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: email@example.com; firstname.lastname@example.org 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. 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BMC Infectious Diseases – Springer Journals
Published: Jun 4, 2018
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