长读长测序在多囊肾患者胚胎植入前遗传学检测中的应用  

Application of long⁃read sequencing in preimplantation genetic testing for patients with polycystic kidney disease

作　　者：李伍高 李哲涛 范莉 唐永梅 唐宁 秦雪[1] LI Wugao;LI Zhetao;FAN Li;TANG Yongmei;TANG Ning;QIN Xue(Department of Clinical Laboratory,Guangxi Medical University,the First Affiliated Hospital of Guangxi Medical University,Nanning,Guangxi,China,530021;Reproductive Centre of Liuzhou Maternity and Child Health Care Hospital,Liuzhou Key Laboratory of Reproductive Medicine,Liuzhou,Guangxi,China,545001;Guangxi Clinical Research Center for Obstetrics and Gynecology,Liuzhou,Guangxi,China,545001)

机构地区：[1]广西医科大学,广西医科大学第一附属医院检验科,广西南宁530021 [2]柳州市妇幼保健院生殖健康助孕中心,柳州市生殖医学重点实验室,广西柳州545001 [3]广西妇产疾病临床医学研究中心,广西柳州545001

出　　处：《分子诊断与治疗杂志》2024年第9期1613-1617,共5页Journal of Molecular Diagnostics and Therapy

基　　金：广西科技计划项目(AD22035223)。

摘　　要：目的探讨通过长读长测序技术构建SNP单体型,为成人常显多囊肾基因(PKD1)致病变异且家系不全患者行胚胎植入前遗传学检测策略提供参考。方法选取2022年1月至2022年9月于柳州市妇幼保健院生殖健康助孕中心就诊的两名患者,分别患有PKD1 c.5976_5978del杂合变异复合PKD1 c.11333C>A杂合变异和PKD1 c.11969 T>G杂合变异导致的常染色体显性遗传多囊肾且家系不全,采集患者夫妻双方外周血提取高质量DNA,通过长读长测序技术构建SNP单体型。活检胚胎滋养外胚层细胞进行全基因组扩增,再利用ASA芯片检测胚胎SNP位点信息,结合长读长测序构建的男女方SNP位点建立连锁分析,分析胚胎变异位点携带情况;同时以Sanger测序检出的受累胚胎作为先证者,利用Karyomapping芯片检测分析胚胎SNP位点单体型;比较两种连锁分析结果的一致性。结果病例1检测5个胚胎,病例2送检4个胚胎。病例1的1号,3号和4号胚胎携带男方PKD1c.5976_5978del致病变异,病例2的1号及4号胚胎均携带有男方PKD1 c.11969T>G致病变异;经Sanger测序均选择1号胚胎作为参照,利用Karyomapping芯片进行连锁分析,病例1的1号,3号和4号,病例2的1号及4号胚胎均携带男方的致病变异,结果与长读长测序家系连锁分析一致。结论对于家系不全PKD1患者,可通过长度长测序进行男女双方的致病位点确定及SNP单体型构建,通过SNP单体型建立连锁分析,从而准确有效的进行胚胎植入前遗传学检测。Objective To explore the construction of single nucleotide polymorphism(SNP)hap-lotype based on the technology of long-read sequencing,to provide reference for preimplantation genetic test-ing strategies for patients with adult polycystic kidney disease(PKD1)pathogenic mutations and incomplete pedigrees.Methods Two patients visited the Reproductive Health Assisted Pregnancy Center of Liuzhou Ma-ternal and Child Health Hospital from January 2021 to September 2022.Both patients had autosomal dominant polycystic kidney disease caused by gene variations of PKD1 c.5976_5978del,PKD1 c.11333C>A and PKD1 c.11969 T>G heterozygous mutations(with incomplete pedigree).Peripheral blood samples were collected from the husband and wife of these patients to extract high-quality DNA.SNP haplotypes were constructed us-ing long-read sequencing technology.Biopsies of trophoblastic ectodermal cells were performed for whole-ge-nome amplification,followed by the detection of embryo SNP site information using the Illumina Asian Screening Array(ASA).Linkage analysis was then established by combining the SNP sites of males and fe-males constructed by long-read sequencing to analyze embryo mutations.Simultaneously,affected embryos de-tected by Sanger sequencing were used as probands for Karyomapping to detect and analyze embryo SNP hap-lotypes Finally,the results of the two linkage analyses were compared for consistency.Results Five embryos were tested for Case 1,and four embryos for Case 2.In case 1,embryos 1,3,and 4 carried the PKD1 c.5976_5978del pathogenic variant from the male side.In case 2,embryos 1 and 4 carried the PKD1 c.11969T>G pathogenic variant from the male side.After Sanger sequencing,Karyomapping chips were used for linkage analysis.Using the affected embryo 1 as the reference.It was found that embryos 1,3,and 4 of case 1,and embryos 1 and 4 of case 2 all carried the pathogenic variant from the male side.This was consistent with the linkage analysis results from the long-read sequencing family.Conclusion For patients with

关 键 词：PKD1基因 多囊肾 单体型构建 植入前遗传学检测 

分 类 号：R714.8[医药卫生—妇产科学] R692[医药卫生—临床医学]