Polymorphisms in sodium taurocholate cotransporting polypeptide are not associated with hepatitis B virus clearance in Chinese Tibetans and Uygurs

    Polymorphisms in sodium taurocholate cotransporting polypeptide are not associated with hepatitis B virus clearance in Chinese Tibetans and Uygurs Zhenzhen Su, Yi Li, Yun Liao, Bei Cai, Jie Chen, Junlong Zhang, Lixin Li, Binwu Ying, Chuanmin Tao, Min Zhao, Zhu Ba, Zhaoxia Zhang, Lanlan Wang PII: DOI: Reference:

S1567-1348(16)30126-5 doi: 10.1016/j.meegid.2016.03.039 MEEGID 2698

To appear in: Received date: Revised date: Accepted date:

29 December 2015 18 March 2016 31 March 2016

Please cite this article as: Su, Zhenzhen, Li, Yi, Liao, Yun, Cai, Bei, Chen, Jie, Zhang, Junlong, Li, Lixin, Ying, Binwu, Tao, Chuanmin, Zhao, Min, Ba, Zhu, Zhang, Zhaoxia, Wang, Lanlan, Polymorphisms in sodium taurocholate cotransporting polypeptide are not associated with hepatitis B virus clearance in Chinese Tibetans and Uygurs, (2016), doi: 10.1016/j.meegid.2016.03.039

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ACCEPTED MANUSCRIPT Polymorphisms in Sodium Taurocholate Cotransporting Polypeptide are not

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Associated with Hepatitis B Virus Clearance in Chinese Tibetans and Uygurs

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Zhenzhen Sua, Yi Lia, Yun Liaoa, Bei Caia, Jie Chena, Junlong Zhanga, Lixin Lia, Binwu Yinga, Chuanmin Taoa, Min Zhaob, Zhu Bac, Zhaoxia Zhangd, Lanlan Wanga,* a

Department of Laboratory Medicine, West China Hospital, Sichuan University,

Department of Laboratory Medicine, People’s Hospital of Tibet Autonomous Region,

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b

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Chengdu 610041, China

Lhasa 850000, China

Lhasa 850000, China

Center of Laboratory Medicine, First Affiliated Hospital of Xinjiang Medical

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d

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Department of Laboratory Medicine, Tibetan Hospital of Tibet Autonomous Region,

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c

University, Urumchi 830054, China

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*Corresponding author Lanlan Wang

Address: No. 37 Guoxue Road, Wuhou District, Chengdu 610041, Sichuan, China Telephone: +86 28 85422752 Fax: +86 28 85422751 E-mail address: [email protected]

Abbreviations: NTCP, sodium taurocholate cotransporting polypeptide; LD, linkage disequilibrium.

ACCEPTED MANUSCRIPT Abstract: An association between polymorphisms in the sodium taurocholate cotransporting

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polypeptide (NTCP) and the natural course of hepatitis B virus (HBV) infection in the

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Chinese Han population has been noted. However, it is not known whether these polymorphisms are associated with the risk of developing chronic HBV infection in other racial or ethnic populations. Accordingly, we conducted a candidate single

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nucleotide polymorphism (SNP) association study in Tibetan and Uygur

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HBV-infected patients. A total of 1302 subjects including 871 Tibetans and 431 Uygurs were recruited. According to their serological and clinical characteristics, each

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ethnic group was divided into two groups comprising spontaneous clearance

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individuals and persistently infected patients. Three SNPs were genotyped by high

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resolution melting curve methodology. Among the SNPs, rs2296651 exhibited a minor allele frequency of <0.01. The frequency of allele A at rs4646287 was much

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higher in Tibetans (9.4% for Tibetans and 4.6% for Uygurs, p<0.001) than in Uygurs, but the frequency of allele A at rs7154439 was the opposite (15.7% for Tibetans and 20.5% for Uygurs, p=0.002). Irrespective of race, no significant differences in the frequency distributions of the three SNP alleles or genotypes were observed between the case and clearance groups. Moreover, none of the NTCP haplotypes were statistically different between the two groups. Data from the Tibetan patients could be grouped by HBeAg status, viral load and HBV genotype; however, no significant differences were found in the SNP genotype distribution for each characteristic. In conclusion, the NTCP polymorphisms we identified tended to be ethnicity-dependent.

ACCEPTED MANUSCRIPT For Tibetans and Uygurs, no association of the three SNPs (rs7154439, rs4646287 and rs2296651) and their haplotypes with HBV chronicity was observed. Examination of

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SNPs in NTCP for their specific associations with the course of HBV infection in

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other ethnic minority groups is now required.

Keywords: Chronicity; HBV; minority; sodium taurocholate cotransporting

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polypeptide; SNP.

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1. Introduction

Persistent hepatitis B virus (HBV) infection remains a serious public health

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problem, and worldwide, approximately 240 million individuals are chronic HBV

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carriers (Ott et al., 2012; Schweitzer et al., 2015). HBV is a major cause of

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progressive liver disease, including chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) (Arzumanyan et al., 2013). Both genetic and environmental factors

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contribute to HBV infection chronicity and its different clinical manifestations. Indeed, the genetic loci of the human leukocyte antigen system, as well as killer cell immunoglobulin-like receptors and cytokines, for example, are known to be associated with HBV clearance from the body, or persistence in the body to become a chronic infection (Thursz et al., 2011). Because human traits are inherited polygenically, person-to-person variation in a few genes or single nucleotide polymorphisms (SNPs) cannot fully explain the spectrum of disease variability in people. Accordingly, associations between HBV infection and host genetic diversity require careful investigation.

ACCEPTED MANUSCRIPT Sodium taurocholate cotransporting polypeptide (NTCP), a bile salt transporter that is expressed exclusively on the basolateral membrane of hepatocytes, has been

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identified recently in a biochemical approach as the receptor of HBV, thereby

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explaining the liver tropism and species specificity of HBV infection (Yan et al., 2012). Not long ago, we found that two SNPs in NTCP (rs7154439 and rs4646287) are possibly associated with the natural course of HBV infection. Individuals carrying

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the rs7154439 AA genotype have a possible tendency towards HBV clearance, while

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individuals carrying the rs4646287 AA genotype have a possible tendency towards HCC (Su et al., 2014). Another association study showed that rs2296651 of NTCP,

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which causes an amino acid change (S267F), may make the carrier more susceptible

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to HBV infection (Li et al., 2014). However, other studies have shown that rs2296651

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is significantly associated with resistance to chronic hepatitis B (Hu et al., 2015; Peng et al., 2015). All of these studies were conducted in the Chinese Han population, and

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no other ethnicities were included. Previous studies have indicated that the HBsAg-positive rate among Tibetans was much higher than that of the Chinese average (Zhao et al., 2001), while the Uygurs have the lowest prevalence of HBsAg positivity among the Han people and other ethnic minority groups (Ji et al., 2014). In addition, differences exist between the Tibetans and Uygurs in regard to the molecular epidemiology of HBV infections. For instance, the HBV genotype distribution was found to vary according to geography and ethnicity (Li et al., 2010; Shen et al., 2014; Yin et al., 2010), with genotype D frequently seen in Uygur HBV patients, while B/C and C/D recombinant genotypes

ACCEPTED MANUSCRIPT were endemic in Tibetans. However, the reasons for these ethnic discrepancies remain unclear. Beyond this, NTCP polymorphisms show wide variation among different

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populations from Asian to American (Ho et al., 2004; Pan et al., 2011), but NTCP

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polymorphisms specific to the various Chinese ethnic minorities have not yet been studied. Therefore, in this study, we examined polymorphisms in the NTCP gene in the two main heterogeneous ethnic minority groups in western China, Tibetans and

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Uygurs. We determined the allele, genotype, and haplotype distributions for NTCP for

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patients displaying HBV spontaneous clearance versus those with persistent infections in each ethnicity, and explored further the potential association of the clinical

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manifestations of HBV infection with this viral receptor at the gene level.

2.1 Subjects

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2. Materials and Methods

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The current study included 1302 subjects, who were either Chinese Tibetan (n=871, consisting of 432 controls and 439 cases), or Uygur (n=431, consisting of 234 controls and 197 cases). All the controls were HBV spontaneous clearance individuals and the cases were HBV persistently infected patients. Genetically, none of these patients were known to be related to any of the other subjects and all of their parents and grandparents were descendants of the aforementioned nationalities. The subjects were consecutively recruited from December 2011 to March 2014 in the following four hospitals: West China Hospital of Sichuan University, People’s Hospital of Tibet Autonomous Region, Tibetan Hospital of Tibet Autonomous Region and First

ACCEPTED MANUSCRIPT Affiliated Hospital of Xinjiang Medical University. Subjects that were persistently infected with HBV were positive for hepatitis B

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surface antigen (HBsAg) and antibodies specific for the hepatitis B core antigen

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(anti-HBc) over a period of at least 6 months. There was no cirrhosis or HCC in these patients, as confirmed by imaging, biopsy and/or α-fetoprotein examination. Specifically, cirrhosis was confirmed by liver biopsy and/or FibroScan (liver stiffness

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value >7.0 kPa); HCC was diagnosed by pathological examination and/or

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α-fetoprotein elevation (>400 ng/mL) combined with typical HCC imaging patterns (using abdominal ultrasound examinations, computed tomography, and/or magnetic

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resonance imaging). We divided the experiment into two stages, with Tibetans as the

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main research subjects and Uygurs as an auxiliary validation. As for the 439 Tibetan

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HBV carriers, clinical data, including HBV genotype (tested using an ABI 3130 Genetic Analyzer, Applied Biosystems, South San Francisco, CA, USA) and viral

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load (tested using a Light Cycler 480 II, Roche Diagnostics, Basel, Switzerland), were obtained from 389 and 241 individuals, respectively. High viral load HBV carriers were defined by HBV DNA levels of >105 copies/mL. All spontaneous clearance population had tested negative for HBsAg and positive for both anti-HBc and antibodies to HBsAg (anti-HBs) within the previous 6 months (as a minimum). Additionally, their liver functions were normal. The exclusion criteria used in this study is as follows: (1) evidence of past or current infection with other hepatitis viruses; (2) a positive test for antibodies to human immunodeficiency virus; (3) the presence of other types of liver diseases such as autoimmune hepatitis, toxic hepatitis,

ACCEPTED MANUSCRIPT hemochromatosis, Wilson’s disease, non-alcoholic steatohepatitis, primary biliary cirrhosis or Budd–Chiari syndrome. The study protocol conformed to the ethical

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guidelines of the 1975 Declaration of Helsinki, which had been reviewed and

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approved by the institutional ethics review committees at all participating hospitals, including the Ethical Committee of the West China Hospital of Sichuan University, the Ethics Committee of the People's Hospital of Tibet Autonomous Region, the

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Ethics Committee of the Tibetan Hospital of Tibet Autonomous Region and the Ethics

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Committee of the First Affiliated Hospital of Xinjiang Medical University. All participants provided informed written consent.

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2.2 Genotyping the NTCP Polymorphisms

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Three SNPs on chromosome 14, namely, rs7154439 (in the 5′ untranslated region

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of the NTCP gene), rs2296651 (in NTCP exon 4) and rs4646287 (in intron 1), were studied (Supplementary Figure 1 and Supplementary Table 1). rs7154439 and

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rs4646287 were selected for this study because we previously found that these two SNPs may be associated with HBV infection outcomes in the Chinese Han population (Su et al., 2014). In contrast, rs2296651, was selected after a priori review of the scientific literature based on its potential functional significance in NTCP-associated physiological function (Peng et al., 2015; Watashi et al., 2014). Whole blood (3 mL) was collected from each participant in EDTA tubes. Genomic DNA was extracted from 200 μL of the whole blood using a DNA extraction kit (Biotake Corporation, Beijing, China) according to the manufacturer’s instructions, and then stored at −80 C.

ACCEPTED MANUSCRIPT The three aforementioned SNPs were genotyped using the touchdown polymerase chain reaction (PCR) method and high resolution melting (HRM) technique in the

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Light Cycler 480 II system (Roche Diagnostics, Basel, Switzerland). Primers,

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designed by Primer Premier (version 5.0, Premier Biosoft International, Palo Alto, CA, USA), were synthesized by Bibliog Technologies Ltd., (Shanghai, China) (Supplementary Table 2). PCR amplification was conducted in a reaction mixture

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containing 1.0 μL of purified genomic DNA, 0.2 μL of 10 μM forward primer, 0.2 μL

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of 10 μM reverse primer, 2.4 μL of 25 mM MgCl2, 10 μL of Master Mix (including FastStart Taq DNA polymerase with ResoLight high-resolution melting dye and

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dNTPs), and 6.2 μL of H2O for each sample. The whole genotyping process

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encompassed four programs: pre-denaturation, amplification, high resolution melting

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and cooling, as has been described previously (Su et al., 2014). The data collected were analyzed by the Light Cycler 480 II Gene Scanning software v1.2 (Roche).

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Some samples genotyped previously by sequencing were used as reference standards. 2.3 Statistical Analyses All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, version 17.0, SPSS Inc., Chicago, IL, USA). Linkage disequilibrium (LD) and haplotypes were assessed using Haploview (version 4.2, Mark Daly’s lab at the Broad Institute, Cambridge, MA, USA). For continuous variables, comparisons between groups were performed using the Mann–Whitney U test. Chi-squared tests with Fisher’s exact test were used to compare the categorical variables. SNP-specific deviations from the Hardy–Weinberg Equilibrium (HWE) were tested using the

ACCEPTED MANUSCRIPT chi-squared goodness-of-fit test. Two genetic models, specifically codominant and recessive were used to test for associations between SNPs and HBV infection

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chronicity. The frequency of genotypes, alleles and haplotypes in the patients and

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clearance individuals were compared using chi-squared tests with Fisher’s exact test where appropriate. The strength of associations was estimated by odds ratio with 95% confidence intervals. The chi-squared test was used also to compare the frequency

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distributions of SNP genotypes among Tibetan patients with different HBeAg

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(hepatitis B e antigen) statuses, viral loads and HBV genotypes. The power of this study was evaluated by Power and Sample Size Calculation (version 3.1.2,

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http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize) (Dupont and Plummer,

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1998). All tests were two-tailed, and p values of <0.05 were regarded as statistically

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3. Results

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significant.

3.1 Baseline Characteristics of the Study Population Table 1 shows the characteristics of the cases and spontaneous clearance subjects for each ethnicity. For the Tibetans, age and sex were not matched (p=0.041 and <0.001, respectively); the patients’ median age was 39 years (interquartile range from 30 to 49 years), 67.4% were male and 66.1% were HBeAg positive, while the median age of the spontaneous clearance persons was 40 years (interquartile range from 30 to 49 years) and 53.9% were male. Two-hundred and twenty-three (57.3%) of the HBV-positive patients were infected with genotype C, 160 (41.1%) were infected with

ACCEPTED MANUSCRIPT genotype D, and 6 were infected with genotype B. Additionally, 65.1% of the Tibetan patients fell into the high viral load group after stratification of the viral load (cut-off

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value, 105 copies/mL). The Uygur patient group contained a higher percentage of men

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and they were somewhat older (p<0.001) than the spontaneous clearance subjects. 3.2 Distribution of NTCP Polymorphisms in each Ethnicity

The HRM results returned a genotype success rate of over 99.0% for the three

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SNPs, and the genotyping failures were ascertained by direct sequencing. In the

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Tibetan spontaneous clearance group, the genotype distribution for rs7154439 deviated from HWE, as did rs4646287 in the Uygur clearances. As a quality control,

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about 10% of the samples were randomly selected and genotyped in duplicate using

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HRM, and the overall concordance rate was over 99.5 % on average. Additionally, we

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verified the homozygous mutant individuals by two methods, HRM and sequencing, and the genotyping results from these methods were 100% consistent with each other.

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For rs2296651, only two genotypes were distinguished: wild-type homozygous GG and heterozygous GA. In this study, the other two SNPs were all classified into three genotypes.

Among the SNPs, rs2296651 showed a minor allele frequency of <0.01. The frequency of allele A at rs4646287 was much higher in the Tibetans (9.4% for Tibetans and 4.6% for Uygurs, p<0.001) than in the Uygurs, but the frequency of allele A at rs7154439 was the opposite (15.7% for Tibetans and 20.5% for Uygurs, p=0.002). Irrespective of race, there were no significant differences in the frequencies of the alleles or genotypes between the case and spontaneous clearance groups (Tables

ACCEPTED MANUSCRIPT 2 and 3, all p>0.05). LD analysis of the SNPs in our data found that three SNPs were in low LD (Figure 1, all r2<0.4). Next, we used Haploview to calculate the number of

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possible haplotypes. We did not observe any significant differences in the haplotype

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frequency distributions between the viral clearance subjects and the HBV persistently infected patients of each racial background (Table 4, all p>0.05).

Polymorphisms in Tibetan Patients

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3.3 Association of HBeAg Status, Viral Load and HBV Genotype with NTCP

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To explore further the association of NTCP polymorphisms with the different clinical features of HBV infection, the persistently infected Tibetan patients were

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divided into two groups according to their HBeAg statuses, viral loads and HBV

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genotypes. We examined the SNP genotype distribution, looking for associations in

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this distribution with HBeAg status, viral load and HBV genotype using codominant and recessive models. Overall, potential correlations between the NTCP

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polymorphisms and the clinical presentations of the patients were not statistically significant (Table 5, all p>0.05).

4. Discussion NTCP, a bona fide HBV receptor, confers hepatocyte susceptibility towards HBV infection (Yan et al., 2012). It is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function (Anwer and Stieger, 2014). We suppose that the variants in NTCP gene may directly influence NTCP protein (especially amino acid 84 to 87 and 157 to 165, which were identified to be

ACCEPTED MANUSCRIPT critical for pre-S1 binding and viral infections) expression, or affect its activity, then influence NTCP function and finally inhibit/promot HBV infection. Hence, the

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current study attempted to evaluate the potential associations of three SNPs in the

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NTCP gene (rs7154439, rs4646287 and rs2296651) and their haplotypes with the natural course of HBV infection in two independent ethnic groups. However, no evidence of associations between any of the SNPs and HBV spontaneous clearance

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was observed. Additionally, no significant differences were found in the distribution

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of SNP genotypes in the patients in terms of their different HBeAg statuses, their viral loads or the HBV genotype groups in the Tibetan patients.

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In terms of the genetic background of NTCP, several SNPs that alter its

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transporter activity have been reported (Watashi et al., 2014). In fact, a recent study

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found that mutation of the NTCP residues that are critical for bile salt binding severely impaired infection with HBV (Yan et al., 2014). However, NTCP-rs2296651,

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when expressed in HEK293-FlpIn cells, conferred a reduction in the normal NTCP-mediated bile acid transport activity via this genetic variant (Pan et al., 2011); nonetheless, we found no association between rs2296651 and HBV infection chronicity in this study. There are two possible explanations for our result. First, two research studies have reported that the likelihood of people being carriers of NTCP polymorphisms is dependent on their ethnicity. rs2296651, which has been identified as an Asian-specific SNP, was found with relatively higher frequency in Chinese (7.4%) and Vietnamese (9.2%) populations than in Korean populations (3.1%), but was not seen in European Americans or African Americans(Ho et al., 2004; Pan et al.,

ACCEPTED MANUSCRIPT 2011). However, all the Chinese subjects recruited for these studies were Han people. In the Tibetan and Uygur populations, the rs2296651 allele A frequencies were 0.2%

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and 0.3%, respectively. Because we found that rs2296651 was extremely rare among

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the Tibetan and Uygur ethnic minorities, this SNP appears not to be an important genetic factor influencing HBV infection progression in these populations. Our previous study also showed that HLA-DP/DQ was the causative gene in that it not

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only contributed to natural clearance of HBV, but also accounted for the discrepancy

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in the global HBV infection prevalence (Liao et al., 2014). Furthermore, because over 70% of viral infections were observed when the rs2296651 variant was cotransfected

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with the wild-type NTCP at a ratio of 1:1, this indicates that heterozygous individuals

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may also be susceptible to HBV infection (Yan et al., 2014). Individuals included in

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the present study carried either a single allele or were homozygous without the NTCP rs2296651 substitution; hence, they are still susceptible to infection by HBV. Despite

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having previously reported that the rs7154439 AA genotype protected patients from chronic HBV infections in the Chinese Han population, no such association was found in the Tibetans and Uygurs studied herein. The reason for this discrepancy is not clear but might be related to ethnic differences and heterogeneity between the patients from the two populations. However, as the sample size for the Uygurs was relatively small, our study possibly did not achieve enough statistical power to discern an intricate association. Nonetheless, consistent with a previous study conducted on the Han population, the rs4646287 genotype had no significant effect on the persistence of HBV infection, which indicates it plays an irrelevant role in susceptibility to infection

ACCEPTED MANUSCRIPT by HBV. Evidence has shown that the clinical and virological manifestations of HBV

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infections are usually correlated with the development of liver disease. Viral load has

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emerged as the most significant factor implicated in the development of cirrhosis or HCC (Chen et al., 2006; Iloeje et al., 2006). In addition, successful HBeAg seroconversion often coincides with a loss of viral replication, which marks the

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transition from the active to inactive phase of infection characterized by the remission

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of disease activity, and possibly even followed by spontaneous HBsAg seroclearance (Hsu et al., 2002; Trepo et al., 2014). Concerning the viral genotypes identified in our

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study, most of the Tibetans were infected with genotypes C and D. With better

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understanding of the relationship between HBV genotypes, progression of hepatitis B

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disease, and clinical outcomes developing over time, differences in the clinical and virological features between genotypes C and D have been found, such as HBeAg

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positivity, the frequency of the PC A1896 mutation and the basal core promoter T1762/A1764 mutation (Liu and Kao, 2013). The tendency towards chronicity after acute HBV infection can be explained partly by the viral genotypes (Mayerat et al., 1999; Wai et al., 2005; Zhang et al., 2008). However, in the present study, no differences in the SNP genotype distributions were observed among patients with different virus genotypes, different HBeAg statuses or different viral loads. Therefore, polymorphisms in NTCP probably do not influence the manifestations of HBV infection, or even the clinical outcomes. We hypothesized that although NTCP functions as a cellular receptor for HBV entry through a specific interaction between

ACCEPTED MANUSCRIPT NTCP and the pre-S1 domain of the HBV large envelope protein, there may be multiple cellular factors required for efficient viral entry. Moreover, viral entry is only

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one step in efficient viral infection and replication in human hepatocytes, and other

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factors, such as the host immune response, are likely to be involved. Accordingly, it is feasible that other factors, such as individual host immune responses, may have impacted on the potential relationships between polymorphisms in NTCP and the

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clinical features of the HBV infections in this study.

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Our study has a few potential limitations. First, all the SNPs involved in the study have been validated to be associated with HBV infection in Han people previously,

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thus novel loci were not be established in this work. Second, the statistical power of

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our study may be limited by the sample size, especially for the Uygur population.

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Additionally, we lacked detailed clinical information for the Uygurs; thus, further analysis to identify any associations between the SNPs and the clinical characteristics

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of these patients could not be done. Third, because no cirrhosis or HCC patients were available, we were unable to appraise whether an association between NTCP polymorphisms and cirrhosis or HCC existed in the ethnic minorities that we studied. Last but not least, we did not test the disease activity related biochemical markers such as AST and ALT levels. Therefore, whether genetic variations in NTCP contribute to disease activity of chronic HBV infection could not be elucidated in this study. In conclusion, our study shows that the three SNPs (rs2296651, rs4646287 and rs7154439) within the NTCP gene and their haplotypes are not associated with

ACCEPTED MANUSCRIPT chronic HBV infections in two Chinese minorities, Tibetan and Uygur. Further study with a larger sample size would provide more definitive information. However, for

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these two races, this study cannot rule out the possible association of NTCP

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polymorphisms with HBV-related liver cirrhosis or hepatocellular carcinoma. Additionally, the ethnicity heterogeneity is strictly related to HBV and NTCP polymorphisms and this might be affected by the different influence of NTCP in

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different populations; therefore, identification of SNPs in NTCP that have a specific

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association with HBV infection in minorities is now required.

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Figure Caption:

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Figure 1. Linkage disequilibrium (LD) map for the three NTCP SNPs in Tibetans

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and Uygurs.

The pair-wise LD between loci (r2) was measured using Haploview 4.2. The strength

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of LD is depicted graphically for each pair-wise comparison (squares), and white represents low levels of LD (r2<0.4). Left: LD map obtained from the Tibetan population; Right: LD map obtained from the Uygur population.

Supplementary Figure 1. Gene structure of NTCP showing the positions of the three candidate SNPs. The exon/intron structure of the gene is indicated by black blocks/lines. The relative distances (in bp) of the three study SNPs in the NTCP gene are shown. White blocks represent 5′ and 3′ untranslated regions.

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Acknowledgements

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This study supported by grants from the Natural Science Foundation of China (No.

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81273256, 81202354). We thank Hong Tang and Enqiang Chen (Center of Infectious Disease, West China Hospital of Sichuan University, China) for their useful advice on case definitions. We also wish to thank all the study participants who generously

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agreed to be tested for this project.

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Peng, L., Zhao, Q., Li, Q., Li, M., Li, C., Xu, T., Jing, X., Zhu, X., Wang, Y., Li, F., Liu, R., Zhong, C., Pan, Q., Zeng, B., Liao, Q., Hu, B., Hu, Z.X., Huang, Y.S., Sham, P., Liu, J., Xu, S., Wang, J., Gao, Z.L., Wang, Y., 2015. The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic

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hepatitis B. Hepatology (Baltimore, Md.) 61, 1251-1260. Schweitzer, A., Horn, J., Mikolajczyk, R.T., Krause, G., Ott, J.J., 2015. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet (London, England) 386, 1546-1555.

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Shen, L., Yin, W., Zheng, H., Cui, F., Zhang, S., Wang, F., Wang, F., Zhang, Y., Liang, X., Bi, S., 2014. Molecular epidemiological study of hepatitis B virus genotypes in Southwest, China. Journal of medical virology 86, 1307-1313. Su, Z., Li, Y., Liao, Y., Cai, B., Chen, J., Zhang, J., Li, L., Ying, B., Tao, C., Wang, L., 2014. Association of the gene polymorphisms in sodium taurocholate cotransporting polypeptide with the outcomes of hepatitis B infection in Chinese Han population. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 27, 77-82. Thursz, M., Yee, L., Khakoo, S., 2011. Understanding the host genetics of chronic hepatitis B and C. Seminars in liver disease 31, 115-127. Trepo, C., Chan, H.L., Lok, A., 2014. Hepatitis B virus infection. Lancet. Wai, C.T., Fontana, R.J., Polson, J., Hussain, M., Shakil, A.O., Han, S.H., Davern, T.J., Lee, W.M., Lok, A.S., 2005. Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States. Journal of viral hepatitis 12, 192-198. Watashi, K., Urban, S., Li, W., Wakita, T., 2014. NTCP and beyond: opening the door to unveil hepatitis B virus entry. International journal of molecular sciences 15, 2892-2905. Yan, H., Peng, B., Liu, Y., Xu, G., He, W., Ren, B., Jing, Z., Sui, J., Li, W., 2014. Viral entry of hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium

ACCEPTED MANUSCRIPT taurocholate cotransporting polypeptide. Journal of virology 88, 3273-3284. Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B., Wang, H., Fu, L., Song, M., Chen, P., Gao, W., Ren, B., Sun, Y., Cai, T., Feng, X., Sui, J., Li, W., 2012. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.

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eLife 1, e00049. Yin, J., Zhang, H., He, Y., Xie, J., Liu, S., Chang, W., Tan, X., Gu, C., Lu, W., Wang, H., Bi, S., Cui, F.,

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Liang, X., Schaefer, S., Cao, G., 2010. Distribution and hepatocellular carcinoma-related viral properties of hepatitis B virus genotypes in Mainland China: a community-based study. Cancer

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epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 19, 777-786. Zhang, H.W., Yin, J.H., Li, Y.T., Li, C.Z., Ren, H., Gu, C.Y., Wu, H.Y., Liang, X.S., Zhang, P., Zhao, J.F., Tan, X.J., Lu, W., Schaefer, S., Cao, G.W., 2008. Risk factors for acute hepatitis B and its

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progression to chronic hepatitis in Shanghai, China. Gut 57, 1713-1720.

Zhao, S.M., Li, H.C., Lou, H., Lu, X.X., Yu, X.F., Gao, D.H., Hu, J., Chiba, H., Takezaki, T., Takeshita, H., Yashiki, S., Fujiyoshi, T., Sonoda, S., Tajima, K., 2001. High Prevalence of HBV in Tibet, China.

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Asian Pacific journal of cancer prevention : APJCP 2, 299-304.

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ACCEPTED MANUSCRIPT Tables Table 1. General characteristics of the subjects from each ethnicity Tibetan population (n=871)

Uygur population (n=431)

Characteristics

Clearance (n=432)

Persistence (n=439)

p Value

Clearance (n=234)

Age, year Male sex, n (%) HBeAg, n (%) Positive Negative Viral load, n (%)* High Low HBV genotype, n (%)* B C D

40 (30, 55) 233 (53.9)

39 (30, 49) 296 (67.4)

0.041 <0.001

48 (39, 58) 140 (59.8)

0 (0.0) 432 (100.0)

290 (66.1) 149 (33.9)

—

— —

157 (65.1) 84 (34.9)

— — —

6 (1.5) 223 (57.3) 160 (41.1)

p Value

T

Persistence (n=197)

<0.001 0.657

— —

— —

—

— —

— —

—

— — —

— — —

—

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29 (23, 42) 122 (61.9)

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—

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Abbreviations: HBeAg, hepatitis B e antigen; HBV, hepatitis B virus. Data are presented as median (interquartile range) or frequency (percentage); —, data were not available. * HBV viral load and genotype information were obtained from 241 and 389 persistently infected Tibetans, respectively. Statistically significant p-values (<0.05) are highlighted in bold.

Table 2. NTCP genotype distributions between HBV spontaneous clearance and persistently infected subjects from each ethnicity Ethnicity

SNP ID

Model

Genotype

Clearance

Persistence

OR (95% CI)

Tibetan

rs2296651

Codominant

GG GA

429 (99.3) 3 (0.7)

438 (99.8) 1 (0.2)

GG GA

361 (83.6) 70 (16.2)

354 (80.6) 79 (18.0)

AA

1 (0.2)

6 (1.4)

GG+GA

431 (99.8)

433 (98.6)

1.00 0.33 (0.03-3.15) 1.00 1.15 (0.81-1.64) 6.12 (0.73-51.08) 1.00

rs4646287

Codominant

Recessive

p Value

Power

0.370

0.175

0.436

0.121

0.068

0.369

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rs4646287

Codominant

Codominant

Recessive Codominant

15 (3.5)

14 (3.2)

GG+GA AA

417 (96.5) 15 (3.5)

425 (96.8) 14 (3.2)

GG GA

233 (99.6) 1 (0.4)

195 (99.0) 2 (1.0)

GG GA

216 (92.3) 15 (6.4)

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AA

178 (90.4) 19 (9.6)

3 (1.3) 231 (98.7) 3 (1.3) 149 (63.7) 71 (30.3)

0 (0.0) 197 (100.0) 0 (0.0) 125 (63.5) 66 (33.5)

AA

14 (6.0)

6 (3.0)

GG+GA AA

220 (94.0) 14 (6.0)

191 (97.0) 6 (3.0)

AA GG+GA AA GG GA

5.97 (0.72-49.82) 1.00 1.24 (0.91-1.69) 0.97 (0.46-2.04) 1.00 0.92 (0.44-1.92) 1.00 2.39 (0.22-26.55) 1.00 1.54 (0.76-3.11) — 1.00 — 1.00 1.11 (0.74-1.67) 0.51 (0.19-1.37) 1.00 0.49 (0.19-1.31)

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308 (70.2) 117 (26.7)

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rs7154439

319 (73.8) 98 (22.7)

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rs2296651

AC

Uygur

GG GA

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Recessive

6 (1.4)

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Codominant

1 (0.2)

D

rs7154439

AA

0.124

0.414

0.180

0.269

0.929

0.050

0.816

0.056

0.595

0.118

0.230

0.227

0.256

0.315

0.254

0.349

0.624

0.078

0.244

0.057

0.149

0.068

Abbreviations: OR, odds ratio; CI confidence interval. Data are presented as frequency (percentage) for each group; —, data were not available. The differences in genotype frequencies between the viral spontaneous clearance controls and the persistently infected patients of each ethnicity were analyzed using chi-squared tests. The power was evaluated by Power and Sample Size Calculation. The ORs calculated include 95% CIs (shown in brackets). A two-sided p value of <0.05 was considered to be statistically significant.

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Ethnicit y

SNP ID

Allel e

Tibetan

rs229665 1

G

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Table 3. NTCP allele distributions between the HBV spontaneous clearance and persistently infected subjects from each ethnicity Frequenc y*

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D

0.998

Clearan ce

Persisten ce

OR (95% CI)

861 (99.7)

877 (99.9)

1.00

3 (0.3)

1 (0.1)

0.33 (0.03-3.15 )

792 (91.7)

787 (89.6)

1.00

72 (8.3)

91 (10.4)

1.27 (0.92-1.76 )

736 (85.2)

733 (83.5)

1.00

128 (14.8)

145 (16.5)

1.14 (0.88-1.47 )

467 (99.8)

392 (99.5)

1.00

1 (0.2)

2 (0.5)

2.38 (0.22-26.3 8)

447 (95.5)

375 (95.2)

1.00

21 (4.5)

19 (4.8)

1.08 (0.57-2.04 )

0.002

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A rs464628 7

G

0.906 0.094

AC

A

rs715443 9

G

0.843 0.157

A

Uygur

rs229665 1

G

0.997 0.003

A rs464628 7

G

0.954 0.046

A

p Valu e

0.37 1

0.14 6

0.32 9

0.59 5

0.81 6

Powe r

0.157

0.303

0.168

0.166

0.063

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G

0.795

369 (78.8)

316 (80.2)

1.00

78 (19.8)

0.92 (0.66-1.28 )

0.205 99 (21.2)

0.108

T

A

0.62 3

D

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Abbreviations: OR, odds ratio; CI confidence interval. Data are presented as frequency (percentage) for each group. The differences in the allele frequencies between the viral spontaneous clearance controls and the persistently infected patients of each ethnicity were analyzed using chi-squared tests. The power was evaluated by Power and Sample Size Calculation. The ORs calculated include 95% CIs (shown in brackets). A two-sided p value of <0.05 was considered to be statistically significant. *The data represent the allele percentages for all the Tibetan or Uygur subjects recruited in this study.

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Clearance

Persistence

OR (95% CI)

0.750 0.154

663 (76.7) 126 (14.6)

644 (73.3) 142 (16.2)

GAG

0.091

70 (8.1)

88 (10.0)

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Table 4. NTCP haplotype distributions between HBV spontaneous clearance and persistently infected subjects from each ethnicity

GGG GGA

0.751 0.200

350 (74.8) 96 (20.5)

297 (75.4) 76 (19.3)

GAG

0.041

18 (3.8)

17 (4.3)

1.00 1.16 (0.89-1.51) 1.29 (0.93-1.80) 1.00 0.93 (0.67-1.31) 1.11 (0.56-2.20)

Haplotype*

Tibetan

GGG GGA

Uygur

Frequency#

CE P

Ethnicity

p Value

Power

0.268

0.187

0.127

0.288

0.688

0.069

0.758

0.059

Abbreviations: OR, odds ratio; CI, confidence interval. Data are presented as frequency (percentage) for each group. The differences in the haplotype frequencies between the viral spontaneous clearance controls and the persistently infected patients of each ethnicity were analyzed using chi-squared tests. The power was evaluated by Power and Sample Size Calculation. The ORs calculated include 95% CIs (shown in brackets). A two-sided p value of <0.05 was considered to be statistically significant. *All frequencies <0.03 were ignored in this analysis. Loci chosen for hap-analysis were in this order: rs2296651, rs4646287, rs7154439. # The

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data represent the haplotype percentage for all the Tibetan or Uygur subjects recruited in this study.

ACCEPTED MANUSCRIPT Table 5. Association of NTCP genotypes with clinical features in Tibetan HBV patients

Genotype

Codominant

157

1

0

(0.3)

(0.0)

234

120

(100.0) —

1.00

GG (80.7)

(80.5)

53

26

1.05 0.867

(18.3)

(17.4)

(0.62-1.76)

3

3

0.51

0.415

AA (2.0)

287

146

(99.0)

(98.0)

3

3

0.413

1

(0.0)

(1.2)

126

73

206

102

(71.0)

(68.5)

78

39

(80.3)

(86.9)

30

10 (11.9)

(0.80-3.76)

1

1

0.58

(1.2)

156

83

(99.4)

(98.8)

1

1 (1.2)

111

61

(70.7)

(72.6)

6

8

0.37 0.083

AA (2.1)

(5.4)

284

141

43

21

(97.9)

(94.6)

6

8 (5.4)

0.37 (0.13-1.09)

1

0

(0.4)

(0.0)

183

131

(82.1)

(81.9)

37

28

1.06

(16.6)

(17.5)

(0.62-1.81)

3

1

0.47

(1.3)

(0.6)

(0.05-4.53)

220

159

(98.7)

(99.4)

3

1

0.46

(1.3)

(0.6)

(0.05-4.48)

0.099

(0.03-8.62)

(25.0)

(0.61-2.07)

3

2

0.82 1.000

(1.9)

(2.4)

154

82

(98.1)

(97.6)

157

112

(70.4)

(70.0)

59

45

1.07

(26.5)

(28.1)

(0.68-1.69)

7

3

0.60

(3.1)

(1.9)

(0.15-2.37)

216

157

(96.9)

(98.1)

7

3

0.59

(3.1)

(1.9)

(0.15-2.32)

0.065

0.060

(0.13-5.07)

3

2

1.000 (1.9)

(2.4)

(0.13-4.88)

0.086

0.840

0.056

0.644

0.077

0.643

0.088

0.774

0.060

0.532

0.086

0.531

0.108

1.00

0.80

0.472

1.000

1.00

1.13

(27.4)

—

0.083

1.00

0.084 (2.1)

(100)

0.284

0.53

0.380

(0.13-1.10)

AA

(99.6)

1.00

0.704

1.00

GG+GA

160

0.341

(0.04-9.40)

0.050

Value

Power

1.00

1.000 (0.6)

(95% CI) 222

1.00

1.000 (0.63-1.56)

0.349

1.000 (0.6)

p

1.00

0.157

(19.1)

OR D

Value

1.74

0.159

0.99

(26.2)

C

1.00

(0.10-2.55)

(26.9)

Power

1.00

1.00

GG

—

0.144

AC

(2.0)

p

1.00

0.053

0.51

GA

Recessive

0.036

HBV genotype *

(98.8)

1.00

GG+GA

(1.0) Codominant

(0.10-2.58)

CE P

(1.0)

AA

rs7154439

0

1.000

83

US

(100.0)

GA

Recessive

(95% CI)

1.00 (99.7)

Low

Value

149

GG

GA

rs4646287

High

IP

289 Codominant

OR

Power (95% CI)

rs2296651

p

Negative

CR

OR Positive

MA N

Model

TE D

SNP ID

Viral load

T

HBeAg

0.065

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Abbreviations: HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; OR, odds ratio; CI, confidence interval. Data are presented as frequency (percentage) for each group; —, data were not available. Differences in the genotype frequencies in respect to HBeAg status, viral load and HBV genotype groups in Tibetans were analyzed using chi-squared tests. The power was evaluated by Power and Sample Size Calculation. The ORs calculated include 95% CIs (in brackets). A two-sided p value of <0.05 was considered to be statistically significant. * As only six subjects were infected with HBV genotype B, we disregarded them in this association analysis.

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Highlights:  It’s the first study about NTCP variations and HBV infection in Chinese minorities.  We find that the NTCP polymorphisms tend to be ethnicity-dependent.  No association of the three SNPs studied herein with HBV chronicity was observed.