Association Between the Telomerase rs2736098_TT Genotype and a Lower Risk of Chronic Hepatitis B and Cirrhosis in Chinese Males

Close to 400 million individuals are chronically infected with hepatitis B virus (HBV) worldwide and approximately one million of them die from HBV-related liver diseases including chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) annually.1 The accumulated evidence has indicated the host-genetic background of susceptibility to HBV infection and CHB. Indeed, early epidemiological studies strongly suggested a genetic context of infection phenotypes, while recent GWAS and genetic analyses further identified a panel of genetic variants associated with HBV infection and CHB susceptibility. Because the host immune system responsive for both innate immune and adaptive immunity plays a key role in controlling HBV infection and outcomes,2, 3 its association with genetic variants of immuno-modulatory factors has been extensively explored. So far, variations in genes encoding IL-6, IL-1, IL-10, PPARγ, HLAs, TNFα, IFNγ, TIM3, CTLA4 and others have been found to significantly influence HBV infection and CHB predisposition.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Telomerase is a RNA-dependent DNA polymerase that elongates telomeric DNA.20 Most normal human somatic cells lack telomerase activity due to the tight transcriptional repression of its rate-limiting, catalytic component telomerase reverse transcriptase (TERT) gene.20, 21, 22 This, together with the end-replication problem, leads to progressive telomere shortening with each round of cell division or with increased age, and when they become too short (dysfunctional) to protect chromosomes, the DNA damage response is activated, thereby triggering the permanent growth arrest of cells (replicative senescence).20, 22 By regulating telomere length and many other biological activities, telomerase or TERT plays critical parts in human health.20 Importantly, it has been shown that abnormal telomere length is associated with chronic liver diseases, and telomerase provides a protection against liver fibrosis.23, 24, 25, 26 In addition, shorter telomere in immune cells is associated with a lower activity against viral infection.27 These findings suggest that TERT or telomerase may be involved in HBV infection-related CHB and LC. There are multiple single-nucleotide polymorphisms in the TERT gene, among which rs2736100 and rs2736098 are most studied. These variants have been shown to be associated with cancer, atherosclerosis, depression and other health problems.28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 Given the findings documented above, we sought to ask whether the rs2736100 and rs2736098 genotypes modify susceptibility to CHB and its LC complication. Our results reveal a significant association between the TERT rs2736098_TT genotype and reduced risk of CHB and LC in Chinese males. We further compared the rs2736098 allele distribution in male and female individuals separately. The highly significant lower TT genotype was observed in male cases (4.8%) compared to that in male controls (13.7%; Table 2, TT vs CT: OR, 4.0, 95% CI, 1.646–9.720, P=0.002; TT vs CC: OR, 2.574, 95% CI, 1.063–6.237, P=0.018). In contrast, a slight difference in the TT genotype was seen between female controls (12.7%) and patients (9.7%), which was not significant (controls vs cases in female, P>0.05 in all the comparisons; Table 2). The variants of the TERT gene have been observed to be associated with a significantly higher susceptibility to cancer and aging-related disorders,29, 33 however, their relationship with chronic virus infection is unclear. In the present study, we investigated the influence of the TERT genetic variants on risk of CHB and LC. Our results reveal significantly higher frequencies of the rs2736098_TT in healthy controls than in CHB patients, which suggest that the TERT rs2736098_TT genotype exerts a protective effect on susceptibility to CHB and LC. To our knowledge, this is the first report demonstrating an association between the rs2736098 variant and CHB or its complications. During HBV infection, complete HBV clearance occurs in more than 90% of infected adults, while 5–10% of them has viral persistence, thereby leading to CHB, and then further development of cirrhosis or even HCC.1 Cell-mediated immune response is believed to contribute to variable clinical outcomes of HBV infection. Telomerase or TERT and telomeres have long been recognized to play pivotal parts in regulating immunological activity.44, 45 Activation of telomerase via induction of TERT expression occurs in activated lymphocytes for their expansion in response to infectious challenge.44, 45 Shorter LTL was associated with increased susceptibility to experimentally induced acute upper respiratory infection and clinical illness in adults.27 Mechanistically, shorter telomeres limit proliferation potentials of immune cells and compromise immune response to pathogens, thereby lowering host resistance to infection.44, 45 It is thus likely that TERT variants modify risk of CHB by influencing host immune function. However, the LTL comparison between healthy controls and CHB patients did not reveal a significant difference, which indicates that telomere homeostasis is not significantly impaired in CHB patients. In addition, the rs2736098 TT and CC genotype-carriers were previously shown to harbor shortest and longest LTL, respectively, while the CT variant was correlated with medium levels of LTL.32 Consistently, Atzmon et al. found a significant association between rs2736098 and LTL.43 However, we did not find this scenario in both healthy controls and CHB patients, indicating that the association between rs2736098_TT allele and a lower CHB risk is unlikely attributable to telomere length regulation. However, the sample size in the present study is relatively small, which might be a potential reason behind the dissociation between the rs2736098 genotype and phenotype (LTL). On the other hand, telomere lengthening is the canonical function of telomerase or TERT, but recent studies have demonstrated its multiple properties independently of telomere homeostasis.46, 47, 48, 49, 50, 51 Ectopic expression of TERT was shown to promote mobilization of stem cells and proliferation of many types of cells via upregulation of stem cell factors, growth factors or their receptor expression;52, 53, 54 telomerase and TERT is capable of protecting cells from apoptosis mediated by various insults.49, 50, 55, 56 Mechanistically, TERT can act as co-factors to stimulate gene transcription.48 It is thus likely that the rs2736098_TT allele boosts host immune response for HBV clearance via a telomere lengthening-independent manner. However, the exact underlying mechanism calls for further investigations. Telomere and telomerase or TERT have been shown to be involved in the pathogenesis of chronic liver diseases including LC.23, 24, 25 Telomerase-deficient mice at late generations exhibit shortened telomeres and diminished capacity for liver regeneration, and undergo accelerated development of cirrhosis after liver injury, whereas re-expression of telomerase activity is shown to improve liver function and protect mice from development of hepatic steatosis and fibrosis.25 Similar pathological alterations including accelerated telomere attrition and regenerative exhaustion of liver cells were observed in patients with loss-of-function of telomerase genes.23, 24 On the other hand, telomere over-erosion also occurs in CHB-related cirrhosis due to a higher turn-over of hepatocytes to compensate for liver damage. Conceivably, HBV infection and shorter telomeres cooperate to accelerate the development of LC. However, it is currently unclear whether this mechanism is involved in the rs2736098_CT/CC-related risk of LC. Intriguingly, we only observed the effect of rs2736098 variants on risk of male CHB patients. It is well-known that estrogen protects females from HBV infection and CHB by directly inhibiting HBV replication.57 On the other hand, however, estrogen is also a strong activator of TERT transcription and telomerase.58, 59 There may thus be a possibility that a potent controlling of TERT expression by estrogen masks the effect of the rs2736098 genotypes in female CHB patients. Further studies are required to elucidate this issue. In summary, the finding presented here demonstrates that the TERT rs2736098 variants significantly contribute to the susceptibility of CHB and its cirrhosis complication in Chinese males, providing new insights into telomerase biology and etiology of CHB and LC. These results also raise a number of intriguing questions: How those different TERT genotypes modify the risk of CHB and LC, and why their influence only occurs in males. In addition, because CHB is a key factor that drives development of HCC, while telomerase and TERT are essential in oncogenesis, it is important to further dissect the relationship between rs22736098 variants and CHB or HBV-related HCC. Elucidation of all these issues will certainly contribute to the management and prevention of CHB and its complications. Guarantor of the article: Feng Kong, MD. Specific author contributions: Planning the study: Guanghui Cheng, Xiaotian Yuan, Feng Kong, Dawei Xu. Conducting the study: Guanghui Cheng, Xiaotian Yuan, Fang Wang, Qing Sun, Qian Xin, Kailin Li, Chao Sun, Zhaomin Lin, Yun Luan, Yiteng Xu, Ping Li. Collecting and/or interpreting data: Guanghui Cheng, Xiaotian Yuan, Fang Wang. Drafting the manuscript: Guanghui Cheng, Xiaotian Yuan, Yiteng Xu, Feng Kong, Dawei Xu. All the authors have approved the final draft submitted. Financial support: This study was supported by grants from the National Basic Research Program of China (Grant No. 2012CB911202), Shandong Provincial Natural Science Foundation, China (No: 2016ZDJS07A09), Swedish Cancer Society, the Swedish Research Council, Cancer Society in Stockholm. The study sponsors have no any roles in the study design, collection, analysis, and interpretation of the data and in the writing of the report. Potential competing interests: None.