Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis

Background: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected em bryos following in vitro fertilisation (NF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. Methods and Results: Using nested PCR, the Delta F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the Delta F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from Delta F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. Conclusion: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.