单精子测序技术在新发突变单基因遗传病家系胚胎植入前遗传学检测中的应用  

Clinical application of single-sperm-based SNP haplotyping for PGT of monogenic hereditary disease associated with de novo mutations

作　　者：周宣佑 陈松长 李淑元[2] 徐晨明 Zhou Xuanyou;ChenSongchang;LiShuyuan;Xu Chenming(Genetic Center,Obstetrics and Gynecology Hospital,Fudan University,Shanghai 200001,China;Department of Reproductive Genetics,International Peace Maternity and Child Health Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200030,China)

机构地区：[1]复旦大学附属妇产科医院妇产科遗传中心,上海200001 [2]上海交通大学医学院附属国际和平妇幼保健院生殖遗传科,上海200030

出　　处：《中华生殖与避孕杂志》2022年第11期1127-1135,共9页Chinese Journal of Reproduction and Contraception

基　　金：国家重点研发计划(2021YFC2701002,2018YFC1004900);国家自然科学基金项目(81971344,82171677,82192860,81901495);上海市科委项目(21Y21901002);上海市卫健委项目(GW-10.1-XK07,202140110);中国医科院胚胎源性疾病创新单元(2019-I2M-5-064)。

摘　　要：目的探讨单精子测序技术在新发突变单基因病家系胚胎植入前遗传学检测(preimplantation genetic testing,PGT)中的应用效果和价值。方法针对3个携带新发突变的常染色体遗传病家系,采用多重置换扩增技术(multiple-displacement amplification,MDA)对单精子进行全基因组扩增(whole genome amplification,WGA),通过检测扩增产物的变异位点以及目的基因上下游2M范围内有效单核苷酸多态性位点(single nucleotide polymorphism,SNP)位点信息,构建携带突变的风险单体型与不携带突变的正常单体型。对待测胚胎进行WGA,产物进行高通量测序,结合单体型信息判断胚胎致病位点的携带状态,选择不携带致病变异的胚胎进行移植。结果共挑取16份有效单精子样本,在原发性高草酸尿症、Kabuki综合征、遗传性大疱性表皮松解症3个新发突变单基因病家系中成功构建单体型。胚胎植入前单基因遗传病检测(PGT for monogenic disorders,PGT-M)结果提示有10枚胚胎携带父源致病变异;6枚胚胎不携带父源致病变异,其中2枚胚胎检出染色体拷贝数变异。除原发性高草酸尿症夫妇外,其余两个家系的夫妇共获得4枚正常胚胎,移植后均未妊娠。结论对于家系中男性携带新发突变的夫妇,可以利用单精子测序技术构建单体型,进而进行PGT。Objective To explore the application value of single-sperm-based single-nucleotide polymorphism(SNP)haplotyping in preimplantation genetic testing of monogenic disorders(PGT-M)associated with de novo mutations.Methods Whole genome amplification(WGA)of the isolated single sperm was performed based on the multiple displacement amplification(MDA).WGA products were tested for the pathogenic mutation site and informative polymorphic SNP loci located within 2M upstream or downstream of the target gene to establish a sperm-based SNP haplotype.Biopsy samples obtained from embryos were subjected to WGA and next-generation sequencing(NGS).All embryos were verified via haplotype analysis and normal embryos were selected for transfer.Results Totally 16 sperm samples were selected.Haplotypes of the affected male in 3 families with monogenic hereditary diseases including primary hyperoxaluria type 1(PH1),Kabuki syndrome and Epidermolysis bullosa(EB)were successfully constructed using single sperm cell sequencing.PGT-M results showed that there were 10 embryos carrying paternal pathogenic variations,while the other 6 embryos did not carry paternal pathogenic variations,2 of them had chromosomal copy number variations(CNVs).Four embryos obtained from 2 families were found to be normal after NGS according to single-sperm-based SNP haplotype analysis.However,no successful pregnancy was obtained.Conclusion For males carrying de novo mutations,single-sperm-based SNP haplotyping can be applied for PGT to construct paternal haplotype.

关 键 词：胚胎植入前遗传学检测 单基因疾病 单精子测序 新发突变 

分 类 号：R714.8[医药卫生—妇产科学]