Genome-wide association study of hepatocellular carcinoma in Southern Chinese patients with chronic hepatitis B virus infection

Abstract

One of the most relevant risk factors for hepatocellular carcinoma (HCC) development is chronic hepatitis B virus (HBV) infection, but only a fraction of chronic HBV carriers develop HCC, indicating that complex interactions among viral, environmental and genetic factors lead to HCC in HBV-infected patients. So far, host genetic factors have incompletely been characterized. Therefore, we performed a genome-wide association (GWA) study in a Southern Chinese cohort consisting of 95 HBV-infected HCC patients (cases) and 97 HBV-infected patients without HCC (controls) using the Illumina Human610-Quad BeadChips. The top single nucleotide polymorphisms (SNPs) were then validated in an independent cohort of 500 cases and 728 controls. 4 SNPs (rs12682266, rs7821974, rs2275959, rs1573266) at chromosome 8p12 showed consistent association in both the GWA and replication phases (OR(combined) = 1.31-1.39; p(combined) = 2.71 × 10(-5)-5.19 × 10(-4); PAR(combined) = 26-31%). We found a 2.3-kb expressed sequence tag (EST) in the region using in-silico data mining and verified the existence of the full-length EST experimentally. The expression level of the EST was significantly reduced in human HCC tumors in comparison to the corresponding non-tumorous liver tissues (P<0.001). Results from sequence analysis and in-vitro protein translation study suggest that the transcript might function as a long non-coding RNA. In summary, our study suggests that variations at chromosome 8p12 may promote HCC in patients with HBV. Further functional studies of this region may help understand HBV-associated hepatocarcinogenesis.

Figure 1. Manhattan plot of GWA study results of testing for association with HBV-related HCC susceptibility.

The dotted line indicates the p-value threshold of 1×10⁻⁵. The circled SNPs are those passing the threshold (Table 1).

Figure 2. Expression of the EST in human HCCs and cell line.

(A) Quantitative expression analysis of the EST transcript in the HBV-associated HCCs showed that the EST transcript was significantly under-expressed in HCC tumors when compared with the corresponding non-tumorous livers (P<0.0001, non-parametric paired t-test). Dots: the ratio of expression level of the transcript versus the level of GAPDH in each testing sample. Horizontal lines: mean of the relative expression level in the HCC tumors and non-tumorous livers. (B) Left panel: Schematic diagram of the FLAG-tagged expression constructs. Right panel: Western blot analysis of FLAG-tagged EST translation in HepG2 cell line. The FLAG-tag could be translated only when tagged at the N-terminus of the ORF but not at the C-terminus. β-actin served as a loading control. EV: empty vector, FLAG-ORF: N-terminal FLAG-tagged ORF, ORF-FLAG: C-terminal FLAG-tagged ORF.