Development of single cell genetic testing strategies : assessment of MHC compatibility, meiotic recombinations and beta-thalassemia in human embryos

Abstract

Single cell molecular biology, a relatively new scientific branch, is promising to study unique questions and leading to novel applications in biology and medicine. Single cell studies have been challenged by difficulties in selection and isolation of appropriate cells, low amplification efficiencies, allele drop outs, PCR contaminations and inefficiency of conventional analysis strategies. This study has explored the possibilities of analyzing multiple genetic conditions particularly concerning the beta globin and the HLA regions in human embryos. The HLA genes, beta-globin gene, and the associated microsatellites have been amplified simultaneously by multiplex PCR. DNA sequencing has been optimized for high resolution genotyping. The real-time PCR and melting curve analysis have been adapted for the first time for rapid and reliable analysis of the HLA compatibility. Use of microsatellites of the extended HLA locus has enabled more accurate and efficient detection of the allele drop outs, contaminations and recombinations. Amplification and informative detection have been obtained for 1012 blastomeres out of 1180 human embryos used in this study, giving a detection rate of 86%. A total of 122 (13%) embryos were found unaffected from beta thalassemia and had identical genotype at ten HLA regions. Transfer of 94 embryos that have resulted into 16 pregnancies with 14 healthy offsprings indicates the feasibility of the single cell applications for preventive medical approaches. Microsatellite typing of the extended HLA locus has enabled to study the characteristics of the meiotic recombinations in human embryos. The recombination rate was determined as 0.44 cM/Mb, 2.1 fold less compared to the general genomic recombination rate of 0.92 cM/Mb. It was 3.83 fold higher in the maternal MHC regions compared to the paternal MHC regions. Upto 14 fold difference was observed among individuals. Breakpoints of recombinations in the class II region were clustered between the DRB1 and DPB1 genes covering the TAP1 and TAP2 genes.