全外显子组测序鉴定七例原发性胆汁性胆管炎家系患者的致病突变基因  

Identification of pathogenic mutant genes in seven families with primary biliary cholangitis patients by whole exome sequencing

作　　者：刘鑫[1] 李燕妮[1] 王一 褚洪玉 张洁[1] 王邦茂[1] 周璐[1] Liu Xin;Li Yanni;Wang Yi;Chu Hongyu;Zhang Jie;Wang Bangmao;Zhou Lu(Department of Gastroenterology,General Hospital of Tianjin Medical University,Tianjin 300052,China;Department of Intensive Medicine,Beijing Youan Hospital,Capital Medical University,Beijing 100069,China)

机构地区：[1]天津医科大学总医院消化科,300052 [2]首都医科大学附属北京佑安医院重症医学科,100069

出　　处：《中华消化杂志》2021年第2期118-124,共7页Chinese Journal of Digestion

基　　金：国家自然科学基金(81860109)。

摘　　要：目的通过全外显子组测序(WES)技术筛查原发性胆汁性胆管炎(PBC)家系共有的低频变异位点,以期发现与PBC相关的新易感基因。方法收集2000年1月至2017年12月于天津医科大学总医院诊断的3个PBC家系的7例PBC患者和2名健康对照者的临床资料,提取血液DNA样本并进行WES,应用SAMtools 1.3软件检测基因单核苷酸多态性(SNP)和得失位变异位点,并于已知数据库1000 Genome、ExAC、ESP6500和诺禾-中华基因数据库筛选低频变异位点。应用Pymol V2.3.2软件模拟主要组织相容性复合体-Ⅱ(MHC-Ⅱ)分子三维结构,观察家系间共有变异位点对应的氨基酸位置。结果7例PBC患者首诊年龄为(61.2±10.2)岁。血清检测结果示7例患者的ALP为(306.9±242.5)U/L,GGT为(121.7±85.9)U/L,ALT为(47.6±33.1)U/L,AST为(55.7±34.1)U/L,免疫球蛋白G为(14.9±3.1)g/L;抗核抗体核型均存在胞质颗粒型特征;抗线粒体抗体均为阳性。5例PBC患者出现腹腔内淋巴结肿大;2例患者合并肝外自身免疫病;2例患者肝脏活组织检查结果均提示界面性肝炎和小胆管病变。3个PBC家系间共有18个SNP位点,分别位于OTOA、OBSCN和人类白细胞抗原-DRB1(HLA-DRBI)基因。OTOA基因的rs200988634是3个家系共有的多态性位点;OBSCN基因的rs746424683、rs545316651、rs553144914、rs533059830和rs56087721分别导致9种氨基酸的改变;基因HLA-DRB1共有12个不同的SNP位点,分别导致12种氨基酸的改变,其中rs16822698、rs112796209和rs11554463核苷酸突变分别导致MHC-Ⅱ分子β链的G154A、Y152C和Y107X氨基酸改变,Y107X氨基酸位于MHC-Ⅱ分子与抗原肽结合凹槽区域。结论对PBC家系进行WES是阐明恶性变异候选基因OBSCN和OTOA较好的策略。HLA-DRB1为PBC的易感基因,可能通过改变氨基酸序列影响MHC-Ⅱ分子介导的抗原提呈过程。Objective To screen the common low-frequency mutation sites in primary biliary cholangitis(PBC)by whole exome sequencing(WES),in order to find PBC-related new susceptibility genes.Methods From January 2000 to December 2017,the clinical data of seven patients with PBC of three PBC families diagnosed at General Hospital of Tianjin Medical University and two healthy controls were collected.The DNA blood samples were extracted and analyzed by WES.SAMtools 1.3 software was used to detect gene single nucleotide polymorphism(SNP)and indel sites,and gene mutation sites were screened from known databases of 1000 Genome,ExAC,ESP6500 and Novo-Zhonghua gene database.Pymol V2.3.2 software was performed to simulate the three-dimensional structure of major histocompatibility complex-Ⅱ(MHC-Ⅱ),and the amino acid position corresponding to the common mutation sites among families were observed.Results The age of first diagnosis of seven PBC patients was(61.2±10.2)years.The results of serum test of seven patients indicated that alkaline phosphatase(ALP)level was(306.9±242.5)U/L,γ-glutamyltranspeptidase(GGT)level was(121.7±85.9)U/L,alanine aminotransferase(ALT)level was(47.6±33.1)U/L,aspartate aminotransferase(AST)level was(55.7±34.1)U/L and immunoglobulin G level was(14.9±3.1)g/L.The antinuclear antibody were all cytoplasmic granule types and anti-mitochondrial antibody were all positive.Five PBC patients developed intra-abdominal lymphadenopathy;two patients had extrahepatic autoimmune diseases and the pathological results of liver biopsy of two patients both showed interface hepatitis and small bile duct lesions.Eighteen SNPs were common in three PBC families,which were located in the gene of OTOA,OBSCN and human leucocyte antigen-DRB1(HLA-DRB1).rs200988634 located in OTOA gene was a common polymorphic locus among the three families.rs746424683,rs545316651,rs553144914,rs533059830 and rs56087721 located in OBSCN caused the changes of nine amino acids of different location.There were 12 SNP variations located in HLA-DRB1

关 键 词：原发性胆汁性胆管炎 家系分析 全外显子组测序 HLA-DRB1基因 

分 类 号：R575.7[医药卫生—消化系统]