A Non-Synonymous Variant of Complement Factor B Associated with Risk of Chronic Hepatitis B in a Korean Population

Background: Hepatitis B is known to cause several forms of liver diseases including chronic hepatitis B (CHB), and hepatocellular carcinoma. Previous genome-wide association study of CHB risk has demonstrated that rs12614 of complement factor B (CFB) was signicantly associated with CHB risk. This association study was conducted in order to identify genetic effects of CFB single nucleotide polymorphisms (SNPs) and to identify additional independent CHB susceptible causal markers within a Korean population. Methods: A total of 10 CFB genetic polymorphisms were selected and genotyped in 1,716 study subjects comprised of 955 CHB patients and 761 population controls. Results: A non-synonymous variant, rs12614 (Arg32Trp) in exon2 of CFB, had signicant associations with risk of CHB (odds ratio = 0.43, P = 5.91×10 -10 ). Additional linkage disequilibrium and conditional analysis conrmed that rs12614 had independent genetic effect on CHB susceptibility with previously identied CHB markers. The genetic risk scores (GRSs) were calculated and the CHB patients had higher GRSs than the population controls. Moreover, OR was found to increase signicantly with cumulative GRS. Conclusions: rs12614 showed signicant genetic effect on CHB risk within the Korean population. As such rs12614 may be used as a possible causal genetic variant for CHB susceptibility.


Statistical analysis
LD was obtained using Haploview v4.2 software downloaded from the Broad Institute (http://www.broadinstitute.org/mpg/haploview), with examination of Lewontin's D' (|D'|) and the LD coe cient r 2 between all pairs of bi-allelic loci [18]. Logistic regression models were used to compare genotype distributions, including MAF and Hardy-Weinberg Equilibrium (HWE), among CHB patients and controls, and to calculate odds ratios (ORs), 95% con dence intervals, and corresponding P-values adjusted for age (continuous value) and sex (male = 0, female = 1) as covariates using SAS, version 9.4 (SAS Inc., Cary, NC, USA). In corrections for multiple comparisons, Bonferroni correction for multiple testing was applied. In order to examine whether the new association signal of investigated SNP is independent or affected by already known CHB susceptible loci, conditional logistic regression analyses were performed. Allele test based on the allele distribution of each SNP was also performed to assess the detailed genetic effects. Ten previously reported CHB susceptible loci in a Korean population (rs9277535 of HLA-DPB1; rs3077 of HLA-DPA1; rs2856718 of HLA-DQB1; rs7453920 of HLA-DQB2; rs1419881 of TCF19; rs1265163 of OCT4; rs652888 and rs35875104 of EHMT2; rs9394021 and rs2517459 of VARS2-SFTA) [5][6][7][8][9] were used for the conditional analysis and allele test. Based on the results from allele test, GRSs were calculated by multiplying the number of minor alleles by effect size (OR) of the SNP. Then, the combined genetic effects for each individual were calculated as the sum of the GRSs. Calculating the distribution of cumulative GRSs, ORs, 95% con dence intervals, and corresponding Pvalues between patients and controls were also performed.

Results
Genotyping of CFB genetic variants A total of 10 CFB SNPs were selected and genotyped in 1,716 Korean subjects, comprised of 955 CHB patients and 761 population controls (Supplementary Table 1). Patients were divided into two subgroups, 659 HCC (-) CHB cases and 296 HCC (+) CHB cases. A gene map and LD among investigated SNPs are shown in Supplementary Fig. 1A and 1B. Detailed information on the investigated SNPs, such as chromosome, position, allele, genotype distribution, heterozygosity, and HWE P, are presented in Supplementary Table 2.

Association of CFB genetic polymorphisms with CHB risk
In order to investigate the association between CFB genetic polymorphisms and risk of CHB, logistic regression analysis under an additive model was conducted. Analysis results indicated that rs12614 was signi cantly associated with risk of CHB even after applying Bonferroni correction for multiple testing (OR = 0.43, P = 5.91 × 10 − 10 , P corr = 2.36 × 10 − 8 ; Table 1). Additional subgroup analysis was performed to investigate the association between CFB SNPs and CHB-related HCC progression. Again, analysis results found that, rs12614 had signi cant associations with risk of CHB in both the HCC (-) CHB and the HCC (+) CHB subgroups (P = 6.60 × 10 − 8 and 3.10 × 10 − 6 , respectively) even after Bonferroni correction was applied for multiple testing (P corr = 2.64 × 10 − 6 and 1.24 × 10 − 4 , respectively). However, rs12614 did not show signi cant genetic effect on CHB-related HCC progression (P > 0.05). Signi cant associations are shown in bold face (P < 0.05).

Independent genetic effect of rs12614 on CHB risk
In order to understand the association between rs12614 and CHB risk, particularly with respect to independent genetic effect on CHB susceptibility, this study conducted LD calculations and conditional analysis on 10 previously identi ed CHB susceptibility markers (rs9277535 of HLA-DPB1; rs3077 of HLA-DPA1; rs2856718 of HLA-DQB1; rs7453920 of HLA-DQB2; rs1419881 of TCF19; rs1265163 of OCT4; rs652888 and rs35875104 of EHMT2; rs9394021 and rs2517459 of VARS2-SFTA). Supplementary Fig. 2 shows the LD plot of rs12614 and the 10 CHB susceptibility markers. The results show that CFB rs12614 did not display tight LDs with any known, nearby CHB-susceptible loci (pairwise r 2 ≤ 0.15; Supplementary Fig. 2). In addition, when adjusting for previously identi ed CHB markers, rs12614 maintained signi cant association with CHB risk (P < 0.05; Table 2), indicating that rs12614 had independent genetic effect on CHB susceptibility to previously identi ed CHB risk markers. Signi cant associations are shown in bold face (P < 0.05).
Cumulative genetic effects of CHB susceptible loci In order to examine the detailed genetic effects of all 11 CHB susceptible loci including rs12614 (rs12614 of CFB; rs9277535 of HLA-DPB1; rs3077 of HLA-DPA1; rs2856718 of HLA-DQB1; rs7453920 of HLA-DQB2; rs1419881 of TCF19; rs1265163 of OCT4; rs652888 and rs35875104 of EHMT2; rs9394021 and rs2517459 of VARS2-SFTA) in a Korean population, an allele test was conducted for each SNP. The GRSs of the genotypes were calculated using the ORs from allele test (Table 3). CHB, chronic hepatitis B; PC, population control; OR, odds ratio; CI, con dence interval; GRS, genetic risk score.
To elucidate the cumulative genetic effects of all 11 CHB loci in the study subjects, the cumulative GRSs were evaluated. The cumulative GRSs ranged from 5.24 (most protected group) to 17.38 (most susceptible group), and CHB patients showed signi cantly higher cumulative GRSs than did the healthy control subjects (Supplementary Table 3 and Fig. 1A). It was found that as cumulative GRSs increased, ORs signi cantly increased as well. In particular, individuals with GRSs of less than 7 showed an OR of 0.17 (log 10 OR = -0.77), while individuals with GRSs of over 14 showed an OR of 3.42 (log 10 OR = 0.53) (Fig. 1B).

Discussion
Hepatitis B, an infectious disease with a high rate of incidence in Asian populations [1], is a major cause of CHB, liver failure, liver cirrhosis, and HCC development, diseases which often result in death [19,20]. Although the mechanisms underlying the different clinical results of HBV infection have not been fully understood, previous studies have linked a diverse range of factors such as viral strain, gender, age of infection, host immune system, and genetic information of the host, with risk of CHB [21]. When viral infection occurs, several immune-related genes are activated, leading to disease outbreak. According to a GWAS conducted on a Chinese population, a CFB genetic variant had signi cant association with risk of CHB [10]. As such, this association study was conducted to investigate the genetic effects of CFB genetic variants on CHB susceptibility within a Korean population.
The complement system is composed of over 30 plasma proteins and is activated by microbes or antibodies which attached to microbes or other antigens [22,23]. This system is an innate immune system that helps operate rapid responses against pathogenic invasions by opsonizing or recruiting in ammatory cells or pathogen lysis [24]. The complement activations occur through three pathways: the classical pathway, the lectin pathway and the alternative pathway.
These pathways are worked through a cascade of enzymes reaction [23,25]. CFB is essential to activate the complement system, particularly the alternative pathway that is against microbe invasion which includes viruses [26].
Previous Chinese studies have identi ed CFB genetic variants which have genetic effect on CHB risk. The most signi cant association was identi ed at rs12614 of CFB (P = 1.28 × 10 − 34 − 4.0 × 10 − 3 ) [10,11]. In this study, rs12614 showed the same direction of genetic effect as found in previous Chinese studies. Moreover, CFB rs12614 was signi cantly associated with risk of CHB in the HCC (-) CHB and the HCC (+) CHB groups (P = 6.60 × 10 − 8 and 3.10 × 10 − 6 , respectively). However, there was no signi cant genetic effect on CHB-related HCC progression. Additionally, the rs12614 C > T T allele was more frequently observed in the PC group than the CHB patients with a signi cance (OR = 0.43, P corr = 2.36 × 10 − 8 ). Considering that individuals with the non-synonymous variant (rs12614 T allele) had signi cantly higher CFB expression than those with the rs12614 C allele in the Chinese study, it can be seen that the rs12614 may affect immune response by in uencing the complement system when viral infection occurs [10].
The rs12614 which is located on coding region of CFB, C to T allele change causes the amino acid change, arginine to tryptophan. To predict the effects of the rs12614 amino acid change, we conducted in silico analysis using the PolyPhen-2 program (http://genetics.bwh.harvard.edu/pph2/index.shtml) [27]. The results predicted this amino acid change is predicted to be probably damaging that means this substitution is predicted to be damaging with high con dence ( Supplementary Fig. 3A, [27]). In addition, amino acid alignment from the program, arginine at position 32 is highly conserved among species ( Supplementary   Fig. 3B). As disease-causing substitutions are more likely to occur at positions that is conserved throughout evolution [28], the rs12614 C to T allele substitution may affect CFB functions. Because the alternative pathway is important to against pathogen invasion, an amino acid change in CFB important in the alternative pathway may affect the immune system to against hepatitis B virus invasion.
Some individuals are more susceptible to diseases while others are less susceptible. Identi cation of the genetic background is key to understand differences in individuals' disease susceptibility, and that can potentially lead to the targeting of preventive measures at those who are at greatest risk [29]. The results of the conditional analysis conducted on the 10 previously identi ed markers indicated that rs12614 can be used as a novel causal variant of CHB susceptibility. To elucidate its cumulative genetic effects, we used odd ratios of rs12614 and previously identi ed 10 CHB markers. Consequently, CHB group showed higher GRSs than the PC group and the higher genetic risk scores range indicated higher odds ratios. These implies CHB patients are more likely to have higher scores than controls.
There is a sampling limitation in this study. While the ideal subjects for the control groups would be the people who are HBsAg (-) and anti-HBc (+) (spontaneously cleared), we used population controls with unknown responses to HBV infection. And some individuals in the control group still have a chance of progression to CHB when exposed to HBV. Although using the population controls in a case-control study may reduce statistical power, it is useful when it is di cult to obtain a su cient number of disease controls.

Conclusions
A non-synonymous variant, rs12614 (Arg32Trp) of CFB was found to have signi cant associations with risk of CHB in a Korean population. Moreover, genetic effect of rs12614 on CHB risk was independent of all known CHB risk loci, and rs12614 can be used as possible causal variant of CHB susceptibility. Therefore, the results from this study may help in understanding and predicting genetic susceptibility to CHB in a Korean population.