Polymorphisms of chemokine receptor genes and clearance of hepatitis C virus infection in Chinese population

Accepted Manuscript Polymorphisms of chemokine receptor genes and clearance of hepatitis C virus infection in Chinese population

Mingzhu Chen, Yinan Yao, Ming Yue, Feng Zang, Mei Liu, Jie Wang, Hongbo Chen, Yun Zhang, Jun Li, Peng Huang, Rongbin Yu PII: DOI: Reference:

S0378-1119(17)30304-9 doi: 10.1016/j.gene.2017.04.042 GENE 41891

To appear in:

Gene

Received date: Revised date: Accepted date:

16 January 2017 19 April 2017 25 April 2017

Please cite this article as: Mingzhu Chen, Yinan Yao, Ming Yue, Feng Zang, Mei Liu, Jie Wang, Hongbo Chen, Yun Zhang, Jun Li, Peng Huang, Rongbin Yu , Polymorphisms of chemokine receptor genes and clearance of hepatitis C virus infection in Chinese population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi: 10.1016/j.gene.2017.04.042

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ACCEPTED MANUSCRIPT Polymorphisms of chemokine receptor genes and clearance of hepatitis C virus infection in Chinese population Running title: CXCR6 variants influence HCV clearance

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Authors: Mingzhu Chen1†, Yinan Yao1†, Ming Yue2, Feng Zang1, Mei Liu1, Jie

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Wang3, Hongbo Chen4, Yun Zhang5, Jun Li2, Peng Huang1*, Rongbin Yu1*

Authors’ affiliations:

Department of Epidemiology and Biostatistics, School of Public Health, Nanjing

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Medical University, Nanjing 211166, China

Department of Infectious Diseases, the First Affiliated Hospital of Nanjing Medical

Department of Basic and Community Nursing, School of Nursing, Nanjing Medical

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University, Nanjing 210029, China

University, Nanjing 211166, China Department of Infectious Diseases, the Jurong Peoples’ Hospital, Jurong 212400,

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China

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Institute of Epidemiology and Microbiology, Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China

† These authors contributed equally to this work *

Corresponding author

Peng Huang: Tel: 86-25-86868437; FAX: 86-25-86868437; E-mail: [email protected]; 1

ACCEPTED MANUSCRIPT Mailing address: Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 211166, China Rongbin Yu: Tel: 86-25-86869187; FAX: 86-25-86869187; E-mail: [email protected];

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Health, Nanjing Medical University, Nanjing 211166, China

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Mailing address: Department of Epidemiology and Biostatistics, School of Public

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ACCEPTED MANUSCRIPT Abstract Background: Chemokine genes play an essential role in both spontaneous clearance in acute infection and therapy of HCV. We investigated whether several CXC family-related genes associated with HCV spontaneous clearance and response to

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treatment.

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Methods: The current study genotyped four SNPs, respectively are CXCR6

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rs2234358, CXCL12 rs1801157, CXCL9 rs10336, rs3733236 to assess their associations with HCV spontaneous clearance and response to treatment in a two

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stage study (668 chronic and 400 resolvers in discovery group, meanwhile 333

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chronic and 199 resolver in replication group), and a treatment cohort of HCV with 282 patients.

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Results: We found that the CXCR6 rs2234358 was associated with HCV spontaneous

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clearance in Chinese Han population (dominant model: adjusted OR = 1.62, 95%CI: 1.30-2.01; additive model: adjusted OR = 1.43, 95%CI: 1.20-1.70). Patients carrying

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GT/TT genotypes had increased sustained virological response compared with

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patients carrying the GG genotype (dominant model: adjusted OR = 2.23, 95%CI: 1.26-3.95).

Conclusion: These results suggest that CXCR6 rs2234358 is associated with spontaneous clearance of HCV and response to IFN-α/RBV therapy, which may be identified as a predictive marker in Chinese Han population of HCV. Key words: Hepatitis C virus; Chemokine; Polymorphism; Spontaneous clearance; Sustained virological response 3

ACCEPTED MANUSCRIPT 1. Introduction Hepatitis C virus (HCV) is a serious global health problem because of its clinical outcomes, a very large proportion of HCV patients will develop to fibrosis or cirrhosis, even hepatocellular carcinoma. In China, it is estimated that up to 29 million people

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are infected with HCV [1], and the prevalence of HCV in mainland is about 1%-1.9%

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[2]. Spontaneous clearance occurs in 10-25% of individuals who are infected with

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HCV [3,4]. Dual therapy with pegylated (PEG) IFN-α and ribavirin (RBV) in most developing countries has been the primary treatment of HCV infection, the rate of

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sustained virological response (SVR) of PEG IFN-α and RBV therapy is around 50%

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for patients infected with HCV genotype 1, and 70-90% in HCV genotype 2 and 3 patients [5]. Environment, viral and host factors have been proved that these factors

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were associated with HCV spontaneous clearance and virological response, and

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studies have discovered that a strong host immune response against HCV is more beneficial to viral clearance [6-9]. Thus, variation of host genes involved in the

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immune response may affect the outcomes of HCV infections and SVR.

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The immune responses of Th cells play an important role in the spontaneous clearance and treatment response of HCV infection. Chemokine receptor and its corresponding ligands will affect the immune response of Th cells, and strength, specificity and diversity of the immune responses can directly affect the clearance and recovery of HCV virus [10]. Nowadays, many studies have proved that chemokine receptor polymorphisms can indeed influence the inflammation and fibrosis progression in patients with chronic hepatitis C [11,12]. CXCL12, CXCL9 and CXCR6 are all the 4

ACCEPTED MANUSCRIPT members of the chemokine CXC subfamily. A study has shown that the CXCL9, together with CXCL10 and CXCL11, are produced in the liver, inducing migration of activated T cells from the periphery to infected liver parenchyma via chemokine receptors in HCV patients, and the CXCL9-11 chemokines are regarded as the pivotal

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role in elimination of HCV [13]. CXCR6 and its ligand CXCL16 support the

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recruitment and survival of NKT cells allowing them to keep high local levels of

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IFN-γ, and promoting the recruitment of Th1 cells therefore [14,15]. Thus in the early phases of HCV infection, chemokines in particular CXCL8 and CXCL16, can promote

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recruitment of innate immune cells to the liver, then initiating the immune response.

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The aim of our study is to investigate whether variants of several chemokine CXC family-related genes might be associated with the outcomes of HCV infection and

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response to treatment. Thus, we selected four single nucleotide polymorphisms (SNPs)

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CXCR6 rs2234358, CXCL9 rs10336, CXCL9 rs3733236 and CXCL12 rs1801157, via

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2. Methods

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a two stage study and a cohort study to research in Chinese Han population.

2.1 Patients

This research includes two kinds of population. The first population refers to 187 drug users and 324 hemodialysis patients extracted from the Nanjing compulsory detoxification center and nine hospital hemodialysis centers in southern China, and 1089 former paid-blood donors were recruited from the cross-sectional study in Jurong of Jiangsu from May 2006 to May 2015. For the analysis, 1068 HCV patients 5

ACCEPTED MANUSCRIPT who were investigated before June 2012 were treated as the discovery group of the study, and the patients who were included in the study between June 2012 and May 2015 were regarded as the replication group to identify the association between SNPs and spontaneous clearance.

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The second population was a treatment cohort of HCV patients. A total of 282 chronic

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HCV patients who were treated with PEG IFN-α and RBV at Jurong People's Hospital

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from January 2011 to December 2015 were included. Patients received PEG IFN-α (180μg) subcutaneously every week and plus oral RBV 600 mg-1000 mg daily for 48

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weeks according to the standard guidelines.

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The inclusion criteria of the two populations were all as follows (1) all the patients were treatment-naive, (2) lack of co-infection with HBV or HIV, (3) lack of other

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causes of liver disease. Every patient was interviewed with a questionnaire to collect

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demographic characteristics and risk factors. Each individual donated 5 ml venous blood for serological tests and DNA extraction after interview.

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2.2 Viral testing and SNP genotyping

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DNA was extracted from peripheral samples by a standard non-enzymatic method [16]. HCV RNA of all patients was isolated using the RT-PCR kit (TaKaRa Biotechnology Co, Ltd, Dalian, China). HCV RNA quantitative examination was made at baseline, weeks 4, 12, 24, 48 during treatment, and at week 24 after stopping therapy. The polymorphisms were Genotyped by the TaqMan allelic discrimination assay on ABI PRISM 7900HT Sequence Detection system (Applied Biosystems, San Diego, CA, USA). CXCR6 rs2234358, CXCL9 rs10336 and rs3733236, CXCL12 6

ACCEPTED MANUSCRIPT rs1801157 were chosen for genotyping. These polymorphisms are all located in 3’untranslated region. Two blank controls and five repeated samples were assigned into each 384-well format for quality control. The genotyping results were determined by the allelic discrimination mode of the SDS 2.3 software package (Applied

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Biosystems, Foster City, CA, USA), and a 100% concordant was achieved. The

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primers and probes used for genotyping were listed in Supplementary Table S1.

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2.3 Statistical analysis

All data were analyzed with Stata/SE (V.12.0 for Windows; StataCorp LP, College

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Station, TX, USA). Demographic characteristics of individuals in each group were

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compared using the chi-squared test or Fisher’s exact test and two-sample t tests. A goodness-of-fit χ2 test was used for Hardy-Weinberg equilibrium (HWE) among the

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controls. The association between genetic with HCV spontaneous clearance and

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clinical data was calculated by multivariate logistic regression analysis, adjusted for age, gender, high-risk population, alanine aminotransferase (ALT), aspartate

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aminotransferase (AST) in first population and age, gender, HCV RNA, albumin,

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platelets, alpha fetal protein (AFP), thyroid stimulating hormone (TSH), fasting blood-glucose (FBG) in clinical population, respectively. The association of genotypes with HCV spontaneous clearance and clinical data were explained by odds ratio (OR) and 95% confidence interval (95%CI). Co-dominant, dominant, recessive, and additive genetic models were used in analysis of each SNP, dominant model stands for (homozygous type + hybrid type) vs. wild type; recessive model stands for homozygous type vs. (hybrid type + wild type) and additive model stands for hybrid 7

ACCEPTED MANUSCRIPT type vs. homozygous type vs. wild type. A stepwise regression model was then fit comprised of all variables and subsequently reduced using forwards elimination to analyze predictive factors for SVR. In all analysis, P-values of less than 0.05 were

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considered as significant. Bonferroni corrections were used for multiple comparisons.

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3.1 Baseline characteristics of the study population

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3 Results

The baseline demographic and clinical characteristics of the first study population in

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two groups were listed in Table 1. The discovery group were including 668 persistent

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HCV cases and 400 spontaneous clearance cases, and 333 persistent HCV cases and 199 spontaneous clearance cases were assigned to the replication group. In all patients

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group, distribution of gender was the same among the persistent and spontaneous

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clearance cases (P > 0.05). The elder and blood donors were more likely to be persistent infection (P < 0.001). Similarly, abnormal rate of AST and ALT was higher

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in the persistent infection group than in the spontaneous clearance group (P < 0.001).

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The baseline demographic and clinical characteristics of the 282 enrolled treatment patients were described in Table 2. After 48 weeks’ treatment, 180 (63.8%) HCV patients achieved SVR, 102 (36.2%) failed to respond to treatment. Treatment response was not related to age and sex (P > 0.05). Baseline levels of viral load, platelets, albumin, AFP, TSH and FBG were different between the SVR group and the non-SVR group (P < 0.05). Patients with high viral load/AFP/FBG and low albumin/platelet/TSH levels at baseline were more likely to fail in PEG IFN-α/RBV 8

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treatment.

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Table 1 Baseline characteristics of HCV persistence and spontaneous clearance patients Discovery group Variables

Persistence (n=668) N%

All patients

Spontaneous clearance (n=400) N%

Replication group Persistence (n=333) N%

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Spontaneous clearance (n=199) N%

Persistence

Age <50 ≥50

191 (28.59) 477 (71.41)

176 (44.00) 224 (56.00)

94 (28.23) 239 (71.77)

84 (42.21) 115 (57.79)

Male Female High-risk population Hemodialysis patient Injecting drug user Paid blood donors ALT <40 ≥40 AST <40 ≥40

224 (33.53) 444 (66.47) 51 (7.63) 104 (15.57) 513 (76.80) 399 (59.73) 269 (40.27)

155 (38.75) 245 (61.25) 63 (15.75) 119 (29.75) 218 (54.50) 305 (76.25) 95 (23.75) 311 (77.75) 89 (22.25)

32 (9.61) 47 (14.11) 254 (76.28)

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379 (56.74) 289 (43.26)

115 (34.53) 218 (65.47)

200 (60.06) 133 (39.94)

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Sex

73 (36.68) 126 (63.32)

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41 (20.60) 54 (27.14) 104 (52.26)

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(n=1001) N% 285 (28.47) 716 (71.53) 339 (33.87) 662 (66.13)

Spontaneous clearance (n=599) N%

P value <0.001

260 (43.41) 339 (56.59) 0.089 228 (38.06) 371 (61.94) <0.001

83 (8.29) 151 (15.08) 767 (76.63)

104 (17.36) 173 (28.88) 322 (53.76) <0.001

158 (79.40) 41 (20.60)

599 (59.84) 402 (40.16)

463 (77.30) 136 (22.70) <0.001

189 (56.76) 144 (43.24)

157 (78.89) 42 (21.11)

Percentages indicate proportion in columns. Abbreviation: AST, aspartate transaminase; ALT, alanine aminotransferase

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568 (56.74) 433 (43.26)

468 (78.13) 131 (21.87)

ACCEPTED MANUSCRIPT Table 2 Baseline characteristics of chronic hepatitis C patients treated with IFN/RBV SVR (n=180) N%

N-SVR (n=102) N%

52.21±8.48

52.76±8.01

Male Female

41 (22.78) 139 (77.22)

27 (26.47) 75 (73.53)

<40 ≥40

75 (41.67) 105 (58.33)

31 (30.39) 71 (69.61)

88 (48.89) 92 (51.11) 6.03±0.85 78.46±6.14 43.62±4.15 137.36±53.95 133.90±15.26 6.30±16.02 20.00±10.72 3.80±7.63 5.79±1.58

40 (39.22) 62 (60.78) 6.25±0.75 77.65±6.00 42.43±4.27 122.37±60.24 132.88±18.22 9.60±11.58 19.31±10.66 2.63±1.36 6.22±1.53

Variables Age Sex

P value 0.669 0.486

ALT

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<40 ≥40 Log HCV-RNA TP (g/L) Albumin (g/L) Platelets (109/L) Hemoglobin (g/L) AFP (ng/mL) Fe (μmol/L) TSH (mIU/L) FBG (mmol/L)

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AST

0.060

0.117 0.034 0.285 0.024 0.033 0.635 0.048 0.731 0.050 0.027

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Percentages indicate proportion in columns. Abbreviation: N-SVR, non-sustained virological response; SVR, sustained virological response; AST, aspartate transaminase; ALT, alanine aminotransferase; TP, total protein; AFP, alpha fetal protein; TSH, thyroid stimulating hormone; FBG, fasting bloodglucose.

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ACCEPTED MANUSCRIPT 3.2 Association of CXCR6 rs2234358 with HCV spontaneous clearance Analysis of the allelic frequencies of the investigated polymorphisms showed that this population was in Hardy-Weinberg equilibrium in discovery group (P = 0.777, 0.171, 0.441 and 0.087 for rs2234358, rs10336, rs3733236 and rs1801157, respectively). As

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shown in Table 3, co-dominant, dominant, recessive, and additive genetic models

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were used in analysis of each SNP. After adjusting for age, gender, high-risk

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population, ALT, AST, and other SNPs, logistic regression analyses showed that the distributions of CXCR6 rs2234358 were associated with spontaneous clearance in

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discovery group. Mutant T allele of CXCR6 rs2234358 was more likely to clear HCV

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virus in the dominant and additive models. The adjusted OR was 1.41 (95% CI = 1.08-1.84), and 1.27 (95% CI = 1.02-1.57), respectively. However, the results did not

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show any association between rs10336, rs373326, and rs1801157 variants with

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spontaneous clearance in discovery group.

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Table 3 Associations of SNPs in gene CXCR6，CXCL12 and CXCL9 with HCV spontaneous clearance in discovery group

208 (52.00) 166 (41.50) 26 (6.50)

620 (92.81) 47 (7.04) 1 (0.15)

373 (93.25) 25 (6.25) 2 (0.50)

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Reference 1.43 (1.08-1.90) 1.27 (0.73-2.20) 1.41 (1.08-1.84) 1.10 (0.65-1.89) 1.27 (1.02-1.57)

0.425 0.258 0.662 0.241 0.756

359 (89.75) 40 (10.00) 1 (0.25)

Reference 1.08 (0.69-1.70) 0.49 (0.05-4.97) 1.14 (0.73-1.77) 0.54 (0.05-5.43) 1.02 (0.69-1.54)

0.734 0.549 0.574 0.598 0.940

178 (44.50) 165 (41.25) 57 (14.25)

Reference 0.84 (0.63-1.12) 1.04 (0.69-1.57) 0.94 (0.73-1.23) 1.17 (0.80-1.71) 0.97 (0.80-1.18)

0.233 0.847 0.665 0.412 0.767

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Reference 0.81 (0.48-1.37) 4.36 (0.34-55.95) 0.89 (0.53-1.49) 4.62 (0.36-59.86) 0.93(0.57-1.50)

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287 (42.96) 295 (44.16) 86 (12.88)

P value

0.012 0.391 0.013 0.717 0.032

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604 (90.42) 61 (9.13) 3 (0.45)

OR (95% CI)

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405 (60.63) 224 (33.53) 39 (5.84)

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Genotype rs2234358 GG GT TT Dominant Recessive Additive rs10336 CC CT TT Dominant Recessive Additive rs3733236 CC CT TT Dominant Recessive Additive rs1801157 GG AG AA Dominant Recessive Additive

(n=668) N%

Spontaneous clearance (n=400) N%

Persistence

Logistic regression analyses adjusted for age, gender, high-risk population, ALT, AST, and other three SNPs.

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ACCEPTED MANUSCRIPT We processed replication group for CXCR6 rs2234358, identifying that rs2234358 was consistently associated with spontaneous clearance in dominant and additive genetic models. The adjusted OR were 1.70 (95% CI = 1.15-2.51) and 1.55 (95% CI = 1.13-2.11), respectively. Then, in all patients, the variant allele of rs2234358 was still

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significantly associated with spontaneous clearance (dominant model: OR = 1.62,

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95% CI = 1.30-2.01, additive model: OR = 1.43, 95% CI= 1.20-1.70) (Table 4).

Table 4 Significant associations of CXCR6 rs2234358 with HCV spontaneous clearance in replication group and all patients Genotype

N%

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201 (60.36) 113 (33.93) 19 (5.71)

606 (60.54) 337 (33.67) 58 (5.79)

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All patients

GG GT TT Dominant Recessive Additive GG GT TT Dominant Recessive Additive

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Replication group

Spontaneous clearance N%

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Group

Persistence

96 (48.24) 89 (44.72) 14 (7.04)

304 (50.75) 255 (42.57) 40 (6.68)

OR (95% CI)

P value

Reference 1.64 (1.09-2.46) 2.15 (0.98-4.72) 1.70 (1.15-2.51) 1.74 (0.81-3.75) 1.55 (1.13-2.11) Reference 1.62 (1.29-2.03) 1.60 (1.02-2.51) 1.62 (1.30-2.01) 1.31 (0.85-2.04) 1.43 (1.20-1.70)

0.018 0.058 0.008 0.156 0.006 <0.001 0.042 <0.001 0.225 <0.001

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Logistic regression analyses adjusted for age, gender, high-risk population, ALT, AST, rs10336, rs3733236, and rs1801157.

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ACCEPTED MANUSCRIPT 3.3 Association of CXCR6 rs2234358 with virological response to treatment The relationship between the CXCR6 rs2234358 and virological response to treatment was similarly performed by four genetic models (co-dominant, dominant, recessive, and additive). We just analyzed the patients that who have completed the course of

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treatment. In the analysis, the distributions of CXCR6 rs2234358 were associated with

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different treatment responses. As shown in table 5, the SVR rate (77.56%) was higher

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in carriers with CXCR6 rs2234358 TT genotype and 69.60% for those carrying the GT variants, only 50.93% for GG patients. So, mutant T allele of CXCR6 rs2234358

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possibly could increase SVR rate in our study. The adjusted ORs were 2.93 (95% CI =

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1.22-7.07), 2.23 (95% CI = 1.26-3.95), 1.80 (95% CI = 1.19-2.72) in co-dominant, dominant, and additive models, respectively. Then, a stepwise regression model

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including all variables was established, the final model only included the CXCR6

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rs2234358 and AFP as independent predictors of SVR. Table 5 Associations of CXCR6 rs2234358 with SVR in chronic hepatitis C patients

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rs2234358 GG GT TT Dominant Recessive Additive

N-SVR (n=102) N%

SVR (n=180) N%

53 (51.96) 38 (37.25) 11 (10.79)

55 (30.56) 87 (48.33) 38 (21.11)

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Genotype

OR (95% CI)

P value

Reference 2.04 (1.11-3.74) 2.93 (1.22-7.07) 2.23 (1.26-3.95) 2.02 (0.89-4.60) 1.80 (1.19-2.72)

0.021 0.016 0.006 0.094 0.006

Abbreviation: N-SVR, non-sustained virological response; SVR, sustained virological response. Logistic regression analyses adjusted for age, gender, HCV RNA, albumin, platelets, AFP, TSH, FBG, rs10336, rs3733236 and rs1801157.

Similarly, the variant allele of rs2234358 was also associated with rapid virological response (RVR) in dominant model (OR = 2.08, 95% CI = 1.17-3.70). 15

ACCEPTED MANUSCRIPT However, we did not find any relationship between rs2234358 and complete early virological response (cEVR) (Supplementary Table S2). 3.4 Effect of the CXCR6 rs2234358 on time-dependent clearance of HCV The influence of CXCR6 rs2234358 on viral kinetics was further analyzed. The all

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HCV treatment patients were stratified by rs2234358 genotypes, and the rate of

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undetectable HCV RNA at 4, 8, 12, 24, and 48 weeks after the start of therapy were

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analyzed, as shown in Fig.1. The rate of undetectable HCV RNA was higher in patients with TT or GT genotypes compared with those with GG genotype. This

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finding might suggest why the patients with CXCR6 rs2234358 T allele have the

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protective effect, and were more likely to achieve SVR.

Figure 1 Effect of CXCR6 rs2234358 mutations on time-dependent clearance of HCV

4. Discussion Our study is the first to demonstrate that CXCR6 rs2234358 was significantly 16

ACCEPTED MANUSCRIPT associated to spontaneous clearance of HCV in Chinese Han population with a comparatively large sample size. Moreover, we explored the connection between rs2234358 and the virological response to treatment, and found CXCR6 rs2234358 TT genotypes had the highest SVR rate to PEG IFN-α/ribavirin therapy. Based on the

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above all, we may be could consider that CXCR6 rs2234358 indeed play a vital role

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on HCV infection outcomes and treatment response in the Chinese Han population.

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HCV as same as the other inflammatory diseases, such as HIV and diabetes mellitus type 2, is likely to be associated with the host's immune response. The CXCR6 is a

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chemokine receptor that is known as the participants in disease progression, and its

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role is a mediator of inflammation in HIV [17]. It is detected major in type 1 polarized T cells, including those in inflamed liver [18,19]. Rs2234358 is located in the three

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prime untranslated regions (3’UTR) of CXCR6, a chemokine receptor on T cells, and

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CXCR6 has been proved associating with colitides [20]. Lymphocyte subsets recruited by CXCL16 display an increased surface expression of CXCR6 in Chronic Hepatitis C

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(CHC) patients and are characteristically Th1-polarized [18,21]. Besides, a GWAS

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study has verified that the rs2234358 polymorphism in the CXCR6 gene was associated with long-term nonprogressors (LTNP) in HIV and this association was proved in three independent European studies (P=9.7-10) [11]. The results of the current study and the findings of the GWAS study make us have the sufficient reason to speculate that CXCR6 rs2234358 were associated with spontaneous clearance and virological response of HCV. The results of our study were consistent with the conjecture. 17

ACCEPTED MANUSCRIPT Studies have found that CXCL9 and CXCL10 were decreased in those for whom treatment had been successful, and the protective genotypes of CXCL9 rs10336 TT were associated with higher SVR rate in HCV genotype 1 and 4 patients already [22,23]. Besides, another study found CXCL9 rs10336 was associated with liver

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fibrosis in HIV/HCV co-infected patients [24]. We could conjecture that rs10336 was

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associated with the outcomes of HCV infection, but in our present study, spontaneous

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clearance and virological response of HCV with the CXCL9 rs10336 and rs3733236 did not show any significant difference. In this case, possible explanation was that the

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allele T of the rs3733236 and the allele T of the rs10336 polymorphisms within

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CXCL9 were found with the much low frequency in Chinese Han population. Our present study is the first to validate the association of rs2234358 in CXCR6 gene

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with the spontaneous clearance and respond treatment of HCV among high-risk

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Chinese Han population, and our sample size is relatively large, particularly in the two stage study. It is rare to study both the association between CXCR6 rs2234358

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with spontaneous clearance and the treatment response to PEG IFN-α/RBV therapy in

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one study. Moreover, patients included in our study were all just infected with HCV, without co-infection with HBV/HIV and other causes of liver disease, so it can reflect the natural history of HCV preferably. However, in the study, still several weaknesses need to be taken into consideration and have to conduct further verification. Firstly, we just determined only one SNP in CXCR6 gene. It suggests the limitation of relying on a single SNP typing to reflect the importance function of gene CXCR6. Secondly, in most developing countries like China, many new treatments are still in clinical 18

ACCEPTED MANUSCRIPT trials and expensive payment lead it will not be available in the future, thus, our study aimed at the response to IFN-based therapy seems to have limitations in the direct-acting antiviral (DAA) era. In conclusion, the present study highlighted the impact of the CXCR6 rs2234358 on

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the spontaneous clearance and the treatment response to PEG IFN-α/RBV therapy in

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HCV patients. CXC related-family polymorphisms might play an important role in

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HCV spontaneous clearance in Chinese Han population.

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Author Contributions: MC, PH and RY participated in the design of the study. YY,

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MY, FZ and ML carried out the surveys and experiments. MC, YY and JW performed the statistical analysis. HC, YZ and JL contributed materials and analysis tools. MC,

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YY and PH wrote the paper. All authors read and approved the final manuscript.

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ACCEPTED MANUSCRIPT Acknowledgment: We thank doctors and the nurses from the Jurong People’s Hospital for helping sample collection and organization of the field investigation. This research would not have been possible without the consent and help of the participants.

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Funding: The current study was supported in part by National Natural Science

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Foundation of China (No.81473029, 81502853 and 81473028), Priority Academic

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Program Development of Jiangsu Higher Education Institutions (PAPD), Medical Reform Project of Health and Family Planning Commission of Jiangsu Province of

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China (YG201413), Science and Technology Program of Zhenjiang, China

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(SH20141).

Conflict of interest: The authors declare that they have no conflict of interest.

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Ethical approval: Our study protocol was approved by the institutional ethics review

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committee of Nanjing Medical University. Informed consent: Written informed consent was obtained from all participants in

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this study.

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ACCEPTED MANUSCRIPT Reference 1.

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Abbreviations list: HCV: Hepatitis C virus PEG: pegylated RBV: ribavirin SNPs: single nucleotide polymorphisms SVR: sustained virological response HWE: Hardy-Weinberg equilibrium ALT: alanine aminotransferase AST: aspartate aminotransferase AFP: alpha fetal protein TSH: thyroid stimulating hormone FBG: fasting blood-glucose OR: odds ratio 95%CI: 95% confidence interval ROC: receiver-operating characteristic AUC: areas under the curve N-SVR: non-sustained virological response TP: total protein 3’UTR : the three prime untranslated regions CHC: Chronic Hepatitis C LTNP: long-term nonprogressors DAA: direct-acting antiviral

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Highlights • It is the first study to validate the association of CXCR6 rs2234358 with spontaneous clearance and virological response of HCV in Chinese Han population. • Both spontaneous clearance and treatment response were studied in one study. • Our sample size is relatively large so that it can provide enough statistical power. • Patients included in our study were all just infected with HCV, without co-infection with HBV/HIV and other causes of liver disease.

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