Liver International ISSN 1478-3223
VIRAL HEPATITIS
Association of HLA-DP/DQ, STAT4 and IL-28B variants with HBV viral clearance in Tibetans and Uygurs in China Yun Liao1, Bei Cai1, Yi Li1, Jie Chen1, Binwu Ying1, Chuanmin Tao1, Min Zhao2, Zhu Ba3, Zhaoxia Zhang4 and Lanlan Wang1 1 2 3 4
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China Department of Laboratory Medicine, People’s Hospital of Tibet Autonomous region, Lhasa 850000, China Department of Laboratory Medicine, Tibetan Hospital of Tibet Autonomous region, Lhasa 850000, China Center of Laboratory Medicine, First Affiliated Hospital of Xinjiang Medical University, Urumchi 830054, China
Keywords HBV – HLA-DP/DQ – IL-28B – STAT4 – Tibetan – Uygur
Correspondence Lanlan Wang, Department of Laboratory Medicine, West China Hospital, Sichuan University, No.37 Guoxue Alley, Wuhou district, Chengdu 610041, China Tel/Fax: +86-28-85422751 e-mail: [email protected] Received 23 April 2014 Accepted 15 July 2014 DOI:10.1111/liv.12643 Liver Int. 2015; 35: 886–896
Abstract Background & Aims: Several genome-wide association studies have revealed that HLA-DP/DQ, STAT4 and IL-28B associated with liver diseases. But because of population heterogeneity, different races would have different causative polymorphisms. Therefore, in this study, we included Chinese Tibetans and Uygurs to examine the roles of these genes on HBV natural clearance. Methods: A total of 1341 subjects including 908 Tibetans and 433 Uygurs were recruited. Seven single nucleotide polymorphisms (SNP) were genotyped. Results: HLA-DP/DQ polymorphisms associated with HBV natural clearance in both ethnicities (Tibetans, rs3077, OR = 0.61, 95% CI = 0.46–0.82; rs9277535, OR = 0.56, 95% CI = 0.41–0.75; rs7453920, OR = 0.64, 95%CI = 0.47–0.85; Uygurs, rs3077, OR = 0.48, 95% CI = 0.24–0.96; rs9277535, OR = 0.43, 95% CI = 0.20–0.91; rs7453920, OR = 0.62, 95% CI = 0.39–0.99), whereas no significant association was observed between IL-28B with HBV natural clearance in neither ethnicities (P > 0.05). STAT4 rs7574865 seemed to be Tibetan specific in HBV natural clearance (OR = 0.76, 95% CI = 0.58–0.99). Moreover, in Tibetan patients, HLA-DQ rs7453920 GG had a higher frequency in HBeAg positive patients (P = 0.032) and STAT4 rs7574865 GG genotype appeared more frequently in Genotype C virus infected patients (P = 0.005). In addition, Uygurs have higher frequencies of HLA-DP/DQ protective alleles (72.5% for rs3077, 76.6% for rs9277535 and 26.8% for rs7453920) than Tibetans (51.7% for rs3077, 52.5% for rs9277535 and 18.5% for rs7453920)(all P < 0.05), and a lower prevalence of HBV infection as previously reported. Conclusions: HLA-DP/DQ but not IL-28B polymorphisms correlate with HBV natural clearance, irrespective of race, and HLA-DP/DQ would be causative genes attributing to varying prevalence in different regions. STAT4 rs7574865 seemed to be population specific in Tibetans and it might synergistically interact with virus contributing to disease progression.
The infection of Hepatitis B virus (HBV) has been a worldwide health problem, which could contribute to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). Globally, more than 2 billion people have been infected with HBV. Among them, 400 million are chronic carriers, which pose great threat to the health worldwide (1, 2). Recently, several Genome-Wide association studies (GWAS) demonstrated that single nucleotide polymorphisms (SNP) of human leucocyte antigen (HLA) might be pertinent to persistent infection after being infected with HBV, especially polymorphisms on HLA-DP/DQ
886
loci (3, 4). Subsequently, these findings were validated in Chinese Han population, comparing patients with chronic hepatitis and those who were deemed as spontaneous clearance persons (antibody to HBsAg positive and antibody to hepatitis core antigen positive, i.e. antiHBs and anti-HBc positive) (5, 6). A prospective study performed in Hongkong investigating pertinence of HLA-DP polymorphisms (rs3077, rs9277378, rs3128917) and HBsAg seroclearance further consolidated the conclusion that HLA-DP polymorphisms correlated with virus clearance after infection with HBV, but posed a question whether HLA-DP polymorphisms Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Liao et al.
would have similar effects on different races (7). Another GWAS study conducted in Chinese Han population substantiated that polymorphisms of Signal Transducer and Activator of Transcription 4 (STAT4) might correlate with progression of HCC (8). STAT4 belongs to STAT family and transmits signals mainly from interleukin-12 (IL-12) to induce interferon-c (IFN-c). Though the GWAS study has explicitly illustrated that STAT4 rs7574865 correlates with the progression of HCC, whether it correlates with spontaneous clearance of HBV still remains elusive. Moreover, recent investigations indicated that IL-28B polymorphisms, which was considered to be correlated with spontaneous clearance of hepatitis C virus and treatment response (9, 10), might associate with hepatitis e antigen (HBeAg) seroconversion and HBsAg seroclearance (11, 12). But some studies denied this (13). Apart from the conflicting results, most of the studies investigating IL-28B were conducted in Han population, no other ethnicities were included. As is known, the prevalence of HBV infection varies greatly based on region disparity. In North American and Europe, the prevalence of HBV infection is rather low, but in East Asia, this number soared up (14). Besides, evidence showed that disease aetiology is common generally, but the risk variants are often population specific. And the difference can be truly originated from ethnic disparity since the degree of genetic variations differs among individuals of different ethnicities (15). Previous study has indicated that the East Asian populations, including Chinese Han, Korean and Japanese, have similarity in the genetic backgrounds. While Uygurs live in the vicinity of Central Asia and the genetic background resembles Hazara, but differ greatly from the Han population (16). As for Tibetans, few studies have involved this ethnicity who lived at a high altitude and their genetic backgrounds as well as the impact on susceptibility of HBV still remain obscure. Therefore, in this study, we recruited Tibetans and Uygurs, attempting to unveil the risk variants of HBV infection in minorities to further attest whether current hot spots of HLA-DP/DQ, STAT4 and IL-28B still correlate with persistent infection of HBV, trying to clarify the importance of these host genetic factors on HBV natural clearance in different races. Methods and materials Subjects
The present study recruited mainly Chinese Tibetans and Uygurs as this study subjects. In total, 1341 subjects, including 908 Chinese Tibetans (422 Tibetan chronic HBV carriers and 486 Tibetans with natural clearance after HBV infection as the control source) and 433 Uygurs (198 Uygur HBV carriers and 235 Uygur controls who naturally cleared the virus), were enrolled. All the subjects were recruited from West China Hospital of Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Genetic variants in HBV in minorities
Sichuan University, People’s Hospital of Tibet Autonomous region, Tibetan Hospital of Tibet Autonomous region and First Affiliated Hospital of Xinjiang Medical University from June 2011 to May 2013. Each race constitutes one case-control study. All the HBV persistent carriers had histories of at least 6 months HBsAg positivity as well as no anti-HBs existed in the serum and with no evidence of cirrhosis or carcinoma by imaging and laboratory testing. Controls were negative for HBsAg, but positive for both anti-HBsAb and antiHBcAb, or subjects who were solely positive for antiHBsAb without history of hepatitis B vaccination, which is also defined as spontaneous clearance subjects. Patients who were co-infected with hepatitis A, C, D virus or human immunodeficiency virus (HIV) were excluded from this study. All the control subjects had no other ailments at the time of enrolment and all had normal liver function. Moreover, the Tibetan subjects were all from Tibet Autonomous Region and his family had no members from other ethnicities, so the genetic backgrounds were pure. So were the Uygurs. We divided the two races into two stages of experimentation: Tibetans as the main research subjects and Uygurs as an auxiliary validation stage. This study was approved by the Institutional Review Board of all participating hospitals, including West China Hospital of Sichuan University, People’s Hospital of Tibet Autonomous region, Tibetan Hospital of Tibet Autonomous region and First Affiliated Hospital of Xinjiang Medical University and consistent with the guidelines of the Helsinki Declaration. All patients signed an informed consent prior to inclusion in this study. HLA-DP/DQ, STAT4 and IL-28B polymorphisms
Recent Genome-Wide Association Study confirmed that HLA-DP (rs3077, rs9277535), HLA-DQ (rs7453920) polymorphisms correlated with persistent HBV infection (3, 4). Subsequent studies further confirmed this correlation in Chinese Han population (6). Thus, single nucleotide polymorphism rs3077, rs9277535 and rs7453920 of HLA-DP, DQ were selected in this study. For STAT4, we chose the most reported rs7574865, which was used to be deemed as correlating with the autoimmune diseases (17), and recently was found to be correlated with increased risk of hepatocellular carcinoma (8). As for IL-28B, we chose the most investigated SNPs (rs12979860, rs8099917 and rs12980275) to appraise its correlation with HBV natural clearance. Primers for the seven SNPs were shown in supplementary Table 1. All the seven SNPs were genotyped using polymerase chain reaction high resolution melting (HRM) analysis performed on Light Cycler480 (Roche Diagnostics, Penzberg, Bavaria, Germany). Genomic DNA kit (Biotake Corporation, Beijing, China) was used to extract the free circulating DNA from the blood sample and the concentration was measured by NanoDrop 2000c spectrophometer (Thermo Scientific, Wilmington,
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Genetic variants in HBV in minorities
Table 1. Characteristics of subjects from each ethnicity Tibetan population
Uygur population
Characteristics
HBV carriers
Control
P value
HBV carriers
Control
P value
Total Age Gender(male/female) HBV genotype B (%) C (%) D (%) HBV DNA (log10 copies/ml) HBsAg+ Anti-HBs HBeAg (positive/negative) Anti-HBc
422 39 (30, 49) 289/148
486 39 (30, 50) 309/177
0.390 0.448
198 29 (23, 42) 122/76
235 48 (39, 58) 141/94
0.05), while the frequency of STAT4 rs7574865 GG genotype was much higher in genotype C infected patients than in genotype D infected patients (56.4% vs. 40.8%), and non-GG genotype carriers are susceptible to genotype D infection (OR = 0.53, 95% CI = 0.34–0.83, P = 0.005) (Table 3). Besides, G allele was still higher in genotype C virus infected patients than in genotype D patients (74.1% vs. 65.5%) (See supplementary Table 2). When stratifying the hepatitis patients according to antigen e status, we found that rs7453920 GG genotype patients are more likely to be HBeAg positive patients, while the non-GG genotype patients are apt to become HBeAg negative patients (dominant model, OR = 0.60, 95% CI = 0.37–0.96, P = 0.032) (Table 3). No other SNPs were observed to be different between HBeAg positive and HBeAg negative patients (P > 0.05). Apart from virus genotypes and e antigen statuses, according to the viral load of HBV, we also divided all the HBV patients into two groups: high viral load group and low viral load group (with the cut-off viral load 105 copies/ml). However, no significant correlation was found between host genetic polymorphisms and viral load. Therefore, HLADP/DQ, STAT4 and IL-28B polymorphisms might not influence the viral load after infection with HBV (See Table 3). Association analysis of HLA-DP/DQ, STAT4 and IL-28B polymorphisms with HBV natural clearance in Uygurs
In the second stage, we simply analysed the polymorphisms of HLA-DP/DQ, STAT4 and IL-28B in Uygurs to attest whether population heterogeneity would influence the causative SNPs. The protective allele found in Tibetans still correlated with decreased risk of HBV persistent infection in Uygurs (rs3077, dominant model: adjusted OR = 0.48, 95% CI = 0.24–0.96, P = 0.038, allelic model: OR = 0.62, 95% CI = 0.47–0.83, P = 0.001; rs9277535, dominant model: adjusted OR = 0.43, 95% CI = 0.20–0.91, P = 0.027, allelic model: OR = 0.57, 95% CI = 0.42–0.76, P = 1.78E-04; rs7453920, dominant model: adjusted OR = 0.62, 95% CI = 0.39–0.99, P = 0.047, allelic model: OR = 0.57, 95% CI = 0.41–0.80, P = 0.001) (Table 4). When appraising the relationship of STAT4 with HBV natural clearance, it seemed that the non-GG genotype inclined to associate with HBV seroclearance (P = 0.079), but allelic model results denied this (P = 0.5) (Table 4). Therefore, this still awaits more powerful studies to draw a confirmative conclusion. As for the correlation of IL-28B polymorphisms with HBV natural clearance, we did not observe any significant association with HBV natural clearance in the Uygurs either (dominant model, rs12979860, OR = 1.00, 95% CI = 0.65–1.53, P = 0.998; rs12980275,
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OR = 0.82, 95% CI = 0.51–1.31, P = 0.397; rs8099917, OR = 1.13, 95% CI = 0.69–1.84, P = 0.634) (Table 4). Allele frequency discrepancy in the two minorities
As mentioned above, HLA-DP/DQ (rs3077, rs9277535 and rs7453920) correlated with HBV natural clearance and rs3077 allele A, rs9277535 allele A as well as rs7453920 allele A favoured HBV clearance. Intriguingly, the frequency of protective allele (allele A) contributing to HBV clearance at rs3077, rs9277535 and even rs7453920 were much higher in Uygurs (72.5% for rs3077, 76.6% for rs9277535 and 26.8% for rs7453920), compared with Tibetans (51.7% for rs3077, 52.5% for rs9277535 and 18.5% for rs7453920) (P < 0.05), meanwhile, the frequency in Uygurs is much higher than Hans, whose frequency was illuminated in previous study (6). Therefore, significant genotype distributions existed among the three ethnicities. While for IL-28B and STAT4, not so conspicuous difference as HLA-DP/ DQ was observed between the two ethnicities (Tables 2 and 3). Haplotype analysis of HLA-DP/DQ and IL-28B with HBV infection
Haplotypes were constructed on the basis of 3 HLA polymorphisms (rs3077, rs9277535 and rs7453920). Both the HLA haplotype blocks in Tibetans and Uygurs were constructed from rs3077 (minor allele A), rs9277535 (minor allele A) and rs7453920 (minor allele A) (Fig. 1A and B). Results showed that haplotype block AAA and AAG significantly correlated with reduced risk of HBV persistent infection (AAA: OR = 0.65, 95% CI = 0.47–0.89; AAG: OR = 0.80, 95% CI = 0.66– 0.97), while haplotype block GGG correlated with an increased risk of chronic HBV infection (OR = 1.36, 95% CI = 1.13–1.63) in Tibetans (See Table 5). While in Uygurs, HLA haplotype block AAA correlated with reduced risk of HBV persistent infection (OR = 0.37, 95% CI = 0.23–0.59) and GGG associated with increased risk of HBV infection (OR = 2.03, 95% CI = 1.34–3.07) (Table 5). IL-28B haplotype was constructed from rs12979860 (minor allele T), rs12980275 (minor allele G) and rs8099917 (minor allele G). We restricted the threshold frequency no. 0.05). STAT4 rs7574865 seemed to be Tibetan specific in HBV natural clearance (OR = 0.76, 95% CI = 0.58–0.99). Moreover, in Tibetan patients, HLA-DQ rs7453920 GG had a higher frequency in HBeAg positive patients (P = 0.032) and STAT4 rs7574865 GG genotype appeared more frequently in Genotype C virus infected patients (P = 0.005). In addition, Uygurs have higher frequencies of HLA-DP/DQ protective alleles (72.5% for rs3077, 76.6% for rs9277535 and 26.8% for rs7453920) than Tibetans (51.7% for rs3077, 52.5% for rs9277535 and 18.5% for rs7453920)(all P < 0.05), and a lower prevalence of HBV infection as previously reported. Conclusions: HLA-DP/DQ but not IL-28B polymorphisms correlate with HBV natural clearance, irrespective of race, and HLA-DP/DQ would be causative genes attributing to varying prevalence in different regions. STAT4 rs7574865 seemed to be population specific in Tibetans and it might synergistically interact with virus contributing to disease progression.
The infection of Hepatitis B virus (HBV) has been a worldwide health problem, which could contribute to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). Globally, more than 2 billion people have been infected with HBV. Among them, 400 million are chronic carriers, which pose great threat to the health worldwide (1, 2). Recently, several Genome-Wide association studies (GWAS) demonstrated that single nucleotide polymorphisms (SNP) of human leucocyte antigen (HLA) might be pertinent to persistent infection after being infected with HBV, especially polymorphisms on HLA-DP/DQ
886
loci (3, 4). Subsequently, these findings were validated in Chinese Han population, comparing patients with chronic hepatitis and those who were deemed as spontaneous clearance persons (antibody to HBsAg positive and antibody to hepatitis core antigen positive, i.e. antiHBs and anti-HBc positive) (5, 6). A prospective study performed in Hongkong investigating pertinence of HLA-DP polymorphisms (rs3077, rs9277378, rs3128917) and HBsAg seroclearance further consolidated the conclusion that HLA-DP polymorphisms correlated with virus clearance after infection with HBV, but posed a question whether HLA-DP polymorphisms Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Genetic variants in HBV in minorities
make a rational comparison between HBeAg positive patients and HBeAg negative patients. Anyway, these conclusions still warrant more studies including prospective studies in different ethnicities to validate. STAT4 is a transcription factor which regulated immune response, transmitted signals from interleukin12 (IL-12) and type I interferon (IFN) to induce IFN-c production (23–25). Recently, a multitude of studies including GWAS studies have confirmed the association of STAT4 variants with autoimmune diseases, especially STAT4 rs7574865 (26–28). Few studies (29) have connected STAT4 with HBV diseases before the GWAS study came into being (8). However, their study did not clarify whether this SNP associate with spontaneous clearance of HBV, let alone in different races. In our study, we recruited chronic HBV patients as well as those who achieved seroclearance after being infected with HBV in a recent period in Tibetan and Uygur population. Intriguingly, we found rs7574865 allele T correlated with a reduced risk of persistent HBV infection, but failed to discern the subtle correlation in Uygurs. Therefore, its role in HBV natural clearance may be population specific and still merits more studies to substantiate. In addition, the frequency of STAT4 rs7574865 risk allele G was much higher in genotype C infected Tibetan patients, given the role of rs7574865 in predicting risk of HCC occurrence (8) and the worse outcome of genotype C infection (30), the two factors would synergistically contribute to HBV progression, and eventually lead to HCC occurrence. But this inference still needs further evidence. IL-28B has been accepted as being correlated with HCV spontaneous clearance, but not so conspicuous in HBV infection. In our study, we investigated the role in HBV patients and found no significant correlation of IL-28BSNP alleles with HBV chronic infection, which indicated that IL-28BSNPs were not the key factor to protect from HBV persistent infection or eradicating HBV. It was once demonstrated that IL-28B polymorphisms might influence the treatment efficacy in genotype D HBeAg negative HBV patients (12) and also in HBeAg positive patients (11), but these positive results also received questions summarized in one recent review (31), and more studies with high-quality are necessitated to clarify this question. The prevalence of HBV varies in different regions. In China, the prevalence in Uygurs living in the northwest is merely around 4–5%, but for Tibetan, Han and other populations dwelling in southwest, central south and east of China reach as high as 7% and even more (>8%) (32). Moreover, it was reported that the prevalence of anti-HBc in Uygurs resembles Han (43.1% vs. 44.2%), but was much higher than it in Tibetans (24.6%), while the prevalence of anti-HBs was similar in the three ethnicities (20.7% for Uygurs, 24.91% for Tibetans, 23.5% for Han). And according to the same study, Uygur still has a lower prevalence of HBsAg than Tibetans (4.7% vs. 5.8%) (33). The higher exposure to HBV infection
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but a lower prevalence of HBV in Uygurs, compared with it in Tibetans, can be well explained by the difference of genetic background, since the influences of vaccine preventing HBV infection in the two races were alike (indicated by the similar prevalence of anti-HBs). In fact, we found that the distribution of HBV prevalence in China just coincided with HLA-DP/DQ protective allele frequency since Uygurs have higher protective frequency, but lower prevalence, while Tibetan and Han have lower protective frequency, but much higher prevalence in the two populations. As is known, Caucasians have low prevalence of HBV infection (
VIRAL HEPATITIS
Association of HLA-DP/DQ, STAT4 and IL-28B variants with HBV viral clearance in Tibetans and Uygurs in China Yun Liao1, Bei Cai1, Yi Li1, Jie Chen1, Binwu Ying1, Chuanmin Tao1, Min Zhao2, Zhu Ba3, Zhaoxia Zhang4 and Lanlan Wang1 1 2 3 4
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China Department of Laboratory Medicine, People’s Hospital of Tibet Autonomous region, Lhasa 850000, China Department of Laboratory Medicine, Tibetan Hospital of Tibet Autonomous region, Lhasa 850000, China Center of Laboratory Medicine, First Affiliated Hospital of Xinjiang Medical University, Urumchi 830054, China
Keywords HBV – HLA-DP/DQ – IL-28B – STAT4 – Tibetan – Uygur
Correspondence Lanlan Wang, Department of Laboratory Medicine, West China Hospital, Sichuan University, No.37 Guoxue Alley, Wuhou district, Chengdu 610041, China Tel/Fax: +86-28-85422751 e-mail: [email protected] Received 23 April 2014 Accepted 15 July 2014 DOI:10.1111/liv.12643 Liver Int. 2015; 35: 886–896
Abstract Background & Aims: Several genome-wide association studies have revealed that HLA-DP/DQ, STAT4 and IL-28B associated with liver diseases. But because of population heterogeneity, different races would have different causative polymorphisms. Therefore, in this study, we included Chinese Tibetans and Uygurs to examine the roles of these genes on HBV natural clearance. Methods: A total of 1341 subjects including 908 Tibetans and 433 Uygurs were recruited. Seven single nucleotide polymorphisms (SNP) were genotyped. Results: HLA-DP/DQ polymorphisms associated with HBV natural clearance in both ethnicities (Tibetans, rs3077, OR = 0.61, 95% CI = 0.46–0.82; rs9277535, OR = 0.56, 95% CI = 0.41–0.75; rs7453920, OR = 0.64, 95%CI = 0.47–0.85; Uygurs, rs3077, OR = 0.48, 95% CI = 0.24–0.96; rs9277535, OR = 0.43, 95% CI = 0.20–0.91; rs7453920, OR = 0.62, 95% CI = 0.39–0.99), whereas no significant association was observed between IL-28B with HBV natural clearance in neither ethnicities (P > 0.05). STAT4 rs7574865 seemed to be Tibetan specific in HBV natural clearance (OR = 0.76, 95% CI = 0.58–0.99). Moreover, in Tibetan patients, HLA-DQ rs7453920 GG had a higher frequency in HBeAg positive patients (P = 0.032) and STAT4 rs7574865 GG genotype appeared more frequently in Genotype C virus infected patients (P = 0.005). In addition, Uygurs have higher frequencies of HLA-DP/DQ protective alleles (72.5% for rs3077, 76.6% for rs9277535 and 26.8% for rs7453920) than Tibetans (51.7% for rs3077, 52.5% for rs9277535 and 18.5% for rs7453920)(all P < 0.05), and a lower prevalence of HBV infection as previously reported. Conclusions: HLA-DP/DQ but not IL-28B polymorphisms correlate with HBV natural clearance, irrespective of race, and HLA-DP/DQ would be causative genes attributing to varying prevalence in different regions. STAT4 rs7574865 seemed to be population specific in Tibetans and it might synergistically interact with virus contributing to disease progression.
The infection of Hepatitis B virus (HBV) has been a worldwide health problem, which could contribute to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). Globally, more than 2 billion people have been infected with HBV. Among them, 400 million are chronic carriers, which pose great threat to the health worldwide (1, 2). Recently, several Genome-Wide association studies (GWAS) demonstrated that single nucleotide polymorphisms (SNP) of human leucocyte antigen (HLA) might be pertinent to persistent infection after being infected with HBV, especially polymorphisms on HLA-DP/DQ
886
loci (3, 4). Subsequently, these findings were validated in Chinese Han population, comparing patients with chronic hepatitis and those who were deemed as spontaneous clearance persons (antibody to HBsAg positive and antibody to hepatitis core antigen positive, i.e. antiHBs and anti-HBc positive) (5, 6). A prospective study performed in Hongkong investigating pertinence of HLA-DP polymorphisms (rs3077, rs9277378, rs3128917) and HBsAg seroclearance further consolidated the conclusion that HLA-DP polymorphisms correlated with virus clearance after infection with HBV, but posed a question whether HLA-DP polymorphisms Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Liao et al.
would have similar effects on different races (7). Another GWAS study conducted in Chinese Han population substantiated that polymorphisms of Signal Transducer and Activator of Transcription 4 (STAT4) might correlate with progression of HCC (8). STAT4 belongs to STAT family and transmits signals mainly from interleukin-12 (IL-12) to induce interferon-c (IFN-c). Though the GWAS study has explicitly illustrated that STAT4 rs7574865 correlates with the progression of HCC, whether it correlates with spontaneous clearance of HBV still remains elusive. Moreover, recent investigations indicated that IL-28B polymorphisms, which was considered to be correlated with spontaneous clearance of hepatitis C virus and treatment response (9, 10), might associate with hepatitis e antigen (HBeAg) seroconversion and HBsAg seroclearance (11, 12). But some studies denied this (13). Apart from the conflicting results, most of the studies investigating IL-28B were conducted in Han population, no other ethnicities were included. As is known, the prevalence of HBV infection varies greatly based on region disparity. In North American and Europe, the prevalence of HBV infection is rather low, but in East Asia, this number soared up (14). Besides, evidence showed that disease aetiology is common generally, but the risk variants are often population specific. And the difference can be truly originated from ethnic disparity since the degree of genetic variations differs among individuals of different ethnicities (15). Previous study has indicated that the East Asian populations, including Chinese Han, Korean and Japanese, have similarity in the genetic backgrounds. While Uygurs live in the vicinity of Central Asia and the genetic background resembles Hazara, but differ greatly from the Han population (16). As for Tibetans, few studies have involved this ethnicity who lived at a high altitude and their genetic backgrounds as well as the impact on susceptibility of HBV still remain obscure. Therefore, in this study, we recruited Tibetans and Uygurs, attempting to unveil the risk variants of HBV infection in minorities to further attest whether current hot spots of HLA-DP/DQ, STAT4 and IL-28B still correlate with persistent infection of HBV, trying to clarify the importance of these host genetic factors on HBV natural clearance in different races. Methods and materials Subjects
The present study recruited mainly Chinese Tibetans and Uygurs as this study subjects. In total, 1341 subjects, including 908 Chinese Tibetans (422 Tibetan chronic HBV carriers and 486 Tibetans with natural clearance after HBV infection as the control source) and 433 Uygurs (198 Uygur HBV carriers and 235 Uygur controls who naturally cleared the virus), were enrolled. All the subjects were recruited from West China Hospital of Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Genetic variants in HBV in minorities
Sichuan University, People’s Hospital of Tibet Autonomous region, Tibetan Hospital of Tibet Autonomous region and First Affiliated Hospital of Xinjiang Medical University from June 2011 to May 2013. Each race constitutes one case-control study. All the HBV persistent carriers had histories of at least 6 months HBsAg positivity as well as no anti-HBs existed in the serum and with no evidence of cirrhosis or carcinoma by imaging and laboratory testing. Controls were negative for HBsAg, but positive for both anti-HBsAb and antiHBcAb, or subjects who were solely positive for antiHBsAb without history of hepatitis B vaccination, which is also defined as spontaneous clearance subjects. Patients who were co-infected with hepatitis A, C, D virus or human immunodeficiency virus (HIV) were excluded from this study. All the control subjects had no other ailments at the time of enrolment and all had normal liver function. Moreover, the Tibetan subjects were all from Tibet Autonomous Region and his family had no members from other ethnicities, so the genetic backgrounds were pure. So were the Uygurs. We divided the two races into two stages of experimentation: Tibetans as the main research subjects and Uygurs as an auxiliary validation stage. This study was approved by the Institutional Review Board of all participating hospitals, including West China Hospital of Sichuan University, People’s Hospital of Tibet Autonomous region, Tibetan Hospital of Tibet Autonomous region and First Affiliated Hospital of Xinjiang Medical University and consistent with the guidelines of the Helsinki Declaration. All patients signed an informed consent prior to inclusion in this study. HLA-DP/DQ, STAT4 and IL-28B polymorphisms
Recent Genome-Wide Association Study confirmed that HLA-DP (rs3077, rs9277535), HLA-DQ (rs7453920) polymorphisms correlated with persistent HBV infection (3, 4). Subsequent studies further confirmed this correlation in Chinese Han population (6). Thus, single nucleotide polymorphism rs3077, rs9277535 and rs7453920 of HLA-DP, DQ were selected in this study. For STAT4, we chose the most reported rs7574865, which was used to be deemed as correlating with the autoimmune diseases (17), and recently was found to be correlated with increased risk of hepatocellular carcinoma (8). As for IL-28B, we chose the most investigated SNPs (rs12979860, rs8099917 and rs12980275) to appraise its correlation with HBV natural clearance. Primers for the seven SNPs were shown in supplementary Table 1. All the seven SNPs were genotyped using polymerase chain reaction high resolution melting (HRM) analysis performed on Light Cycler480 (Roche Diagnostics, Penzberg, Bavaria, Germany). Genomic DNA kit (Biotake Corporation, Beijing, China) was used to extract the free circulating DNA from the blood sample and the concentration was measured by NanoDrop 2000c spectrophometer (Thermo Scientific, Wilmington,
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Liao et al.
Genetic variants in HBV in minorities
Table 1. Characteristics of subjects from each ethnicity Tibetan population
Uygur population
Characteristics
HBV carriers
Control
P value
HBV carriers
Control
P value
Total Age Gender(male/female) HBV genotype B (%) C (%) D (%) HBV DNA (log10 copies/ml) HBsAg+ Anti-HBs HBeAg (positive/negative) Anti-HBc
422 39 (30, 49) 289/148
486 39 (30, 50) 309/177
0.390 0.448
198 29 (23, 42) 122/76
235 48 (39, 58) 141/94
0.05), while the frequency of STAT4 rs7574865 GG genotype was much higher in genotype C infected patients than in genotype D infected patients (56.4% vs. 40.8%), and non-GG genotype carriers are susceptible to genotype D infection (OR = 0.53, 95% CI = 0.34–0.83, P = 0.005) (Table 3). Besides, G allele was still higher in genotype C virus infected patients than in genotype D patients (74.1% vs. 65.5%) (See supplementary Table 2). When stratifying the hepatitis patients according to antigen e status, we found that rs7453920 GG genotype patients are more likely to be HBeAg positive patients, while the non-GG genotype patients are apt to become HBeAg negative patients (dominant model, OR = 0.60, 95% CI = 0.37–0.96, P = 0.032) (Table 3). No other SNPs were observed to be different between HBeAg positive and HBeAg negative patients (P > 0.05). Apart from virus genotypes and e antigen statuses, according to the viral load of HBV, we also divided all the HBV patients into two groups: high viral load group and low viral load group (with the cut-off viral load 105 copies/ml). However, no significant correlation was found between host genetic polymorphisms and viral load. Therefore, HLADP/DQ, STAT4 and IL-28B polymorphisms might not influence the viral load after infection with HBV (See Table 3). Association analysis of HLA-DP/DQ, STAT4 and IL-28B polymorphisms with HBV natural clearance in Uygurs
In the second stage, we simply analysed the polymorphisms of HLA-DP/DQ, STAT4 and IL-28B in Uygurs to attest whether population heterogeneity would influence the causative SNPs. The protective allele found in Tibetans still correlated with decreased risk of HBV persistent infection in Uygurs (rs3077, dominant model: adjusted OR = 0.48, 95% CI = 0.24–0.96, P = 0.038, allelic model: OR = 0.62, 95% CI = 0.47–0.83, P = 0.001; rs9277535, dominant model: adjusted OR = 0.43, 95% CI = 0.20–0.91, P = 0.027, allelic model: OR = 0.57, 95% CI = 0.42–0.76, P = 1.78E-04; rs7453920, dominant model: adjusted OR = 0.62, 95% CI = 0.39–0.99, P = 0.047, allelic model: OR = 0.57, 95% CI = 0.41–0.80, P = 0.001) (Table 4). When appraising the relationship of STAT4 with HBV natural clearance, it seemed that the non-GG genotype inclined to associate with HBV seroclearance (P = 0.079), but allelic model results denied this (P = 0.5) (Table 4). Therefore, this still awaits more powerful studies to draw a confirmative conclusion. As for the correlation of IL-28B polymorphisms with HBV natural clearance, we did not observe any significant association with HBV natural clearance in the Uygurs either (dominant model, rs12979860, OR = 1.00, 95% CI = 0.65–1.53, P = 0.998; rs12980275,
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OR = 0.82, 95% CI = 0.51–1.31, P = 0.397; rs8099917, OR = 1.13, 95% CI = 0.69–1.84, P = 0.634) (Table 4). Allele frequency discrepancy in the two minorities
As mentioned above, HLA-DP/DQ (rs3077, rs9277535 and rs7453920) correlated with HBV natural clearance and rs3077 allele A, rs9277535 allele A as well as rs7453920 allele A favoured HBV clearance. Intriguingly, the frequency of protective allele (allele A) contributing to HBV clearance at rs3077, rs9277535 and even rs7453920 were much higher in Uygurs (72.5% for rs3077, 76.6% for rs9277535 and 26.8% for rs7453920), compared with Tibetans (51.7% for rs3077, 52.5% for rs9277535 and 18.5% for rs7453920) (P < 0.05), meanwhile, the frequency in Uygurs is much higher than Hans, whose frequency was illuminated in previous study (6). Therefore, significant genotype distributions existed among the three ethnicities. While for IL-28B and STAT4, not so conspicuous difference as HLA-DP/ DQ was observed between the two ethnicities (Tables 2 and 3). Haplotype analysis of HLA-DP/DQ and IL-28B with HBV infection
Haplotypes were constructed on the basis of 3 HLA polymorphisms (rs3077, rs9277535 and rs7453920). Both the HLA haplotype blocks in Tibetans and Uygurs were constructed from rs3077 (minor allele A), rs9277535 (minor allele A) and rs7453920 (minor allele A) (Fig. 1A and B). Results showed that haplotype block AAA and AAG significantly correlated with reduced risk of HBV persistent infection (AAA: OR = 0.65, 95% CI = 0.47–0.89; AAG: OR = 0.80, 95% CI = 0.66– 0.97), while haplotype block GGG correlated with an increased risk of chronic HBV infection (OR = 1.36, 95% CI = 1.13–1.63) in Tibetans (See Table 5). While in Uygurs, HLA haplotype block AAA correlated with reduced risk of HBV persistent infection (OR = 0.37, 95% CI = 0.23–0.59) and GGG associated with increased risk of HBV infection (OR = 2.03, 95% CI = 1.34–3.07) (Table 5). IL-28B haplotype was constructed from rs12979860 (minor allele T), rs12980275 (minor allele G) and rs8099917 (minor allele G). We restricted the threshold frequency no. 0.05). STAT4 rs7574865 seemed to be Tibetan specific in HBV natural clearance (OR = 0.76, 95% CI = 0.58–0.99). Moreover, in Tibetan patients, HLA-DQ rs7453920 GG had a higher frequency in HBeAg positive patients (P = 0.032) and STAT4 rs7574865 GG genotype appeared more frequently in Genotype C virus infected patients (P = 0.005). In addition, Uygurs have higher frequencies of HLA-DP/DQ protective alleles (72.5% for rs3077, 76.6% for rs9277535 and 26.8% for rs7453920) than Tibetans (51.7% for rs3077, 52.5% for rs9277535 and 18.5% for rs7453920)(all P < 0.05), and a lower prevalence of HBV infection as previously reported. Conclusions: HLA-DP/DQ but not IL-28B polymorphisms correlate with HBV natural clearance, irrespective of race, and HLA-DP/DQ would be causative genes attributing to varying prevalence in different regions. STAT4 rs7574865 seemed to be population specific in Tibetans and it might synergistically interact with virus contributing to disease progression.
The infection of Hepatitis B virus (HBV) has been a worldwide health problem, which could contribute to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). Globally, more than 2 billion people have been infected with HBV. Among them, 400 million are chronic carriers, which pose great threat to the health worldwide (1, 2). Recently, several Genome-Wide association studies (GWAS) demonstrated that single nucleotide polymorphisms (SNP) of human leucocyte antigen (HLA) might be pertinent to persistent infection after being infected with HBV, especially polymorphisms on HLA-DP/DQ
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loci (3, 4). Subsequently, these findings were validated in Chinese Han population, comparing patients with chronic hepatitis and those who were deemed as spontaneous clearance persons (antibody to HBsAg positive and antibody to hepatitis core antigen positive, i.e. antiHBs and anti-HBc positive) (5, 6). A prospective study performed in Hongkong investigating pertinence of HLA-DP polymorphisms (rs3077, rs9277378, rs3128917) and HBsAg seroclearance further consolidated the conclusion that HLA-DP polymorphisms correlated with virus clearance after infection with HBV, but posed a question whether HLA-DP polymorphisms Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Genetic variants in HBV in minorities
make a rational comparison between HBeAg positive patients and HBeAg negative patients. Anyway, these conclusions still warrant more studies including prospective studies in different ethnicities to validate. STAT4 is a transcription factor which regulated immune response, transmitted signals from interleukin12 (IL-12) and type I interferon (IFN) to induce IFN-c production (23–25). Recently, a multitude of studies including GWAS studies have confirmed the association of STAT4 variants with autoimmune diseases, especially STAT4 rs7574865 (26–28). Few studies (29) have connected STAT4 with HBV diseases before the GWAS study came into being (8). However, their study did not clarify whether this SNP associate with spontaneous clearance of HBV, let alone in different races. In our study, we recruited chronic HBV patients as well as those who achieved seroclearance after being infected with HBV in a recent period in Tibetan and Uygur population. Intriguingly, we found rs7574865 allele T correlated with a reduced risk of persistent HBV infection, but failed to discern the subtle correlation in Uygurs. Therefore, its role in HBV natural clearance may be population specific and still merits more studies to substantiate. In addition, the frequency of STAT4 rs7574865 risk allele G was much higher in genotype C infected Tibetan patients, given the role of rs7574865 in predicting risk of HCC occurrence (8) and the worse outcome of genotype C infection (30), the two factors would synergistically contribute to HBV progression, and eventually lead to HCC occurrence. But this inference still needs further evidence. IL-28B has been accepted as being correlated with HCV spontaneous clearance, but not so conspicuous in HBV infection. In our study, we investigated the role in HBV patients and found no significant correlation of IL-28BSNP alleles with HBV chronic infection, which indicated that IL-28BSNPs were not the key factor to protect from HBV persistent infection or eradicating HBV. It was once demonstrated that IL-28B polymorphisms might influence the treatment efficacy in genotype D HBeAg negative HBV patients (12) and also in HBeAg positive patients (11), but these positive results also received questions summarized in one recent review (31), and more studies with high-quality are necessitated to clarify this question. The prevalence of HBV varies in different regions. In China, the prevalence in Uygurs living in the northwest is merely around 4–5%, but for Tibetan, Han and other populations dwelling in southwest, central south and east of China reach as high as 7% and even more (>8%) (32). Moreover, it was reported that the prevalence of anti-HBc in Uygurs resembles Han (43.1% vs. 44.2%), but was much higher than it in Tibetans (24.6%), while the prevalence of anti-HBs was similar in the three ethnicities (20.7% for Uygurs, 24.91% for Tibetans, 23.5% for Han). And according to the same study, Uygur still has a lower prevalence of HBsAg than Tibetans (4.7% vs. 5.8%) (33). The higher exposure to HBV infection
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but a lower prevalence of HBV in Uygurs, compared with it in Tibetans, can be well explained by the difference of genetic background, since the influences of vaccine preventing HBV infection in the two races were alike (indicated by the similar prevalence of anti-HBs). In fact, we found that the distribution of HBV prevalence in China just coincided with HLA-DP/DQ protective allele frequency since Uygurs have higher protective frequency, but lower prevalence, while Tibetan and Han have lower protective frequency, but much higher prevalence in the two populations. As is known, Caucasians have low prevalence of HBV infection (
