Leukocyte Telomere Length-Related rs621559 and rs398652 Genetic Variants Influence Risk of HBV-Related Hepatocellular Carcinoma

Recent genome-wide association studies (GWAS) have identified eleven leukocyte telomere length (LTL)-related single nucleotide polymorphisms (SNPs). Since LTL has been associated with risk of many malignancies, LTL-related SNPs may contribute to cancer susceptibility. To test this hypothesis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we genotyped these eleven LTL-related SNPs in a case-control set including 1186 HBV-related HCC cases, 508 chronic HBV carriers and 1308 healthy controls at the discovery stage. The associations of HCC risk with these SNPs were further confirmed in an independent case-control set. We found that 1p34.2 rs621559 and 14q21 rs398652 were significantly associated with HBV-related HCC risk (both P<0.005 after Bonferroni corrections). There was no significant difference of either rs621559 or rs398652 genotypes between chronic HBV carriers and healthy controls, demonstrating that the association was not due to predisposition to HBV infection. In the pooled analyses (1806 HBV-related HCC cases and 1954 controls), we observed a decreased HCC risk, 0.72-times, associated with the 1p34.2 rs621559 AA genotype compared to the GG genotype (P = 1.6×10−6). Additionally, there was an increased HCC risk, 1.27-fold, associated with the rs398652 GG genotype (P = 3.3×10−6). A statistical joint effect between the rs621559 GG and rs398652 GG genotypes may exist in elevating risk of HBV-related HCC. We show, for the first time, that rs398652 and rs621559 might be marker genetic variants for risk of HBV-related HCC in the Chinese population.

It has been reported that LTL is genetically heritable, with heritability ranging from 44% to 80% [19][20][21]. Recently, several genome-wide association studies (GWAS) identified eleven single nucleotide polymorphisms (SNPs) which are associated with LTL in different ethnic populations [9,[22][23][24][25]. For example, Gu J et al. found that four SNPs (rs398652 on 14q21, rs621559 on 1p34.2, rs6028466 on 20q11.22 and rs654128 on 6q22.1) were associated with LTL in Caucasian populations (pooled P, 10 25 ). In a large case-control study, they observed that subjects with the variant allele of rs398652 has a significantly reduced risk of bladder cancer [Odds ratio (OR) = 0.81; 95% Confidence interval (CI) = 0.67-0.97; P = 0.025], consistent with the correlation of this variant allele with longer telomeres. In a previous study, we also investigated whether these four genetic variants are associated with LTL and risk of esophageal squamous cell carcinoma (ESCC) in Chinese populations [26]. After measuring LTL of 550 healthy individuals, we verified that both rs621559 and rs398652 genetic variants are significantly associated with LTL. On the basis of analyzing 1550 ESCC patients and frequency-matched 1620 controls from 4 medical centers in China, we found that the rs621559 AA genotype is associated with a decreased risk of ESCC, 0.71-times, compared to the rs621559 GG genotype (P = 5.9610 26 ). We also detected a moderately increased OR for ESCC that was associated with the 14q21 rs398652 G allele (P = 6.5610 24 ). The aforementioned results indicate that these LTL-related SNPs identified by GWAS [9,[22][23][24][25], might contribute to cancer susceptibility.
As the third third-most cause of cancer mortality in the world, hepatocellular carcinoma (HCC) shows unbalanced distribution, with the highest morbidity in Asia and Sub-Saharan Africa [27,28]. Notably, China alone accounts for approximately half of all HCC patients [27]. Epidemiological evidences indicate that chronic infections with the hepatitis B or C viruses (HBV or HCV), exposure to dietary aflatoxin B as well as alcohol abuse are major risk factors. In China, HBV infection is prevalent and particular important, due to its coherent distribution with HCC [27,28]. However, only a fraction of HBV chronic carriers developed HCC, suggesting that genetic makeup may also contribute to development of HBV-related HCC [27,28]. The existence of genetic polymorphisms influencing the development of HBV-related HCC has also been proved by several GWAS on HBV-related HCC in eastern Asians [29][30][31][32].
Taken together, we hypothesized that the LTL-related SNPs identified by GWAS [9,[22][23][24][25], may influence HBV-related HCC risk in Chinese populations. To test this hypothesis, we conducted a two-stage case-control study of HBV-related HCC from different regions of China.

Study case-control sets
This study consisted of two case-control sets: (a) Shandong set (Discovery set): 1186 patients with HBV-related HCC, sex-and age-matched (65 years) 508 chronic HBV carriers as well as sexand age-matched 1308 healthy controls. HBV-related HCC patients and chronic HBV carriers were recruited between June 2009 and October 2012 at Shandong Cancer Hospital, Shandong Academy of Medical Sciences (Jinan, Shandong Province, China). Control subjects were randomly selected from a pool of 4500 individuals from a community cancer-screening program for cancer early detection conducted in Jinan city during the same time period as the patients were collected. Part of the case-control set has been reported previously [29]. (b) Jiangsu set (Validation set): 560 HBV-related HCC patients from Huaian No. 2 Hospital (Huaian, Jiangsu Province, China) and sex-and age-matched 566 controls. Patients were consecutively recruited between January 2009 and September 2012 at Huaian No. 2 Hospital. Controls were cancer-free individuals selected from a community cancerscreening program (3000 individuals) for early detection of cancer conducted in Huaian city during the same time period as the patients were collected. Part of the case-control set has been reported previously [33,34]. The diagnosis of all patients was confirmed by a pathological examination combined with positive imaging (magnetic resonance imaging and/or computerized tomography). All participants were negative for antibodies to HCV, hepatitis D virus or HIV since we excluded all participants with HCV, hepatitis D virus or HIV infection. Chronic HBV carriers and HBV-related HCC cases were defined as individuals with the following serological parameters [HBsAg(+) for.6 months, anti-HBc(+), and anti-HBs(-)]. Healthy controls are ones without HBV infection. Individuals who smoked one cigarette per day for over 1 year were considered as smokers. Subjects were considered as alcohol drinkers, if they drank at least once per week. All subjects were ethnic Han Chinese. At recruitment, the written informed consent was obtained from each subject. This study was approved by the institutional Review Boards of Shandong Cancer Hospital and Huaian No. 2 Hospital.

Polymorphism genotyping
During the discovery stage, all eleven LTL-related SNPs (rs398652 ref. 9 25) were firstly analyzed in the Shandong case-control set (Discovery set) by the MassArray system (Sequenom Inc., San Diego, California, USA). A 15% blind, random sample of study subjects was genotyped in duplicates and the reproducibility was 100%. To reduce experimental costs, we developed a PCR-based restriction fragment length polymorphism (RFLP) method to determine genotypes of two SNPs (rs398652 and rs621559) in the validation case-control set (Jiangsu set). In PCR-RFLP genotyping, the primers used for amplifying DNA segments containing the rs398652 and rs621559 site (mismatch bases are underlined) were 59-GAAACTAATTCTTGCTGTCTCGA-39/59-CCTTCACTC-CTGCTCATTTCC-39, or 59-AGTGTTCCTTTGCCTCATC-TA-39/59-ACTCTGTGGAACAGTTGGGTA-39.
Restriction enzymes XhoI and RsaI (New England Biolabs) were used to distinguish the rs398652 A.G and rs621559 G.A genotypes. A 15% random sample was reciprocally tested by different person, and the reproducibility was 99.5%.

Statistical analyses
Pearson's x 2 test was used to examine the differences in demographic variables, smoking status, drinking status, and genotype distributions of eleven LTL-related polymorphisms between patients and controls. The associations between genotypes of these genetic variants and HBV-related HCC risk were estimated by ORs and their 95% CIs computed by logistic regression models. All ORs were adjusted for age, sex, smoking or drinking status, where it was appropriate. A P value of less than 0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All analyses were performed using SPSS 16.0 (SPSS Inc.).

Results
No statistically significant differences were found between HBVrelated HCC patients, chronic HBV carriers and healthy controls for Shandong set and Jiangsu set in terms of median age and sex distribution (P.0.05). This indicates that the frequency matching was adequate (Table 1). However, smokers were over-represented among patients compared with controls in both Shandong and Jiangsu case-control sets (P,0.05). In addition, we observed that there are more subjects who drink alcohol among cases than those among controls in both case-control sets (P,0.001).
Firstly, unconditional logistic regression analysis was utilized to calculate associations between genotypes of eleven LTL-related SNPs (rs398652, rs621559, rs6028466, rs654128, rs12696304, rs3772190, rs4452212, rs4387287, rs2162440, rs7235755, and rs16847897) and HBV-related HCC risk in Shandong discovery set (Table 2). Among eleven SNPs, rs6028466 showed no frequency in Chinese and was excluded in further analyses. In Shandong set consisting of 1186 patients with HBV-related HCC, 508 chronic HBV carriers as well as 1308 healthy controls, we found that two common SNPs (14q21 rs398652 and 1p34.2 rs621559) were significantly associated with HBV-related HCC risk (both P,0.005 after Bonferroni corrections; HCC cases vs. healthy controls). However, none of these LTL-related SNPs were associated with chronic HBV infection when different genotypes were compared between chronic HBV carriers and healthy controls. Therefore, we believe that the positive association between rs398652 or rs621559 SNPs and HBV-related HCC was not due to predisposition to HBV infection. Interestingly, these two SNPs were associated with LTL in healthy Chinese subjects in our previous study [26]. The associations of HBV-related HCC risk with these two SNPs were further verified in an independent case-control set. Genotyping results showed that these two SNPs were both significantly associated with HBV-related HCC risk in Jiangsu Chinese population (Table 3 and 4). Carriers of rs621559 AA genotype showed significantly and consistently decreased risks to develop HBV-related HCC compared with rs621559 GG carriers (OR = 0.69, 95% CI = 0.54-0.88, P = 0.003) ( Table 3). Significantly reduced HBV-related HCC risk was observed among rs621559 AG carriers compared to individuals with GG rs621559 genotype in Jiangsu case-control set (OR = 0.73, 95% CI = 0.57-0.94, P = 0.015) ( Table 3). Individuals with rs398652 GG genotype also had significantly increased HBV-related HCC risk compared with those with rs398652 AA genotype in the validation set (OR = 1.23, 95% CI = 1.01-1.50, P = 0.039) ( Table 4). Logistic regression analyses also revealed that individuals with rs398652 AG genotype were associated with an increased risk, 1.27-fold, to develop HBV-related HCC in the validation set (P = 0.064) ( Table 4).
Because 14q21 rs398652 or 1p34.2 rs621559 polymorphism alone was respectively associated with risk of HBV-related HCC, we further examined whether there was a statistically joint effect between rs621559 and rs398652 genotypes on HCC risk (Table 5). We found that patients who carried the rs621559 GG genotype were also more likely to carry the rs398652 GG genotype than controls (9.6% versus 5.9%; P = 3.1610 28 ). The presence of the rs621559 GG genotype or rs398652 GG genotype alone was associated with an increased risk of HBV-related HCC (OR = 2.52, 95% CI = 1.57-4.05, P = 1.2610 24 ; or OR = 3.34, 95% CI = 2.07-5.37, P = 7.0610 27 ) compared with the absence of such a genotype. However, the presence of both rs621559 GG and rs398652 GG genotypes was associated with an even higher risk of HBV-related HCC (OR = 4.16, 95% CI = 2.51-6.90, P = 3.1610 28 ) compared with the lack of both genotypes. These results indicate that a statistical joint effect between the rs621559 GG and rs398652 GG genotypes may exist in elevating risk of HBV-related HCC.
In a previous study, Gu J et al. showed that subjects with the variant allele of rs398652 (associated with longer telomeres) has a significantly reduced risk of bladder cancer [9]. In addition, we found that both rs621559 and rs398652 genetic variants have obvious impact on LTL and ESCC susceptibility in Chinese [26]. In line with these findings, our current study also confirmed that only rs621559 and rs398652 influence risk of HBV-related HCC among all eleven GWAS-identified LTL-related SNPs. These findings demonstrate that rs621559 and rs398652 might be common genetic components in intensifying risk of different malignancies.
Another possible explanation of this inconsistence is that rs621559 and rs398652 SNPs may also contribute to HCC risk through influence function and/or expression of genes in a transregulation way. rs398652 locates about 60 kb from the PELI2 gene, encoding the pellino2 protein which plays a pivotal role in inflammatory response and production of cytokines. Interestingly, it has been shown that chronic inflammation is associated with up to 25% of malignancies and also accelerates telomere attrition [35][36][37]. Therefore, it is possible that chronic inflammation caused by HBV infection can partly elucidates associations between rs398652 and telomere length as well as HCC risk. For rs621559, there is no report on its host, the WDR65 gene, shows its involvement in either telomere maintenance or carcinogenesis.
Our results are obtained from two independent case-control analyses derived from different Han Chinese populations. Having relative large sample sizes, significantly increased/decreased ORs with small P values, these results are unlikely to be attributable to selection bias or unknown confounding factors. The fact that genotype frequencies of all SNPs fit Hardy-Weinberg equilibrium and are identical in the three populations further supports the  randomness of our subject selection. Therefore, further studies of HCC and other cancers would be helpful to verify our results. In all, to the best of our knowledge, this is the first study to reveal that the LTL-related 1p34.2 rs621559 and 14q21 rs398652 polymorphisms influence development of HBV-related HCC.