Antiviral Therapy 2012; 17: (doi: /IMP2018)

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1 ntiviral Therapy 12; 17: (doi: /IMP18) Short communication The vitamin D receptor gene bt (CC) haplotype impairs the response to pegylated-interferon/ ribavirin-based therapy in chronic hepatitis C patients Katharina Baur 1, Joachim C Mertens 1, Johannes Schmitt 1, Rika Iwata 1, Bruno Stieger 2,3, Pascal Frei 1, Burkhardt Seifert 4, Heike Bischoff Ferrari 5,6, rnold von Eckardstein 7, Beat Müllhaupt 1,8, ndreas Geier 1,3,8 *, the Swiss Hepatitis C Cohort Study Group 1 Division of Gastroenterology & Hepatology, University Hospital Zurich, Zurich, Switzerland 2 Division of Clinical Pharmacology, University Hospital Zurich, Zurich, Switzerland 3 Zurich Centre for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland 4 Division of Biostatistics, Institute of Social und Preventive Medicine, University of Zurich, Zurich, Switzerland 5 Centre on ging and Mobility, University of Zurich, Zurich, Switzerland 6 Department of Rheumatology, University of Zurich, Zurich, Switzerland 7 Institute for Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland 8 Swiss Hepatopancreatobiliary (HPB)-Centre, University Hospital Zurich, Zurich, Switzerland *Corresponding author andreas.geier@usz.ch These authors made an equal contribution to this work list of members of the Swiss Hepatitis C Cohort Study Group can be accessed via dditional file 1 Background: Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients. Methods: total of 155 chronic hepatitis C patients were recruited from the Swiss Hepatitis C Cohort Study for NR1I1 genotyping and plasma 25-OH vitamin D level measurement. NR1I1 genotype data and combined effects of plasma 25-OH vitamin D level were analysed regarding therapy response (sustained virological response). Results: strong association was observed between therapy non-response and the NR1I1 CC (bt) haplotype consisting of rs (BsmI) C, rs (pai) C and rs (TaqI) alleles. Of the HCV patients carrying the CC haplotype, 5.3% were non-responders (odds ratio [OR] 1.69, 95% CI 1.7, 2.67; P=.28). similar association was observed for the combinational CCCC genotype (OR 2.94, 95% CI 1.36, 6.37; P=.7). The combinational CCCC genotype was confirmed as an independent risk factor for non-response in multivariate analysis (OR 2.5, 95% CI 1.7, 5.87; P=.34). nalysing combined effects, a significant impact of low 25-OH vitamin D levels on sustained virological response were only seen in patients with the unfavourable NR1I1 CC (bt) haplotype (OR for non-svr 3.55; 95% CI 1.5, 12.57; P=.49). Conclusions: NR1I1 vitamin D receptor polymorphisms influence response to pegylated-interferon/ribavirinbased therapy in chronic hepatitis C and exert an additive genetic predisposition to previously described low 25-OH vitamin D serum levels. Introduction significant proportion of end-stage liver disease is caused by chronic hepatitis C infection. The current recommended therapy is a combination of pegylated interferon-a and ribavirin [1]. Triple therapy regimens including protease inhibitors have led to an improvement of sustained virological response (SVR) in HCV genotype 1 but impose a burden of additional adverse effects and are not helpful in genotype non-1 patients and those with prior null-response to standard therapy [2]. Thus, future research needs to further define subgroups of patients that respond to optimized standard treatment, resulting in an overall response rate beyond 12 International Medical Press (print) -58 (online) 541

2 K Baur et al. 55% as is achieved today [1]. Genome-wide association studies led to the identification of IL28B polymorphisms as the most predictive patient-related genetic factor influencing therapy response that might affect therapy decisions in the future [3] and recent data suggest that vitamin D may be an important determinant of treatment response [4]. The association between vitamin D status and chronic liver disease has come into focus for different disease entities [5]. Recently, a significant correlation between 25-OH vitamin D deficiency and therapy non-response to pegylated-interferon/ribavirin has been observed in a population with HCV genotype 1 [6] and later confirmed in genotype 2 and 3 patients [7]. Interestingly, the CYP27B1-126 promoter polymorphism rs had a substantial effect on 1,25-(OH) 2 vitamin D serum levels and SVR in HCV genotype 1, 2 and 3 patients in this study [7]. s the physiological target, common genetic variations of the vitamin D receptor (NR1I1) gene, such as the bt haplotype, offer a Table 1. Patient characteristics Characteristic Value Gender Male, n (%) 12 (65.8) Female, n (%) 53 (34.2) Median age, years (range) 5 (25 77) Ethnicity Caucasian, % 93 Black, % 1 sian, % 4 Other, % 2 HCV genotype 1, % and 3, % 47.1 Mean body mass index, ±.33 and ±3.76 kg/m² ±se and ±sd a Liver histology Mean fibrosis, ±se and ±sd b 2.1 ±.1 and ±1.22 Mean activity score, ±se and ±sd b 1.79 ±.7 and ±.81 Mean fibrosis progression rate,.111 ±.1 and ±.118 ±se and ±sd c Mean LT, U/l ±se and ±sd d ±9.6 and ±1.46 Mean GGT, U/l ±se and ±sd e ±14.7 and ±152.9 Therapy response SVR, n (%) 87 (56.1) Non-SVR, n (%) 68 (43.9) 25-OH vitamin D F 2 Mean, ng/ml ±se and ±sd f 21.7 ±1.59 and ±11.71 <12 ng/ml, % 24.1 < ng/ml, % 48.1 <3 ng/ml, % 75.9 Baseline characteristics of the 155 patients who underwent NR1I1 genotyping after meeting the inclusion and exclusion criteria. a n=154. b n=134. c n=11. d n=18. e n=16. f n= 54. LT, alanine aminotransferase; GGT, g-glutamyltransferase; SVR, sustained virological response. possibility to further analyse vitamin D effects on HCV therapy outcome as vitamin D receptor gene variants affect vitamin D effects independently of its ligand and have no known influence on vitamin D plasma levels [8,9]. Therefore, we aimed to investigate the effect of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma 25-OH vitamin D levels in a well-described cohort of hepatitis C patients. Methods Patient population total of 155 patients were recruited from the Swiss Hepatitis C Cohort Study for plasma 25-OH vitamin D measurement and NR1I1 genotyping (Table 1). HCV antibody- and RN-positive patients >18 years previously treated with pegylated-interferon/ribavirin were included. Exclusion criteria were coinfection with HBV or HIV (positive hepatitis B surface antigen or anti-hiv antibody), alcohol consumption > g/day and morbid obesity (body mass index > kg/m 2 ). cohort of 15 HCV-negative patients that underwent liver resection served as independent control cohort [1]. The study was approved by the local ethics committee (EK-695) following the guidelines of the Declaration of Helsinki and written informed consent was provided. DN isolation and genotyping of vitamin D receptor (NR1I1) polymorphisms DN was isolated from whole blood/peripheral blood mononuclear cells using the QIamp DN Minikit (Qiagen, Hilden, Germany). NR1I1 genotyping was performed using standard TaqMan technology (pplied Biosystems, Darmstadt, Germany). Fluorogenic 5 -nuclease (TaqMan) assays were performed on an BI PRISM 77 sequence detection system (pplied Biosystems) to determine human NR1I1 single nucleotide polymorphisms (SNPs; rs731236, rs and rs , formerly termed TaqI, BsmI and pai) as described [11 14]. IL28B rs genotyping was performed either with melting curve analysis using FRET probes in a LightCycler (Roche Diagnostics, Rotkreuz, Switzerland) or with TaqMan SNP genotyping assays (pplied Biosystems) as described [15]. ll genotyping data were analysed with the SDS 2.3 software (pplied Biosystems). Haplotype reconstruction was performed using the Haploview software. Serum 25-OH vitamin D quantification Plasma 25-OH vitamin D levels were determined for a subset of 54 patients using standard radio-immuno assay (DiaSorin, Saluggia, Italy) with a measuring range of ng/ml and analytical sensitivity <1. ng/ ml. Patients with higher stages of fibrosis >F2 were excluded from this subgroup analysis due to known International Medical Press

3 VDR gene bt haplotype influences HCV therapy response interference of advanced liver fibrosis and cirrhosis per se with 25-hydroxylation [1]. Statistical analysis Statistical analysis was performed using SPSS 17. (SPSS Inc., Chicago, IL, US). Statistical associations were calculated for therapy response and either vitamin D plasma level or NR1I1 genotype. Statistical analyses to compare groups include Fisher s exact test or χ 2 test where appropriate. Effects of age, gender, HCV genotype, NR1I1 haplotype and IL28B genotype on SVR were analysed using univariate and multivariate logistic regression. Odds ratios (ORs) with 95% CIs were computed using logistic regression. correction for multiple comparisons according to Bonferroni was performed where appropriate. The patient number within the two different response groups was estimated to yield potential significance (power=.821 and a=.1 for combinational CCCC genotype; power=.697 and a=.16 for CC haplotype). Results Patient characteristics Characteristics of 155 patients fulfilling the inclusion and exclusion criteria are detailed in Table 1. Mean 25-OH vitamin D plasma levels were 21.7 ±1.59 ng/ml for a subgroup of 54 F 2 patients. Impact of NR1I1 haplotype on therapy response in HCV-infected patients To investigate a possible association between therapy response and polymorphisms in the vitamin D receptor gene NR 1I1 genotyping was successfully performed in 155 patients. The allelic frequency in this HCV-cohort was compared to a control group of 15 HCV-negative individuals undergoing liver resection. No significant differences in the distribution of geno- and haplotypes between the HCV cohort and this established control cohort [1] could be identified (dditional file 2). strong association was observed between therapy non-response and the CC (bt) haplotype consisting of rs (formerly BsmI) C, rs (pai) C and rs (TaqI) alleles (Figure 1). Overall, 5.3% of HCV-infected patients carrying the CC haplotype were non-responders resulting in an OR of 1.69 (95% CI 1.7, 2.67; P=.28) compared to any other haplotype, TG (BaT) or C (bt). similar association was observed for the combinational CCCC genotype contributing to the bt (CC) haplotype (OR 2.94, 95% CI 1.36, 6.37; P=.7). Multivariate analysis confirmed the combinational CCCC genotype (OR 2.5, 95% CI 1.7, 5.87; P=.34) among other risk factors including HCV and IL28B genotype as an independent risk factor for non-svr (Table 2). No significant association between the bt (CC) haplotype and HCV RN level could be detected. The association with non-svr was also significant for the individual SNPs rs (pai; P=.37 for CC versus C versus and P=.13 for CC versus C and ; OR 2.67, 95% Cl 1.24, 5.7) and rs (TaqI; P=.18 for versus G versus GG and P=.2 for and G versus GG; OR 6.5, 95% CI 1.71, 21.43). s shown in Figure 1B, 62.2% of the patients with rs (pai) CC and 53.4% with rs (TaqI) had non-svr. No significant association with regard to therapy response could be identified for rs (BsmI). Combined effect 25-OH vitamin D levels and NR 1I1 haplotype on therapy response in patients without relevant fibrosis In a subgroup of 54 patients with early stage fibrosis (F 2) plasma 25-OH vitamin D levels within 3 months before starting therapy were quantified. In a combined analysis, 25-OH vitamin D levels significantly affect non-svr only in patients with the unfavourable NR1I1 CC (bt) haplotype (P=.9; Figure 2). strong trend was observed for the combinational CCCC genotype, which did not reach statistical significance after Bonferroni correction (P=.44; dditional file 3). Patients carrying the CC (bt) haplotype with vitamin D levels < mg/l had an SVR rate of 54.2% compared to 8.8% in other haplotypes (OR for non-svr 3.55; 95% CI 1.5, 12.57; P=.49; Figure 2B). For patients with vitamin D levels > mg/l only a weak association could be observed. s for the NR1I1 CC (bt) haplotype, no correlation of HCV RN levels to 25-OH vitamin D levels could be detected. Discussion This study contributes important new findings regarding genetic variants in the vitamin D receptor (NR1I1) and combined effects of 25-OH vitamin D levels on therapy outcome with pegylated-interferon/ ribavirin standard therapy in chronic hepatitis C. Our findings highlight a genetic predisposition in carriers of the NR1I1 bt (CC) haplotype towards nonresponse to therapy and extend previous findings on the influence of 25-OH vitamin D levels on HCVtherapy [1,2,7,15]. HCV-positive patients carrying the bt (CC) haplotype involving rs (pai) and rs (TaqI) variants display ORs between 1.7 and 6.5 for non-response. Interestingly, the observed effects were independent of IL28B genotype. This finding is in-line with another recent study showing vitamin D effects ntiviral Therapy

4 K Baur et al. Figure 1. Effect of vitamin D receptor (NR1I1) haplotype and genotype on interferon therapy response 8 Non-SVR OR 2.94; P=.7 P=.1 6 OR 1.69; P=.28 P=.45 6 Bsml pai TaqI B CC b CC t CT b/b CT b/b CC b C /a a C /a G t/t G t/t t Haplotype Bsm pa Taq CC CC CT C G CT G CC C TT GG OR 2.66; P=.13 P=.37 TT B a GG T Bsml pai TaqI C C = b t C b T B = a = a t G T Haplotype Bsm pa Taq CC C TG OR 6.5; P=.2 P=.18 C pai (rs ) CC G GG TaqI (rs731236) C CC G GG 1 8 P=.85 6 CC CT TT BsmI (rs154441) CC CT TT () The CC (bt) haplotype (rs [formerly BsmI] C, rs [pai] C and rs [TaqI] alleles) shows a significant association with non-response to interferon-based HCV therapy with an odds ratio (OR) of 1.69 (95% CI 1.7, 2.67; P=.28) compared to other common haplotypes. similar association was observed for the combinational CC CC genotype contributing to the bt (CC) haplotype (OR 2.94, 95% CI 1.36, 6.37; P=.7). (B) The individual polymorphisms rs (pai) CC and rs (TaqI) per se are similarly associated with non-sustained virological response (SVR) International Medical Press

5 VDR gene bt haplotype influences HCV therapy response Table 2. Univariate and multivariate analysis 95% Cl Parameter OR Lower Upper P-value Univariate analysis, non-svr ge, >45 versus <45 years Gender, male versus female HCV genotype, 1 versus 2 and Combinational genotype, CC CC versus other Il28_rs127986, CC versus CT/TT Multivariate analysis, non-svr HCV genotype, 1 versus 2 and Combinational genotype, CC CC versus other Il28_rs127986, CC versus CT/TT Univariate and multivariate analysis were performed using logistic regression. The combinational CC CC genotype contributing to the bt (CC) haplotype (odds ratio [OR] 2.5, 95% CI 1.7, 5.87; P=.34) was identified as an independent risk factor for non-sustained virological response (SVR) in uni- and multivariate analysis. Significant parameters from univariate analysis (HCV genotype, NR1I1 haplotype and IL28B genotype) were included into the multivariate analysis. Figure 2. Combined influence of 25-OH vitamin D plasma levels and vitamin D receptor (NR1I1) haplotype on interferon therapy response OR 3.77; P=.9 5 P=.49 3 P=.8 1 B VitD> others VitD> CC VitD< others Haplotype Bsm pa Taq VitD< CC Vitamin D < ng/ml Vitamin D > ng/ml Parameter Other haplotypes, n (%) Non SVR SVR Non SVR SVR 5 (19.2) 21 (8.8) 5 (16.7) 25 (83.3) CC haplotype, n (%) 11 (45.8) 13 (54.2) 5 (19.2) 21 (8.8) Non-SVR, OR (95% CI) 3.55 (1.5, 12.57) 1.19 (.3, 4.67) P-value of OR.49.8 () The 25-OH vitamin D (vitd) serum levels within 3 months prior to therapy were quantified in 54 patients with early stage fibrosis (F 2). combined analysis using c 2 test showed that only in patients with the unfavourable NR1I1 CC (bt) haplotype are low 25-OH vitd levels significantly associated with non-sustained virological response (SVR; P=.9). (B) Only 54.2% of patients with a combination of the CC (bt) haplotype and vitd levels < mg/l reached SVR, compared to 8.8% SVR for other haplotypes (odds ratio [OR] for non-svr 3.55, 95% CI 1.5, 12.57; P=.49). ntiviral Therapy

6 K Baur et al. are complementary to the IL28B genotype [16]. s our data are based on vitamin D receptor (NR1I1) polymorphisms, the study circumvents confounding effects of HCV-associated liver fibrosis on vitamin D serum levels [6]. Of note, variants in the vitamin D receptor gene, a pivotal receptor in the vitamin D signalling cascade, alter vitamin D effects at the gene regulation level and have no relevant influence on vitamin D plasma levels [8,9]. Expression of the vitamin D receptor is independent of these gene variants (unpublished data). dditive effects of the genetic predisposition in the NR1I1 gene and vitamin D deficiency are plausible from our data, since 25-OH vitamin D levels significantly affect SVR in patients with the unfavourable NR1I1 CC (bt) haplotype but are less pronounced in others. Moreover the described effects of vitamin D receptor polymorphisms fit with the concept of a most likely immunomodulatory role of vitamin D in HCV therapy. Recent data confirm a role of vitamin D in T-cell antigen receptor signalling and activation [17] and a recent genome-wide association study has identified vitamin D receptor binding sites in several genes involved in interferon- signalling pathways [18]. The underlying immunological and molecular mechanisms are still poorly understood but may not involve direct effects on HCV replication since no correlation of HCV RN levels to NR1I1 haplotypes and 25-OH vitamin D levels have been observed in this study. s chronic liver disease is characteristically associated with disturbed 25-OH vitamin D homeostasis, the supplementation of vitamin D prior to pegylated- interferona-based therapy may offer an attractive approach to improve therapy outcomes with no additional side effects, especially in those patients who have no benefit from future triple-therapy regimens. Disclosure statement This study was funded by Hoffmann-La-Roche Pharmaceuticals (to G and BM). Hoffmann-La- Roche had no role in study design, analysis and interpretation of the data. ll other authors declare no competing interests. dditional files dditional file 1: list of members of the Swiss Hepatitis C Cohort Study Group can be found at SC-81_Baur_dd_file1.pdf dditional file 2: Comparisons of the allelic frequency and distribution of genotypes and haplotypes between patients in the study and a control group of HCV-negative individuals undergoing liver resection can be found at documents/vt-11-sc-81_baur_dd_file2.pdf dditional file 3: Results of the combined influence of 25-OH vitamin D plasma levels and vitamin D receptor (NR1I1) combinational genotypes on interferon therapy response can be found at com/uploads/documents/vt-11-sc-81_baur_ dd_file3.pdf References 1. Ghany MG, Strader DB, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 9; 49: Hézode C, Forestier N, Dusheiko G, et al. 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7 VDR gene bt haplotype influences HCV therapy response 17. von Essen MR, Kongsbak M, Schjerling P, Olgaard K, Odum N, Geisler C. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nat Immunol 1; 11: Ramagopalan SV, Heger, Berlanga J, et al. ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution. Genome Res 1; : ccepted 6 July 11; published online 14 December 11 ntiviral Therapy