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.
Zhonghua Nan Ke Xue. 2022 May;28(5):408-414.
To investigate the value of single-sperm sequencing technology in preimplantation genetic testing.
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.3815T>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.
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.3815T>G mutation in the fetuses.
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.
探讨单精子测序技术在植入前基因检测中的价值。
对于一名因PKD1基因c.3815T>G新发突变导致常染色体显性多囊肾病（ADPKD）的患者，通过单精子分离结合单精子测序构建单倍型。通过机械制动分离50个单精子样本，进行全基因组扩增，并设计突变位点及其上下游187个单核苷酸多态性（SNP）。对扩增产物进行验证，以确定携带或不携带致病突变的染色体单倍型。对7个胚胎滋养外胚层细胞活检样本进行全基因组扩增后，通过高通量测序鉴定携带致病突变的胚胎。选择可用的囊胚进行胚胎移植，并在妊娠18周时采集羊水样本，以确定胎儿是否携带致病突变。
通过单精子测序共鉴定出30个SNP，并成功构建了单倍型。植入前单倍型分析表明，5个胚胎携带致病突变，2个不携带。妊娠中期羊水基因检测显示胎儿中未检测到PKD1基因c.3815T>G突变。
对于携带新发致病突变的男性，可通过单精子测序鉴定SNP，并通过连锁分析构建单倍型，用于胚胎植入前基因检测。
