极体测序技术在Van der Woude综合征患者胚胎植入前遗传学检测中的应用  

Application of polar body sequencing for preimplantation genetic testing of a female patient with Van der Woude syndrome

作　　者：陈佳[1] 吴兴武[1] 田莉峰[1] 陈格 黄志辉[1] 辛才林[1] 赵琰[1] 刘艳秋[3] 伍琼芳[1] Chen Jia;Wu Xingwu;Tian Lifeng;Chen Ge;Huang Zhihui;Xin Cailin;Zhao Yan;Liu Yanqiu;Wu Qiongfang(Reproductive Medicine Center,Jiangxi Provincial Maternal and Child Health Hospital,Nanchang 330006,China;Central Laboratory,Jiangxi Provincial Maternal and Child Health Hospital,Nanchang 330006,China;Medical Genetics Center,Jiangxi Provincial Maternal and Child Health Hospital,Jiangxi Provincial Key Laboratory of Birth Defect for Prevention and Control,Nanchang 330006,China)

机构地区：[1]江西省妇幼保健院生殖医学中心,南昌330006 [2]江西省妇幼保健院中心实验室,南昌330006 [3]江西省妇幼保健院医学遗传中心江西省出生缺陷防控重点实验室,南昌330006

出　　处：《中华生殖与避孕杂志》2023年第1期78-83,共6页Chinese Journal of Reproduction and Contraception

基　　金：江西省卫生健康委科技计划项目(202130825)。

摘　　要：目的探讨极体测序技术在Van der Woude综合征患者胚胎植入前单基因遗传病检测中的应用价值。方法对1例因IRF6基因新发变异导致的Van der Woude综合征女性患者,应用极体测序技术进行胚胎植入前遗传学检测。6枚卵经卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)受精后,分别顺序活检第一极体、第二极体和囊胚滋养外胚层细胞。活检细胞经全基因组扩增后,利用PCR联合Sanger测序的方法检测极体和胚胎的致病变异携带状态,推断对应胚胎的致病性。为预防因囊胚培养失败造成无可移植胚胎的情况,在囊胚形成前对1枚致病性低的优质胚胎进行玻璃化冷冻。此外,通过在夫妇双方以及特定基因型的极体和胚胎样本中对变异位点及其上下游1M区域内的175个单核苷酸多态性(single nucleotide polymorphism,SNP)位点进行靶向捕获高通量测序,连锁分析构建单体型。选择致病性低的胚胎移植,成功妊娠后进行产前诊断以及跟踪随访。结果成功获得第一极体和第二极体各6枚,其中11枚极体的变异位点检测成功。6枚胚胎中,1枚预测致病性低的胚胎在患者知情同意后于第4日(day 4,D4)玻璃化冷冻;1枚胚胎成功发育至囊胚,但致病性高;4枚胚胎囊胚培养失败。连锁分析成功构建出与致病变异紧密连锁的SNP单体型,胚胎单体型分析与Sanger测序结果吻合。移植致病性低的D4期冷冻胚胎,成功妊娠后,患者夫妇拒绝有创产前诊断。出生后新生儿随访未发现唇腭裂,脐血基因检测不携带致病变异。结论本研究利用卵母细胞极体测序的检测方法,为1例因IRF6基因新发变异导致的Van der Woude综合征女性患者进行胚胎植入前单基因遗传病检测,成功阻断了该病向子代传递。Objective To explore the value of polar body sequencing in preimplantation genetic testing(PGT)for monogenic disease of a female patient with Van der Woude syndrome.Methods PGT based on polar body sequencing was performed for a female patient with Van der Woude syndrome caused by a de novoIRF6 pathogenic variant.Totally six oocytes were fertilized by intracytoplasmic sperm injection(ICSI).The first,second polar bodies and the trophoblast ectoderm cells of blastocysts were biopsied respectively.Sanger sequencing was used to detect the pathogenic variant in the biopsied cells after genome-wide amplification.The genotypes and pathogenic possibilities of the embryos were inferred according to the genotypes of corresponding tested polar bodies.In order to prevent the absence of transplantable embryos due to the failure of blastocyst culture,vitrification was performed on an embryo with good morphology and low pathogenic possibility before blastocyst formation.The 175 single nucleotide polymorphisms(SNPs)within the 1M region upstream and downstream from the pathogenic variant location were tested by targeted capture sequencing in the couple and selected polar bodies and embryos to construct the haplotypes.An embryo with low pathogenic possibility was transferred.Prenatal diagnosis was strongly recommended after successful pregnancy.Prenatal and postnatal follow-up were performed.Results Totally six first polar bodies and six second polar bodies were obtained.The pathogenic variant was successfully sequenced in 11 polar bodies.Among the six embryos,one embryo with low pathogenic possibility was vitrified on day 4(D4)after fully informed consent of the couple;one embryo developed to blastocyst was detected with high pathogenic possibility;the other four embryos were degenerated during blastocyst culture.The SNP haplotypes closely linked to the pathogenic variant location were successfully constructed by linkage analysis.The haplotype analysis of the embryos was in consistent with Sanger sequencing.The D4 embryo with low

关 键 词：Van der Woude综合征 新发变异 胚胎植入前遗传学检测 极体测序 等位基因脱扣 

分 类 号：R714.8[医药卫生—妇产科学] R440[医药卫生—临床医学]