Author Affiliations: 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)

 

Corresponding Author: 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.

doi: 10.1016/S1499-3872(13)60009-0

 

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 first 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 benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

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 non-cancer 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 significantly 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 here-ditary 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)

 

KEY WORDS: human telomerase reverse transcriptase; single nucleotide polymorphism; hepatocarcinogenesis; hereditary susceptibility

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 indefinitely (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) have been 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 significantly increasing risk of lung cancer compared with the rs2735940C/rs2736098G haplotype.^[14] Savage et al^[7] however identified 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.

 

 

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 defined with no previous medical diagnosis or suspected diagnosis of HCC or other cancers. The diagnosis of HCC was confirmed pathologically or by two positive findings 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 defined as consuming an average of more than 2 drinks per day and for women, heavy drinking is typically defined 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 enzyme-linked 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]}

 

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 amplification was performed at annealing temperature of 65 �� in a PTC-200 thermal cycler (BioRad, USA). The fragment was amplified 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 fluorescence imaging system. Two amplification 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 purified using UNIQ-5 column PCR products purification 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.

 

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 significant differences in genotype and allelic frequency between the two groups were determined using the Chi-square test. Odds ratio (OR) and 95% confidence 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, and P<0.05 was considered statistically significant.^[20]

 

Demographic variables and laboratory parameters of the two groups are shown in Table 1. There were significant 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).

 

The genotype at rs2736098 was identified 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 significantly 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 the A allele was 0.41. Thus, A allele genotypic frequencies in the HCC patients were significantly different from those of the control group (P=0.040).

 

In the HCC patients, the genotype distribution was not significantly 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).

 

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 stratified 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. The association 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 significant (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 significant effect of rs2736098[A] on the onset of HCC in the patients and controls.

 

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 significantly 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 significantly 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 significantly associated with smoking and drinking among the NV/?S-A HCC patients (HCC patients without hepatitis virus infection).

 

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, significant association was not observed between GA genotype (OR=1.47; P=0.054) and HCC. In addition, family history of cancer was not significantly related to HCC.

 

 

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 specificity. 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 significantly 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 significant 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 find 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 inflammation 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 influence the results of our study.

 

This is the first study to investigate rs2736098 polymorphism with the HCC risk. Although independent studies with a large number of patients would be necessary to confirm 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.

 

 

1 Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006;6:674-687. PMID: 16929323

2 Molmenti EP, Klintmalm GB. Hepatocellular cancer in liver transplantation. J Hepatobiliary Pancreat Surg 2001;8:427-434. PMID: 11702252

3 El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557-2576. PMID: 17570226

4 Wang J, Ni H, Chen L, Liu YX, Chen CB, Song WQ. Preparation and analysis of cSNP chip on hepatocellular carcinoma-related genes. Hepatobiliary Pancreat Dis Int 2005;4:398-402. PMID: 16109524

5 Fabris C, Toniutto P, Falleti E, Fontanini E, Cussigh A, Bitetto D, et al. MTHFR C677T polymorphism and risk of HCC in patients with liver cirrhosis: role of male gender and alcohol consumption. Alcohol Clin Exp Res 2009;33:102-107. PMID: 18945219

6 Artandi SE, DePinho RA. Telomeres and telomerase in cancer. Carcinogenesis 2010;31:9-18. PMID: 19887512

7 Savage SA, Chanock SJ, Lissowska J, Brinton LA, Richesson D, Peplonska B, et al. Genetic variation in five genes important in telomere biology and risk for breast cancer. Br J Cancer 2007;97:832-836. PMID: 17848914

8 Ozturk M, Arslan-Ergul A, Bagislar S, Senturk S, Yuzugullu H. Senescence and immortality in hepatocellular carcinoma. Cancer Lett 2009;286:103-113. PMID: 19070423

9 Dharel N, Kato N, Muroyama R, Moriyama M, Shao RX, Kawabe T, et al. MDM2 promoter SNP309 is associated with the risk of hepatocellular carcinoma in patients with chronic hepatitis C. Clin Cancer Res 2006;12:4867-4871. PMID: 16914573

10 Tanabe KK, Lemoine A, Finkelstein DM, Kawasaki H, Fujii T, Chung RT, et al. Epidermal growth factor gene functional polymorphism and the risk of hepatocellular carcinoma in patients with cirrhosis. JAMA 2008;299:53-60. PMID: 18167406

11 Wang J, Ni H, Chen L, Song WQ. Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population. Hepatobiliary Pancreat Dis Int 2006;5:60-64. PMID: 16481285

12 Zhai Y, Zhou G, Deng G, Xie W, Dong X, Zhang X, et al. Estrogen receptor alpha polymorphisms associated with susceptibility to hepatocellular carcinoma in hepatitis B virus carriers. Gastroenterology 2006;130:2001-2009. PMID: 16762623

13 Rafnar T, Sulem P, Stacey SN, Geller F, Gudmundsson J, Sigurdsson A, et al. Sequence variants at the TERT-CLPTM1L locus associate with many cancer types. Nat Genet 2009;41: 221-227. PMID: 19151717

14 Choi JE, Kang HG, Jang JS, Choi YY, Kim MJ, Kim JS, et al. Polymorphisms in telomere maintenance genes and risk of lung cancer. Cancer Epidemiol Biomarkers Prev 2009;18: 2773-2781. PMID: 19773453

15 Bruix J, Sherman M; Practice Guidelines Committee, American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma. Hepatology 2005;42:1208-1236. PMID: 16250051

16 Department of Health and Human Services and the Department of Agriculture, Alcoholic Beverages. In: U.S. Department of Health and Human Services and U.S. Department of Agriculture. Dietary Guidelines for Americans, 2005. 6th Edition, Washington, DC: U.S. Government Printing Office, 2005:44. Available from: http://www.health.gov/dietaryguidelines/dga2005/document/

17 Eberl MM, Sunga AY, Farrell CD, Mahoney MC. Patients with a family history of cancer: identification and management. J Am Board Fam Pract 2005;18:211-217. PMID: 15879569

18 Akkiz H, Bayram S, Bekar A, Ozdil B, Akgöllü E, Sümbül AT, et al. G-308A TNF-alpha polymorphism is associated with an increased risk of hepatocellular carcinoma in the Turkish population: case-control study. Cancer Epidemiol 2009;33: 261-264. PMID: 19683483

19 Ezzikouri S, El Feydi AE, Afifi R, El Kihal L, Benazzouz M, Hassar M, et al. MDM2 SNP309T>G polymorphism and risk of hepatocellular carcinoma: a case-control analysis in a Moroccan population. Cancer Detect Prev 2009;32:380-385. PMID: 19233569

20 Qi P, Wang H, Chen YM, Sun XJ, Liu Y, Gao CF. No association of EGF 5'UTR variant A61G and hepatocellular carcinoma in Chinese patients with chronic hepatitis B virus infection. Pathology 2009;41:555-560. PMID: 19900104

21 Nakayama J, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998; 18:65-68. PMID: 9425903

22 Kyo S, Inoue M. Complex regulatory mechanisms of telomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene 2002;21:688-697. PMID: 11850797

23 Grochola LF, Greither T, Taubert HW, Möller P, Knippschild U, Udelnow A, et al. Prognostic relevance of hTERT mRNA expression in ductal adenocarcinoma of the pancreas. Neoplasia 2008;10:973-976. PMID: 18714398

24 Cairney CJ, Keith WN. Telomerase redefined: integrated regulation of hTR and hTERT for telomere maintenance and telomerase activity. Biochimie 2008;90:13-23. PMID: 17854971

25 Calado RT, Young NS. Telomere diseases. N Engl J Med 2009;361:2353-2365. PMID: 20007561

26 Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, et al. A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science 2007;315:525-528. PMID: 17185560

27 Duan J, Wainwright MS, Comeron JM, Saitou N, Sanders AR, Gelernter J, et al. Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Hum Mol Genet 2003;12:205-216. PMID: 12554675

28 Komar AA. Genetics. SNPs, silent but not invisible. Science 2007;315:466-467. PMID: 17185559

29 Varadi V, Brendle A, Grzybowska E, Johansson R, Enquist K, Butkiewicz D, et al. A functional promoter polymorphism in the TERT gene does not affect inherited susceptibility to breast cancer. Cancer Genet Cytogenet 2009;190:71-74. PMID: 19380022

30 Wang SS, Cozen W, Severson RK, Hartge P, Cerhan JR, Davis S, et al. Cyclin D1 splice variant and risk for non-Hodgkin lymphoma. Hum Genet 2006;120:297-300. PMID: 16783567

31 Bréchot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004;127:S56-61. PMID: 15508104

32 Koike K. Molecular basis of hepatitis C virus-associated hepatocarcinogenesis: lessons from animal model studies. Clin Gastroenterol Hepatol 2005;3:S132-135. PMID: 16234061

33 Henson JD, Neumann AA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene 2002;21:598-610. PMID: 11850785

34 Saini N, Srinivasan R, Chawla Y, Sharma S, Chakraborti A, Rajwanshi A. Telomerase activity, telomere length and human telomerase reverse transcriptase expression in hepatocellular carcinoma is independent of hepatitis virus status. Liver Int 2009;29:1162-1170. PMID: 19627485