Single Nucleotide Polymorphisms of Toll-Like Receptor 7 in Hepatitis C Virus Infection Patients from a High-Risk Chinese
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Single Nucleotide Polymorphisms of Toll-Like Receptor 7 in Hepatitis C Virus Infection Patients from a High-Risk Chinese Population Xing-xin Xue,1,2 Jian-ming Gong,1,2 Shai-di Tang,1,2 Chun-fang Gao,3 Jia-jia Wang,1,2 Li Cai,1,2 Jie Wang,4 Rong-bin Yu,1 Zhi-hang Peng,1 Nai-jun Fan,3 Chang-jun Wang,2 Jin Zhu,2 and Yun Zhang1,2,5
Abstract—Hepatitis C virus (HCV) infection varies in the outcomes depending on both viral and host factors. This study aims to investigate the associations of three single nucleotide polymorphisms (SNPs) of Tolllike receptor 7 (TLR7), rs179016, rs5743733, and rs1634323, with susceptibility to HCV infection and clearance. The three SNPs were genotyped in a high-risk Chinese population, including 444 HCV spontaneous clearance cases, 732 persistent infection cases, and 1107 healthy controls. The G allele of rs1634323 was related to the protection from persistent infection among females (dominant model: odds ratio (OR)=0.558, 95 % confidence interval (CI)=0.348–0.894, P=0.015). This protective effect was more evident in blood donation and HCV non-1 genotype-infected subgroups (all P50 years), hemodialysis (HD), and HCV-1 and HCV non-1 genotypes-infected subjects (all PG (rs5743733), and -7926A>G (rs1634323) were selected as candidates for the present study. The positions and regions of these selected SNPs were represented in Table S2. Genotyping Assays Genomic DNA was extracted from leucocytes in peripheral blood by sodium dodecyl sulfate lysis and protease K digestion and followed by phenol-chloroform extraction and ethanol precipitation. Genotyping of the three SNPs was performed by the TaqMan allelic discrimination assay on the ABI PRISM 7900HT Sequence Detection System (SDS) (Applied Biosystems, Foster City, CA, USA). The primers and probe sequences for the selected SNPs were listed in Table S2. Reactions were carried out in a 384-well format with a final volume of 5 μL, containing 1 μL
genomic DNA (10 ng/μL), 2.5 μL TaqMan Master Mix, 0.225 μL forward and reverse primer (20 pmol/μL) with a final concentration of 900 nM, 0.125 μL VIC and FAM (10 pmol/μL) with a final concentration of 250 nM, and 0.8 μL sterile double distilled H2O (ddH2O). All the genotyping was performed without knowing the subjects’ case or control status. Each plate consisted of three blank samples as negative controls and two samples from the same individual as positive controls for the confirmation of genotyping quality. The accordance rate of each SNP was 100 % for the repeated experiments of 10 % random samples. Statistical Analysis Student’s t test, χ2 test, and Kruskal-Wallis test were used to evaluate the distributions of the general demographic, clinical, and virological features between cases and controls when appropriate. Hardy-Weinberg equilibrium (HWE) test for each SNP among females was conducted by using a goodness-of-fit χ2 test. PHASE 2.0 software was used to estimate the haplotype frequencies of the three polymorphisms [19]. The associations between HCV infection and SNPs were determined by computing th
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