Identification of pathogenic mutant genes in seven families with primary biliary cholangitis patients by whole exome sequencing / 中华消化杂志

ABSTRACT

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 gene, which leaded to the changes of 12 amino acids of different location, among them rs16822698, rs112796209 and rs11554463 mutation induced G154A, Y152C and Y107X amino acid variation of MHC-Ⅱ beta chain, and Y107X amino acid was located in the groove region of MHC-Ⅱ binding with peptide. Conclusions:WES in PBC families is a good strategy to elucidate the candidate deleterious mutation genes OBSCN and OTOA. HLA- DRB1 which is a susceptible gene of PBC may affect MHC-Ⅱ mediated antigen presentation process by the changing amino acid sequence.