单精子测序技术在常染色体显性遗传多囊肾患者植入前遗传学检测中的应用  

Application of single-sperm sequencing technology in preimplantation genetic testing for men with autosomal dominant polycystic kidney disease

作　　者：邓天勤 谢雨莉 张华坤[1] 陈海燕[1] 焦淑静 李雪梅[1] DENG Tian-qin;XIE Yu-li;ZHANG Hua-kun;CHEN Hai-yan;JIAO Shu-jing;LI Xue-mei(Center of Reproductive Medicine,Shenzhen Maternity and Child Healthcare Hospital of South Medical University,Shenzhen,Guangdong 518028,China;Center for Neonatal Disease Screening,Shenzhen Maternity and Child Healthcare Hospital of South Medical University,Shenzhen,Guangdong 518028,China;Yikon Genomics Company,Suzhou,Jiangsu 215021,China)

机构地区：[1]南方医科大学附属深圳市妇幼保健院,生殖医学中心,广东深圳518028 [2]南方医科大学附属深圳市妇幼保健院,新生儿疾病筛查中心,广东深圳518028 [3]苏州亿康医学检验有限公司,江苏苏州215021

出　　处：《中华男科学杂志》2022年第5期408-414,共7页National Journal of Andrology

基　　金：深圳市医学重点学科建设经费资助(SZXK031)。

摘　　要：目的:探讨单精子测序技术在植入前遗传学检测中的应用价值。方法:针对1例PKD1基因c.3815T>G新发变异导致的常染色体显性多囊肾患者,应用单精子分离结合单精子测序技术完成单倍型的构建,用机械制动法分离50份单精子样本并进行全基因组扩增,设计变异位点及其上下游共187个单核苷酸多态性(SNP)位点的扩增引物,对扩增产物进行检测,确定未携带以及携带致病变异的染色体单倍型,将7份胚胎滋养层细胞活检样本作为对象,在完成全基因组扩增后,通过高通量测序进行检测,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:通过单精子测序共筛选出47个SNP位点,成功构建单体型,植入前单体型分析提示5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PKD1基因c.3815T>G变异。结论:对于携带新发致病变异的男性,可通过单精子测序筛选SNP位点,通过连锁分析构建单体型,准确有效的进行胚胎植入前遗传学检测。Objective:To investigate the value of single-sperm sequencing technology in preimplantation genetic testing.Methods:Haplotypes were constructed by single-sperm isolation combined with single-sperm sequencing for a patient with autosomal dominant polycystic kidney disease(ADPKD)caused by de novo mutation of the PKD1 gene c.3815 T>G.50.Single-sperm samples were isolated by mechanical braking,whole-genome amplification was performed,and mutation loci and their 187 upstream and downstream single nucleotide polymorphisms(SNP)were designed.The amplified products were verified for determination of the chromosome haplotypes carrying or not carrying pathogenic mutations.The embryos carrying pathogenic mutations were identified in 7 embryonic trophectoderm cell biopsy samples by high-throughput sequencing after whole-genome amplification.Available blastocysts were selected for embryo transfer,and amniotic fluid samples were collected at 18 weeks of gestation to determine whether the fetuses carried pathogenic mutations.Results:A total of 30 SNPs were identified by single-sperm sequencing,and haplotypes were successfully constructed.Preimplantation haplotype analysis indicated that 5 embryos carried pathogenic mutations and 2 did not.mid-gestation amniotic fluid genetic testing revealed no PKD1 gene c.3815 T>G mutation in the fetuses.Conclusion:SNPs can be identified by single-sperm sequencing in males carrying de novo pathogenic mutation,and haplotypes can be constructed by linkage analysis for preimplantation genetic testing of embryos.

关 键 词：常染色体显性多囊肾 植入前遗传学检测 单精子测序 

分 类 号：R715.5[医药卫生—妇产科学]