单精子测序技术在软骨发育不良患者植入前遗传学检测中的应用  

Application of single sperm sequencing for preimplantation genetic testing of male patient with achondroplasia

作　　者：吕远[1] 李闯 周飞飞[2] 李岭 谭季春[2] 刘彩霞[1] 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;Center of Reproduction,Shengjing Hospital Affiliated to China Medical University,Shenyang,Liaoning 110022,China;State Key Laboratory of Biotherapy,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]中国医科大学附属盛京医院妇产科,辽宁省母胎医学重点实验室,沈阳110004 [2]中国医科大学附属盛京医院生殖中心,沈阳110022 [3]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《中华医学遗传学杂志》2020年第9期929-933,共5页Chinese Journal of Medical Genetics

基　　金：国家重点研发计划(2018YFC1002900);国家自然科学基金(81701462);中央引导地方科技发展专项资金(2016007014);盛京自由研究者基金(201501)。

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FGFR3基因新发变异导致的软骨发育不良患者,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20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25个单核苷酸多态性(single nucleotide polymorphism,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12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19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8个SNP位点,成功构建单倍型。植入前单倍型分析提示5枚胚胎携带致病变异,7枚未携带。妊娠中期羊水基因检测证实胎儿未携带FGFR3基因c.1138G>A变异。结论对于携带新发致病变异的男性患者,可通过单精子测序筛选SNP位点,通过连锁分析构建单倍型,进行胚胎植入前遗传学检测。Objective To assess the value of single sperm sequencing in preimplantation genetic diagnosis.Methods 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.Results Eight SNPs 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.Conclusion 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.

关 键 词：软骨发育不良 植入前遗传学检测 单精子测序 等位基因脱扣 

分 类 号：R714.8[医药卫生—妇产科学]