单精子测序技术在植入前胚胎染色体结构异常遗传学检测中区分罗氏易位携带者的应用  

Application of single-sperm sequencing in resolving the carrier status of preimplantation genetic testing for chromosomal structural

作　　者：廖宝琼 赖丽丹 刘如天 张琪 连文昌 谢武明 LIAO Bao-qiong;LAI Li-dan;LIU Ru-tian;ZHANG Qi;LIAN Wen-chang;XIE Wu-ming(Ganzhou Maternal and Child Health Hospital,Ganzhou,Jiangxi 341000,China;Yikon Genomics Company,Suzhou,Jiangsu 215021,China;Ganzhou Peoples Hospital,Ganzhou,Jiangxi 341000,China)

机构地区：[1]赣州市妇幼保健院,江西赣州341000 [2]苏州亿康医学检验有限公司,江苏苏州215021 [3]赣州市人民医院,江西赣州341000

出　　处：《中华男科学杂志》2024年第6期499-506,共8页National Journal of Andrology

基　　金：江西省卫生健康科技计划项目(202312033)。

摘　　要：目的:探讨单精子测序技术在植入前胚胎结构异常遗传学检测中区分携带者的应用价值。方法:针对1例罗氏易位携带者45,XY,der(13;14)(q10;q10)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(WGA),再利用ASA基因芯片对WGA产物进行全基因组183708个单核苷酸多态性(SNP)位点检测,通过CNV测序检测出与易位相关且可作为单体型推断的单精子,推断亲本正常及携带罗氏易位的染色体单倍型。将3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,判断胚胎携带易位染色体的情况,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6037个SNP位点,挑选出30个可区分正常与易位单体型位点成功构建单体型,植入前单体型分析提示3枚胚胎均为不携带罗氏易位染色体的整倍体胚胎,妊娠中期羊水基因检测证实胎儿核型为46,XN,未携带罗氏易位染色体。结论:对于男性罗氏易位携带者,可通过单精子测序筛选SNP位点构建单体型,用于区分正常和罗氏易位携带者胚胎,为胚胎植入前染色体结构遗传学检测胚胎选择提供依据。Objective:To investigate the application value of single-sperm sequencing in resolving the carrier status of preimplantation genetic testing(PGT)for chromosomal structural rearrangements in Robertsonian translocations.Methods:Haplotypes were constructed by single-sperm isolation combined with single-sperm sequencing for a patient with 45,XY,der(13;14)(q10;q10).Twenty single-sperm samples were isolated by mechanical braking and subjected to whole-genome amplification(WGA),and then the Asian Screening Array(ASA)gene chip was used to detect the 183708 single nucleotide polymorphisms(SNP)of the WGA products.The single sperm associated with the translocation that could be used as haplotype inference was detected by copy number variation(CNV)sequencing,and the chromosomal haplotypes with normal and Robertsonian translocations were inferred.Three biopsy samples of embryonic trophoblast cells were used as the objects.After whole-genome amplification,high-throughput sequencing was employed to determine the status of the translocation chromosome carried by the embryos.The available blastocysts were selected for transfer,and the amniotic fluid samples were taken at 18 weeks of gestation to confirm whether the fetus carried the pathogenic mutation.Results:A total of 6037 SNP sites were screened by single-sperm sequencing,and 30 sites selected to distinguish normal and translocation haplotypes.Preimplantation haplotype analysis showed that all the three embryos were euploids without Robertsonian translocation chromosome.Genetic testing of amniotic fluid in the second trimester confirmed that the karyotype of the fetus was 46,XN,carrying no Robertsonian translocation chromosome.Conclusion:For male carriers of Robertsonian translocation,single sperm sequencing can be used to screen SNP sites to construct haplotypes for distinguishing normal and Robertsonian translocation embryos,and to provide a basis for embryo selection by preimplantation chromosomal structural genetic testing.

关 键 词：罗氏易位 植入前染色体结构异常遗传学检测 单精子测序 

分 类 号：R715.5[医药卫生—妇产科学]