摘要
目的在一个肝豆状核变性(WD)家系中进行致病突变鉴定和产前基因诊断。方法酚-氯仿法提取外周全血或妊娠13周胎儿绒毛组织基因组DNA;利用PCR-Sanger测序技术对先证者ATP7B基因外显子及外显子/内含子衔接区序列进行突变分析;针对先证者携带的突变位点,应用聚合酶链反应(PCR)-高分辨熔解曲线(HRM)方法对先证者家系4名成员进行突变鉴定,并在此基础上为患儿母亲提供产前基因诊断。结果先证者ATP7B基因第8外显子存在纯合致病突变c.2333G>T(p.R778L);该家系5名成员和4名群体正常样本分属于3种不同熔解曲线。HRM分析结果与测序结果一致,即:患儿本人为R778L的纯合子,其父母、姐姐和母亲产前诊断的胎儿均为突变杂合子,4名群体正常对照为纯合野生型。结论在一个WD家系中检测到ATP7B基因热点突变c.2333G>T(p.R778L),并针对该突变建立了一种基于PCR-HRM技术的快速突变鉴定方法,成功为家系提供产前基因诊断。
Objective To identify the ATP7 B mutation and perform the prenatal gene diagnosis in a family with Wilson’s disease（ WD）. Methods Genomic DNA was extracted from peripheral blood and the fetal villi tissue by standard phenol / chloroform method. PCR and Sanger DNA sequencing were used to analyze the causative mutation in the proband; Mutation identification was performed in other 4 familial members including a fetus with high risk of WD by PCR-HRM. In the same way,a prenatal gene diagnosis was completed successfully in the mother of the proband. DNA sequencing was used to validate the genotype of the samples in different HRM-curves. Results The proband was a homozygote of the missense mutation c. 2333 G 〉 T（ p. R778L）,located in the exon 8 of gene ATP7 B. HRM analysis displayed three different curves,representing three different genotypes. The curve types for the 5 familial members and 4 normal control samples were consistent with genetic sequencing： the proband himself was a mutant homozygote,all the other family members were the heterozygote of the mutation,and the 4 normalcontrols were wild type. Conclusions The authors found a known mutation of p. R778 L in a WD family,and the prenatal gene diagnosis was achieved in a fetus with high risk by PCR-HRM.
出处
《基础医学与临床》
CSCD
2016年第2期218-221,共4页
Basic and Clinical Medicine
基金
"十一五"国家科技支撑计划(2006BAI05A03)
