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【目的】采用多重置换扩增建立一种可靠、准确的植入前遗传学诊断方法,应用于X-连锁严重联合免疫缺陷病(X-SCID)的植入前遗传学诊断(PGD)。【方法】选择5个位于致病基因IL-2受体γ链(IL2RG)基因两侧的短串联重复序列(STR)位点对X-SCID家系进行单体型分析,采用多重置换扩增(MDA)对单细胞进行全基因组扩增,对扩增产物采用在家系分析中具有多态性的STR位点进行单体型分析,结合位点Amel进行性别诊断,STR位点均采用单重荧光PCR,同时对IL2RG基因exon5进行测序分析。【结果】对家系分析中,共有3个STR位点具有多态性。对10个单淋巴细胞及10个单卵裂球进行了预实验:MDA扩增成功率为100%,对致病基因IL2RG exon5的行基因测序的检测效率为100%;对于3个有多态性的STR位点和AMEL,PCR扩增效率为96.3%(77/80),等位基因脱扣率(ADO)为11.5%(7/61)。对该家系进行了一个周期的PGD,对7个胚胎进行了诊断,其中2个为正常胚胎,移植后获得双胎妊娠,早产活产一个健康男婴及一个健康女婴。【结论】采用多重置换扩增结合致病基因特异性扩增测序检测及单体型分析在单细胞水平对X-连锁严重联合免疫缺陷病进行检测,两者相结合可避免污染、等位基因脱扣等导致的误诊,提高了PGD诊断效率。
【Objective】 To establish a reliable and accurate method of preimplantation genetic diagnosis using multiple displacement amplification and to evaluate the preimplantation genetic diagnosis (PGD) of X-linked severe combined immunodeficiency disease (X-SCID). 【Method】 Five haplotypes of X-SCID pedigrees were selected from five STR loci located on both sides of the IL-2 receptor gene (IL2RG) gene of disease-causing gene. Multiplexed amplification MDA) genome-wide amplification of single cells, the amplified products were analyzed by family members of the STR polymorphisms with haplotype analysis, combined with locus Amel sex diagnosis, STR loci were used single fluorescence PCR, at the same time IL2RG gene exon5 sequencing analysis. 【Result】 Three pedigree STR loci were polymorphic in pedigree analysis. Pre-experiments on 10 single lymphocytes and 10 single blastomeres were carried out. The success rate of MDA amplification was 100%. The detection efficiency of line gene sequencing of IL2RG exon5 was 100% Sex STR loci and AMEL, PCR amplification efficiency was 96.3% (77/80), allele rate (ADO) was 11.5% (7/61). A one-cycle PGD was performed on this pedigree. Seven embryos were diagnosed. Two of them were normal embryos, twin pregnancies were obtained after transplantation, one healthy baby boy was born alive and one healthy baby girl was born. 【Conclusion】 The detection of X-linked severe combined immunodeficiency disease at single cell level by multiplexed amplification combined with pathogen-specific sequencing and haplotypic analysis can avoid contamination and allele Triage caused by misdiagnosis, improve the PGD diagnostic efficiency.