Lyu Yuan, Li Chuang, Zhou Feifei, Li-Ling Jesse, Tan Jichun, Liu Caixia
Department of Gynecology and Obstetrics, Shengjing Hospital Affiliated to China Medical University, Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Shenyang, Liaoning 110004, China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2020 Sep 10;37(9):929-933. doi: 10.3760/cma.j.cn511374-20190705-00338.
To assess the value of single sperm sequencing in preimplantation genetic diagnosis.
A male patient with achondroplasia due to a de novo FGFR3 variant was subjected to single sperm isolation and sequencing. Twenty single sperm samples were isolated by mechanical immobilization, and their whole genome was amplified. PCR primers were designed for the variant site and 25 flanking single nucleotide polymorphism (SNP) loci, and the PCR products were sequenced to determine the chromosomal haplotype which did not harbor the pathogenic variant. Biopsy samples of 12 embryonic trophoblasts were taken. Following whole genome amplification, high-throughput sequencing was carried out to detect the carrier status of the embryos. Wild type blastocysts were selected for transplantation. Amniotic fluid samples were taken at 19 weeks of gestation to confirm the status of the fetus.
Eight SNP were selected by single sperm sequencing, with which the haplotypes were successfully constructed. Preimplantation genetic testing indicated that 5 embryos have carried the pathogenic variant and 7 did not. Testing of amniotic fluid sample during the second trimester of pregnancy confirmed that the fetus did not carry the FGFR3 gene c.1138G>A variant.
For male patients carrying de novo pathogenic variants, SNP sites can be selected through single sperm sequencing, and haplotypes can be constructed by linkage analysis for preimplantation genetic diagnosis.
评估单精子测序在植入前遗传学诊断中的价值。
对一名因新发FGFR3变异导致软骨发育不全的男性患者进行单精子分离和测序。通过机械固定法分离出20个单精子样本，并对其全基因组进行扩增。针对变异位点和25个侧翼单核苷酸多态性（SNP）位点设计PCR引物，对PCR产物进行测序以确定不携带致病变异的染色体单倍型。采集12个胚胎滋养层的活检样本。全基因组扩增后，进行高通量测序以检测胚胎的携带状态。选择野生型囊胚进行移植。在妊娠19周时采集羊水样本以确认胎儿状态。
通过单精子测序选择了8个SNP，成功构建了单倍型。植入前遗传学检测表明，5个胚胎携带致病变异，7个未携带。妊娠中期羊水样本检测证实胎儿未携带FGFR3基因c.1138G>A变异。
对于携带新发致病变异的男性患者，可通过单精子测序选择SNP位点，并通过连锁分析构建单倍型用于植入前遗传学诊断。
