基于单精子SNP单体型的1例遗传性球形红细胞增多症患者植入前遗传学检测

Clinical application of single-sperm-based SNP haplotype for preimplantation genetic testing of a patient with hereditary spherocytosis

目的探讨单精子单核苷酸多态性(SNP)单体型在植入前遗传学检测(PGT)中的应用价值。方法收集1例ANK 1基因c.3641delC杂合突变导致的常染色体显性遗传性球形红细胞增多症患者的精液样本,用机械制动法分离单精子样本并进行全基因组扩增,通过ASA基因芯片分析基因上下游1 Mb范围内SNP位点,确定未携带以及携带致病变异的染色体单体型。将获得的4份胚胎滋养层细胞活检样本作为研究对象,在完成全基因组扩增后,通过高通量测序检测判断胚胎携带致病变异的情况,选取可用囊胚进行移植,于孕18周抽取羊水样本,确认胎儿是否携带致病变异。结果通过单精子SNP位点检测共筛选出30个SNP位点,成功构建突变型及野生型单精子SNP单体型,植入前单体型分析提示2枚胚胎携带致病变异,2枚未携带;妊娠中期羊水基因检测证实胎儿未携带ANK1基因c.3641delC杂合突变。结论对于携带新发致病变异的男性,通过单精子SNP单体型构建连锁分析,可为患者夫妇提供准确有效的单基因病植入前遗传学检测(PGT-M),筛选正常胚胎进行移植,从而阻断致病基因垂直传递。

Objective:To investigate the value of single-sperm-based SNP haplotype in preimplantation genetic testing(PGT).Methods:The semen sample was collected from a patient with autosomal dominant hereditary spherocytosis caused by heterozygous mutation of ANK1 gene c.3641delC.Single sperm was isolated by mechanical braking method and conducted by whole-genome amplification.Single nucleotide polymorphism(SNP)loci within 1 Mb range upstream and downstream of the gene were analyzed by ASA gene chip to determine haplotypes that did not carry or carry pathogenic variation.Four biopsy samples of embryonic trophoblast cells were obtained and taken as the research objects.After the whole genome amplification was completed,pathogenic variants carried by embryos were detected by high-throughput sequencing,and available blastocysts were selected for transplantation.Then amniotic fluid samples were taken at 18 gestation weeks to confirm whether the fetuses did or not carry pathogenic variants.Results:A total of 30 SNP loci were screened through single sperm SNP loci detection.The mutant and wild-type single sperm SNP haplotype were constructed successfully.Preimplantation haplotype analysis showed that two embryos carried pathogenic variants and two did not.Amniotic fluid was detected in the second trimester,which was confirmed that the fetus did not carry the heterozygous mutation of ANK1 gene c.3641delC.Conclusions:For males with de novo pathogenic mutation,linkage analysis based on single sperm SNP haplotype can provide accurate and effective preimplantation genetic testing for monogenic diseases(PGT-M)for couples,which can screen normal embryos for transfer.Therefore the vertical transmission of pathogenic genes could be blocked.