Association Of The GLB1 Rs4678680 Genetic Variant With Risk Of HBV-related Hepatocellular Carcinoma

Objective:Although ranking the fifth most common cancer,hepatocellular carcinoma(HCC)is one of the most dangerous diseases,accounts for thirdly in cancer-related mortality worldwide.There is a high incidence of HCC in China and other Asia-Pacific region.Particularly in China,it accounts for about 50%of the world's entire HCC cases.There were several epidemiological features,such as marked variations between geographical regions,racial and ethnic groups,and sex.It has been revealed that men have a higher prevalence of HCC than women,i.e.the male:female ratio of HCC patients is-2.65:1 in high-risk Chinese populations.Major environmental risk factors of HCC includes chronic infections with the hepatitis B or C viruses(HBV or HCV),excessive alcohol drinking and exposure to dietary aflatoxin B.Since about 75%cases of HCC are associated with HBV infection in China,HBV infection is considered as one major risk factor for HCC.However,only 15%chronic HBV carriers suffer from HCC in their lifetime,indicating that genetic factors of the host may also be connected with the development of HBV-related HCC.Both several candidate gene and genome-wide association study(GWAS)on HBV-related HCC have proved this notion in eastern Asians.To identify genetic factors associated with HCC,Clifford et al.conducted a comprehensive,genome-wide variation analysis(both genetic polymorphisms and copy number variations)in a population of unrelated Asian individuals.After genotyping 386 Korean HCC cases and 587 Korean controls with the Affymetrix SNP6.0 assay,they found that the 3p21.33 GLB1 rs4678680 G>T polymorphism is most significantly associated with HCC risk(odds ratio[OR]= 2.27,95%confidence intervals[CI]= 1.68-3.08,P = 6 × 10-7).GLB1 encodes a member of the glycosyl hydrolase 35 family of proteins,which catalyzes the hydrolysis of a terminal betalinked galactose residue from ganglioside substrates and other glycoconjugates.Accumulated evidences demonstrated that GLB1 is involved in cell senescence and cancer development.Senescence phenotype could be induced by increased SA-b-galactosidase staining and elevated GLB1 mRNA expression,indicating the important role of GLB1 in cell senescence.During the long latency of tumor development,oncogene-induced senescence has emerged as a barrier to tumorigenesis including HCC.Chen et al.conducted a replication case-control study on the association of 1p36.22,2q32.2-q32.3,3p21.33,8p12,14q32.11 and 21q21.3 with HCC among 507 Chinese HCC patients and 3014 Chinese controls.However,they did not included GLB1 rs4678680 G>T single nucleotide polymorphism(SNP)in the study.As a result,it is still largely unclear if GLB1 rs4678680 polymorphism plays a part in etiology of HCC in Chinese.Also,little or nothing has been known about functional significance of the GLB1 rs4678680 SNP in HBV-related HCC.Methods:Therefore,in order to examine the connection between the GLB1 rs4678680 genetic polymorphism and risk for developing HBV-related HCC,we conducted two large independent case-control studies.We investigated the association in GLB1 rs4678680 genotypes and its mRNA expression levels to validate the biological function of the rs4678680 SNP in vivo in normal liver tissues.This study consisted of two case-control sets(Supplementary Table S1):(a)Shandong set:1186 individuals with HBV-related HCC,sex-and age-matched(±5 years)508 chronic HBV carriers were recruited at Shandong Cancer Hospital affiliated to Shandong University,Shandong Academy of Medical Sciences(Jinan,Shandong Province,China),(b)Jiangsu set:620 HBV-related HCC individuals from Huaian No.2 Hospital(Huaian,Jiangsu Province,China)and sex-and age-matched 1200 chronic HBV carriers as controls.Cases and controls were recruited at Huaian No.2 Hospital.The case-control sets has been reported previously[17].A total of 56 pairs of HCC tissue specimens from 56 HCC individuals recruited in this study.All HCC individuals received curative resection in Huaian No.2 Hospital or Qianfoshan Hospital,Shandong University.Prior to the surgery,no HCC individuals received any local or systemic anticancer treatments.All subjects of the study were the Han Chinese.The informed consent has been obtained from each subject at recruitment.The institutional Review Boards of Shandong Cancer Hospital,Qianfoshan Hospital and Huaian No.2 Hospital have approved this study.We used the MassArray system(Sequenom Inc.,San Diego,California,USA)to analyze the GLB1 rs4678680 polymorphism.GLB1 rs4678680 PCR primers are 5'-ACGTTGGATGAGTCCAAGCCTGCTTTCTTC-3'(Forward)and 5'-ACGTTGGATGTCTGCCGAGTTGT TGCAAAG-3'(Reverse).GLB1 rs4678680 UEP_SEQ primer is 5'-cctcaTGCTTTCTTCCCTTTTCT-3'.GLB1 rs4678680 EXT1_SEQ primer is 5'-cctcaTGCTTTCT TCCCTTTTCTG-3'.GLB1 rs4678680 EXT2_SEQ primer is 5'-cctcaTGCTTTCTTCCCTTTTCTT-3'.A 15%random,blind random sample of study subjects was genotyped in duplicates and the reproducibility was 100%.We examined GLB1 mRNA levels by SYBR-Green real-time quantity PCR method in normal liver tissues as described previously[18-20].We used the ReverTra Ace qPCR RT Kit(TOYOBO)to isolated and converted total cellular RNA to cDNA.At the same time,we used the ABI 7500 real-time PCR system in triplicates to carry out the relative gene expression quantitation for GLB1 and ?-actin as an internal reference gene.The primers of GLB1 were 5'-GTCTATTCTTCTCCGCTCCT-3' and 5'-TGTGCTCCATCAGTGGTAA-3';and the primers of ?-actin were 5'-GGCGGCACCACCATGTACCCT-3' and 5'-AGGGGCCGGACTCGTCATACT-3'.The expression of individual GLB1 mRNA measurements was measured relative to expression of ?-actin mRNA using the method as described previously.We via Pearson's ?2 test to examine the differences between genotype distributions of the GLB1 rs4678680 SNP and demographic variables in cases and controls.By logistic regression models,we estimated the associations in HBV-related HCC risk and genotypes of GLB1 rs4678680 by ORs and their 95%CIs computed.it was appropriate that all ORs were adjusted for age,sex,smoking or drinking status.We calculated GLB1 mRNA expression differences in different rs4678680 genotype carriers by Kruskal-Wallis one-way analysis of variance tests.The criterion of statistical significance was a P value of less than 0.05,and all data analysis tests were two-sided.We used SPSS 16.0(SPSS Inc.)to perform all analyses.Results:Allele frequencies and genotype distributions of GLB1 rs4678680 SNP in cases and controls from the Shandong and Jiangsu sets are showed in Table 1.The allele frequencies for rs4678680 G were 0.087 or 0.094 in cases and 0.059 or 0.058 in controls in Shandong or Jiangsu case-control set.In both cases and controls,all observed genotype frequencies conform to Hardy-Weinberg equilibrium.And we compared distributions of the rs4678680 genotypes in HCC cases and controls.Frequencies of rs4678680 TT,TG and GG genotypes among HCC cases differed significantly from those among controls in either Shandong set(?2 = 7.593,P = 0.022,df= 2)or Jiangsu set(?~2 = 16.14,P = 3.12 × 10-4,df= 2).We examined associations between the GLB1 rs4678680 SNP and HBV-related HCC risk in Shandong and Jiangsu sets(Table 1)by unconditional logistic regression analysis.We found the rs4678680 G allele was risk allele,and individuals with the TG genotype had an OR of 1.51(95%CI = 1.10-2.07,P = 0.010,Shandong set)or 1.49(95%CI= 1.11-1.99,P = 0.008,Jiangsu set)for developing HBV-related HCCcompared with individuals with the TT genotype respectively(Table 1).It was also found that carriers of the rs4678680 TG or GG genotype showed significantly and consistently increased risk to develop HBV-related HCC compared with the TT carriers in both case-control sets(Shandong set;OR = 1.53,95%CI = 1.12-2.10,P =0.007;Jiangsu set:OR = 1.57,95%CI = 1.18-2.09,P = 0.002).we observed that the odds of having the rs4678680 TG genotype in cases was 1.56(95%CI = 1.24-1.97,P=1.76 × 10-4)compared with the TT genotype in the pooled analyses.Similarly,the rs4678680 TG or GG genotype carriers showed a 1.52-fold increased HCC risk compared with the TT genotype carriers(95%CI = 1.19-1.94,P = 0.001).We further examined the risk of HBV-related HCC associated with the GLB1 rs4678680 genotypes by stratifying for sex(Table 2).A significantly increased risk of HCC associated with the rs4678680 TG or GG genotype compared with the TT genotype was observed in males(Shandong set:OR =1.60,95%Cl = 1.15-2.23;P =0.006;Jiangsu set:OR = 1,59,95%CI = 1.17-2.16;P=0.003).However,this genetic polymorphism was not significantly associated with HCC risk in females(all P>0.05).In the stratification analyses with age,elevated risk of HCC associated with the GLB1 rs4678680 TG or GG genotype was only observed among individuals aged older than 57 years(Shandong set:OR = 1.63,95%CI = 1.01-2.62;P = 0.047),but not among individuals aged 57 years or younger(Shandong set:OR = 1.46,95%CI =0.96-2.21;F = 0.076)(Table 3),Similar results were observed among individuals aged older than 57 years in Jiangsu set(rs4678680 TG or GG genotype:OR-2.10,95%CI = 1.37-3.21;P = 0.001).Interestingly,the rs4678680 TG or GG genotype was also significantly associated with increased HCC risk in drinkers compared to the TT genotype in both sets(Shandong:OR = 1.90,95%CI = 1.26-2.86,P = 0.002;Jiangsu:OR = 1.76,95%CI = 1.21-2.57,P = 0.003)(Table 4).However,no such associations was observed among non-drinkers(all P>0.05).We next examined whether the HCC susceptibility SNP rs4678680 has an allele-specific impact on GLB1 expression using HCC tissues since it locates in a 18 kb upstream region of GLB1.As shown in Figure 1,we found that there were significantly higher GLB1 mRNA levels(mean ± SD)in HCC tissues compared to normal tissues(313.3 ± 33.93 vs.189.9 ± 21.1;P = 0.003).Subjects with the rs4678680 TG or GG genotype had significantly higher GLB1 mRNA levels than those with the TT genotypes in normal tissues(TG or GG:367.5 ± 40.2[n = 9],TT:155.9 ± 20.6[n = 47];P=0.001).Similar results were observed when the GLB1 mRNA levels were compared between rs4678680 TG or GG and TT genotypes in HCC tissues(TG or GG:590.7 ± 32.2[n=9],TT:260.1 ± 35.0[n=47];P=0.001)(Figure 1).Conclusions:In the current study,we examined the association between the GLB1 rs4678680 SNP and risk of developing HBV-related HCC in a case-control design.Although the genetic predisposition to HCC of the GLB1 rs4678680 polymorphism was firstly identified in Korean populations via GWAS,this is still the first validation study with relative large sample size in different ethnic populations.We found significantly increased HCC risk among individuals with the GLB1 rs4678680 TG or GG genotype compared with those with TT genotype in Chinese.In the genotype-phenotype correlation analyses of fifty-six human liver tissue samples,rs4678680 TG or GG was associated with a statistically significant increase of GLB1 mRNA expression.The GLB1 gene provides instructions for producing an enzyme called P-galactosidase.This enzyme is located in lysosomes,which are compartments within cells that break down and recycle different types of molecules.Within lysosomes,GLB1 helps break down certain molecules,including substances called GM1 ganglioside and keratan sulfate.Caldwell et al.found that GLB1 activity is the only biomarker that accurately identifies a small and heterogeneous population of non-proliferating premalignant cells in the pancreas,indicating the utility of GLB1 to predict the senescent state in pancreatic preneoplasia.Additionally,increased GLB1 is a valuable marker in formalin-fixed paraffin-embedded tissues for the senescence-like phenotype and associates with improved prostate cancer outcomes.All these evidences support the involvement of GLB1 in carcinogenesis,possibly through regulating cell senescence.Significance:our study elucidated that the GLB1 rs4678680 polymorphism was associated with risk of HBV-related HCC in Chinese populations,highlighting the involvement of GLB1 and cell senescence in etiology of HCC.