hTERT rs2736098 genetic variants and susceptibility of hepatocellular carcinoma in the Chinese population: a case-control study

Tianjin, China

hTERT rs2736098 genetic variants and susceptibility of hepatocellular carcinoma in the Chinese population: a case-control study

Chao Zhang, Ya-Ping Tian, Yue Wang, Feng-Hua Guo, Jun-Fang Qin and Hong Ni

Tianjin, China

BACKGROUND:The human telomerase reverse transcriptase (hTERT) gene encodes the catalytic subunit of telomerase, which mediates pleiotropic effects, including the regulation of senescence and proliferation and plays an important role in carcinogenesis. This study attempts to clarify the genetic predisposition to hepatocellular carcinoma (HCC), focusing on the hTERT gene rs2736098 polymorphism.

METHOD:Four hundred patients with HCC and 400 noncancer controls were genotyped to elucidate the potential association between hTERT rs2736098 polymorphism and HCC risks.

RESULTS:Compared with the controls, the patients with HCC had a lower frequency of G/G genotype (33.3% vs 44.3%,P=0.001) and a higher frequency of G/A (51.5% vs 39.5%,P=0.001). Allele genotypic frequencies in the patients differed from those of the controls (P=0.040). The data of this study rs2736098[A] allele contributed signif i cantly to HCC risk in female patients (OR=1.78, 95% CI, 1.17-2.72,P=0.007), patients with HCV infection (OR=2.89, 95% CI, 1.08-7.70,P=0.031), non-drinker patients (OR=1.32, 95% CI, 1.06-1.65,P=0.015), and patients not affected by HBV (OR=1.77, 95% CI, 1.30-2.40,P<0.001).

CONCLUSIONS:rs2736098[A] may be an independent hereditary parameter in HCC, but some risk factors would cover up the association by more powerful hepatocarcinogenesis. These results are important guidance for further studies in detecting HCC-associated single nucleotide polymorphisms.

(Hepatobiliary Pancreat Dis Int 2013;12:74-79)

human telomerase reverse transcriptase; single nucleotide polymorphism; hepatocarcinogenesis; hereditary susceptibility

Introduction

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide and represents more than 5% of all cancers.[1]There are 350 000 new liver cancer patients in China every year, representing nearly half of all new cases worldwide.[2]Hepatitis B and C virus infection and abuse of alcohol as oncogenic factors have been recognized as genetic factors to alter the susceptibility to HCC.[3-5]

Telomeres are nucleoprotein structures that protect the end of the chromosome from deterioration or from fusion with neighboring chromosomes.[6]Telomeres shorten in part because of the end replication problem that is exhibited during DNA replication in eukaryotes only, and eventually reach a critical state, at which cellular senescence and/or apoptosis is normally triggered.[7]As a cell begins to become cancerous, it would require not only the bypass of senescence, but also a way that can prevent telomeres from getting shorter and even elongate them. Additionally, cancer cells require a mechanism to maintain their telomeric DNA at a minimal length to maintain intact chromosomal structures in order to continue dividing inde fi nitely (immortalization).[8]In most advanced cancers, telomerase is reactivated and serves to maintain telomere length, and emerging data have also documented the capacity of telomerase directly to regulate cancer-promoting pathways.[6]Telomerase activity is regarded as a key event in 80%-90% of HCCs.[8]

The analysis of genetic risk to HCC may be helpful to understand the occurrence of hepatocarcinoma and develop the effective methods for early intervention. Several single nucleotide polymorphisms (SNPs) havebeen found to play important roles in HCC.[9-12]A study[13]of Icelandic and European sample sets showed that the carriers of rs2736098[A] allele were at a high susceptibility of lung cancer, bladder cancer and prostate cancer. The human telomerase reverse transcriptase (hTERT) rs2735940[T]/rs2736098[A] haplotype was associated with a signif i cantly increasing risk of lung cancer compared with the rs2735940C/rs2736098G haplotype.[14]Savage et al[7]however identif i ed that rs2736098 (Ex2-659G>A) may be associated with a lower risk of breast cancer among individuals with a family history of breast cancer. As yet, genetic screens of large populations for hTERT gene rs2736098 polymorphism associated with increasing susceptibility to HCC have not been conducted. The current study investigated the relationship between hTERT rs2736098 genetic variants and risk of HCC by PCR-RFLP genotype analysis technique.

Methods

Study population

The study was approved by the institutional ethics committee of Nankai University. Informed consent was obtained from all patients. Blood samples were taken from 400 Han Chinese patients with HCC at the General Hospital of PLA from September 2008 to October 2010. At the same time, blood samples were taken from 400 non-cancer patients with the same age, gender and ethnicity as the HCC patients who were recruited as controls. The non-cancer patients were def i ned with no previous medical diagnosis or suspected diagnosis of HCC or other cancers. The diagnosis of HCC was conf i rmed pathologically or by two positive fi ndings of diagnostic imaging, including CT, ultrasonography, and angiography.[15]The following oncogenic factors of HCC were investigated in the form of questionnaire interview by well-trained researchers, that included heavy alcohol intake (For men, heavy drinking is typically def i ned as consuming an average of more than 2 drinks per day and for women, heavy drinking is typically def i ned as consuming an average of more than 1 drink per day),[16]smoking status (current smoking or no-smoking) and family history of cancer (2 or more relatives on the same side of the family with the same or a related cancer, earlier than 50 years of age, and the presence of more than 1 primary cancer in a family member).[17]Anti-HCV was determined by third-generation enzymelinked immunosorbent assay (ELISA). Serum HBsAg was checked using ELISA. Samples of peripheral blood were frozen at -20 ℃ until use. All samples were processed by technicians blinded to the identity of samples.[18,19]

Genotyping of polymorphisms

DNA extracts from peripheral blood were analyzed with the Qiagen DNA Blood Kit (Qiagen, Germany). The DNA was dissolved in ddH2O and frozen at -20 ℃ until analysis. 659G>A polymorphism (rs2736098) in the hTERT gene was genotyped by PCR and RFLP analysis. A step-down amplif i cation was performed at annealing temperature of 65 ℃ in a PTC-200 thermal cycler (BioRad, USA). The fragment was amplif i ed in a reaction volume of 25 µL with 25 pmol of each primer, 100 ng of patient DNA, 1.5 mmol/L MgCl2, 200 mmol/L total dNTP, 1 × PCR buffer, and 2.5 U Taq DNA polymerase. The polymerase chain reaction was performed with a set of primers: forward 5′-GCC AGA CCC GCC GAA GAA G-3′ and reverse 5′-GCG CGT GGT TCC CAA GCA G-3′). Two µL of 379-bp PCR product was digested for 6 hours at 37 ℃ in 2 µL of 10× digestion buffer containing 1 unit of Psp OMI (New England Biolabs, Schwalbach, Germany). Digestion products were loaded on 8% polyacrylamide gel stained with ethidium bromide and observed on an ultraviolet fl uorescence imaging system. Two amplif i cation products (289bp and 90bp) were generated from the wild-type allele, whereas the G>A polymorphism allele generates only one fragment with the size of 379bp. We selected randomly 500 blood samples to identify the PCR-RFLP restriction patterns by sequence. PCR products were purif i ed using UNIQ-5 column PCR products purif i cation kit (Sangon Bio Co., Shanghai, China) and sequencing was performed with an applied biosystems automatic sequencer (ABI3730XL) at Sangon Sequencing Center (Shanghai, China). The sequencing results were 100% concordant with genotyping of PCR-RFLP.

Statistical analysis

Analyses were performed using commercially available software (SPSS, version 11.0; SPSS Inc., Chicago, Illinois, USA). The Chi-square test was used for deviation from the Hardy-Weinberg equilibrium. The signif i cant differences in genotype and allelic frequency between the two groups were determined using the Chi-square test. Odds ratio (OR) and 95% conf i dence interval (CI) were calculated to provide an estimate of the statistical association between different genotypes. Demographic variables (smoking status, alcohol use, family history of cancer), laboratory markers of hepatitis (HBsAg and HCV targeted antibodies) and genotypes of rs2736098 were examined in a multiple logistic regression model to assess the association with HCC susceptibility. All statistical tests were two-sided, andP<0.05 was considered statistically signif i cant.[20]

Results

Population characteristics

Demographic variables and laboratory parameters of the two groups are shown in Table 1. There were signif i cant differences between patients of HCC and non-cancer controls in demographic variables such as drinking or smoking status, family history of cancer, and markers of hepatitis (HBsAg and HCV targeted antibodies).

Allele and genotype distribution of rs2736098

The genotype at rs2736098 was identif i ed in the non-cancer and HCC groups. The allele and genotype distributions are shown in Table 2. Genotype distributions at rs2736098 conformed to the Hardy-Weinberg equilibrium in all groups.

Compared with the control group, the HCC patients had a lower frequency of G/G genotype (33.3% vs 44.3%,P=0.001) and a higher frequency of G/A (51.5% vs 39.5%,P=0.001). Homozygous A/A genotype frequency was not signif i cantly different between the two groups (15.3% vs 16.3%,P=0.698). The frequency of the G allele in the control group was estimated to be 0.64 while the variant A allele was 0.36. In the HCC patients, the frequency of the G allele was 0.59 while theA allele was 0.41. Thus, A allele genotypic frequencies in the HCC patients were signif i cantly different from those of the control group (P=0.040).

Table 1.Characteristics of HCC patients and controls

Table 2.Allele and genotype distribution of rs2736098 in HCC patients and controls

In the HCC patients, the genotype distribution was not signif i cantly different from that expected for a population in the Hardy-Weinberg equilibrium (χ2=1.664, df=1,P=0.197). But the genotype distribution of the control group was not in the Hardy-Weinbery equilibrium (χ2=8.156, df=1,P=0.004).

Association of rs2736098[A] with HCC adjusted by risk factors

HCC is known for its genetic heterogeneity. Thus, we examined the effect of the rs2736098[A] on different subclasses of the HCC patients. The hTERT rs2736098 genotype distributions in the HCC patients and controls were stratif i ed by age, gender, smoking status, alcohol use, family history of cancer and HBV or HCV infection (Table 3).

Subgroup analysis revealed that the effect of rs2736098[A] was higher in women. Female patients with rs2736098[A] had an odds ratio of 1.78 (95% CI: 1.17-2.72,P=0.007). The HCC patients infected by HCV with rs2736098[A] allele had a 2.89-fold (95% CI: 1.08-7.70,P=0.031) risk than the controls. Theassociation was observed between rs2736098A] and HCC with a OR of 1.32 (95% CI: 1.06-1.65,P=0.015) for the non-drinkers. A similar association was also observed in non-smokers although it was not signif i cant (OR=1.28, 95% CI: 0.99-1.64,P=0.057). rs2736098[A] allele carriers in patients without HBV infection had a 1.77-fold (95% CI: 1.30-2.40,P<0.001) susceptibility in the HCC patients than in the controls. There was no signif i cant effect of rs2736098[A] on the onset of HCC in the patients and controls.

Table 3.Association of rs2736098[A] with HCC adjusted by risk factors

Table 4.Association of rs2736098 genotype with risk factors in HCC patients

The association of rs2736098 genotype with risk factors (chronic hepatitis virus, smoking, and alcohol consumption) was analyzed in HCC patients (Table 4). The rate of AA genotype in HCV/NS-NA HCC patients (HCC patients with HCV infection and without smoking and alcohol consumption,P=0.041) was signif i cantly higher than that in NV-NS-NA HCC patients (HCC patients without hepatitis virus infection and without smoking and alcohol consumption). However, the frequency of patients with GG genotype among the HBV/NS-NA HCC patients (HCC patients with hepatitis B virus infection, and without smoking and alcoholic consumption,P<0.001) was signif i cantly higher than that of the NV-NS-NA HCC patients. The association of rs2736098 genotype with smoking and drinking among the HCC patients without hepatitis virus infection was also analyzed. rs2736098 genotype was not signif i cantly associated with smoking and drinking among the NV/ S-A HCC patients (HCC patients without hepatitis virus infection).

Multivariate analysis of risk factors for HCC

Logistic regression analysis was performed to assess such variables as rs2736098 genotype, smoking and alcohol consumption, HBsAg and HCV targeted antibodies, family history of cancer. Multivariate analysis indicated that rs2736098 AA genotype (OR=1.74;P=0.048), smoking (OR=1.90;P=0.005), alcohol consumption (OR=2.16;P=0.008), HBsAg positivity (OR=17.85;P<0.001), and anti-HCV positivity (OR=10.66;P<0.001) were independent risk factors for HCC (Table 5). However, signif i cant association was not observed between GA genotype (OR=1.47;P=0.054)and HCC. In addition, family history of cancer was not signif i cantly related to HCC.

Table 5.Multivariate analysis of risk factors for HCC

Discussion

Telomerase activity is undetectable in most normal adult human somatic cells, but in about 85% of human malignant tumors and immortalized cell lines, telomerase is highly activated.[21]The activation of hTERT gene transcription is a dominant, rate-limiting step in telomerase activation.[22]hTERT is strongly expressed in most HCCs, and is the key component regulating telomerase activity in human liver.[23]hTERT plays a critical role in the regulation of telomere biological effects by activating telomerase and thus contributes to the development of malignancy.[6,8,24,25]

The hTERT gene has limited nucleotide diversity in humans, and rs2736098 is a kind of synonymous SNPs. However, synonymous variants may also affect the function of the gene. Kimchi-Sarfaty et al[26]found that silent polymorphisms particularly C3435T in MDR1 could alter P-glycoprotein conformation and protein activity/substrate specif i city. They concluded that silent SNPs may affect protein translation, protein folding and activity, and silent SNPs which do not change the coding sequence of the protein might contribute to the altered pharmacokinetics and disease. Not only the silent SNPs can lead to changes in protein folding and function, but also can alter mRNA stability. Duan et al[27]found that C957T in human DRD2 could alter the predicted mRNA folding and lead to a decrease in mRNA stability and translation, thus changing dopamine-induced up-regulation of DRD2 expression. Synonymous SNPs could bring up transcriptional changes of mRNA and alter the levels of protein expression.[28]Our data indicate that rs2736098[A] synonymous polymorphism increases the susceptibility to HCC. The rs2736098 polymorphism may be susceptible to other cancers as the genotype distribution in the controls differed from that expected from the Hardy-Weinberg equilibrium.

Controversy results that rs2736098[A] allele may contribute to the susceptibility to cancer. The rs2736098[A] allele was a high risk factor for lung, bladder and prostate cancer among Icelandic and European sample sets.[13]The hTERT rs2735940[T]/rs2736098[A] haplotype was associated with a signif i cantly increased risk of lung cancer compared with the rs2735940[C]/rs2736098[G] haplotype.[14]However, Savage et al[7]stated that rs2736098 may be associated with reduced risk of breast cancer in subjects with a family history of breast cancer. Some studies[29,30]found no signif i cant association of rs2736098 with familial or sporadic breast cancer risk as well as with the risk for non-Hodgkin lymphoma. Although it is hard to explain controversy conclusions in different studies, different genetic backgrounds in the study populations and cancers or interactive effects of hereditary factors and environmental factors might contribute to the discrepancy.[14]

We speculated that hereditary factors would increase the risk of HCC in the study population together with such environmental factors as HBV, HCV, cigarette smoking and alcohol consumption. Unexpectedly, we did not fi nd the joint effect of rs2736098[A] polymorphism with HBV, smoking or heavy drinking on the risk of HCC in the present study (Table 3). It is one of the reasons why these environmental factors have more powerful hepatocarcinogenesis than rs2736098[A]. rs2736098[A] increased the risk of HCC only in women because of their less exposure to alcohol intake and heavy smoking.

An important factor is the integration of HBV DNA and hepatocyte chromosomal DNA, which leads to rearrangement and instability of the host DNA. In addition, chronic inf l ammation and expression of HBV proteins may have a direct effect on cellular functions, and some of these can favor malignant transformation. A recent study showed that the telomerase-encoding gene (hTERT) was targeted by HBV DNA integration in several independent HCCs and in HCC-derived cell lines.[31]The involvement of HCV in hepatocarcinogenesis can be explained by involvement of the HCV core protein, leading to the overproduction of oxidative stress, which may yield changes of cellular gene expression, intracellular signal transduction, and even genetic aberration. Therefore, unlike other carcinogenesis, HCV infection can elicit HCC in the absence of the multiple steps of genetic aberration in hepatocytes.[32]The different process of hepatitis virus for the induction of HCC is considered, and thus a plausible explanation might be given for the correlation of rs2736098[A] with HCC in our study. The susceptibility to HCC with a single nucleotide polymorphism (minor alterations in the genetic code) may be covered up by integration of large genomic HBV DNA fragments, but it would enhance the carcinogenesis of HCV core protein. Immortalized mammalian cell lines and tumors may maintain or increase the overall length of their telomeres in the absence of telomerase activity by one or more mechanisms such as alternative lengthening of telomeres.[33]Saini et al[34]found the telomerase activity of HBsAg-positive HCC patients was less likely to express telomerase (70.6%) than non-HBV and non-HCV HCC patients (91.7%). Possibly, HBV HCC patients are suspected to have alternative lengthening of telomeres mechanisms for telomere maintenance of HCC.

However, a number of limitations must be considered. Firstly, our analytic approach selected only one SNP site related to hTERT. Secondly, related functional assay was absent in our study, which leads to limited discussion on biological mechanism of the observed associations between rs2736098 polymorphism and liver cancer risk. Thirdly, bias of case selection and information might inf l uence the results of our study.

This is the fi rst study to investigate rs2736098 polymorphism with the HCC risk. Although independent studies with a large number of patients would be necessary to conf i rm the relationship between rs2736098 polymorphism and HCC risk, our data present an interesting result that rs2736098[A] may be an independent hereditary parameter in HCC, but oncogenic factors such as HBV, cigarette smoke and alcohol consumption would cover it up by more powerful hepatocarcinogenesis. Our study would guide further studies on the detection of HCC-associated single nucleotide polymorphisms.

Acknowledgements:We thank Drs. Ying Xing, Li Liang and Yang Mu for their assistance in recruiting the subjects and Ying Hu, Tian-Hao Dong, and Bei-Lei Ma for their laboratory assistance.

Contributors:NH proposed the study. ZC and NH performed research and wrote the fi rst draft. ZC collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. NH is the guarantor.

Funding:This study was supported by a grant from the Natural Science Funds of Nankai University (06-h83)

Ethical approval:The study was approved by the institutional ethics committee of Nankai University.

Competing interest:No benef i ts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received September 26, 2011

Accepted after revision July 12, 2012

AuthorAff i liations:Tianjin Medical University General Hospital, Tianjin 300071, China (Zhang C); Department of Clinical Biochemistry, General Hospital of PLA, Beijing 100853, China (Tian YP); Nankai University College of Medicine, Tianjin 300071, China (Wang Y, Guo FH, Qin JF and Ni H)

Hong Ni, MD, Departmeat of Pathophysiology, Nankai University College of Medicine, Tianjin 300071, China (Tel: 86-22-23507728; Fax: 86-22-23502554; Email: hongni@nankai.edu.cn)

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(13)60009-0