Genetic Variants Influence Susceptibility to HCV
Genetic Variants Influence Susceptibility to HCV
All enrolled participants were divided into three groups based on their anti-HCV and HCV RNA status, including 482 anti-HCV negative subjects (uninfected subjects), 193 spontaneous clearance cases (anti-HCV positive and HCV RNA negative, also called resolvers), and 532 persistent HCV cases (both anti-HCV and HCV RNA positive, also called chronic cases). The later two groups were combined as HCV infected subjects. Some demographic and selected characteristics were shown in Table 1. Distribution of age and gender was not different among groups. Consistent with current knowledge of HCV transmission pattern, experience of plasma donation was associated with HCV infection and the risk of HCV chronicity was increased with the times of plasma donation. The level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicated that HCV infected subjects had worse liver function compared with non-infected subjects.
Three genetic models (dominant, recessive, and additive models) were used in analysis of each SNP. Significance in any model was considered as a possible association of these SNPs with HCV infection (Additional file 1: Table S2 http://www.biomedcentral.com/1471-2334/14/716/additional). After adjustment for age, gender, experience of blood/plasma donation, and times of blood/plasma donation, logistic regression analyses showed that variants in HLA-DMA, HLA-DOA, HLA-DOB, LMP2, and LMP7 were associated with outcomes of HCV infection.
The allelic frequencies of candidate genes were firstly compared among the uninfected controls and infected cases (including both natural clearance cases and chronic cases). HLA-DMA rs1063478-T mutant had a decreased frequency in infected cases compared with C wild type. HLA-DOA rs2284191-A (vs. G) and HLA-DOB rs11244-T (vs. C) variants were positively related with anti-HCV (Table 2a). Conditional logistic regression analysis was performed to test the independence of these SNPs. The effect of rs11244 was weakened (P = 0.078) after being conditioned on the other two SNPs, so it was excluded from further analysis of combined effect (Additional file 1: Table S3a http://www.biomedcentral.com/1471-2334/14/716/additional). The combined effect of two independent SNPs (rs1063478-T and rs2284191-G) was analyzed by Cochran-Armitage's trend test. The results showed an increased protection effect with more favorable alleles (P = 0.037). Carrying three favorable alleles offered the highest protective effect (OR = 0.46, 95% CI = 0.27–0.78), as showed in Table 3a.
The ability of viral clearance was then compared among the spontaneous infection subjects and the persistent infection subjects. LMP2 rs17587-A (vs. G), LMP7 rs2071543-A (vs. C), HLA-DOA rs2284191-A (vs. G), rs376892-T (vs. C), rs416622-A (vs. G), and HLA-DOB rs7383287-G (vs. A) were positively associated with HCV clearance (Table 2b). The effect of rs17587 and rs7383287 on HCV clearance remained obvious after being conditioned on the other five SNPs (P = 0.03 for rs17587 and P = 0.007 for rs7383287) (Additional file 1: Table S3b http://www.biomedcentral.com/1471-2334/14/716/additional). Therefore, the independent SNPs of LMP2 rs17587 and HLA-DOB rs7383287 were analyzed in Cochran-Armitage's trend test. There was also an increased protection effect with more favorable alleles (P = 0.001). Subjects carrying two favorable alleles had a 58% decrease in risk of HCV persistent infection (OR = 0.42, 95% CI = 0.26–0.66) (Table 3b).
The association between the combined effect of the independent SNPs and HCV infection was further evaluated by adjustment for confounding factors including age, gender, experience of blood/plasma donation, and times of blood/plasma donation. The results were shown in Additional file 1: Table S4 http://www.biomedcentral.com/1471-2334/14/716/additional.
The combined protective effect of HLA-DMA rs1063478-T and HLA-DOA rs2284191-G was more prominent in female subgroup (OR = 0.68, 95% CI = 0.49–0.94). Subjects < 60 years favored more from the protective effect of rs1063478-T and rs2284191-G as compared with subjects ≥ 60 years (OR = 0.62, 95% CI = 0.43–0.88). The protective effect of carrying rs1063478-T and rs2284191-G was also more obvious in subjects with experience of blood donation (OR = 0.69, 95% CI = 0.51–0.94), subjects with blood donation ≥ 30 times (OR = 0.61, 95% CI = 0.40–0.93), subjects with experience of plasma donation (OR = 0.66, 95% CI = 0.49–0.90), and subjects with plasma donation ≥ 50 times (OR = 0.67, 95% CI = 0.47–0.95). Heterogeneity test showed that heterogeneity in every two strata was significant for gender and plasma donation (P = 0.023 and 0.024, respectively).
The combined protective effect of LMP2 rs17587-A and HLA-DOB rs7383287-G was more pronounced in female subjects (OR = 0.63, 95% CI = 0.43–0.93), subjects < 60 years (OR = 0.49, 95% CI = 0.31–0.78), subjects with experience of blood donation (OR = 0.67, 95% CI = 0.47–0.97), subjects with blood donation < 30 times (OR = 0.50, 95% CI = 0.31–0.81), subjects with experience of plasma donation (ever vs. never, OR = 0.61, 95% CI = 0.43–0.87), and subjects with plasma donation ≥ 50 times (OR = 0.46, 95% CI = 0.27–0.78). No obvious evidence of heterogeneity associations was observed.
The interaction between the meaningful SNPs and potential risk factors was also analyzed. The results were shown in Table 4. Significant multiplicative interactions on HCV susceptibility were found between the combined effects of rs1063478 and rs2284191 and plasma donation (Pinteraction = 0.020). Compared to subjects carrying 0–2 favorable alleles and with experience of plasma donation, subjects carrying 3–4 favorable alleles but without plasma donation had a decreased detection rate of anti-HCV (OR = 0.18, 95% CI = 0.10–0.32). There was also a multiplicative interaction between rs7383287 genotypes and exposure of blood donation (Pinteraction = 0.040). Compared to subjects carrying rs7383287AA genotypes and with experience of blood donation, subjects carrying AG/AA genotypes but without blood donation had a 66% decrease in risk of HCV persistent infection (OR = 0.34, 95% CI = 0.15–0.78).
Because high linkage disequilibrium exists in HLA region, we also analyzed the LD among the candidate SNPs (detailed information showed in Additional file 1: Table S5 http://www.biomedcentral.com/1471-2334/14/716/additional) and then we performed haplotype analysis.
In the above analyses, rs1063478, rs2284191, and rs11244 were identified to be related with anti-HCV status. Compared with the most frequent CGC haplotype, the haplotype with rs1063478-T (TGC) was associated with a protective effect (P < 0.001), while the haplotype carrying rs2284191-A (CAC) indicated a risk effect of HCV infection (P < 0.001) (Table 5a).
Six SNPs were identified to be related with chronicity of HCV infection, including rs17587, rs2071543, rs2284191, rs7383287, rs376892 and rs416622. The most frequent haplotype was GCGACG. No difference of viral clearance was found between reference haplotype GCGACG and haplotype carrying rs2071543 (GAGACG). Haplotypes carrying rs7383287-G (GCGGCG) and rs17587-A (ACGACG) were more frequently found in spontaneous clearance group than in chronic infection group (P < 0.001 and P = 0.001, respectively). Haplotypes carrying the other alleles enhanced the risk of chronic HCV infection. The results were shown in Table 5b.
Results
Demographic and Selected Variables of Participants
All enrolled participants were divided into three groups based on their anti-HCV and HCV RNA status, including 482 anti-HCV negative subjects (uninfected subjects), 193 spontaneous clearance cases (anti-HCV positive and HCV RNA negative, also called resolvers), and 532 persistent HCV cases (both anti-HCV and HCV RNA positive, also called chronic cases). The later two groups were combined as HCV infected subjects. Some demographic and selected characteristics were shown in Table 1. Distribution of age and gender was not different among groups. Consistent with current knowledge of HCV transmission pattern, experience of plasma donation was associated with HCV infection and the risk of HCV chronicity was increased with the times of plasma donation. The level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicated that HCV infected subjects had worse liver function compared with non-infected subjects.
Association of Candidate SNPs With HCV Infection Outcomes
Three genetic models (dominant, recessive, and additive models) were used in analysis of each SNP. Significance in any model was considered as a possible association of these SNPs with HCV infection (Additional file 1: Table S2 http://www.biomedcentral.com/1471-2334/14/716/additional). After adjustment for age, gender, experience of blood/plasma donation, and times of blood/plasma donation, logistic regression analyses showed that variants in HLA-DMA, HLA-DOA, HLA-DOB, LMP2, and LMP7 were associated with outcomes of HCV infection.
The allelic frequencies of candidate genes were firstly compared among the uninfected controls and infected cases (including both natural clearance cases and chronic cases). HLA-DMA rs1063478-T mutant had a decreased frequency in infected cases compared with C wild type. HLA-DOA rs2284191-A (vs. G) and HLA-DOB rs11244-T (vs. C) variants were positively related with anti-HCV (Table 2a). Conditional logistic regression analysis was performed to test the independence of these SNPs. The effect of rs11244 was weakened (P = 0.078) after being conditioned on the other two SNPs, so it was excluded from further analysis of combined effect (Additional file 1: Table S3a http://www.biomedcentral.com/1471-2334/14/716/additional). The combined effect of two independent SNPs (rs1063478-T and rs2284191-G) was analyzed by Cochran-Armitage's trend test. The results showed an increased protection effect with more favorable alleles (P = 0.037). Carrying three favorable alleles offered the highest protective effect (OR = 0.46, 95% CI = 0.27–0.78), as showed in Table 3a.
The ability of viral clearance was then compared among the spontaneous infection subjects and the persistent infection subjects. LMP2 rs17587-A (vs. G), LMP7 rs2071543-A (vs. C), HLA-DOA rs2284191-A (vs. G), rs376892-T (vs. C), rs416622-A (vs. G), and HLA-DOB rs7383287-G (vs. A) were positively associated with HCV clearance (Table 2b). The effect of rs17587 and rs7383287 on HCV clearance remained obvious after being conditioned on the other five SNPs (P = 0.03 for rs17587 and P = 0.007 for rs7383287) (Additional file 1: Table S3b http://www.biomedcentral.com/1471-2334/14/716/additional). Therefore, the independent SNPs of LMP2 rs17587 and HLA-DOB rs7383287 were analyzed in Cochran-Armitage's trend test. There was also an increased protection effect with more favorable alleles (P = 0.001). Subjects carrying two favorable alleles had a 58% decrease in risk of HCV persistent infection (OR = 0.42, 95% CI = 0.26–0.66) (Table 3b).
Stratified Analysis of Independent SNPs
The association between the combined effect of the independent SNPs and HCV infection was further evaluated by adjustment for confounding factors including age, gender, experience of blood/plasma donation, and times of blood/plasma donation. The results were shown in Additional file 1: Table S4 http://www.biomedcentral.com/1471-2334/14/716/additional.
The combined protective effect of HLA-DMA rs1063478-T and HLA-DOA rs2284191-G was more prominent in female subgroup (OR = 0.68, 95% CI = 0.49–0.94). Subjects < 60 years favored more from the protective effect of rs1063478-T and rs2284191-G as compared with subjects ≥ 60 years (OR = 0.62, 95% CI = 0.43–0.88). The protective effect of carrying rs1063478-T and rs2284191-G was also more obvious in subjects with experience of blood donation (OR = 0.69, 95% CI = 0.51–0.94), subjects with blood donation ≥ 30 times (OR = 0.61, 95% CI = 0.40–0.93), subjects with experience of plasma donation (OR = 0.66, 95% CI = 0.49–0.90), and subjects with plasma donation ≥ 50 times (OR = 0.67, 95% CI = 0.47–0.95). Heterogeneity test showed that heterogeneity in every two strata was significant for gender and plasma donation (P = 0.023 and 0.024, respectively).
The combined protective effect of LMP2 rs17587-A and HLA-DOB rs7383287-G was more pronounced in female subjects (OR = 0.63, 95% CI = 0.43–0.93), subjects < 60 years (OR = 0.49, 95% CI = 0.31–0.78), subjects with experience of blood donation (OR = 0.67, 95% CI = 0.47–0.97), subjects with blood donation < 30 times (OR = 0.50, 95% CI = 0.31–0.81), subjects with experience of plasma donation (ever vs. never, OR = 0.61, 95% CI = 0.43–0.87), and subjects with plasma donation ≥ 50 times (OR = 0.46, 95% CI = 0.27–0.78). No obvious evidence of heterogeneity associations was observed.
Interaction Analysis
The interaction between the meaningful SNPs and potential risk factors was also analyzed. The results were shown in Table 4. Significant multiplicative interactions on HCV susceptibility were found between the combined effects of rs1063478 and rs2284191 and plasma donation (Pinteraction = 0.020). Compared to subjects carrying 0–2 favorable alleles and with experience of plasma donation, subjects carrying 3–4 favorable alleles but without plasma donation had a decreased detection rate of anti-HCV (OR = 0.18, 95% CI = 0.10–0.32). There was also a multiplicative interaction between rs7383287 genotypes and exposure of blood donation (Pinteraction = 0.040). Compared to subjects carrying rs7383287AA genotypes and with experience of blood donation, subjects carrying AG/AA genotypes but without blood donation had a 66% decrease in risk of HCV persistent infection (OR = 0.34, 95% CI = 0.15–0.78).
Haplotype Analysis
Because high linkage disequilibrium exists in HLA region, we also analyzed the LD among the candidate SNPs (detailed information showed in Additional file 1: Table S5 http://www.biomedcentral.com/1471-2334/14/716/additional) and then we performed haplotype analysis.
In the above analyses, rs1063478, rs2284191, and rs11244 were identified to be related with anti-HCV status. Compared with the most frequent CGC haplotype, the haplotype with rs1063478-T (TGC) was associated with a protective effect (P < 0.001), while the haplotype carrying rs2284191-A (CAC) indicated a risk effect of HCV infection (P < 0.001) (Table 5a).
Six SNPs were identified to be related with chronicity of HCV infection, including rs17587, rs2071543, rs2284191, rs7383287, rs376892 and rs416622. The most frequent haplotype was GCGACG. No difference of viral clearance was found between reference haplotype GCGACG and haplotype carrying rs2071543 (GAGACG). Haplotypes carrying rs7383287-G (GCGGCG) and rs17587-A (ACGACG) were more frequently found in spontaneous clearance group than in chronic infection group (P < 0.001 and P = 0.001, respectively). Haplotypes carrying the other alleles enhanced the risk of chronic HCV infection. The results were shown in Table 5b.