U-shaped association between telomere length and esophageal squamous cell carcinoma risk: a case-control study in Chinese population

Abstract

Telomeres play a critical role in biological ageing by maintaining chromosomal integrity and preventing chromosome ends fusion. Epidemiological studies have suggested that inter-individual differences of telomere length could affect predisposition to multiple cancers, but evidence regarding esophageal squamous cell carcinoma (ESCC) was still uncertain. Several telomere length-related single nucleotide polymorphisms (TL-SNPs) in Caucasians have been reported in genome-wide association studies. However, the effects of telomere length and TL-SNPs on ESCC development are unclear. Therefore, we conducted a case-control study (1045 ESCC cases and 1433 controls) to evaluate the associations between telomere length, TL-SNPs, and ESCC risk in Chinese population. As a result, ESCC cases showed overall shorter relative telomere length (RTL) (median: 1.34) than controls (median: 1.50, P<0.001). More interestingly, an evident nonlinear U-shaped association was observed between RTL and ESCC risk (P<0.001), with odds ratios (95% confidence interval) equal to 2.40 (1.84–3.14), 1.36 (1.03–1.79), 1.01 (0.76–1.35), and 1.37 (1.03–1.82) for individuals in the 1st (the shortest), 2nd, 3rd, and 5th (the longest) quintile, respectively, compared with those in the 4th quintile as reference group. No significant associations were observed between the eight reported TL-SNPs and ESCC susceptibility. These findings suggest that either short or extremely long telomeres may be risk factors for ESCC in the Chinese population.

Keywords

esophageal squamous cell carcinoma / telomere length / genetic variants / susceptibility / genome-wide association study

1 Introduction

Telomeres are special DNA sequences with tandem repeats of TTAGGG nucleotides at the linear ends of chromosomes that play an important role in maintaining chromosomal integrity by preventing chromosome end fusion [1]. Telomere length shows significant inter-individual variation, and different telomere lengths may result in different predispositions to diseases, including cancer [2]. In recent years, multiple studies reported that short telomeres were associated with elevated risk of cancers in head and neck [3,4], lung [3,5−7], renal cell [3,8], bladder [3,9,10], ovarian [3,11], breast [12−14], and colon or rectum [12]. However, several studies observed long telomeres in melanoma [15], breast cancer [16−18], lung cancer [19,20], hepatocellular carcinoma [21], and non-Hodgkin lymphoma [22]. Recently, several studies have verified that telomere length may have a nonlinear association with the risk of specific cancer types. For instance, a U-shaped association pattern was observed in pancreatic cancer [23], breast cancer [24], and glioma [25]. Our recent study also identified that both short and extremely long telomeres may contribute to the development of gastric cancer [26]. The inter-individual variation of telomere length may be partly explained by genetic factors [27]. Recent genome-wide association studies (GWAS) have identified several loci (2p16.2 [ACYP2], 3q26 [TERC], 5p15.33 [TERT], 4q32.2 [NAF1], 10q24.33 [OBFC1], 19p12 [ZNF208], and 20q13.3 [RTEL1]) that were associated with telomere length in populations of European descent [28−31], and some of these telomere length-related single nucleotide polymorphisms (TL-SNPs) were associated with cancer susceptibility [31−34]. However, these findings were mainly observed in European populations. Our recent replicative study suggested that TL-SNPs were not implicated in gastric cancer susceptibility in the Chinese population [26].

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. ESCC incidence is much higher in East Asia than in western countries, and more than half of newly diagnosed ESCC cases occurred in China [35]. Currently, the potential role of telomere length in ESCC etiology remains to be clarified. Some retrospective case-control studies suggest that short telomeres are associated with increased risk of ESCC [36,37]. However, a recent prospective study reported that short telomeres are not associated with esophageal cancer [38]. The small sample size of these studies may have restricted the analysis of association between telomere length and ESCC risk. Thus, the effects of telomere length and TL-SNPs on ESCC risk are still uncertain. Therefore, we conducted a case-control study with 1045 ESCC cases and 1433 age- and sex-matched controls in a Chinese population to investigate the association between telomere length and ESCC risk, as well as the contribution of TL-SNPs to ESCC susceptibility in European descent.

2 Materials and methods

2.1 Study subjects

In this study, ESCC patients were recruited between January 2007 and June 2010 at the Jiangsu Tumor Hospital and the First People’s Hospital of Huai’an, Jiangsu. All of the cases were histopathologically confirmed, and those having a history of treatment prior to recruitment were excluded. Finally, 1045 newly diagnosed ESCC cases were included in this study. Controls were randomly selected from Jiangsu Province based on a non-infectious diseases community screening project for more than 30 000 individuals, and were frequency-matched to cases according to age (5-year interval) and sex. After the patients submitted signed informed consent documents, we conducted a face-to-face interview for each subject. The interview included a standard questionnaire with information on demographics and related risk factors, such as cigarette smoking and alcohol consumption. After the interview, a ~5 ml venous blood sample was collected from each subject. Individuals who smoked at least once a day for more than one year were defined as smokers and those who drank twice or more per week for at least one year were considered as drinkers; otherwise, they were treated as non-smokers or non-drinkers. This study was approved by the Institutional Review Board of Nanjing Medical University.

2.2 Measurement of relative telomere length

The method for relative telomere length measurement has been previously described [39]. In brief, relative telomere lengths (RTLs) were determined by the ratio of telomere repeat copy number (T) to single copy gene copy number (S) and expressed as T/S ratio in individual samples relative to a reference of DNA pool from five healthy donors. The reference DNA was first used to generate a standard curve, with concentrations ranging from 0.25 ng/μl to 8 ng/μl, and linearity (r ²>0.99) over this range of input DNAs was measured. Samples with threshold cycle numbers that fell outside the range defined by the standard curves were rerun at a different concentration to ensure that they were amplified within the linear range. The primer sequences for telomere and single-copy gene (36B4) quantitative PCR (qPCR) amplification are listed in Supplementary Table 1. Reference pooled DNA was then assayed in each 384-well plate to account for variations between plates. Each well contained 10 μl SYBR^® Green PCR Master Mix (Applied Biosystems) with a final DNA concentration of 5 ng/μl. All samples were run in duplicate, and the mean of two measurements was used in the statistical analyses. In general, equal cases and controls were assayed on each 384-well plate, and technicians were blinded to the case-control status.

2.3 Selection and genotyping of TL-SNPs

TL-SNPs were selected from published GWAS [28,29,31] by following the steps below: (1) searching the reported TL-SNPs that reached significant genome-wide association (i.e., P≤5 × 10⁻⁸); (2) filter out the TL-SNPs with minor allele frequency (MAF) less than 5% in the Chinese population; (3) only one variant was selected as the others were in linkage disequilibrium (LD) at r ²>0.5. MAF and LD information were obtained from the CHB+JPT subjects of the 1000 Genomes Project (Phase I interim release). We included seven TL-SNPs, including rs10936599 at 3q26.2 (TERC), rs11125529 at 2p16.2 (ACYP2), rs2736100 at 5p15.33 (TERT), rs4387287 (OBFC1) at 10q24.33, rs755017 at 20q13.33 (RTEL1), rs7675998 at 4q32.2(NAF1), and rs8105767 at 19p12 (ZNF208). In addition, the SNP rs2736108 at 5p15.33 (TERT) that was associated with telomere length and breast cancer risk in a GWAS [31] was also included in the current study, though the reported P value was 5.8 × 10⁻⁷ for the association with telomere length.

Genotyping was performed using the TaqMan allelic discrimination assay on an ABI PRISM 7900HT Sequence Detection System. Detailed information for the primers and probes are shown in Supplementary Table 1. Two negative controls were included in each 384-well reaction plate, and the genotyping results were determined using the SDS 2.3 Allelic Discrimination Software (Applied Biosystems). Technicians were blinded to the status of cases and controls during genotyping.

2.4 Calculation of weighted genetic scores

To evaluate the integrated effect of the eight TL-SNPs, we calculated the weighted genetic score (WGS) based on the genotypes of these SNPs. For each subject, WGS was calculated by counting the number of alleles associated with long telomeres and weighted by the telomere length associated beta (β _j), which was derived from the regression analysis of telomere length and genotype in the reported source literature. WGS was calculated using the following formula:

WG{S}_i={\displaystyle \sum _{\mathrm{1}}^j{\mathrm{X}}_{ij}{\beta }_j}

In this formula, x _ij is the number of long telomere length related alleles for the j-th variant in the i-th subject (x _ij = 0, 1, or 2) and β _j is the weight for the j-th variant.

2.5 Statistical methods

Pearson’s χ² test was used to compare the differences between cases and controls for categorical variables (i.e., sex, age, smoking, and drinking status). Rank-sum test was used to assess the differences in RTL between subgroups divided by sex, age groups, smoking, and drinking status. Generalized linear models were used to conduct tests for correlation between RTL and age. To examine the association between RTL or WGS and ESCC risk, we categorized RTL or WGS into five groups based on its quintile distribution in controls. Unconditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (95% CI) for ESCC risk in each genetic variant, RTL or WGS quintile, with an adjustment for age, sex, smoking, and drinking status. A restricted cubic spline curve was plotted in the logistic regression model to evaluate the shape of the association between RTL and ESCC risk. Wald test was used for nonlinearity estimation. The restricted cubic spline curve was plotted using the “RCS_Reg Version 1.0” macro in SAS version 9.1.3 (SAS Institute) [40]. General analyses were performed with Stata version 9.2 (StataCorp LP).

3 Results

Comparative analysis of relative telomere length (RTL) between case-control groups and characteristics subgroups are summarized in Table 1. A total of 1045 ESCC cases and 1433 cancer-free controls were included in this study. The age and sex were fully matched between cases and controls (P>0.05). The smoking and drinking rates of patients were both significantly higher than those of healthy controls (P<0.05). As expected, peripheral blood leukocyte RTL was inversely correlated with age in both cases and controls (P<0.001, Supplementary Fig. 1). In addition, women showed significantly longer telomeres than men among controls (P = 1.04 × 10⁻⁵), but no apparent differences were observed between subgroups divided by smoking or drinking status (P>0.05).

The overall RTL in ESCC cases (median: 1.34) was significantly shorter than that of controls (median: 1.50, P = 7.39 × 10⁻¹³), which was consistent in subgroups divided by age, sex, smoking, and drinking status (Table 1). When the details of association between RTL and ESCC risk were further evaluated through restricted cubic spine function in the logistic regression model, we observed an evident U-shaped association for ESCC risk (P<0.001 for nonlinearity test, Fig. 1). To further survey the relationship between RTL and ESCC, we categorized subjects into five groups based on quintile distribution of RTL in controls (Table 2). Compared with the lowest risk subjects in the 4th quintile, a significant dose-response effect was observed between the increased risk of ESCC and decreasing telomeres of the 3rd, 2nd, and 1st quintiles, with ORs (95% CIs) ranging from 1.01 (0.76−1.35), 1.36 (1.03−1.79), and 2.40 (1.84−3.14), respectively. More interestingly, a significant elevated risk was clearly shown in the 5th quintile (the longest RTL) (OR= 1.37, 95% CI: 1.03−1.82) compared with the 4th quintile (Table 2). These findings suggest that either short or extremely long telomeres are associated with an increased risk of ESCC. Furthermore, subgroup analysis stratified by age and sex were conducted for the association of quintile grouped RTL and ESCC risk (Supplementary Table 2). As a result, similar U-shaped associations were observed in subgroups.

We further investigated whether those TL-SNPs can affect susceptibility to ESCC. No significant association was observed between the eight TL-SNPs and ESCC risk (Table 3). After integrating the eight SNPs by computing the WGS, ESCC cases (median: 0.426) and controls (median: 0.429; P = 0.824) were found to have similar WGS. Logistic regression analysis based on quintile groups (Table 4) did not reveal any notable association between WGS and ESCC risk. These results suggest that the known TL-SNPs may not affect the ESCC susceptibility.

4 Discussion

In this large case-control study, we not only investigated the relationship between telomere length and ESCC risk but also analyzed the association between eight TL-SNPs of European descent and ESCC susceptibility in the Chinese population. Our results first showed that telomere length has a nonlinear U-shaped association with ESCC risk, providing novel epidemiological evidence for the effect of telomeres on the development of ESCC. However, no significant association was observed between TL-SNPs and ESCC susceptibility.

Two recent case-control studies evaluated the relationship between telomere length and ESCC risk and found that subjects with shorter telomere length had an elevated risk of ESCC compared with those that had longer telomeres [36,37]. We also observed that ESCC cases had shorter telomeres than controls, which was consistent with results of previous studies. Interestingly, we observed a U-shaped association between telomere length and ESCC risk, suggesting that both short and extremely long telomeres may contribute to the development of ESCC. This finding was similar with our recent published study on the association between gastric cancer and telomere length, in which subjects with either short or extremely long telomeres have increased gastric cancer risk [26]. Moreover, this U-shaped association pattern was also indicated in other cancers, including pancreatic cancer [23], breast cancer [24], and glioma [25]. Therefore, previously inconsistent associations between telomere length and cancer risk may be partially attributed to the nonlinear U-shaped association pattern. Nevertheless, large, well-designed studies, especially prospective ones, are warranted to confirm our findings in diverse populations.

Emerging evidence demonstrated that the U-shaped association between telomere length and cancer risk was biologically plausible. Telomeres play an important role in the evolution of cancer cell genome, acting as a double-edged sword in cancer development. In general, the wear and tear of telomeres can lead to chromosomal fusion and genome instability, which may accelerate the process of carcinogenesis [3]. By contrast, extremely long telomeres may allow for more cell divisions and promote cell immortalization, and thus may cause cancer development [41]. Genetic association study also supported this hypothesis. Alleles associated with both short and long telomere length have been implicated in elevated cancer susceptibility [28,29]. In combination with these evidences, our findings indicate that telomere length needs to be balanced between shortening and elongation to produce an optimal length for cell growth.

Researchers have reached a consensus that telomere length is involved in cancer risk. However, whether TL-SNPs can modify cancer susceptibility has not been widely investigated. Evidence supporting this hypothesis is flimsy at best. For example, the short telomere allele A of rs2736098 and rs2736100 were associated with susceptibility of lung cancer, bladder cancer, and prostate cancer [32,33,42]. In our study, no significant association was found between those eight TL-SNPs and ESCC susceptibility. This negative association can be interpreted as follows: first, the cancer heterogeneity attenuated the pathogenicity of these SNPs, and more precise molecular classification in cancer susceptibility study is needed; second, the U-shaped relationship between telomere length and ESCC may restrict the association study to investigate the indirect pathogenic effect from SNPs to ESCC development.

Our study has important strengths and limitations. First, we systematically investigated the contribution of telomere length and known TL-SNPs to ESCC risk simultaneously. Second, the large sample size in this study allowed us to analyze the nonlinear association between telomere length and ESCC risk more accurately. Third, we measured the telomere length using advanced technology under strict quality controls. However, the drawback in this retrospective case-control design is that because the DNA was collected after diagnosis, we could not confirm causal relationship between telomere length attrition and ESCC risk. Further prospective cohort study may confirm our findings.

In summary, the current study indicates that telomere length is associated with ESCC risk in a U-shaped pattern and demonstrates that TL-SNPs may not be important in ESCC carcinogenesis. Our study provides valuable clues for better understanding the underlying mechanism of ESCC development.

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