SNP Screening of Five Candidate Genes and Study of Their Genetic Association with Chronic Hepatitis B Virus Infection

Abstract: Hepatitis B virus (HBV) infection is one of the major infectious diseases with more than 350 million chronic carriers worldwide, which causes a broad spectrum of liver diseases ranging from asymptomatic carrier, fulminant hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Persistent HBV infection has been considered as a multifactorial and polygenic disorder with viral, environmental and genetic components. HBV genomic variability and a number of conventional risk factors, including age, gender, concurrent infection with the hepatitis C virus (HCV), hepatitis D virus (HDV) and human immune deficiency virus (HIV), are clearly the important factors contributing to the incidence of persistent HBV infection. However, segregation analysis and twin studies strongly support the role of host genetic components in determining the chronicity of HBV infection. Genetic association analyses based on the Gambia, European and Asian cohorts have implicated the HLA allele DRB1*1302, tumor necrosis factor α, mannose binding protein and vitamine D receptor were associated with the persistent HBV infection or HBV clearance. An unknown number of other unidentified genes are likely to modify the susceptibility to persistent HBV infection. For genetic susceptibility studies of common complex diseases, the population-based linkage disequilibrium / association study has much higher power than classic family-base linkage analysis. Single nucleotide polymorphism (SNP) is a powerful tool for identifying genetic susceptibilities to common complex diseases in recent years. Several studies have shown the important role of estrogen receptor α (ESR1), androgen receptor (AR), MHC-II transactivator (C2TA), CD209 and CXCL10 in chronic HBV infection and HBV-related liver desease, however, the importance between their genetic polymorphisms and chronic liver diseases has not been studied. In this study, we systematically identify polymorphisms in these 5 genes by sequencing, examine the relationship between their polymorphisms and chronic HBV infection in a large HBV-infected population of Chinese and further validate the biological functions of positive associated SNP loci.DNA samples from 27 unrelated Chinese individuals unselected for disease status and 5 DNA pools were used for SNP discovery. 2489 unrelated Chinese were screened for this study from 4112 individuals. 1405 patients with chronic HBV infection (421 with asymptomatic carrier, 544 with chronic hepatitis B, 312 with liver cirrhosis and 92 with hepatocellular cancer) and 749 unrelated convalescents who recovered from self-limited HBV infection were used as case control population. 74 individuals who had been exposed to HBV but remained uninfected (EUI) and 261 blood donors were used for general reference population. 315 nuclear families were enrolled for transmission disequilibrium test (TDT), each nuclear family comprised one proband and his (her) kindred parents. The leukocytes genomic DNA was isolated and DNA panels for genotyping were made. Overlapping primer sets covering genomic regions were designed, and the 5 candidate genes mentioned above were screened for SNPs by PCR-based re-sequencing. Assembled contigs were reviewed in Consed and SNPs were identified by PolyPhred. Haplotypes were constructed by PHASE program. Pair-wise linkage disequilibrium (LD) between SNPs was analysed by LDA. Haplotype tagging SNPs (htSNPs) which resolve existing common haplotypes were infered by software htSNP2. SNPs were selected for genotyping in case, control populations and nuclear families using PCR-restriction fragment length polymorphism (PCR-RFLP), allelic-specific PCR or direct sequencing. (2 tests and unconditional logistic regression models were used for calculating odds ratios (95% confidential intervals) and corresponding P values controlling age and sex. A within-family Monte Carlo permutation procedure and (2 test were performed by TDT/S-TDT program1.1. To further validate the biological functions of positive associated SNP loci, electrophoresis mobilit…
Key words: hepatitis B virus; single nucleotide polymorphism; linkage disequilibrium; estrogen receptor; androgen receptor; CXCL10; CD209; C2TA; haplotype; transmission disequilibrium test; electrophoresis mobility shift essay; allele