COL7A1 gene and suitability for preimplantation genetic diagnosis of hallopeau-siemens recessive dystrophic epidermolysis bullosa

Background: Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (HSRDEB) is a severe inherited blistering skin disorder caused by mutations in the anchoring fibril type VII collagen gene, COL7A1. There is currently no effective treatment but DNA-based prenatal testing in families at risk of recurrence is possible, mostly involving chorionic villus sampling at 10-11 weeks' gestation. Objectives: An alternative method, for avoiding recurrence of HS-RDEB, is preimplantation genetic diagnosis (PGD). This involves DNA analysis of single blastomeres extracted from late cleavage stage embryos following in vitro fertilisation. Methods: To establish PGD for HS-RDEB, we designed and optimised a sensitive single cell semi-duplex polymerase chain reaction (PCR) assay for two highly polymorphic dinucleotide repeat microsatellite markers, D3S1581 (telomeric) and D3S1289 (centromeric), close to the COL7A1 gene. Results: We demonstrated high PCR efficiency, low allele drop out rates and no contamination in testing this assay on 50 single buccal cells of known heterozygous genotype and 13 research blastomeres from donated embryos. Conclusions: This semi-duplex PCR method provides robust, reproducible and informative amplification results for single cells. Moreover, this test has now been approved for clinical application by the UK Human Fertilisation and EmbryologyAuthority (HFEA). As such, the development of PGD for HS-RDEB broadens the range of prenatal testing options and personal choice for couples at reproductive risk of this severe genetic skin disease.