A Pilot Two-stage Genome-wide Association Study Of Hepatocellular Carcinoma

A pilot two-stage genome-wide association study of hepatocellular carcinomaBackground:Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality. China is a region of high prevalence of hepatitis B virus (HBV) infection, and has a high incidence of HCC. Over the world, more than 50% new HCC cases come from China. The main aetiological factor is chronic hepatitis B virus (HBV), however, only a small fraction of the HBsAg positive populations may advance to HCC, indicating that genetic factors play important role in hepatocarcinogenesis.Methods:In this study, we collected 660 male samples with chronic HBV infection (HBsAg positive) from southeast region of China and did a pilot two-stage genome-wide association study (GWAS) of hepatocellular carcinoma by case-control design. The genotyping process of the fisrt stage was conducted in Affymetrix 500K genechip by scanning 50 HCC cases and 50 controls. We selected 1152 SNPs from the results of the first stage and did genotying in 282 cases and 278 controls with Illumina GoldenGate iselect platform. We used false positive report probability (FPRP) method to adjust the results. we further detected the expression of mRNA and protein of three genes (VEPH1, FZD4 and PCDH9) in HCC tissuses, tumor-adjacent tissuses and nomal liver tissues by quantitative Real-time PCR analysis (qRT-PCR) and immunohistochemistry. Results:In the first stage of GWAS, there were 357 SNPs with a miniP (decided from allele_P, genotype_P and Cochran-Armitage_P) less than 1×10-3. In the second stage, there were 26 SNPs whose allele P were less than 0.05. The combined analysis of association results from the first stage and the second stage showed there were eight SNPs (rs2120243, rs1350171, rs7116140, rs1048338, rs4480667, rs4417097, rs9893681 and rs4561519) passed the FPRP criteria (FPRP<0.20). The strongest statistical evidence for an association signal wss with rs2120243 (combined ORallele=1.76,95%CI:1.39-2.22, p=2.00×10-6), which maps within the fourth intron of VEPH1. The second strongest statistical evidence for an association was for rs1350171 (combined ORallele=1.66,95%CI:1.33-2.07, p=6.48×10-6), which maps to the downstream of the FZD4 gene. The third strongest statistical evidence for an association was for rs4480667 (combined ORallele=0.64,95%CI:0.51-0.81, p=1.42×10-4), which maps to the uptream of the PCDH9 gene. The other SNP related genes were PRMT6, LHX1 and KIF2B. qRT-PCR and immunohistochemistry showed the mRNA expression of VEPH1 and FZD4 was down-regulated in HCC tissues than in tumor-adjacent and nomal liver tissues. The protein levels of the VEPH1, FZD4 and PCDH9 all down-regulated in HCC tissues than in tumor-adjacent tissues and in nomal liver tissues.Conclusion:This pilot two-stage GWAS (332 case and 328 controls) and gene expression study found some susceptibility loci for HCC. GO classification and protein network analysis showed the related genes involved in TGFB, PI3K/Insulin, Wnt and EGFR signal pathway. The association need replication and validation in lager samples with diverse genetic backgrounds and the related gene functions need to be studied further.Part one. The first stage of GWASIn the first stage, we genotyped 500447 (single nucleotide polymorphisms) SNPs in 50 HBsAg sera-positive male HCC cases and 50 HBsAg sera-positive male controls using Affymetrix genechip 500k array set.The aim of the first stage was to select promising SNPs to do the replication and validation research in the second stage.The samples in this study were all male Chineses Han collected from QiDong City, JiangSu Province. There were 357 SNPs with a miniP (decided from allele_P, genotype_P and Cochran-Armitage trend_P) less than 1×10-3, of which 26 SNPs had a miniP less than 1×10-4。Part two. The second stage of the association study using Illumina GoldenGate PlatformWe selected 1152 SNPs (Supplementary data 3) from the results of stage1 for genotyping in Illumina goldenGate platform in 560 samples in stage2. Samples included 282 HCC patients and 278 controls all came from the southeastern region and were all male Chineses Han with HBsAg sera-positive.Of the selected 1152 SNPs, there were 643 SNPs passed quality filter (MAF> 0.01 and HWE disequilibrium P> 0.001, call frequency> 0.95). Of which, there were 26 SNPs whose unadjusted allele P< 0.05. Combined analysis showed there were eight SNPs (rs2120243, rs1350171, rs7116140, rs1048338, rs4480667, rs4417097, rs9893681 and rs4561519) with combined allele P<0.05 and FPRP<0.20. The six genes adjoint to the eight SNPs were VEPH1, FZD4, PCDH9, PRMT6, LHX1 and KIF2B.Part three. The study of gene expressions and gene annotation qRT-PCR was employed to measure mRNA levels of VEPH1 and FZD4 for 11 HCC tissues, tumor-adjacent tissues and two nomal liver tissues. Immunohistochemistry was employed to detect VEPH1, FZD4 and PCDH9 protein levels in 22 HCC tissues, tumor-adjacent tissues and 11 normal liver tissues.The experimemt showed the mRNA and protein levels of VEPH1 and FZD4 both down-regulated in HCC tissuese than in tumor-adjacent tissuese and nomal liver tissues(the detection of PCDH9 mRNA is under way). The protein levels of the VEPH1, FZD4 and PCDH9 proteins were down-regulated in in HCC tissuese than in tumor-adjacent tissuese and nomal liver tissues. We explored the bioinformatics of the six related genes of the eight SNPs and found that most of genes were involved in carcinogenesis. The related SNPs locates in the gene intron, or upstream or downstream or is a deletious nonsynonymous SNP. These SNPs may be involved in carcinogenesis by altering genes'expressions or impairing the protein's function. These hypotheses metrit further studies.Part four. Gene-gene and gene-environment interactions analysis We used multifactor dimensionality reduction (MDR)and multivariate logit regress to explore gene-gene and gene-environment interactions and to explore the risk factors of HCC. We collected six SNPs (rs2120243, rs4480667, rs1350171, rs4417097, rs9893681, rs4561519) genotype data of 560 samples of the second stage to analyze the gene-gene interactions by using MDR. The results showed that rs4480667, rs2120243 and rs1350171 had gene-gene interactions.The combined genotype classification made a distinction between cases and controls, indicating these SNPs might become the diagnostic biomarkers of HCC.The multivariate logit regression analysis including six SNPs (rs2120243, rs4480667,rs1350171, rs4417097, rs9893681,rs4561519)and age, family HCC history, smoking and alcohol drinking showed family history, rs4480667, rs2120243, rs1350171, rs4561519 and rs9893681 were independent risk factors of HCC and related to HCC occurrence.Part five. Gene classification and protein network configurationIn order to explore the HCC related genes and the interactions of the genes, we collected all SNPs whose combined allele_P or allele_P of the second stage were less than 0.05. Then, we searched the adjacent genes in NCBI MAP and collected 71 genes. We then added the 71 genes to do gene classification analysis in MAS 3.0 and DAVID server. The results showed the genes involved in forebrain development, cell migration, cell proliferation, alternative splicing, signal transductor, member protein and cell adhesion were enriched compared to the human genome. STRING analysis found a lot of crutial nodes in the network:FYN, EGFR, SRC, PTK2B, BDNF, IL2, PRKCQ and CCND1, etc., of which many genes were related to cancer.Conclusion In conclusion, by using a two stage GWAS strategy and mining the data of bioinformatics, we have found some potential susceptibility loci for HCC in male Han Chinese with HBsAg seropositive. The experiment verified that three genes (VEPH1, FZD4 and PCDH9) were diffenrtly expressed in HCC and tumor-adjacent tissues and nomal liver tissues. Gene classification and protein network showed some potential susceptibility genes for HCC. The association and gene function should be validated in larger studies.The novelty of the studyThe study first used GWAS stratige to scan the susceptible genes of HCC in 660 suscetable male populations with HBsAg sero-positive in a case-control design. The study found several potential HCC related genes. Potential merits for clinical application The searching and validation of HCC related genes is a major task of the study on hepatocarcinogenesis. The validated HCC related genes may be used in HCC risk prediction and as gene therapy target, so it may have some important clinical values.