家族性脂蛋白脂酶缺乏症脂蛋白脂酶基因未报道变异及功能学分析  

Discovery of a novel variant of lipoprotein lipase gene in familial lipoprotein lipase deficiency based on the whole exome sequencing and its functional study

作　　者：陈腊梅 郝婵娟 陈元颖 胡旭昀 巩纯秀[2] 李巍[1] CHEN Lamei;HAO Chanjuan;CHEN Yuanying;HU Xuyun;GONG Chunxiu;LI Wei(Department of Beijing Key Laboratory for Genetics of Birth Defects,Beijing Pediatric Research Institute,MOE Key Laboratory of Major Diseases in Children,Genetics and Birth Defects Control Center,National Center for Children's Health,Beijing Children's Hospital,Capital Medical University,Beijing 100045,China)

机构地区：[1]首都医科大学附属北京儿童医院国家儿童医学中心北京市儿科研究所出生缺陷遗传学研究室儿科重大疾病研究教育部重点实验室出生缺陷遗传学研究北京市重点实验室,100045 [2]首都医科大学附属北京儿童医院内分泌遗传代谢科,100045

出　　处：《心肺血管病杂志》2020年第8期999-1005,1015,共8页Journal of Cardiovascular and Pulmonary Diseases

基　　金：科技部重点研发计划(2016YFC1000306);北京市科委医药协同科技创新研究项目(Z181100001918003);北京市首发基金(首发2018-2-1141);北京市医管中心儿科协同项目(XTCX201807,XTZD201805)。

摘　　要：目的:探讨功能学研究在明确高脂血症致病基因未报道变异致病性判读中的有效性。方法:收集患儿及其父母的外周血DNA,利用全外显子组测序(WES)发现致病基因并进行一代测序验证,通过美国医学遗传学会的变异位点解读指南对检测到的变异位点进行分析;针对未报道的变异,分别构建野生型和携带有未报道变异位点的突变型基因的真核表达载体,转染COS-7细胞后,检测两组细胞中脂蛋白脂酶(LPL)的表达、分泌及酶活性。结果:该患者核心家系的WES结果提示,患者LPL基因存在复合杂合变异c.590G>A(p.R197H)和c.940G>A(p.G314S)。通过变异位点解读指南评估LPL c.590G>A(p.R197H)为可能致病变异。未报道变异LPL c.940G>A(p.G314S)的功能学实验表明,体外过表达LPL-G314S突变体蛋白,其胞浆内LPL蛋白表达量与野生型相比差异无统计学意义,但分泌量显著减少;LPL酶活性检测显示,LPL-G314S突变体的细胞裂解液和细胞培养液中LPL酶活性均比野生型显著降低。综合证据提示该未报道变异为致病性变异。结合患儿临床表现和遗传诊断结果最终诊断为家族性脂蛋白脂酶缺乏症。结论:功能学实验增强了未报道变异位点致病性判读的证据级别,为遗传诊断和临床确诊提供重要依据。Objective:To explore the effectiveness of functional studies to be applied accurately and consistently in variant interpretation of candidate genes on hyperlipidemia.Methods:DNA was collected from peripheral blood of patients and family members.Whole exome sequencing(WES)was used to detect the core family exomes and selected variants were further validated by Sanger sequencing.The detected variants were filtered and analyzed according to the standards and guidelines for the interpretation of sequence variants recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.The novel variant was verified by functional assays.Wild-type and mutant genes with the novel variant site were constructed to eukaryotic expression vectors,respectively.After transient transfection of COS-7 cells,the expression and secretion of lipoprotein lipase(LPL)were detected by western blot,and the lipoprotein lipase activity was measured by the lipoprotein lipase activity assay kit.Results:Trio-based sequencing Results:indicated that the compound heterozygous variants c.590G>A(p.R197H)and c.940G>A(p.G314S)in LPL gene were found.The variant LPL c.590G>A(p.R197H)was classified as likely pathogenic variant by interpretation guidelines.Functional study of the novel variant LPL c.940G>A(p.G314S)showed that cytoplasmic expression of LPL was not significantly different between the wild-type and mutant protein(LPL-G314S),but the secretion level of the mutant LPL protein(LPL-G314S)was significantly lower than that of wild-type.The lipoprotein lipase activity from both the cell lysate and cell culture medium was significantly lower than that of wild-type.The novel variant was classified as pathogenic variant by interpretation guidelines.The patient was diagnosed as familial lipoprotein lipase deficiency.Conclusions:Functional studies can provide powerful insights into the effects of a novel variant on LPL protein function,underscoring the need for genetic and clinical diagnosis.

关 键 词：家族性脂蛋白脂酶缺乏症 脂蛋白脂酶基因 全外显子组测序 功能学分析 

分 类 号：R54[医药卫生—心血管疾病]