Vitamin D deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy

BACKGROUND: Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in patients with chronic hepatitis C. We therefore performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS: Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and the 1α- hydroxylase were determined in a cohort of 468 HCV genotype 1, 2 and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS: Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D3<10 ng/mL in 25% versus 12%, p<0.00001), which was in part reversible after HCV eradication. 25(OH)D3 deficiency correlated with SVR in HCV genotype 2 and 3 patients (63% and 83% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.001). In addition, the CYPB27-1260 promoter polymorphism rs10877012 had substantial impact on 1-25- dihydroxyvitamin D serum levels and SVR rates in HCV genotype 1, 2 and 3 infected patients. CONCLUSIONS: Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25- hydroxyvitamin D levels and CYPB27-1260 promoter polymorphism are associated with failure to achieve SVR in HCV genotype 1, 2, 3 infected patients.

HLA Class I alleles associated with HCV polymorphisms and response to antiviral therapy in HCV genotype 1-infected patients

Aims: The adaptive immune response against hepatitis C virus (HCV) is significantly shaped by the host's composition of HLA alleles. Thus, the HLA phenotype is a critical determinant of viral evolution during adaptive immune pressure. Potential associations of HLA class I alleles with polymorphisms of HCV immune escape variants are largely unknown. Methods: Direct sequence analysis of the genes encoding the HCV proteins E2, NS3 and NS5B in a cohort of 159 patients with chronic HCV genotype 1 infection who were treated with pegylated interferon-alfa 2b and ribavirin in a prospective controlled trial for 48 weeks was exhibited. HLA class I genotyping was performed by strand-specific reverse hybridization with the INNO-LiPA line probe assays for HLA-A and HLA-B and by strand-specific PCR-SSP. We analyzed each amino acid position of HCV proteins using an extension of Fisher's exact test for associations with HLA alleles. In addition, associations of specific HLA alleles with inflammatory activity, liver fibrosis, HCV RNA viral load and virologic treatment outcome were investigated. Results: Separate analyses of HCV subtype 1a and 1b isolates revealed substantially different patterns of HLA-restricted polymorphisms between subtypes. Only one polymorphism within NS5B (V2758x) was significantly associated with HLA B*15 in HCV genotype 1b infected patients (adjusted p=0,048). However, a number of HLA class I-restricted polymorphisms within novel putative HCV CD8+ T cell epitopes (genotype 1a: HLA-A*11 GTRTIASPK1086-1094 [NS3], HLA-B*07 WPAPQGARSL1111-1120 [NS3]; genotype 1b: HLA-A*24 HYAPRPCGI488-496 [E2], HLA-B*44 GENETDVLL530-538 [E2], HLA-B*15 RVFTEAMTRY2757-2766 [NS5B]) were observed with high predicted epitope binding scores assessed by the web-based software SYFPEITHI (>21). Most of the identified putative epitopes were overlapping with already otherwise published epitopes, indicating a high immunogenicity of the accordant HCV protein region. In addition, certain HLA class I alleles were associated with inflammatory activity, stage of liver fibrosis, and sustained virologic response to antiviral therapy. Conclusions: HLA class I restricted HCV sequence polymorphisms are rare. HCV polymorphisms identified within putative HCV CD8+ T cell epitopes in the present study differ in their genomic distribution between genotype 1a and 1b isolates, implying divergent adaptation to the host's immune pressure on the HCV subtype level.

Systematic molecular and cytogenetic screening of 100 patients with marfanoid syndromes and intellectual disability.

The association of marfanoid habitus (MH) and intellectual disability (ID) has been reported in the literature, with overlapping presentations and genetic heterogeneity. A hundred patients (71 males and 29 females) with a MH and ID were recruited. Custom-designed 244K array-CGH (Agilent®; Agilent Technologies Inc., Santa Clara, CA) and MED12, ZDHHC9, UPF3B, FBN1, TGFBR1 and TGFBR2 sequencing analyses were performed. Eighty patients could be classified as isolated MH and ID: 12 chromosomal imbalances, 1 FBN1 mutation and 1 possibly pathogenic MED12 mutation were found (17%). Twenty patients could be classified as ID with other extra-skeletal features of the Marfan syndrome (MFS) spectrum: 4 pathogenic FBN1 mutations and 4 chromosomal imbalances were found (2 patients with both FBN1 mutation and chromosomal rearrangement) (29%). These results suggest either that there are more loci with genes yet to be discovered or that MH can also be a relatively non-specific feature of patients with ID. The search for aortic complications is mandatory even if MH is associated with ID since FBN1 mutations or rearrangements were found in some patients. The excess of males is in favour of the involvement of other X-linked genes. Although it was impossible to make a diagnosis in 80% of patients, these results will improve genetic counselling in families.

A Genetic Validation Study Reveals a Role of Vitamin D Metabolism in the Response to Interferon-Alfa-Based Therapy of Chronic Hepatitis C.

BACKGROUND: To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C. METHODOLOGY/PRINCIPAL FINDINGS: Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D(3) (25[OH]D(3)) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061-2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D(3)<20 ng/mL) during all seasons, but 25(OH)D(3) serum levels were not associated with treatment outcome. CONCLUSIONS/SIGNIFICANCE: Our study suggests a role of bioactive vitamin D (1,25[OH](2)D(3), calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D(3) is not a suitable predictor of treatment outcome.

Importance of IL28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients.

BACKGROUND & AIMS: Genetic variation in the interleukin 28B (IL28B) gene has been associated with the response to interferon-alfa/ribavirin therapy in hepatitis C virus (HCV) genotype 1-infected patients. The importance of three IL28B single nucleotide polymorphisms (rs8099917, rs12980275 and rs12979860) for HCV genotype 2/3-infected patients is unknown. METHODS: In patients with chronic hepatitis C genotype 2/3 (n=267), IL28B host genotypes (rs8099917, rs12980275 and rs12979860) were analyzed for associations with sustained virologic response (SVR) to antiviral therapy with (pegylated) interferon-alfa and ribavirin and with respect to epidemiological, biochemical, and virological parameters. For comparison, hepatitis C genotype 1 patients (n=378) and healthy controls (n=200) were included. RESULTS: The rs12979860 CC genotype, lower age, and genotype 2 were significantly associated with SVR in HCV genotype 2/3-infected patients (p=0.01, p=0.03 and p=0.03, respectively). No association was observed for rs8099917 and rs12980275. In addition, an SVR in patients with rapid virologic response (RVR) was associated with the rs12979860 CC genotype (p=0.05), while for non-RVR no association was found. Furthermore, a significant association with a higher baseline viral load was observed for all three IL28B genotypes in genotype 1/2/3-infected patients. Finally, increasing frequencies of the rs12979860 CC genotypes were observed in genotype 1- (33.9%), genotype 3- (38.9%), and genotype 2-infected (51.9%) patients in comparison with healthy controls (49.0%) (p<0.01). CONCLUSIONS: In genotype 2/3-infected patients, rs12979860 was significantly associated with SVR. The frequency of the rs12979860 CC genotype is lower in HCV genotype 1 vs. genotype 2/3 patients. All major IL28B genotypes are associated with HCV-RNA concentration.

Nucleos(t)ide analogue treatment reduces apoptotic activity in patients with chronic hepatitis B.

Background: Reduction of necroinflammatory activity is a major goal of antiviral therapy of patients with chronic hepatitis B. Serum ALT does not detect all forms of cell death.Objectives: To analyze dynamics of novel serum cell death markers for apoptosis and necrosis in association with virologic response to nucleos(t)ide (Nuc) analogue treatment.Study design: Quantification of the M30-apoptosis neoepitope and the cytokeratin-18 (M65-necrosis) serum levels before and during treatment of patients with chronic hepatitis B with Nuc (n = 26).Results: Before treatment, M30-apoptotic activity was significantly correlated with M65-necrosis and fibrosis but not with serum ALT. During therapy with Nucs, cell death parameters M30-apoptosis, M65-necrosis, and ALT declined in association with virologic response. The most frequent cell death pattern was simultaneous decline of ALT and M30-apoptosis which occurred more frequently in patients with HBs-Antigen decline than in patients with HBs-Antigen increase during treatment (87.5% vs. 40.0%; p = 0.024). ALT decline in association with increase of M30 apoptosis was frequent in patients with HBs-Antigen increase during treatment (36.3%) but was not observed in patients with HBs-Antigen decline during treatment.Conclusion: Decline of cell death parameters in association with decline of HBV-DNA and HBs-Antigen indicates a reduction in overall cell death activity during Nuc treatment supporting the concept that response to Nuc therapy reduces necroinflammatory activity and progression of liver disease. (C) 2011 Elsevier B. V. All rights reserved.

A large-scale genetic validation study coupled with in vitro analyses reveal a role for vitamin Dsignaling in the pathogenesis and response to treatment of hepatitis C virus infection

Background and Aims: Vitamin D is an important modulatorof numerous cellular processes. Some of us recently observedan association of the 1a-hydroxylase promoter polymorphismCYP27B1-1260 rs10877012 with sustained virologic response (SVR)in a relatively small number of German patients with chronichepatitis C. In the present study, we aimed to validate thisassociation in a large and well characterized patient cohort, theSwiss Hepatitis C Cohort Study (SCCS). In addition, we examinedthe effect of vitamin D on the hepatitis C virus (HCV) life cyclein vitro.Methods: CYP27B1-1260 rs10877012 and IL28B rs12979860 singlenucleotide polymorphisms (SNPs) were genotyped in 1049 patientswith chronic hepatitis C from the SCCS, of whom 698 were treatedwith pegylated interferon-a (PEG-IFN-a) and ribavirin. In addition,112 patients with spontaneous clearance of HCV were examined.SNPs were correlated with variables reflecting the natural courseand treatment outcome of chronic hepatitis C. The effect of1,25-(OH)2D3 (calcitriol) on HCV replication and viral particleproduction was investigated in vitro using human hepatoma celllines (Huh-7.5) harbouring subgenomic replicons and cell culturederivedHCV.Results: The CYP27B1-1260 rs10877012 genotype was notassociated with SVR in patients with the good-response IL28Brs1279860 CC genotype. However, in patients with poor-responseIL28B rs1279860 genotype CT and TT, CYP27B1-1260 rs10877012was a significant independent predictor of SVR (15% difference inSVR between rs10877012 genotype AA vs. CC, p = 0.030, OR = 1.495,95% CI = 1.038-2.152). The CYPB27-1260 rs10877012 genotype wasneither associated with spontaneous clearance of HCV, nor withliver fibrosis progression rate, inflammatory activity of chronichepatitis C, or HCV viral load. Physiological doses of 1,25-(OH)2D3did not significantly affect HCVRNA replication or infectiousparticle production in vitro.Conclusions: The results of this large-scale genetic validationstudy reveal a role of vitamin D metabolism in the responseto treatment in chronic hepatitis C, but 1,25-(OH)2D3 does notexhibit a significant direct inhibitory antiviral effect. Thus, theability of vitamin D to modulate immunity against HCV shouldbe investigated.

Extended treatment of treatment-naive patients with chronic hepatitis c virus (HCV) genotype 1-infection and slow response to pegylated interferon-alfa and ribavirin: a meta-analysis

Aims: Recently, several clinical trials analyzed if extended duration of treatment with pegylated interferon-alfa and ribavirin over 48 weeks can improve sustained virologic response (SVR) rates in HCV genotype 1-infected patients with slow virologic response. Because results of these clinical trials are conflicting, we performed a metaanalysis to determine the overall impact of extended treatment compared to standard treatment on virologic response rates in treatment-naive HCV genotype 1 slow responders. Methods: Literature search was performed independently by two observers using Pub Med, EMBASE, CENTRAL and abstracts presented in English at international liver and gastroenterology meetings. Randomized controlled clinical trials (RCTs; but studies that re-analyzed data retrospectively RCTs were also allowed) were considered if they included monoinfected treatment-naive HCV genotype 1 patients and compared treatment with pegIFN-alfa 2a or 2b in combination with ribavirin for 48 weeks versus extended treatment (up to 72 weeks) in slow responders. Primary and secondary end points were SVR rates and end-of-treatment (EOT) and relapse rates, respectively. In the present meta-analysis, study endpoints were summarized with a DerSimonian-Laird estimate for binary outcome basing on a random effects model. Results: Literature search yielded seven RTCs addressing the benefit of extended treatment with pegylated interferon-alfa and ribavirin in treatment-naive HCV genotype 1 slow responders. In total, 1330 slow responders were included in our meta-analysis. We show that extended treatment duration compared to the standard of care significantly improves SVR rates in HCV genotype 1 slow responders (12.4% improvement of overall SVR rate, 95% CI 0.055- 0.193, P = 0.0005). In addition, we show that rates of viral relapse were significantly reduced by extended treatment (24.1% reduction of relapse, 95% CI −0.3332 to −0.1487, P < 0.0001), whereas no significant impact of extended treatment on EOT response rates was found. Though extended treatment was burdened with an enhanced rate of premature treatment discontinuation due to interferonalfa- and ribavirin-related side effects, the frequency of serious adverse events was not increased. Conclusions: Treatment extension in HCV genotype 1 slow responders can improve SVR rates in difficult to treat patients and should be considered in patients who need to be treated before specific antivirals will be approved.

Cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis C correlates with a reduced activation of the endogenous interferon system.

Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-alpha. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta. The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS.