DNA Pooling Identify A Variant In The GRIN2A Gene That Affects Disease Progression Of Chronic HBV Infection In Han Chinese Population

Background:Chronic hepatitis B virus (HBV) infection is a serious global public health problem. There are approximately 350 million people suffering from chronic HBV infection in the world and more than one million deaths annually attribute to HBV-related disease. The prevalence of chronic HBV infection is approximately 5% globally, but differs greatly from area to area. The prevalence of hepatitis B antigen (HBsAg) carriage is high in China (7.18%), that is there are 97 million HBsAg carriers in China and the number of chronic HBV carriers is 2 million in China. Most HBV carriers are considered to have been infected through maternal transmission in the neonatal period or infancy in high prevalent areas particularly in China. Persistent HBV infection has been considered a multifactorial and polygenic disequilibrium with viral, environmental, and host genetic components, including HBV genomic variability, age at infection, sex, chronic alcohol abuse, and co-infection with hepatitis C virus (HCV), hepatitis D virus (HDV) and human immunodeficiency virus (HIV). Though viral factors such as viral load, viral genotype and mutations in the viral genome itself are known risk factors for HBV infection, twin and ethical studies of HBV infection strongly support the role of genetic susceptibility to HBV persistence and the course of infection.Aims:The objective of this study was to identify novel susceptibility loci to HBV progression in Han Chinese chronic HBV carriers, we conducted a two-stage genome-wide association study for HBV infection and progression in Han Chinese population.Methods:Affymetrix Genome-Wide Human Mapping SNP6.0 Array with DNA pooling was used in the screening stage, and ten high-ranked single nucleotide polymorphisms (SNPs) were included in this panel. All HBV carriers were genotyped by using TaqMan method in the second stage. A total of 2798 unrelated Han Chinese HBV carriers were recruited from Hubei province (n=2357) in south of China (Wuhan Tongji hospital, Wuhan Union hospital and Traditional Chinese Medicine hospital of Hubei province) and Shandong province (n=441) in north of China (Qingdao hospital for Infection Disease and the Affiliated hospital of Binzhou Medical College) between September 2007 and March 2010 in our hospital-based case-control study. All HBV carriers were positive for both HBsAg and antibody to HBV core antigen of the immunoglobulin G type for at least 6 months. Among the 2798 HBV carriers,854 asymptomatic HBV carriers (AsC) (628 from Hubei province and 226 from Shandong province) and 1944 progressed HBV carriers (1729 from Hubei and 215 from Shandong) were included. Of the 1944 progressed HBV carriers,263 individuals were patients with fulminant hepatitis B (FHB),786 subjects with liver cirrhosis (LC) and 895 were patients with hepatocellular carcinoma (HCC). The 420 unrelated healthy Han Chinese were recruited as naive control (210 from Hubei and 210 from Shandong), who were HBV serum markers negative and no HB vaccination history. The AsC and naive controls were all over 35 years old to provide adequate time for symptoms to manifest. All subjects had no serologic evidence for co-infection with HCV, HDV and HIV. The criteria for selection of SNPs for further investigation was Silhouette width exceeding 0.7 and minor allele frequency (MAF)> 0.05. They were all tagSNPs using data from the International HapMap project (www. hapmap.org; CHB+JPT population). TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, Calif.) was used for genotyping according to the manufacture's instruction. Statistical analysis was performed using SPSS software (version 17.0; SPSS Inc, Chicago, IL).χ2 tests were used to examine the differences in clinical characteristics of participants. The adjusted Odds ratio (OR) and 95% confidence interval (95% CI) were tested by multivariable unconditional logistic regression under genetic models with adjustment for gender, age and alcohol consumption.Results:In Hubei population, on the basis of multivariable logistic regression with adjustment for gender, age and alcohol consumption, variant rs346473 in the ARHGAP24 gene, SNP rs11866328 in the GRIN2A gene, SNP rs7861010 in the BNC2 gene and variant rs12206945 in the ASCC3 gene were found having increased susceptibility with disease progression of HBV infection in south of China (1729 progressed HBV carriers vs 628 AsC). At variant rs346473 in the ARHGAP24 gene, TT genotype carriers were found having significant associations with disease progression of HBV infection (OR 1.849,95% CI 1.498-2.283, P=1.1 x 10-8; additive model). At variant rs346482 in the ARHGAP24 gene, TT genotype carriers were found having significant associations with disease progression of HBV infection (OR 1.516,95% CI 1.233-1.865, P=7.7×10-5; additive model). SNP rs11866328 in the GRIN2A gene, subjects bearing GG homozygote were susceptive to HBV progression compared with GT heterozygote carriers (OR,1.645; 95% CI, 1.340-2.020; P=1.96×10-6; additive model). HBV patients carrying GG genotype at SNP rs7861010 in the BNC2 gene displayed a strong correlation with disease progression of HBV infection (OR,1.305; 95% CI,1.059-1.607; P=1.2×10-2; additive model). Variant rs12206945 in the ASCC3 gene, A allele carriers performed an elevated effect for disease progression of HBV infection (OR,1.742; 95% CI,1.210-2.509; P=3.0×10-3; dominant model). Using genotypes of 420 naive controls, we defined these two SNPs (rs346482 and rs346473) in strong linkage disequilibrium with D'=0.99 and r2= 0.951 generated by software HaploView 4.1.When haplotype CC was chosen as a baseline, haplotype TT displayed a significant increased risk for developing into progressed hepatitis (OR,1.980; 95% CI,1.538-2.545; P= 8.1×10-8). To validate the results of the association test, we carried out a replication using population recruited from Shandong province (215 progressed HBV carriers vs.226 AsC) in north of China. The three noticeably correlated SNPs (rs11866328, rs7861010 and rs12206945) were genotyped using the same TaqMan assay. SNP rs11866328 in the tenth intron of GRIN2A gene was confirmed to have an elevated effect on diverse outcomes of HBV infection (OR 1.727; 95% CI,1.140-2.646; P= 1.0×10-2; additive model). Combining all population, subjects bearing GG homozygote of rs11866328 had an increased susceptibility to HBV progression (OR 1.684,95%CI 1.404-2.016, P=1.6×10-8). In stratification analysis of SNP rs11866328 in all studied HBV carriers, the results remained significant in male patients (OR,1.610; 95% CI,1.302-1.992; P= 1.1×10-5; additive model), female patients (OR,1.776; 95% CI,1.253-2.525; P= 1.0×10-3; additive model) and patients 35 years or older (OR,1.634; 95% CI,1.353-1.972; P= 3.1×10-7; additive model). Using data from the International HapMap project (www.hapmap.org; CHB+JPT population) and HaploView (version 4.1) software, we found SNP rs11866328 having linkage disequilibrium (LD) within a 10kb region with r2≥0.8Conclusions Performing Genome-wide association (GWA) studies on pools of DNA samples has been shown to be an efficient method to select candidate susceptibility loci for follow-up by individual genotyping. SNP rs11866328 in the GRIN2A gene could be a part of the genetic variants underlying the susceptibility of individuals to disease progression of chronic HBV infection.