Background: Female gender and favorable IFNL3 genotypes are the primary independent predictors of spontaneous clearance of HCV infection. However, chronic hepatitis C infection occurs in numerous women carrying favorable IFNL3 genotypes, indicating that other host and/or virological factors contribute to the prognosis of infection. Methods: A cohort of 137 anti-HCV-positive female Han Chinese cases, including 64 chronic HCV carriers and 73 HCV spontaneous resolvers, was recruited in the study. 111 SNPs in 23 genes encoding HCV co-receptors, transcription factors, Toll-like receptors, co-stimulating molecules, and cytokines were selected for SNP analysis. Results: After comparison of genotypes and allelotype frequencies of 111 SNPs in 23 genes in the primary cohort, the SNPs rs9826 (P = 0.024 for CC/TT/CT; P = 0.015 for C allele/T allele) and rs1521177 (P = 0.017 for GG/TT/GT; P = 0.006 for G allele/T allele) in the RORC gene were significantly associated with spontaneous HCV clearance. In the sub-cohort carrying favorable IFNL3 genotypes (rs12979860CC, rs8099917 TT, rs12980275 AA), rs1521177 (genotype: P= 0.040; allelotype: P= 0.021) remained significantly associated with spontaneous HCV clearance. Importantly, the most common RORC haplotype rs9826-T/rs1521177-T was presented at significantly different frequencies in resolvers and carriers in both the primary cohort (P = 0.0027) and the IFNL3 favorable sub-cohort (P= 0.0117). Conclusions: This study indicates that genetic polymorphisms in human Th17-related RORC gene are associated with different natural prognosis of HCV infection. The RORC haplotype, rs9826-T/rs1521177-T, was favorable for spontaneous clearance of HCV infection. Keywords: HCV, IFNL3, Polymorphism, RORC, Spontaneous clearance Background their infection and recover spontaneously . It is re- Chronic hepatitis C infection is a global healthcare ported that among host factors, gender, age, IFNL3 (previ- burden and associated with the development of liver ously IL28B) genotypes, KIR/HLA alleles, ethnicity, and cirrhosis, hepatocellular carcinoma, and death if untreated HIV co-infection are associated with spontaneous recov- . In China, the prevalence of HCV in the general popu- ery [5–15]. In particular, female sex and favorable IFNL3 lation is estimated as 1.6% [2, 3], which equals to approxi- genotypes (rs12979860 CC, rs8099917 TT and rs12980275 mately 20 million HCV carriers. A minority of acutely AA) are primary independent predictors of spontaneous infected individuals (approximately 15–40%) will resolve clearance [5–9, 12, 13, 16, 17]. In the general Han Chinese ethnic population, the three favorable IFNL3 genotypes are very common (~ 90%) [12, 16–18]. However, many * Correspondence: firstname.lastname@example.org; email@example.com Zhe Xie and Yuantao Li contributed equally to this work. females carrying favorable IFNL3 genotypes suffer from Department of Infectious Diseases, Guangzhou Eighth People’s Hospital, chronic hepatitis C in China, indicating that besides Guangzhou Medical University, Guangzhou 510060, Guangdong, China IFNL3 genotypes and possibly some KIR/HLA alleles, Department of Microbiology and Center of Infectious Diseases, School of Basic Medical Sciences, Peking University, Beijing 100191, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xie et al. BMC Infectious Diseases (2018) 18:254 Page 2 of 8 other immune-related factors associated with HCV spon- excluded from the study, as HIV infection could affect taneous clearance still remain unknown. the ability of some individuals to spontaneously resolve T helper 17 cells (Th17), a subset of pro-inflammatory their HCV infection. The final case number of primary T helper cells, play an important role in adaptive immun- cohort was 137, which consisted of a “Chronic” group neg ity, protecting hosts against pathogens primarily through (HIV HCV carriers, n = 64) and a “Resolved” group (n neg pos secreting cytokines(such as IL-17A, IL-17F, IL-21, and = 73, including 45 HIV HCV resolvers and 28 HIV IL-22) . Receptor retinoic acid-related orphan receptor HCV resolvers). Overall, the cohort exhibited a similar C (RORC), a DNA-binding transcription factor, is the high frequency of IFNL3 favorable genotypes to Han master regulator of Th17 differentiation [20, 21]. Th17 Chinese nationality (Additional file 1: Table S1), indicat- cells may have dual roles (both harmful and beneficial) in ing that the study was based on a representative cohort HCV-related disease . Specific HCV-Th17 cells are in- of the Chinese population [12, 16]. To eliminate the in- volved in immune response modulation, and their levels fluence of IFNL3 SNPs on viral clearance, a sub-cohort are associated with severity of fibrosis and intrahepatic in- carrying favorable IFNL3 genotypes (rs12979860CC, flammatory status [23, 24]. On the other hand, several re- rs8099917TT, rs12980275 AA) was constructed and ana- ports indicate that elevated Th17 responses could be lyzed. The IFNL3 favorable sub-cohort included 54 and neg associated with spontaneous HCV clearance [25, 26]. 67 participants in the “Chronic” and “Resolved” (HIV , pos In this study, two single nucleotide polymorphisms n = 43; HIV , n = 24) groups, respectively. A flow dia- (SNPs) located in the RORC gene were identified to be gram for cohort construction was provided as Fig 1. The associated with spontaneous clearance of HCV infection. clinical and biochemical characteristics of the primary cohort were presented in Table 1. Methods Participants SNP selection and genotyping In the present study, 190 HBsAg-negative, anti-HCV-positive, A total of 111 SNPs in 23 genes encoding HCV Han Chinese females were identified by screening a total co-receptors (CD81, SCARB1, CLDN1, OCLN, NPC1L1, of 1252 residents (80% of the local population) in APOE, LDLR), transcription factors(IRF3, RORC, TBX21, Wangying Village, Shangcai County, Henan province, in FOXP3, BCL6), Toll-like receptors (TLR3, TLR7, TLR9), August 2009. More than 90% of patients were former co-stimulating molecules (ICOS, CXCR5, CD40LG), and plasma donors, and the remaining individuals composed cytokines(IL4, IFNG, IL21, IFNL3, CXCL13) were of their parents, spouses, or children. Samples were tested selected for SNP analysis (Additional file 1: Table S2). The for anti-HCV antibody using the Architech anti-HCV sys- selected SNPs met at least one of the following criteria:1) tem (Abbott Diagnostics, USA), and those with signal/ a reported minor allele frequency > 10% in the Han cut-off ratios between 1.0 and 5.0 were confirmed by Chinese population according to SNP browser software RIBA assay (HCV BLOT 3.0, MP diagnostics, USA). 4.0 (Applied Biosystems), with reference to the NCBI SNP Plasma HCV viral load was determined using the Abbott database (http://www.ncbi.nlm.nih.gov/projects/SNP/); 2) RealTime HCV Amplification Kit (Abbott Molecular Inc., published evidence that the SNP was associated with USA) according to the manufacturer’s instructions. disease. Spontaneous resolvers were defined as subjects who were Genomic DNA extracted from whole blood samples positive for HCV antibody and negative for plasma HCV usingaDNeasy Blood&Tissuekit (Qiagen, USA) RNA with no history of HCV-specific treatment. HCV was dissolved in sterile double distilled water and RNA-negative status was confirmed in a second sample stored at − 20 °C until use. DNA purity was checked and collected in 2012 and/or 2013. Chronic HCV carriers were DNA samples with 260/280 ratios< 1.7 were re-purified. defined as those patients positive for both HCV antibody SNP genotyping was performed using the iPLEX andplasmaHCV viralloadand also confirmedbythe Sequenom MassARRAY system (Sequenom Inc., USA). follow-up detection. No participants had received any type of HCV-specific antiviral therapy. All subjects were Statistical analyses screened for HIV-1 infection status. Anti-HIV-1 antibody Statistical and graphical analyses were performed using status was initially tested by ELISA assay (GBI biotech Co., GraphPad Prism 5.0, Microsoft Excel 2007, or SPSS Ltd., Beijing, China) and confirmed by HIV Blot 2.2 WB 20.0. The allele frequency and genotype distributions of assay (Genelabs Diagnostics, Singapore). All HIV-positive each SNP were descriptively summarized as numbers of patients had received first-line antiretroviral therapy for 6~ cases and frequencies. Chi-square (χ ) and Fisher’s exact 8 years, regularly or intermittently. None of the participants tests were used to examine differences in frequencies of received any types of anti-HCV treatment. individual SNPs between HCV carriers and spontaneous pos Of 190 female anti-HCV-positive individuals, HIV resolvers. SNP-specific deviation from Hardy–Weinberg women with chronic HCV infection (n = 53) were Equilibrium (HWE) in the whole study population was Xie et al. BMC Infectious Diseases (2018) 18:254 Page 3 of 8 Fig. 1 Flow diagram of study cohort selection. From a total of 1252 residents of Wangying Village, Shangcai County, 190 anti-HCV-positive female Chinese Han individuals were initially recruited to the study after screening. Of these 190 subjects, 53 HIV-positive women with chronic HCV infections were excluded. The remaining 137 females constituted the primary cohort, which consisted of “Chronic” (HIV- HCV carriers, n = 64) and “Resolved” (total n = 73: HIV+ HCV resolvers, n = 28, and HIV- HCV resolvers, n = 45) groups. An IFNL3 favorable sub-cohort was also constructed by exclusion of women with unfavorable IFNL3 genotypes (rs12979860 TC/TT, rs80999917 TG/TT, and rs12980275AG/GG; “Chronic”, n = 54, and “Resolved”, n = 67) tested using a χ test in SHEsis software, and SNPs with genotypes were higher in “Resolved” group than HWE violation (P < 0.01) were excluded. Pairwise D′ “Chronic” group (resolved vs. chronic: rs12979860, CC and r measures of linkage disequilibrium for RORC 96% vs. 86%; rs8099917, TT 95% vs. 91%; and SNPs were calculated using SHEsis (http://analysis.- rs12980275, AA 96% vs. 86%), although only the differ- bio-x.cn/myAnalysis.php). Specific parameters were set ence in the frequency of the rs12979860 C allele was sta- as previously described . P-values, odds ratios (ORs), tistically significant (P = 0.043) (Table 2). and 95% confidence intervals (95% CIs) were used for Of the 22 remaining genes tested (excluding IFNL3), association analysis. All P-values were two-tailed, and only SNPs rs9826 and rs1521177, located in non-coding were considered significant at < 0.05. regions of RORC, exhibited frequency differences be- tween the “Resolved” and “Chronic” groups (rs9826: P = Results 0.024 for CC/TT/CT, P = 0.015 for C allele/T allele, OR Associations between target gene SNPs and spontaneous = 1.97, 95% CI: 1.14-3.41; rs1521177: P = 0.017 for GG/ HCV clearance in the primary cohort TT/GT, P = 0.006 for G allele/T allele, OR = 2.18, 95% Genotype distributions and allele frequencies were cal- CI: 1.24-3.82)(Table 2). The rs9826 TT and rs1521177 culated for 111 candidate SNPs in both the “Chronic” TT genotypes were associated with improved viral clear- and “Resolved” groups of the primary cohort (Additional ance. Overall, the primary cohort exhibited a similar dis- file 1: Table S2). The frequencies of favorable IFNL3 tribution of RORC genotypes to Han Chinese nationality Xie et al. BMC Infectious Diseases (2018) 18:254 Page 4 of 8 Table 1 Characteristics of individuals in the primary cohort sub-cohort indicated that significant differences between the two groups remained for rs1521177 (P = 0.040 for Characteristic Chronic HCV Resolved HCV (n = 73) (n = 64) neg pos GG/TT/GT, P = 0.021 for G allele/T allele, OR = 2.00, HIV (n = 45) HIV (n = 28) a 95% CI: 1.11-3.55), although no significant differences Age (years) 50 (47–59) 59 (49–67) 54 (47–64) were identified for rs9826 (P = 0.092 for CC/TT/CT, BMI 23.5 (21.1–26.1) 23.2 (21.3–26.3) 22.1 (20.5–24.5) P = 0.062 for C allele/T allele, OR = 1.74, 95% CI: Anti-HCV Positive Positive Positive 0.97-3.19) (Table 3). These data suggested that SNP al- Anti-HIV Negative Negative Positive leles in RORC had an independent effect on HCV viral HCV RNA 6.17 (5.82–6.54) Negative Negative clearance, which couldnot be explained by the distribu- (log IU/mL) tion of IFNL3 polymorphisms. HCV genotype 1b (n,%) 39 (60.9%) –– Analysis of the RORCrs9826/rs1521177 haplotype Linkage disequilibrium tests for RORC and IFNL3 SNPs 2a (n,%) 25 (39.1%) –– in the primary cohort were shown in Additional file 1: Others None –– Figure S3. The RORC SNPs, rs9826 and rs1521177, were ALT (n,%) determined to be in linkage disequilibrium (Primary co- > 40 (IU/L) 28 (43.7%) 4 (8.9%) 7 (25.0%) 2 hort: D’ = 0.831, r = 0.639; IFNL3 favorable sub-cohort: ≤40 (IU/L) 36 (56.3%) 41 (91.1%) 21 (75.0%) D’ = 0.812, r = 0.645) (Additional file 1: Table S7). Two AST (n,%) main haplotypes were present in the primary cohort (rs9826-T/rs1521177-T: 62% vs. 79%, chronic vs. > 40 (IU/L) 30 (46.9%) 1 (2.2%) 7 (25.0%) resolved; rs9826-C/rs1521177-G: 27% vs. 16%, chronic ≤40 (IU/L) 34 (53.1%) 44 (97.8%) 21 (75.0%) vs. resolved) and the IFNL3 favorable sub-cohort CD4+ T cells/μL 882 (599–1088) 888 (658–1119) 445 (346–587) (rs9826-T/rs1521177-T: 64% vs. 78%, chronic vs. re- a b Data presented as medians (inter-quartile range) or number of cases (%) solved; rs9826-C/rs1521177-G: 26% vs. 17%, chronic vs. BMI body mass index, ALT alanine aminotransferase, AST aspartate aminotransferase resolved). Overall, the presence of the rs9826-T/ rs1521177-T haplotype was associated with a higher like- (Additional file 1: Table S3). The Hardy–Weinberg lihood of spontaneous resolution in both the primary co- Equilibrium (HWE) test of three IFNL3 SNPs and two hort (P = 0.0027, OR = 0.445, 95% CI: 0.26–0.76) and the RORC SNPs in the primary cohort were tested, and no IFNL3 favorable sub-cohort (P = 0.0117, OR = 0.485, 95% SNPs with HWE violation were presented in the study CI:0.28–0.86). In addition, the frequency of the (Additional file 1: Table S4). rs9826-C/rs1521177-G haplotype was significantly differ- In the present study, a minority of “Resolved” individ- ent between the chronic and resolved groups in the pri- pos uals were co-infected with HIV (HIV resolved, n = 28) mary cohort (P = 0.0315, OR = 1.893, 95% CI:1.05–3.40), and very similar IFNL3 (rs12979860, rs8099917, and suggesting that individuals carrying this haplotype rs12980275) and RORC (rs9826 and rs1521177) SNP was difficult in spontaneous resolution of HCV in- neg genotype distributions were identified among HIV , fection (Table 4). pos HIV , and total resolved individuals (Additional file 1: Figure S1). No association was observed between RORC Discussion SNPs (rs9826 and rs1521177) and HCV RNA levels in Genetic polymorphisms of innate immunity-related HIV-uninfected “Chronic” individuals (Additional file 1: genes were reported to be associated with HCV clear- Figure S2). In addition, we did not find any differences ance [28, 29]. Specially, SNPs of the IFNL3 (IL-28B) have in genotypes and allele frequency distributions of RORC shown strong association with spontaneous clearance pos SNPs (rs9826 and rs1521177) between HIV HCV of HCV and with the response to anti-HCV therapy pos carriers and HIV HCV resolvers (Additional file 1: [30–33]. Another important correlation of HCV spon- Table S5), and no associations between RORC SNPs and taneously viral clearance was a robust and sustained type of HCV genotypes (HCV 2a vs. HCV 1b) was found HCV-specific T cell response. Cumulative studies indi- neg in HIV HCV carriers (Additional file 1: Table S6). cated that HLA-associated viral polymorphisms were closely correlated with immune recognition of T cells Re-evaluation of RORC SNP associations with to virus (both HIV and HCV) [34–37]. spontaneous HCV clearance in the IFNL3 favorable Exploration of adaptive immune factors involved in sub-cohort spontaneous clearance of viral infection will help to In the IFNL3 favorable sub-cohort, cases with unfavor- understand HCV pathogenesis. Besides IFNL genotypes, able IFNL3 genotypes in the “Chronic” (n = 10) and “Re- HCV-specific T cells are typically detectable 5–9 weeks solved” (n = 6) groups were excluded. Analysis of the after acute infection [38, 39]. Recently, Grebely et al. Xie et al. BMC Infectious Diseases (2018) 18:254 Page 5 of 8 Table 2 Genotype and allele frequency distributions of IFNL3and RORCSNPs in the primary cohort a a SNP Genotype Chronic HCV (n = 64) Resolved HCV (n = 73) P-value OR (95% CI) rs12979860 CC 55 (0.85) 70(0.96) 0.105 TT 1 (0.02) 0 (0.00) CT 8 (0.13) 3 (0.04) C allele 118 (0.92) 143 (0.98) 0.043 0.25 (0.07–0.87) T allele 10 (0.08) 3 (0.02) 4.04 (1.15–13.89) rs8099917 TT 58(0.91) 69 (0.95) 0.514 GG 0 (0.00) 0 (0.00) GT 6 (0.09) 4 (0.05) T allele 122 (0.95) 142 (0.97) 0.523 0.57 (0.16–2.08) G allele 6 (0.05) 4 (0.03) 1.75 (0.48–6.33) rs12980275 AA 55 (0.85) 69 (0.96) 0.189 GG 1 (0.02) 0 (0.00) AG 8 (0.13) 4 (0.04) A allele 118 (0.92) 142 (0.97) 0.096 0.33 (0.10–1.09) G allele 10 (0.08) 4 (0.03) 3.01 (0.92–9.84) RORC gene rs9826 CC 7 (0.11) 1 (0.01) 0.024 (3′ UTR) TT 29 (0.45) 45 (0.62) CT 28 (0.44) 27 (0.37) C allele 42 (0.33) 29 (0.20) 0.015 1.97 (1.14–3.41) T allele 86 (0.67) 117 (0.80) 0.51 (0.29–0.88) rs1521177 GG 6 (0.10) 1 (0.01) 0.017 (intron) TT 29 (0.45) 48 (0.66) GT 29 (0.45) 24 (0.33) G allele 41 (0.32) 26 (0.18) 0.006 2.18 (1.24–3.82) T allele 87 (0.68) 120 (0.82) 0.46 (0.26–0.81) Number of cases (frequency). SNP genotypes were determined using the iPLEX MassARRAY system (Sequenom Inc., USA), and allele frequencies and genotype distributions were calculated. Chi-square (χ ) and Fisher’s exact tests were used to evaluate differences in SNP frequencies between HCV carriers and spontaneous resolvers. P-values, odds ratios, (ORs), and 95% confidence intervals, (95% CIs) were determined for association analysis. P-values (two-tailed) < 0.05 were considered significant (bold) reported that the median time for acute HCV clearance with self-limiting infection than in those with chronically among 173 spontaneous resolvers undergoing follow-up evolving hepatitis . Moreover, Seetharam et al. observation was 16.5 weeks , indicating that a strong, described a transient IL-17 response followed by a sub- broad, and persistent HCV-specific adaptive immune sequent reactivation of Th1 responses, resulting to spon- response is required during acute infection for viral taneous recovery in a liver transplant recipient with clearance . recurrent hepatitis C . It is possible that Th17 The role of Th17 responses in HCV pathogenesis is responses in HCV-associated disorders may act as a intriguing but not well characterized. Numerous studies double-edged sword, playing different roles in different have focused on its destructive potentials, since they disease courses. could aggravate the severity of fibrosis and intrahepatic In this study, a number of the HCV resolvers had a inflammatory status [22, 41, 42]. However, some studies background of HIV infection, which could be primarily have also reported a role for Th17 responses in spontan- ascribed to historical contaminated commercial blood eous HCV clearance. Kared et al. reported that the donations in the late 1990s. As HCV is more efficiently differentiation of IL-17A-producing CD4+ T cells was transmitted through blood contamination or infected correlated with prognosis of infection . In addition, needles than HIV, self-limiting acute HCV infection in the concentration of plasma IL-17A was significantly these individuals may usually precede, or be coincident higher in the acute phase of HCV infection in patients with, HIV infection [6, 43, 44]. Regardless of whether Xie et al. BMC Infectious Diseases (2018) 18:254 Page 6 of 8 Table 3 Genotype and allele distributions of SNPs in the RORC surrounding environment, ethnicity, and income level, gene in the IFNL3 favorable sub-cohort which strengthed the comparison of genetic immune b b RORC SNPs Chronic HCV Resolved HCV P-value OR (95% CI) factors between HCV carriers and resolvers. Among the (n = 54) (n = 67) two RORC SNPs (rs9826 and rs1521177) identified in rs9826 this study, the rs9826 polymorphism was associated with CC 5 (0.09) 1 (0.01) 0.092 severity of rheumatoid arthritis in the Polish population . Further functional studies to explore the associa- TT 26 (0.48) 41 (0.61) tions between RORC SNPs genotypes and Th17 re- CT 23 (0.43) 25 (0.37) sponses, as well as Th17 responses and viral clearance, C allele 33 (0.31) 27 (0.20) 0.062 1.74 (0.97–3.19) should be performed in the future. T allele 75 (0.69) 107 (0.80) 0.57 (0.31–1.03) Our cohort design precluded the influence factors of rs1521177 ethnicity, gender and IFNL-3. However, the sample size GG 6 (0.11) 1 (0.01) 0.040 of our cohort was still limited, and future larger-scale queues were needed for better verification of our TT 26 (0.48) 43 (0.64) conclusion. In addition, the RORC SNPs rs9826-T and GT 22 (0.41) 23 (0.34) rs1521177-T were validated in only Chinese Han popu- G allele 34 (0.31) 25 (0.19) 0.021 2.00 (1.11–3.55) lation in this study. The adaptation of these SNPs in T allele 74 (0.69) 109 (0.81) 0.50 (0.28–0.90) other ethnic and national populations needs to be Participants in the, IFNL3 favorable sub-cohort were screened for, IFNL3 further analyzed. genotypes (rs12979860CC/rs8099917TT/rs12980275AA) Number of cases (frequency) SNP genotyping was conducted using the iPLEX MassARRAY system (Sequenom Inc., USA), and allele and genotype frequency distributions were Conclusions calculated. Chi-square (χ ) and Fisher’s exact tests were used to evaluate The present study determined that polymorphisms in differences in, SNP frequencies between, HCV carriers and spontaneous resolvers. P-values, odds ratios, (ORs) and 95% confidence intervals, (95% CIs) human Th17-related RORC gene were associated with were determined for association analysis. P-values (two-tailed) < 0.05were differing natural prognosis for Chinese Han females with considered significant (bold) HCV infection. The most common RORC haplotype in this cohort, rs9826-T/rs1521177-T, was favorable for spontaneous recovery from HCV occurred before or spontaneous clearance of HCV infection. after HIV infection, it remains conceivable that genetic immune characteristics of these individuals could be as- sociated with spontaneous HCV eradication. By contrast, Additional file we excluded chronically HIV/HCV-co-infected patients from the study, due to the possibility that some Additional file 1: Figure S1. Comparison of distributions of IFNL3 and HIV-co-infected individuals who could clear HCV RORC SNP genotypes among HIVneg, HIVpos, and total resolved individuals. spontaneously in the absence of HIV became chronic (a) Distribution of genotypes (%) of three IFNL3 SNPs (rs12979860, rs8099917, and rs12980275). (b)Distributionofgenotypes (%)oftwo RORC SNPs (rs9826, HCV carriers. In addition, all the participants in this and rs1521177). Chi-square (χ2) and Fisher’sexact testswereusedto evaluate study came from the same village and shared similar the differences in SNP distributions between two groups. P-values (two-tailed) characteristics of viral contamination mode, daily diet, < 0.05 were considered significant (n.s., not significant). Figure S2. HCV viral neg load levels of RORC SNP genotypes (rs9826, rs1521177) among HIV HCV carriers of the primary cohort. Figure S3. Linkage disequilibrium tests for RORC SNPs (rs9826 and rs1521177) and IFNL3 SNPs (rs12979860, rs8099917 and Table 4 Common RORC SNP haplotypes (rs9826/rs1521177) and rs12980275) in the primary cohort (a)D’ value. (b)r value. Table S1. Allele their association with spontaneous viral clearance in the primary frequencies of IFNL3 gene SNPs in different populations. Table S2. Allele, cohort and IFNL3 favorable sub-cohort genotype, and carrier frequencies and percentages of tested SNPs in the a a Chronic HCV Resolved HCV P-value OR (95% CI) primary cohort. Table S3. RORC gene SNP allele frequencies in different populations. Table S4. The Hardy–Weinberg Equilibrium (HWE) test of all Primary cohort SNPs in the whole study population. Table S5. Genotype and allele frequency pos T/T 0.62 0.79 0.0027 0.445(0.26–0.76) distributions of RORC SNPs in HIV group. Table S6. Genotype and allele neg frequency distributions of RORC SNPs in HIV HCV carriers of the primary C/G 0.27 0.16 0.0315 1.893(1.05–3.40) cohort. Table S7. Linkage disequilibrium tests for RORC rs9826/rs1521177 in the primary cohort and the IFNL3 favorable sub-cohort. (DOC 1671 kb) IFNL3 favorable sub-cohort T/T 0.64 0.78 0.0117 0.485(0.28–0.86) C/G 0.26 0.17 0.1042 1.672(0.90–3.12) a 2 Abbreviations Haplotype frequency. Chi-square (χ ) tests were used to evaluate differences 95% CIs: 95% confidence intervals; HAART: High active antiretroviral therapy; in haplotype frequencies between, HCV carriers and spontaneous resolvers, HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; HIV: Human using SHEsis software. Haplotypes with frequencies < 0.05 (C/T, C/G, C/T, T/G) immunodeficiency virus; IFNL3: Interferon lambda 3; LD: Linkage are not shown. P-values, odds ratios, (ORs) and 95% confidence intervals (95% CIs) were determined for association analysis. P-values (two-tailed) < 0.05 were disequilibrium; OR: Odds ratios; RORC: Receptor retinoic acid-related orphan considered significant (bold) receptor C; SNP: Single nucleotide polymorphisms; Th17: T helper 17 cells Xie et al. BMC Infectious Diseases (2018) 18:254 Page 7 of 8 Acknowledgements synergism for spontaneous viral clearance in hepatitis C virus (HCV) We are grateful to all who provided blood samples for this study. We also seroconverters from a community-based cohort. PLoS One. 2011;6(11):e27555. thank Dr. Weidong Zhang and Dr. Yuanlin Xi for their assistance for 7. Alric L, Bonnet D, Fort M. Association between female sex, IL28B genotype, construction of the study cohort. but also DQB1*0301 allele and the outcome of acute hepatitis C virus infection. Hepatology. 2014;60(6):2127. Funding 8. Ikezaki H, Furusyo N, Hiramine S, Ura K, Mitsumoto-Kaseida F, Takayama K, This work was financially supported by the National Science and Technology Shimizu M, Toyoda K, Ogawa E, Kainuma M, et al. Association of IL28B Major Project of China (2017ZX10202101-003, 2018ZX10731101-001-008 and rs8099917 genotype and female sex with spontaneous clearance of hepatitis C 2018ZX09739002-003), State Key Laboratory of Infectious Disease Prevention virus infection: a Japanese cross-sectional study. Arch Virol. 2016;161(3):641–8. and Control (2015SKLID506) and the National Natural Science Foundation of 9. Grebely J, Page K, Sacks-Davis R, van der Loeff MS, Rice TM, Bruneau J, China (81271826, 81201286).The funding bodies had no role in the design of Morris MD, Hajarizadeh B, Amin J, Cox AL, et al. The effects of female sex, the study and collection, analysis, and interpretation of data and in writing viral genotype, and IL28B genotype on spontaneous clearance of acute the manuscript. hepatitis C virus infection. Hepatology. 2014;59(1):109–20. 10. Moqueet N, Infante-Rivard C, Platt RW, Young J, Cooper C, Hull M, Walmsley S, Klein MB. Favourable IFNL3 genotypes are associated with spontaneous Availability of data and materials clearance and are differentially distributed in aboriginals in Canadian HIV- All data generated and analyzed during this study are included in this hepatitis C co-infected individuals. Int J Mol Sci. 2015;16(3):6496–512. published article and its Additional files. The datasets analyzed during the 11. Peng J, Chen X, He J, Zheng J, Qin B, Jiang Y. Relationship between current study are available from the corresponding author on request. interleukin 28B, equilibrative nucleoside transporters 1 gene polymorphisms and spontaneous clearance of HCV in HIV/HCV co-infectors. Zhonghua liu Authors’ contributions xing bing xue za zhi = Zhonghua liuxingbingxue zazhi. 2015;36(4):379–82. ZX, YL and LL evaluated the data, performed analyses, and contributed to 12. Shi X, Pan Y, Wang M, Wang D, Li W, Jiang T, Zhang P, Chi X, Jiang Y, Gao writing the manuscript. YL contributed the cellular total DNA isolation and Y, et al. IL28B genetic variation is associated with spontaneous clearance of purification. WC and HL participated in the study design. TS conceived of the hepatitis C virus, treatment response, serum IL-28B levels in Chinese study, also involved in the evaluation and interpretation of data, and wrote population. PLoS One. 2012;7(5):e37054. the manuscript. All authors read and approved the final manuscript. 13. Rao HY, Sun DG, Jiang D, Yang RF, Guo F, Wang JH, Liu F, Zhang HY, Zhang HH, Du SC, et al. IL28B genetic variants and gender are associated with spontaneous Ethics approval and consent to participate clearance of hepatitis C virus infection. J Viral Hepat. 2012;19(3):173–81. The study was approved by the institutional review authorities of Peking 14. Sagnelli E, Santantonio T, Coppola N, Fasano M, Pisaturo M, Sagnelli C. University Health Science Center (Approval ID: PKUPHLL20090011). All Acute hepatitis C: clinical and laboratory diagnosis, course of the disease, patients provided written informed consent before enrollment in the study. treatment. Infection. 2014;42(4):601–10. 15. Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, Cheng J, Competing interests Goedert JJ, Vlahov D, Hilgartner M, et al. HLA and NK cell inhibitory receptor The authors declare that they have no competing interests. genes in resolving hepatitis C virus infection. Science. 2004;305(5685):872–4. 16. Dong ZX, Zhou HJ, Xiang XG, Guo SM, Zhuang Y, Zhao GD, Xie Q. IL28B genetic variations are associated with treatment response of patients with Publisher’sNote chronic hepatitis C in a Chinese Han population. J Dig Dis. 2015;16(2):90–7. Springer Nature remains neutral with regard to jurisdictional claims in 17. Jin G, Kang H, Chen X, Dai D. Evaluation of the relationship between IL28B, published maps and institutional affiliations. IL10RB and IL28RA single-nucleotide polymorphisms and susceptibility to hepatitis C virus in Chinese Han population. Infec Genet Evol. 2014;21:8–14. Author details 18. Liao XW, Ling Y, Li XH, Han Y, Zhang SY, Gu LL, Yu DM, Yao BL, Zhang DH, Department of Microbiology and Center of Infectious Diseases, School of Jin GD, et al. Association of genetic variation in IL28B with hepatitis C Basic Medical Sciences, Peking University, Beijing 100191, China. State Key treatment-induced viral clearance in the Chinese Han population. Antivir laboratory of Infectious Disease Prevention and Control, National Center for Ther. 2011;16(2):141–7. AIDS/STD Control and Prevention, China CDC, Collaborative Innovation 19. Zambrano-Zaragoza JF, Romo-Martinez EJ, Duran-Avelar Mde J, Garcia- Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China. Magallanes N, Vibanco-Perez N. Th17 cells in autoimmune and infectious Department of Infectious Diseases, Guangzhou Eighth People’s Hospital, diseases. Int J Inflamm. 2014;2014:651503. Guangzhou Medical University, Guangzhou 510060, Guangdong, China. 20. Benoit G, Cooney A, Giguere V, Ingraham H, Lazar M, Muscat G, Perlmann T, Renaud JP, Schwabe J, Sladek F, et al. International Union of Pharmacology Received: 19 July 2017 Accepted: 18 May 2018 LXVI. Orphan nuclear receptors. Pharmacol Rev. 2006;58(4):798–836. 21. Sun Z, Unutmaz D, Zou YR, Sunshine MJ, Pierani A, Brenner-Morton S, Mebius RE, Littman DR. Requirement for RORgamma in thymocyte survival References and lymphoid organ development. Science. 2000;288(5475):2369–73. 1. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, Barnes 22. Balanescu P, Ladaru A, Voiosu T, Nicolau A, Ene M, Balanescu E. Th17 and IL-17 E. Global distribution and prevalence of hepatitis C virus genotypes. immunity in chronic hepatitis C infection. Rom J Intern Med. 2012;50(1):13–8. Hepatology. 2015;61(1):77–87. 23. Chang Q, Wang YK, Zhao Q, Wang CZ, Hu YZ, Wu BY. Th17 cells are 2. Kao JH, Ahn SH, Chien RN, Cho M, Chuang WL, Jeong SH, Liu CH, Paik SW. increased with severity of liver inflammation in patients with chronic Urgency to treat patients with chronic hepatitis C in Asia. J Gastroenterol hepatitis C. J Gastroenterol Hepatol. 2012;27(2):273–8. Hepatol. 2017;32(5):966–74. 24. Sun HQ, Zhang JY, Zhang H, Zou ZS, Wang FS, Jia JH. Increased Th17 cells 3. Bennett H, Waser N, Johnston K, Kao JH, Lim YS, Duan ZP, Lee YJ, Wei L, contribute to disease progression in patients with HBV-associated liver Chen CJ, Sievert W, et al. A review of the burden of hepatitis C virus cirrhosis. J Viral Hepat. 2012;19(6):396–403. infection in China, Japan South Korea and Taiwan. Hepatol Int. 2015;9(3): 25. Seetharam AB, Borg BB, Subramanian V, Chapman WC, Crippin JS, 378–90. Mohanakumar T. Temporal association between increased virus-specific 4. Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology Th17 response and spontaneous recovery from recurrent hepatitis C in a of hepatitis C virus infection: new estimates of age-specific antibody to HCV liver transplant recipient. Transplantation. 2011;92(12):1364–70. seroprevalence. Hepatology. 2013;57(4):1333–42. 5. Huang J, Huang K, Xu R, Wang M, Liao Q, Xiong H, Li C, Tang X, Shan Z, 26. Kared H, Fabre T, Bedard N, Bruneau J, Shoukry NH. Galectin-9 and IL-21 Zhang M, et al. The associations of HLA-A*02:01 and DRB1*11:01 with mediate cross-regulation between Th17 and Treg cells during acute hepatitis C virus spontaneous clearance are independent of IL28B in the hepatitis C. PLoS Pathog. 2013;9(6):e1003422. Chinese population. Sci Rep. 2016;6:31485. 27. Chen J, Liang Z, Lu F, Fang X, Liu S, Zeng Y, Zhu F, Chen X, Shen T, Li J, et al. 6. van den Berg CH, Grady BP, Schinkel J, van de Laar T, Molenkamp R, van Toll-like receptors and cytokines/cytokine receptors polymorphisms associate Houdt R, Coutinho RA, van Baarle D, Prins M. Female sex and IL28B, a with non-response to hepatitis B vaccine. Vaccine. 2011;29(4):706–11. Xie et al. BMC Infectious Diseases (2018) 18:254 Page 8 of 8 28. Romero-Gomez M, Eslam M, Ruiz A, Maraver M. Genes and hepatitis C: susceptibility, fibrosis progression and response to treatment. Liver Int. 2011; 31(4):443–60. 29. Selvarajah S, Tobler LH, Simmons G, Busch MP. Host genetic basis for hepatitis C virus clearance: a role for blood collection centers. Curr Opin Hematol. 2010;17(6):550–7. 30. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, Heinzen EL, Qiu P, Bertelsen AH, Muir AJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature. 2009;461(7262):399–401. 31. Suppiah V, Moldovan M, Ahlenstiel G, Berg T, Weltman M, Abate ML, Bassendine M, Spengler U, Dore GJ, Powell E, et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet. 2009;41(10):1100–4. 32. Tanaka Y, Nishida N, Sugiyama M, Kurosaki M, Matsuura K, Sakamoto N, Nakagawa M, Korenaga M, Hino K, Hige S, et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet. 2009;41(10):1105–9. 33. Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O'Huigin C, Kidd J, Kidd K, Khakoo SI, Alexander G, et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature. 2009;461(7265):798–801. 34. Gaudieri S, Rauch A, Park LP, Freitas E, Herrmann S, Jeffrey G, Cheng W, Pfafferott K, Naidoo K, Chapman R, et al. Evidence of viral adaptation to HLA class I-restricted immune pressure in chronic hepatitis C virus infection. J Virol. 2006;80(22):11094–104. 35. Ansari MA, Pedergnana V, LCI C, Magri A, Von Delft A, Bonsall D, Chaturvedi N, Bartha I, Smith D, Nicholson G, et al. Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus. Nat Genet. 2017;49(5):666–73. 36. Moore CB, John M, James IR, Christiansen FT, Witt CS, Mallal SA. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science. 2002;296(5572):1439–43. 37. von Delft A, Humphreys IS, Brown A, Pfafferott K, Lucas M, Klenerman P, Lauer GM, Cox AL, Gaudieri S, Barnes E. The broad assessment of HCV genotypes 1 and 3 antigenic targets reveals limited cross-reactivity with implications for vaccine design. Gut. 2016;65(1):112–23. 38. Thimme R, Bukh J, Spangenberg HC, Wieland S, Pemberton J, Steiger C, Govindarajan S, Purcell RH, Chisari FV. Viral and immunological determinants of hepatitis C virus clearance, persistence.and disease. Proc Nati Acad Sci USA. 2002;99(24):15661–8. 39. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med. 2001;194(10):1395–406. 40. Klenerman P, Thimme R. T cell responses in hepatitis C: the good the bad and the unconventional. Gut. 2012;61(8):1226–34. 41. Basha HI, Subramanian V, Seetharam A, Nath DS, Ramachandran S, Anderson CD, Shenoy S, Chapman WC, Crippin JS, Mohanakumar T. Characterization of HCV-specific CD4+Th17 immunity in recurrent hepatitis C-induced liver allograft fibrosis. Am J Transplant Off J Am Soc Transplant Am Soc Transplant Surg. 2011;11(4):775–85. 42. Wang JM, Shi L, Ma CJ, Ji XJ, Ying RS, Wu XY, Wang KS, Li G, Moorman JP, Yao ZQ. Differential regulation of interleukin-12 (IL-12)/IL-23 by Tim-3 drives T(H)17 cell development during hepatitis C virus infection. J Virol. 2013; 87(8):4372–83. 43. Shen T, Chen X, Zhang W, Xi Y, Cao G, Zhi Y, Wang S, Xu C, Wei L, Lu F, et al. A higher correlation of HCV core antigen with CD4+ T cell counts compared with HCV RNA in HCV/HIV-1 coinfected patients. PLoS One. 2011; 6(8):e23550. 44. Jauncey M, Micallef JM, Gilmour S, Amin J, White PA, Rawlinson W, Kaldor JM, van Beek I, Dore GJ. Clearance of hepatitis C virus after newly acquired infection in injection drug users. J Infect Dis. 2004;190(7):1270–4. 45. Paradowska-Gorycka A, Stypinska B, Pawlik A, Romanowska-Prochnicka K, Haladyj E, Manczak M, Olesinska M. RORC2 genetic variants and serum levels in patients with rheumatoid arthritis. Int J Mol Sci. 2016;17(4):488.
BMC Infectious Diseases – Springer Journals
Published: Jun 4, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera