Preimplantation Genetic Diagnosis of X-linked Severe Combined Immunodeficiency / 中山大学学报(医学科学版)

ABSTRACT

[Objective]To establish a reliable and accurate preimplantation genetic diagnosis (PGD)method using multiple dis?placement amplification (MDA), which can be applied to the diagnosis of X-linked severe combined immunodeficiency disease (X-SCID).[Methods]Haplotype analysis for the X-SCID family was performedusing five short tandem repeats (STR) markers flanking the both sides of the interleukin-2 (IL-2) receptor gamma chain (IL2RG) gene. MDA technique was used for single-cell whole genomic amplification. The products were used as template in polymerase chain reaction (PCR) of informative STR markers found by linkage analysis for haplotype analysis as well as sequencing of the IL2RG gene exon 5.The amelogenin (AMEL) locus was used to do sex diag?nosis.[Results]Linked analysis revealed 3 STR markers were informative. The method was evaluated with 10 single lymphocytes and 10 single blastomeres. MDA was successful in all single cell. The detection efficiency of gene sequencing of pathogenic IL 2RG exon5 was 100%. The PCR efficiency of 3 STR informative markers and AMEL was 96.3%(77/80)and the average allele drop-out (ADO) rate was 11.5%(7/61). A cycle of PGD was performed on the family, and seven embryos were diagnosed, two of which were normal embry?os. Twin pregnancy occurred after transplantation which were given a healthy baby boy and a healthy baby girl.[Conclusion]In this study, multiple displacement amplification combined with specific amplification/sequencing of pathogenic gene and haplotype analysis in the single cell level of X-linked severe combined immunodeficiency disease were performed. The protocol can avoid misdiagnosis caused by contamination and ADO, and improve the diagnostic efficiency of PGD.