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.