Preimplantation Genetic Diagnosis Of X-linked Disorders And Hla Genotyping

In this study, we developed a new PGD protocol for gender determination and HLA genotyping in preimplantation embryos by the analysis of multiple loci or multiple genes using isothermal multiple displacement amplification from single blastomere as a preliminary step to yield large amounts of DNA of high quality.1-2 blastomeres were removed from the cleavage stage embryos by micromanipulation techniques and lysed by Proteinase K (n=21) or Alkaline (n=17) randomly. The whole blastomere genomic amplification by MDA was achieved using bacteriophage 29 DNA polymerase and random hexamer oligonucleotide primers in a 30℃reaction. There was no statistical differences were found between the Proteinase K and Alkaline methods (85.7% vs.82.4%, p>0.05) and so was the one blastomere and the two blastomeres group (80% vs.87.0%, p>0.05). It is demonstrated that MDA could be successfully used to amplify single blastomere DNA. In the subsequent study, one or two blastomeres were collected from one preimplantation embryo and lysed by proteinase K method then used directly for MDA. After that, the amplified products of MDA were used in follow up various gene analyses.Our research consists of two parts of which the first was the preimplantation gender determination by fluorescent PCR after multiple displacement amplification. 86.7%(13/15) multiple displacement amplification were successfully amplified. Fluorescent PCR was secondly performed to amplify the SRY,XHPRT,AMEL and X22 sex gene or STR loci markers. The amplification products were sized using Capillary electrophoretogram to determine the numbers of sex chromosomes. There were 7 male embryos and 6 female embryos in the 13 embryos and the fluorescent PCR efficiency was 100%. ADO only occurred in one embryo in the AMEL PCR reaction and the ADO rate was 7.7%, however, no ADO occurred in the other three markers. Therefore this was an efficient and accurate protocol for preimplantation gender determination using SRY,XHPRT,AMEL and X22 markers by fluorescent PCR after multiple displacement amplification, which could be used in the sex-linked disorders.The second part of this research was the preimplantation HLA genotyping. Four couples who donated their peripheral blood and discarded embryos voluntarily were collected in this study. Six embryos were subjected to MDA and five were amplified successfully with the amplification rate was 83.3%. Then the alleles of HLA-A,B,DR loci were detected from the MDA product by the PCR-SSP method. HLA genotypes were obtained in three embryos from two families. Then the parents' peripheral blood samples were withdrawn and the HLA genotypes were analyzed by the same PCR-SSP protocol. Each genotype of the specific HLA region was evaluated to distinguish the segregation of each haplotype of the family members and the primary HLA matching was done between the preimplantation embryos. Preimplantation genetic diagnosis of HLA matching combined with or without a single gene defects in families with previous siblings requiring HLA-identical bone marrow transplantation with the selection and transfer of HLA-identical embryos so that the HLA-compatible baby to be born can be a donor of umbilical cord blood or the bone marrow stem cells to transplant and eventually cure the affected sibling. So the preimplantation HLA matching could be a new tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder.