64个常染色体显性多囊肾病家系的基因检测及产前诊断

Genetic testing and prenatal diagnosis of 64 pedigrees with autosomal dominant polycystic kidney disease

目的检测和分析64个无亲缘关系的常染色体显性多囊肾病(autosomal dominant polycystic kidney disease,ADPKD)家系的基因变异类型,探讨多种基因分析技术的检测效能和基因变异特点。方法该研究为横断面研究,回顾性分析2017年12月至2020年8月就诊于郑州大学第一附属医院肾脏内科或遗传与产前诊断中心的64个ADPKD家系的临床资料,采集先证者和家系成员血样,应用二代测序对先证者进行基因检测,对筛选出的可疑变异通过多重连接依赖式探针扩增或长片段PCR结合Sanger测序进一步验证。抽取高风险家系胎儿绒毛或羊水样本,排除母源污染后进行产前基因诊断。结果64个ADPKD家系中有57个家系(89.06%)检测到PKD1/PKD2基因变异,其中51个家系(79.69%)共检出49种PKD1/PKD2基因致病性/可能致病性变异,包含14种(28.57%)无义变异、14种(28.57%)移码变异、11种(22.45%)错义变异、5种(10.20%)剪接变异和5种(10.20%)缺失变异,PKD1和PKD2基因变异分别占87.76%(43/49)和12.24%(6/49)。共检测到14种PKD1/PKD2基因新变异,包含7种移码变异、3种剪接变异、2种无义变异、1种缺失变异和1种错义变异,其中11种为PKD1基因变异,3种为PKD2基因变异。来自17个家系的20例高风险胎儿接受了产前基因诊断,6例胎儿检出PKD1基因杂合变异,14例胎儿未检出PKD1/PKD2基因变异。结论联合应用二代测序、多重连接依赖式探针扩增及长片段PCR结合Sanger测序可对ADPKD家系进行快速、高效和准确的基因诊断。PKD1/PKD2基因变异均以点突变最为常见。14种PKD1/PKD2基因新变异的检出丰富了其基因变异谱,可为ADPKD家系的遗传咨询和产前诊断提供依据。

Objective To detect and analyze the gene variation types of 64 unrelated pedigrees affected with autosomal dominant polycystic kidney disease(ADPKD),and explore the detection efficiency of multiple gene analysis techniques and variation characteristics.Methods It was a cross-sectional study.The clinical data of 64 pedigrees with ADPKD from Nephrology Department or Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from December 2017 to August 2020 were retrospectively analyzed.The blood samples of probands and other family members were collected.Genetic analysis was carried out by next generation sequencing,and suspected mutations were verified by multiplex ligation-dependent probe amplification,or long-range PCR combined with Sanger sequencing.Prenatal diagnosis for high-risk fetuses was performed by fetal villi or amniotic fluid cells after genotyping without maternal genomic DNA contamination.Results Among detected 64 pedigrees,57 pedigrees(89.06%)had genetic variants in PKD1/PKD2.A total of 49 pathogenic/likely pathogenic variants in PKD1/PKD2 were identified in 51 pedigrees(79.69%),including 14 nonsense variants(28.57%),14 frameshift variants(28.57%),11 missense variants(22.45%),5 splicing variants(10.20%)and 5 deletion variants(10.20%).Of these variants,87.76%(43/49)were in PKD1 and 12.24%(6/49)were in PKD2.Totally,14 novel variants in PKD1/PKD2 were identified,including 7 frameshift variants,3 splicing variants,2 nonsense variants,1 deletion variant and 1 missense variant,of which 11 variants were in PKD1 and 3 variants were in PKD2.Twenty high-risk fetuses from 17 pedigrees received prenatal diagnosis,in whom 6 fetuses had PKD1 variation,and other 14 fetuses had no PKD1/PKD2-genetic variation.Conclusions The combination of next-generation sequencing,multiplex ligation-dependent probe amplification,and long-range PCR combined with Sanger sequencing can be helpful for rapid,efficient and accurate genetic diagnosis of ADPKD pedigrees.Point mutations are the most common types in PKD1/PKD2.Fourteen novel variants in PKD1/PKD2 extend its pathogenic variant spectrum and can provide basis for genetic counseling and prenatal diagnosis of ADPKD pedigrees.