Genome-wide association study identifies new loci associated with risk of HBV infection and disease progression

Abstract

Background: Recent studies have identified susceptibility genes of HBV clearance, chronic hepatitis B, liver cirrhosis, hepatocellular carcinoma, and showed the host genetic factors play an important role in these HBV-related outcomes.

Methods: Collected samples from different outcomes of HBV infection and performed genotyping by Affymetrix 500 k SNP Array. GCTA tool, PLINK, and Bonferroni method were applied for analysis of genotyping and disease progression. ANOVA was used to evaluate the significance of the association between biomarkers and genotypes in healthy controls. PoMo, F_ST, Vcftools and Rehh package were used for building the racial tree and population analysis. F_ST statistics accesses 0.15 was used as a threshold to detect the signature of selection.

Results: There are 1031 participants passed quality control from 1104 participants, including 275 HBV clearance, 92 asymptomatic persistence infection (ASPI), 93 chronic hepatitis B (CHB), 188 HBV-related decompensated cirrhosis (DC), 214 HBV-related hepatocellular carcinoma (HCC) and 169 healthy controls (HC). In the case-control study, one novel locus significantly associated with CHB (SNP: rs1264473, Gene: GRHL2, P = 1.57 × 10^-6) and HCC (SNP: rs2833856, Gene: EVA1C, P = 1.62 × 10^-6; SNP: rs4661093, Gene: ETV3, P = 2.26 × 10^-6). In the trend study across progressive stages post HBV infection, one novel locus (SNP: rs1537862, Gene: LACE1, P = 1.85 × 10^-6), and three MHC loci (HLA-DRB1, HLA-DPB1, HLA-DPA2) showed significant increased progressive risk from ASPI to CHB. Underlying the evolutionary study of HBV-related genes in public database, the derived allele of two HBV clearance related loci, rs3077 and rs9277542, are under strong selection in European population.

Conclusions: In this study, we identified several novel candidate genes associated with individual HBV infectious outcomes, progressive stages, and liver enzymes. Two SNPs that show selective significance (HLA-DPA1, HLA-DPB1) in non-East Asian (European, American, South Asian) versus East Asian, indicating that host genetic factors contribute to the ethnic disparities of susceptibility of HBV infection. Taken together, these findings provided a new insight into the role of host genetic factors in HBV related outcomes and progression.

Keywords: Disease progression; GWAS; HBV infection; Host genetic factors; SNPs.

Fig. 1

Regional plots shown –log10 P-values of SNPs in association study. Marker SNPs are shown as purple diamonds, other SNPs are shown as dots. R-square of Marker SNPs and other SNPs are shown against dark blue, blue, green, yellow and red colors, indicating the linkage disequilibrium. The structure of genes within the region are shown as rectangles and arrows. Abbreviation: ASPI, asymptomatic persistence infection; CHB, chronic hepatitis B; DC, decompensated cirrhosis; HCC, hepatocellular carcinoma

Fig. 2

The racial tree (Left) was based on the SNPs in HBV-infection related genes, including HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1. The genotype and minor allele frequency of each SNPs were accessed from 1000 Genome Project. EAS, AFR, SAS, EUR, AMR refer to East Asian, African, South Asian, European and American of 1000 Genome Project, respectively. The racial tree indicated a genetic difference in HBV-infection related genes among EAS, AFR, SAS, EUR, AMR. The genetic difference (Right) of each SNPs was evaluated by F_ST value. X-axis refer to physical position in chromosome 6. Y-axis refer to F_ST value of paired SNP. F_ST values of all paired SNPs of AFR, SAS, AMR, EUR versus EAS were displayed in grey bar. F_ST values accessed 0.15 (Red horizontal line) indicated the signal of selective event. Red bars and rs IDs showed the reported HBV infection-related SNPs. The F_ST values of European versus East Asian showed the genetic difference in HLA-DPA1 and HLA-DPB1, indicating a genetic selection against the HBV infection. The racial tree showed that the East Asian population is at the root, indicating that why we used East Asian population as a comparative population but not the other population, and compared other four populations with East Asian population

Fig. 3

Haplotype bifurcation diagrams of infection-related SNPs, including rs3077 on HLA-DPA1 (upper) and rs9277542 on HLA-DPB1 (lower), in European (left) and East Asian (right). EUR and EAS in plot title refer to European and East Asian. DA and AA in plot title refer to derived allele (red) and ancestral allele (blue). Black dash line refers to the position of core SNP. Each node refers to a haplotype. The edge width reflects the population-specific frequency. Haplotype bifurcation diagrams were displayed via Rehh package. Haplotype bifurcation diagrams showed that the derived allele led to a long-range high frequency haplotype in European population and the ancestral allele led to a high frequency haplotype in East Asian population; the ancestral allele led to more haplotypes than the derived allele. The long-range high frequency haplotype confirms the genetic selection in HLA-DPA1 and HLA-DPB1 in European population

Fig. 4

The raising allele frequency in HBV related outcomes during the progression. Four SNPs with increased resistance in CHB, ASPI, HBV clearance during HBV infection (a) and two SNPs with increased risk in the CHB, DC, HCC during the development of CHB (b). Abbreviation: ASPI, asymptomatic persistence infection; CHB, chronic hepatitis B; DC, decompensated cirrhosis; HCC, hepatocellular carcinoma

Fig. 5

The association between HBV related loci and serum liver enzyme levels in health controls. P values were calculated by ANOVA test. White-circle refer to the mean liver enzymes level with different genotypes. The significant differences indicate that these SNPs contribute to liver enzyme activity