Studies on Human Genomic Variations Associated with Responses to HCV Infection and Therapies

Abstract

Hepatitis C virus is a major cause of chronic liver disease, with more than 150 million people are infected worldwide. HCV infection is highly prevalent in developing countries in comparison to developed countries. About 10 million Pakistanis are chronically infected with HCV. Acute HCV infection is resolved within six months without any treatment in about 20-30% of infected individuals due to host immune response against the infection, while remaining develop chronicity. Untreated chronic HCV infection leads to liver cirrhosis in 20-30% of infected individuals, which progresses to hepatocellular carcinoma, ultimately leading to death. According to the World Health Organization 0.4 million people die annually worldwide because of HCV related chronic liver diseases. Host genetic variants have significant role in the resolution of HCV infection and prediction of response to antiviral therapies. The genetic structure of racial and ethnic populace influences the elimination of HCV infection. African populations exhibit poor response, while Asian populations demonstrate high SVR rate to antiviral therapies. The aim of this study is to analyze the frequency distributions of different alleles associated with HCV infection and therapeutic response in global populations and to evaluate the SNPs associated with response to HCV 3a infection and peg IFN and RBV antiviral therapies in Pakistani population. During the course of the present study, a list of 203 genes associated with HCV infection was prepared by using different manually curated databases including PharmGKB (Pharmacogenomics Knowledgebase), Pharmacogenomic Mutation Database (PGMD), Disease Ontology Annotation Framework (DOAF), and literature survey. Moreover, 263 genetic variants were enlisted by literature searches, with significant contribution in the resolution of HCV infection and the modulation of the side effects of therapeutic agents. Whole genome and exome sequence data of Pakistani individuals from 1000 Genomes Project (PJL, n=96) and ExAC Project (SAS, n=8,256), was analyzed by using different bioinformatics tools against the enlisted genes. Functional annotation of common variants by ANNOVAR and CADD tools revealed 37 deleterious variants from PJL and 49 from ExAC-SAS database in protein coding region of the enlisted genes. Filtration of non coding variants with CADD (phred score ≥15) showed 788 deleterious variants from PJL while 387 from ExAC-SAS data. xiv The comparative analysis of allele frequencies of associated alleles revealed that Pakistani population exhibits similar allele frequencies pattern as compared to other South Asian populations, and less diverse with European and American populations, while highly diverse with East Asian and African population. The allele frequencies of associated variants from IFNL3 gene were found higher among East Asian while lower in African populations. Pairwise linkage disequilibrium analysis of IFNL3 variants showed higher linkage in EAS, SAS, EUR, and AMR, while population from Africa showed poor linkage pattern. The population genetic differentiation highlighted that Pakistani population is well differentiated with East Asian and African populations in comparison to other populations. The principle component analysis showed that Pakistani and other SAS subpopulations are grouped with European and American populations in terms of common genetic variants associated with HCV infection and therapeutic responses. To evaluate the genetic variants reported to be associated with the prediction of treatment response and spontaneous HCV clearance; genome wide genotyping of 48 Pakistani individuals treated with peg IFN and RBV and representing treatment responders (n=21) and non-responders (n=21), along with cases of spontaneous HCV clearance (n=6) was carried out using infinium global screening BeadChip array. The functional annotation using ANNOVAR revealed most of the variants to be intergenic followed by intronic, exonic, downstream and upstream. Polyphen-2, SIFT and CADD based annotation predicted 1811 variants as deleterious. All genotyped variants were filtered with ClinVar database. 16 Variants were identified as likely pathogenic, 36 as pathogenic, 4 variants were associated with the prediction of therapeutic response and 2 were associated with toxicity against peg IFN and RBV therapies. Finally, 82 genetic variants, reported to be associated with HCV infection and therapeutic response, were analyzed to evaluate their association with response to peg IFN and RBV therapy and spontaneous HCV clearance in Pakistani population. A total of 28 SNPs, mainly from IFNL3 locus (n=12) were associated with the prediction of SVR. It is observed that rs8103142-TT (Dominant model: OR=0.41, CI 95%; 0.11 to 1.56, p=0.19), rs28416813-CC (Dominant model: OR=0.38, CI 95%; 0.11 t o1.31, p=0.15), rs74597329- TT (Dominant model: OR=0.55, CI 95%; 0.16 to 1.92, p=0.35), rs12979860 CC (Dominant model: OR=0.55, CI 95%; 0.16 to 1.92, p=0.35), and rs12980275-AA (Dominant model: OR=0.55, CI 95%; 0.16 to 1.92, p=0.35) from IFNL3 are significantly involved in the prediction of SVR against HCV 3a infection as well as associated with spontaneous HCV clearance. Variants from HLA gene such as rs4273729 GG, andxv rs9469220 AA; rs2229857 GG from ADAR, rs11730582 CC from SPP1 and rs3775290 GG from TLR3 are associated with the prediction of SVR in HCV infected patients. Few variants from all patients were validated using Sanger sequencing to cross check the accuracy of genotyping. To conclude, this study presents a comprehensive picture of genetic variants associated with HCV infection across the global population. Genetic variants associated with prediction of SVR can be set as the markers for Pakistani and South Asian patients.