Characterization of polymorphisms in TNF-α, HLA-DRB1 and HLA DQB1 in patients with Hepatitis C and relevance to treatment response and outcome

Abstract

Hepatitis C virus (HCV) is a major cause of chronic liver infection and is an important public health challenge in Pakistan. Host immunological factors are accountable for the differential outcome of HCV infection and response to anti-HCV therapy. However, not much is known about the spontaneous recovery and response to anti-HCV therapy for the patients under treatment. Moreover, prior to this report, none of the studies in Pakistan focused on the role of genetic polymorphism in key immunological factors TNF-α, HLA DRB1 and DQB1 genes. This study was carried out to determine the relationship of polymorphisms in TNF-α, HLA-DRB1, DQB1 genes and their possible association with response to anti-HCV therapy. Sampling for this study was done by collecting blood samples from the patients who were visiting Nuclear Medicine Oncology and Radiotherapy Institute (NORI) Islamabad. Patients under scrutiny were positive for anti-HCV and information regarding each patient’s demography, signs and symptoms and risk factors were obtained using a designed proforma. Consent was obtained from all the patients whereas, ethical approval for this study was granted by Institutional Review Board of Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad. Samples were subjected to viral RNA quantification, along with genotyping of HCV. After viral RNA analysis, human DNA extraction was done by using phenol chloroform method. The extracted DNA was transported to the Laboratory of Virology and Infectious Disease, The Rockefeller University, New York USA. Three genes (TNF-α, HLA-DRB1, DQB1) were targeted for single nucleotide variation analysis. Primers were designed for HLA-DRB1, DQB1genes, whereas already reported primers were used for the amplification of TNF-α gene. PCR conditions were optimized for each gene and amplicons were confirmed on agarose gel by comparing the desired product size with a DNA marker. After PCR amplicons were purified using gel purification kit. DNA sequencing was done by Macrogen New York. After sequencing peak analysis of the sequences were carried out followed by patient's genotype and allele distributions and mapping of the identified SNPs. Haplotype determination was carried out for the studied SNPs and statistical analysis was performed. Gender and demographic data analysis for the studied patients revealed that the distribution of male and female patients in this study was almost equal, 49% vs 51%. The most common signs and symptoms distributed in studied group of patients were jaundice, fatigue, abdominal discomfort, anorexia and malaise and revealed a significant association with HCV infection in studied patients (p<0.05). Demographic analysis showed that most of the patients analyzed in this study belonged to Rawalpindi and Islamabad region, followed by Punjab and Khyber Pakhtunkhwa. Similarly analysis for age groups revealed that most of the patients fell into age group, 31-40 years. Distribution of the risk factors in examined individuals revealed that the most prevalent risk factors were injections, intravenous infusions, barbers visits and skin piercing respectively (p<0.05). HCV genotype 3a was established to be the most prevalent genotype in the studied group of patients. Similarly literacy rates analysis depicted that the 86% of the patients were literate whereas, 14% of the patients were illiterate with matriculation being the most common education level followed by primary and middle education. Occupational information for the studied patients showed that the most of the male patients had Government jobs, laborer and shopkeeper respectively, whereas, most of the female patients were housewives followed by teachers. Viral RNA detection analysis depicted that viral RNA was detected in total of 51% of the studied patients, whereas the peak load in males was found to be 51,809,739 IU/ml, similarly the maximum HCV RNA load in female patients was reported to be 31,874,791 IU/ml respectively. Average viral load in both male and female patients was reported to be 5,315,791 IU/ml and 3,180,912 IU/ml, while the cut off value for viral RNA detection were 12 IU/ml. All the included patients in the study were categorized into two group that included therapy group and non-therapy group. Therapy group was further divided into responsive to interferon/ribavirin group (R) and non-responsive to interferon/ribavirin therapy group (NR). The non-therapy group was divided into chronically infected (CI) group and spontaneously recovered groups (SR). After sequencing analysis the obtained traces were analyzed for the presence of SNPs using different softwares, a total of twenty SNPs were examined in the analyzed set of patients. Three SNPs in Tumor necrosis factor alpha (TNF-α) were studied, whereas seven SNPs were analyzed in HLA-DRB1 gene. In total ten SNPs were studied in HLA-DQB1 gene and it was found that five of the total ten SNPs were novel. Mapping of the SNPs was carried out and we showed that SNPs in TNF-α gene mapped to the promoter region, similarly SNPs in HLA-DRB1gene mapped to the exon 2 of the gene. HLA-DQB1 SNPs mapped to the promoter region of HLA-DQB1 gene. After mapping genotyping of the studied SNPs were carried out followed by allelic and haplotype determination. Genotype analysis showed that genotypes at TNF-α were not significantly associated with therapy or HCV disease outcome. However, differences in genotype distributions at -8362 position in the HLA-DQB1 gene were statistically significant among spontaneously recovered and chronically infected patients. Similarly genotypes, TT and CC at position 6151 (rs1064663) in the HLA-DRB1 gene were significantly associated in both male and female patients whereas, genotype distribution at 6231 (rs3167799) position in HLA-DRB1 gene was found to be significantly associated with HCV infection in both male and female patients. Allelic distributions at the analyzed position showed that that allele T at 6228 (rs230382) position in HLA-DRB1 gene was significant predictor of response to anti-HCV therapy (IFN/RBV combinatorial therapy). Similarly a novel allele in the HLA-DQB1 gene at -8447 position was also responsible for response to interferon/ribavirin therapy in studied patients. Allele C/T distribution in both male and female patients in HLA-DRB1 gene at 6151 (rs1064663) position was found to be statistically significant in male and female patients. Similarly Allele T/C variation at 6228 (rs230382) position in the HLA-DRB1 gene was found to have a statistically significant association with infection outcomes in male and female patients, whereas allelic distribution at 6231 (T/C/G) (rs3167799) position revealed that allele G and T were significantly associated with defining the fate of HCV infection in male and female patients. Multivariate logistic regression analysis for HLA-DRB1 (rs2308802) and HLA-DQB1 (-8471) showed that these variations were responsible for IFN/RBV therapy response. Haplotype analysis showed that none of the haplotypes were associated with therapy response or outcome of HCV infection. Linkage disequilibrium analysis for the genotypes revealed that there was genetic linkage among various genotypes at identified SNPs in HLA-DRB1 and HLA-DQB1gene.