Impact of human genetic polymorphisms and HCV genome variation on the outcome of HCV-induced liver fibrosis

Abstract

Hepatitis C virus (HCV) infection is a major health problem causing nearly 350,000 deaths per year. Almost 71 million individuals are estimated to be infected worldwide, with around 750,000 new infections occurring annually. Viral hepatitis is the 7th leading cause of mortality globally, and half of this mortality is attributed to HCV, which is the primary cause for liver fibrosis, cirrhosis and liver cancer. Egypt has the highest HCV prevalence worldwide (estimated 14.8%). Genotype 4 and more specifically subtype 4a dominate the HCV epidemic in Egypt. The main cause of this high prevalence of HCV is the mass parenteral antischistosomal therapy (PAT) campaigns in the late years of the 20th century. HCV is a blood borne infection, where parenteral transmission remains the principle route of transfer. Parenteral transmission includes sharing needles during injection, unsafe intravenous injections. The liver is the principal site of HCV replication and the infection outcome is heterogeneous. Acute phase (first 6 months after infection), only 20% of patients spontaneously clear the virus during this phase. When the viral replication persists for more than six months after acute infection, the hepatitis is considered chronic. This chronic infection leads to hepatic inflammation, which often stimulates liver fibrosis. Liver fibrosis directly modulates signaling and metabolic pathways by viral proteins. Moreover, it indirectly induces host antiviral immune responses leading to chronic inflammation. Liver fibrosis can progress to cirrhosis and hepatocellular carcinoma (HCC). Most of hepatitis C patients have asymptomatic or nonspecific symptoms. Liver biopsy is considered the most common method used for the diagnosis and follow up of progressive hepatic fibrosis. Also, transient elastography is used nowadays as non-invasive methods for accurate assessment of liver fibrosis. Multiple host genetic variations may affect progression of fibrosis in HCV infection. SNPs are the most common and simple form of genetic polymorphism in the human. Genes influencing fibrosis include immune, metabolic, oxidative stress, and viral entry genes, such as interleukin 10 (IL-10), microsomal triglyceride-transfer protein (MTP), superoxide dismutase-2 (SOD2), and apolipoprotein E (ApoE)-encoding genes, respectively. This study aims to test whether polymorphism in IL-10 -627 C/A, MTP -400 A/T, SOD2 -1183 C/T, and ApoE (E3/E2/E4) genes are associated with differential fibrosis induced by HCV genotype 4 (HCV-gt4) in a cohort of Egyptian chronic patients. A hundred blood samples were collected from fibrotic chronic HCV-gt4 patients diagnosed at National Hepatology & Tropical Medicine Research institute, and genomic DNA was extracted and tested for polymorphisms by PCR-RFLP. The patients were categorized into two groups: group 1, 59 HCV patients with mild stages of fibrosis (thereafter, indicated as the Mild group), and group 2, 41 HCV patients with severe stages of fibrosis (thereafter, indicated as the Severe group). In this study, the Egyptian cohort of patients with HCV showed significant association between severe fibrosis stages and high BMI (obesity), low albumin level, high AFP level, low TSH level and high ALP level. Genotyping for IL-10 -627 showed that the most common genotype was CC, accounting for 59%, followed by CA accounting for 30% and then AA accounting for 11%. In mild fibrosis group the frequency of CC, CA, and AA genotypes was 54.2%, 27.1% and 18.6%, respectively. While in severe fibrosis group the frequency of CC, CA, and AA genotypes were 65.9%, 34.1%, and 0%, respectively. Comparing association between genotype/allele frequencies and severity of liver fibrosis using chi-square test and odds ratio revealed that, there was no significant association of any genotypes of IL-10 on the development of severe fibrosis in both groups. While, the low risk (protective) of developing severe fibrosis was associated with A allele (p-value=0.016, OR 0.433) in both groups. Genotyping for MTP -400 showed that the most common genotype was AT, accounting for 62%, followed by AA accounting for 32% and then TT accounting for 6%. In mild fibrosis group, the frequency of AA, AT, and TT genotypes were 33.9%, 55.9% and 10.2%, respectively. While in severe fibrosis group the frequency of AA, AT, and TT genotypes were 29.3%, 70.7%, and 0%, respectively. Comparing association between genotype/allele frequencies and severity of liver fibrosis using the chi-square test and odds ratio revealed that, there was no significant difference in effect of any genotypes and alleles of MTP with the development of severe fibrosis in both groups. Genotyping for SOD2 -1183 showed that the most common genotype was CT, accounting for 48%, followed by TT accounting for 31% and then CC accounting for 21%. In mild fibrosis group, the frequency of CC, CT, and TT genotypes were 11.9%, 52.5% and 35.6%, respectively. While in severe fibrosis group, the frequency of CC, CT, and TT genotypes were 34.1%, 41.5%, and 24.4%, respectively. Comparing association between genotype/allele frequencies and severity of liver fibrosis using the chi-square test and odds ratio revealed that, lower risk for developing severe fibrosis were associated with TT genotype (p-value=0.015, OR 0.238) and AT genotype (p-value=0.016, OR 0.274). While comparing the allele distribution, the low risk (protective) of developing severe fibrosis was associated with T allele (p-value=0.019, OR 0.507) in both groups. Genotyping for ApoE showed that the most common genotype was E2E3, accounting for 52%, followed by E2E4 accounting for 21% and then E2E2, E3E3, and E4E4 accounting for 15%, 6%, and 6%, respectively. In mild fibrosis group the frequency of E3E3, E2E2, E2E3, E2E4 and E4E4 genotypes were 1.7%, 18.6%, 42.4%, 30.5% and 6.8%, respectively. While in severe fibrosis group the frequency of E3E3, E2E2, E2E3, E2E4 and E4E4 genotypes were 12.2%, 9.8%, 65.9%, 7.3% and 4.9%, respectively. Comparing association between genotype/allele frequencies and severity of liver fibrosis using the chi-square test and odds ratio revealed that, lower risk for developing severe fibrosis associated with E2E4 genotype (p-value=0.004, OR 0.033) and E2E4 genotype (p-value=0.046, OR 0.072). While comparing the allele distribution, the lowest risk (protective) of developing severe fibrosis was associated with E4 allele (p-value=0.001, OR 0.197), compared with E2 allele (p-value=0.009, OR 0.427) in both groups. Combined haplotypes of candidate SNPs in IL10, MTP, SOD2 and Apo E-encoding gene could provide more significance in predicting the degree of HCV-gt4 induced liver fibrosis. Five out of 2 haplotype combinations were associated mild fibrosis with high statistically significance value: CATE2 (p-value= 0.006), AATE2 (p-value= 0.010), ATTE4 (p-value=0.014), CTTE4 (p-value=0.032), and CTTE2 (p-value=0.035). The univariate analysis showed that, multiple factors including BMI, albumin, TSH, Icam, ALP, A allele of IL-10 -627, T allele of SOD2 -1183, E3 and E4 of ApoE were designated as significant predictors associated with the severe fibrosis. While in multivariate analysis, BMI, TSH, ALP, and T allele of SOD2 were designated as independent significant variables that could be used as predictors for the risk of fibrosis severity in HCV infections. In this study the correlation between combined alleles for SNPs from multiple genes implicated in liver fibrosis represents a novel approach that could be of significant prognostic value in predicting the degree of fibrosis. In addition this study can help in determination of new genetic markers becomes crucial in therapeutics decision prior to the onset of therapy for liver fibrosis in patients with chronic hepatitis C.