No association between TERT-CLPTM1L SNP rs401681 and mean telomere length or cancer risk

Abstract

Background

A recent study reported genetic variants in the TERT-CLPTM1L locus to be associated with mean telomere length, and with risk of multiple cancers.

Methods

We evaluated the association between SNP rs401681 [c>t] and mean telomere length, using quantitative Real Time PCR, in blood-extracted DNA collected from 11,314 cancer-free participants from the Sisters in Breast Screening (SIBS) study, the MAPLES melanoma family study, and the SEARCH Breast, Colorectal, Melanoma studies. We also examined the relationship between rs401618 genotype and susceptibility to breast cancer (6800 cases, 6608 controls), colorectal cancer (2259 cases, 2181 controls) and melanoma (787 cases, 999 controls).

Results

The ‘per t allele’ change in mean telomere length (ΔCt), adjusted for age, study plate, gender and family, was 0.001 (95%CI -0.01 – 0.02), p-trend=0.61. The ‘per t allele’ Odds Ratio (OR) for each cancer was: breast cancer OR=1.01 (95% Confidence Intervals 0.96-1.06), p-trend=0.64; colorectal cancer OR=1.02 (95%CI 0.94-1.11), p-trend=0.66; and melanoma OR=0.99 (95%CI 0.84-1.15), p-trend=0.87.

Conclusion

We found no evidence that this SNP is associated with mean telomere length, or with risk of breast cancer, colorectal cancer or melanoma.

Impact

Our results indicate that observed associations between rs401681 and several cancer types may be weaker than previously described. The lack of an association in our study between this SNP and mean telomere suggests that any association with cancer risk at this locus is not mediated through TERT.

Introduction

Telomeres are repetitive (ttaggg)_n sequences, present on the ends of chromosomes, which protect against coding sequence erosion and consequent DNA damage repair, resulting in genome instability, chromosomal fusions and rearrangements^1-3. The relationship between telomere length and cancer risk has been investigated in several publications, but the results have been inconclusive, and few truly prospective studies have, thus far, been carried out^4-6. Many common sequence variants associated with susceptibility to cancer have been discovered by genome wide association studies (GWAS)^7-11. One such study has found sequence variants on chromosome 5p15, in the TERT-CLPTM1L genomic region associated with the risk of basal cell carcinoma^{12, 13}. TERT (Telomerase Reverse Transcriptase) encodes the protein subunit of telomerase, responsible for telomere synthesis and, hence, the maintenance of telomere length. Subsequent analysis demonstrated that the major (c) allele of rs401681 was associated with an increased risk of various cancers, including lung, bladder, prostate and cervix, but with a decreased risk of colorectal cancer and melanoma. The c allele was also associated with shorter mean telomere length in lymphocytes. No association between rs401681 and breast cancer risk was observed.

To confirm and extend these observations, we have investigated the association between rs401681 and risk of three types of cancer, and with mean telomere length, as measured by quantitative Real Time PCR, in almost 10,000 individuals with cancer and over 11,000 disease-free controls.

Materials and Methods

Study summaries

SEARCH

The SEARCH Study is an ongoing population based study in Eastern England. Cases were ascertained through the Eastern Cancer Registration and Information Centre (ECRIC, http://www.ecric.org.uk/) and were aged between 18 and 70 years at diagnosis. Controls were drawn from SEARCH and EPIC-Norfolk. Details of these studies have been previously published^8,14. In total, 6800 breast cancer cases and 6608 controls; 2259 colorectal cancer cases and 2248 controls, and 378 melanoma cases and 380 controls were genotyped for the polymorphism studied here.

MAPLES

Additional melanoma association study subjects (404 cases, 619 controls) were recruited via the Melanoma And Pigmented Lesions Evaluative Study (MAPLES), whose aim was the identification of genetic mutations responsible for moliness and, consequently, to identify individuals at high risk of melanoma. Cases and controls were ascertained through pigmented lesion clinics and general practices in the Cambridge area.

SIBS

The Sisters in Breast Screening (SIBS) study is an ongoing investigation of intermediate phenotypes related to breast cancer (http://www.srl.cam.ac.uk/genepi/sibs/sibs_aims.html). Its aim is the mapping of genes underlying these quantitative traits, specifically mammographic density and sex steroid hormone levels. 1740 cancer-free subjects were genotyped.

Ethical approval for the SEARCH and SIBS participants was obtained from the Eastern Multicentre Research Ethics Committee. Ethical approval for the MAPLES participants was obtained from the Huntingdon Local Research Ethics Committee, Cambridge Local Research Ethics Committee, the Norwich District Ethics Committee and the East Norfolk and Waveney Research Governance Committee.

Informed consent was obtained from each patient.

Taqman® genotyping

Genotyping was performed by Taqman® Assay as previously described¹⁴. The call rate was >98% for all studies. Failed genotypes were not repeated. 3.2% of the SEARCH samples and 2.9% of the combined melanoma study were duplicated for assessment of quality control. The concordance between duplicate calls was 100%.

Real Time PCR

Relative mean telomere length was ascertained by high-throughput SYBR® Green Real Time PCR, the method for which has been previously described^{4, 15-16}. The disease-free samples from each study were assayed; SIBS (n=1655), MAPLES (n=619) and SEARCH (n=9050). 21% of the combined Melanoma study, 22% of the SIBS study and 12% of the SEARCH breast and colorectal studies were duplicated for quality control. Failed PCR reactions were not repeated.

Statistical Methods

Analyses were performed using Intercooled Stata 10.1 statistical package (Stata, College Station, TX). Methods are detailed in the legends of Table 1 and Table 2.

Table 1.

TERT SNP genotype and mean telomere length.

	δΔCt (95%CI)
TERT rs401681	Breast Cancer 6434 controls	Melanoma 979 controls	SIBS 1655 controls	Colorectal Cancer 2246 controls	Combined 11314 controls
CC	0.00 ref	0.00 ref	0.00 ref	0.00 ref	0.00 ref
CT	−0.002 (−0.02 - 0.02)	0.06 (0.003 - 0.1)	−0.002 (−0.04 - 0.04)	0.01 (−0.02 - 0.05)	0.02 (−0.005 - 0.04)
TT	−0.02 (−0.03 - 0.02)	0.03 (−0.05 - 0.1)	0.03 (−0.02 - 0.08)	0.004 (−0.04 - 0.05)	0.04 (−0.03 - 0.04)
Per T allele	p-trend=0.86	p-trend=0.38	p-trend=0.29	p-trend=0.78	p-trend=0.61

Genotype frequencies and mean telomere length, as represented by the continuous ΔCt variable, were analysed in control samples using linear regression, for the ‘per T allele’ change in mean telomere length (δΔCt), with associated 95% confidence intervals (95% CI). Analyses were adjusted for 384-well plate and age in all the studies and for gender and family where applicable.

Table 2.

TERT SNP genotype and cancer risk.

	OR (95% CI), p-het
TERT rs401681	Breast Cancer 6800 cases, 6608 controls	Colorectal Cancer 2259 cases, 2246 controls	Melanoma 782 cases, 999 controls
CC	1.00 ref	1.00 ref	1.00 ref
CT	1.02 (0.94 - 1.10),0.49	1.09 (0.96 - 1.25), 0.19	1.01 (0.79 - 1.29), 0.95
TT	1.01 (0.92 - 1.12),0.70	1.02 (0.86 - 1.21), 0.80	0.98 (0.70 - 1.37), 0.90
Per T allele	1.01 (0.96 - 1.06) p-trend = 0.64	1.02 (0.94 - 1.11) p-trend = 0.66	0.99 (0.84 - 1.17) p-trend = 0.91

Genotype frequencies in cases and controls were compared using a 2 degree of freedom (df) χ² test for heterogeneity (p-het) and a 1 df Cochran-Armitage χ² test for trend in risk by T allele dose (p-trend). Genotype-specific risks were estimated as odds ratios (OR), with associated 95% confidence intervals (95% CI), using unconditional logistic regression. For each study, the deviation of genotype distribution in controls from Hardy-Weinberg equilibrium was assessed by a χ² test with one degree of freedom (data not shown).

Results

We found no association between rs401681 genotype and mean telomere length in a combined sample set of 11,314 cancer-history-free study participants ages 18-81 years (mean = 57 years) (Table 1). Similarly, none of the individual studies showed any significant effect when analysed separately. As a validation of the assay, we examined the association of mean telomere length with age. There was a significant decrease in mean telomere length with age; ‘per annum’ increase in ΔCt, adjusted for study, gender and 384-well plate = 0.014 (95%CI 0.012–0.016), p-trend=1.5 × 10⁻³⁹.

There was no significant association between rs401681 genotype and risk of any of the three cancers assessed (Table 2). The ‘per T allele’ OR was 1.01 (95%CI 0.92–1.06), p-trend=0.64, for breast cancer, OR=1.02 (95%CI 0.94–1.11), p-trend=0.66, for colorectal cancer, and OR=0.99 (95%CI 0.84–1.15), p-trend=0.87, for melanoma. There was no heterogeneity in the per-allele OR between the two melanoma studies.

Discussion

We found no association between mean telomere length and rs401681 genotype in ~11,000 cancer-free individuals, using an assay that successfully detects the known reduction of mean telomere length with increasing age in the same subjects. Rafnar et al.¹ had reported an association between genotype and age- and plate-corrected mean telomere length in 276 healthy controls, aged between 85yrs and 95 yrs (p=0.017). These findings were not significant in a group of 260 younger women in the same study (60-70yrs), (p=0.081).

Furthermore, we found no association between rs401681 genotype and risk of three different cancers. Rafnar et al.¹ also reported no association between this SNP and breast cancer risk. However, the major c allele was associated with protection against both colorectal cancer and cutaneous melanoma (CM), in the same study. The CM risk association was subsequently replicated in a larger study by the same group². Our colorectal cancer analysis was sufficiently large to exclude any substantial risk (95%CI 0.94-1.11). Our melanoma association analysis was based on a smaller sample size and our estimated ‘per t allele’ OR was 0.99 (95%CI 0.84-1.15), p=0.91. In comparison with the published ‘per c allele’ estimates, (0.82-0.95)¹ and (0.81-0.91)², our ‘per c allele’ data shows a significant overlap; OR=1.01 (95%CI 0.86-1.19), p-trend=0.91. Thus, while we did not observe an association, our results are consistent with the published association.

The previous publications examined a second variant in the region, rs2736098, and found a significant association with cancer risk and with mean telomere length, independent of rs401681 genotype. We were unable to manufacture a Taqman® Assay to interrogate the second SNP, which is correlated with rs401681 (r²=0.39 and D’=0.94 in Europeans¹⁷). We would have less power to detect an association at this locus, however, assuming that rs2736098 is more strongly correlated with a putative causal variant, our results would still exclude any substantial association with breast or colorectal cancer.

Our results indicate that observed associations between rs401681 and several cancer types do not extend to breast or colorectal cancer, and that the melanoma association may be weaker than previously described. The lack of an association in our study between this SNP and mean telomere length in lymphocytes in middle-aged adults suggests either that any association with cancer risk at this locus is not mediated through TERT, or that the modification of TERT expression does materially affect telomere length in lymphocytes.