Background
Telomeres are DNA-protein complexes that protect the ends of chromosomes. Telomerase maintains telomere ends during DNA replication by catalyzing the addition of short telomere repeats (TTAGGG). The enzyme is comprised of a protein with reverse transcriptase activity that is encoded by the telomerase reverse transcriptase (
TERT) gene, and a telomerase RNA component (TERC) which serves as a template for the telomere repeat after recognition of a single stranded G-rich primer [
1]. Expression of telomerase is normally repressed in somatic cells leading to a gradual shortening of telomeres and cellular senescence with aging [
2,
3]. Heritability of telomere length in humans has been reported to range from 36 % - 90 % [
4,
5].
Leukocyte telomere length has been reported to be associated with susceptibility to cardiovascular disease [
6]. When mean telomere length was measured in 10 patients with severe coronary artery disease and compared to that observed for 20 controls, the size was significantly reduced and equivalent to that found in individuals without heart disease who were 9 years older [
7]. Significantly shorter telomeres were also detected in leukocyte DNA from 203 subjects who had had a myocardial infarction (MI) before the age of 50 [
8], from 620 chronic heart failure patients [
9], and from 150 stroke patients [
10] when compared to controls. Shorter telomere length has also been reported to be associated with a higher prevalence of both atherothrombotic and hemorrhagic stroke in a Chinese case–control study [
11]. Finally, in four prospective studies that evaluated disease incidence, there was an increased risk of coronary artery disease [
12], MI [
13‐
15], and stroke [
13] associated with shorter telomere length, while no association was found with ischemic stroke in either the Nurses’ Health Study or the Physicians’ Health Study [
16,
17]. Taken together, these results suggest that variation in telomere length may play a role in the risk and progression of cardiovascular disease. An association between survival and leukocyte telomere length has also been observed in several previous epidemiological studies [
18‐
20].
Recently, a single nucleotide polymorphism (SNP) (rs2736100) located within an intron of
TERT was significantly associated with mean leukocyte telomere length in a genome-wide association (GWA) study in which 37,684 individuals from fifteen cohorts were included in the discovery set. The estimated per-allele effect of addition of the A allele was 94.2 base pairs, equivalent to 3.14 years of age-related telomere shortening [
21]. The aim of the present study was to determine whether six haplotype tagging SNPs located within the
TERT gene or the 5’ promoter region including rs2736100 were associated with risk of incident coronary heart disease (CHD), ischemic stroke, and all-cause mortality in participants in the large biracial population-based ARIC cohort. To date, there have been few previous investigations of the role of telomerase variants in cardiovascular disease and its risk factors that have included individuals of African ancestry [
22].
Results
The allele and genotype frequencies for six
TERT polymorphisms evaluated in this study (Table
1) were in accordance with Hardy-Weinberg expectations for both white and African-American study subjects (all p > 0.05). When LD was estimated for these variants, the SNPs were not highly correlated for either white or African-American study participants (all r
2 < 0.15) (Table
2). A description of the study sample at the first clinical visit stratified by race is shown in Table
3. There were 403 incident CHD cases (13.3 %) and 287 ischemic stroke cases (9.5 %) ascertained in African-American subjects during an average follow-up period of 20.0 years, and 933 CHD cases (10.4 %) and 452 stroke cases (5.0 %) identified in whites during an average follow-up period of 20.4 years. All of the clinical and demographic characteristics differed significantly between white and African-American participants with the exception of the levels of total and LDL cholesterol.
Table 1
TERT genotype and allele frequencies stratified by race. ARIC study (1987–1989)
rs2736122 | | | | | | | |
GG | 1,857 | 56.1 | 0.25 | 5,283 | 53.8 | 0.27 | 0.04 |
AG | 1,246 | 37.7 | | 3,835 | 39.1 | | |
AA | 205 | 6.2 | | 698 | 7.1 | | |
rs4246742 | | | | | | | |
TT | 1,444 | 43.5 | 0.35 | 7,037 | 71.6 | 0.15 | <0.01 |
AT | 1,453 | 43.8 | | 2,549 | 25.9 | | |
AA | 421 | 12.7 | | 241 | 2.5 | | |
rs6863494 | | | | | | | |
TT | 2,950 | 90.3 | 0.05 | 9,823 | 100..0 | 0.00 | <0.01 |
CT | 305 | 9.3 | | 2 | 0.0 | | |
CC | 13 | 0.4 | | 0 | 0.0 | | |
rs4975605 | | | | | | | |
CC | 1,012 | 30.6 | 0.45 | 2,742 | 27.9 | 0.47 | <0.01 |
AC | 1,619 | 48.9 | | 4,861 | 49.5 | | |
AA | 679 | 20.5 | | 2,218 | 22.6 | | |
rs2736100* | | | | | | | |
CC | 683 | 20.6 | 0.54 | 2,533 | 25.8 | 0.49 | <0.01 |
CA | 1,661 | 50.0 | | 4,932 | 50.2 | | |
AA | 975 | 29.4 | | 2,361 | 24.0 | | |
rs2853668 | | | | | | | |
GG | 841 | 25.3 | 0.50 | 5,432 | 55.3 | 0.26 | <0.01 |
TG | 1,647 | 49.6 | | 3,707 | 37.7 | | |
TT | 831 | 25.1 | | 688 | 7.0 | | |
Table 2
Linkage disequilibrium between TERT single nucleotide polymorphisms
r2, White | | rs2736122 | rs4246742 | rs6863494* | rs4975605 | rs2736100 | rs2853668 |
| rs2736122 | x | 0.053 | --- | 0.062 | 0.058 | 0.021 |
| rs4246742 | 0.053 | x | --- | 0.09 | 0.005 | 0.006 |
| rs6863494 | | | x | | | |
| rs4975605 | 0.062 | 0.09 | --- | x | 0.04 | 0.034 |
| rs2736100 | 0.058 | 0.005 | --- | 0.04 | x | 0.13 |
| rs2853668 | 0.021 | 0.006 | --- | 0.034 | 0.13 | x |
r2, African-American | | rs2736122 | rs4246742 | rs6863494 | rs4975605 | rs2736100 | rs2853668 |
| rs2736122 | x | 0.001 | 0.011 | 0.052 | 0.001 | 0.016 |
| rs4246742 | 0.001 | x | 0.097 | 0.047 | 0.015 | 0.001 |
| rs6863494 | 0.011 | 0.097 | x | 0.032 | 0.024 | 0.004 |
| rs4975605 | 0.052 | 0.047 | 0.032 | x | 0.021 | 0.011 |
| rs2736100 | 0.001 | 0.015 | 0.024 | 0.021 | x | 0.023 |
| rs2853668 | 0.016 | 0.001 | 0.004 | 0.011 | 0.023 | x |
Table 3
Race-specific clinical and demographic characteristics. ARIC participants free of CVD (1987 – 1989)
Male | 3,022 | 1,081 (35.8) | 8,987 | 4,030 (44.8) | <0.001 |
Current smokers | 3,020 | 857 (28.4) | 8,984 | 2,145 (23.9) | <0.001 |
Current alcohol | 2,994 | 951 (31.8) | 8,975 | 5,909 (65.8) | <0.001 |
Hypertension | 3,009 | 1,613 (53.6) | 8,953 | 2,250 (25.1) | <0.001 |
Diabetes | 2,953 | 532 (18.0) | 8,972 | 715 (8.0) | <0.001 |
Incident MI/Fatal CHD | 3,022 | 403 (13.3) | 8,987 | 933 (10.4) | <0.001 |
Incident ischemic stroke | 3,022 | 287 (9.5) | 8,987 | 452 (5.0) | <0.001 |
| | Mean (SD) | | Mean (SD) | |
Age (years) | 3,022 | 53.1 (5.7) | 8,987 | 54.1 (5.7) | <0.001 |
DBP, mm Hg | 3,022 | 79.6 (11.8) | 8,983 | 71.6 (10.0) | <0.001 |
SBP, mm Hg | 3,022 | 127.8 (20.3) | 8,984 | 118.2 (16.9) | <0.001 |
Glucose (mmol/L) | 2,941 | 6.4 (3.0) | 8,980 | 5.8 (1.6) | <0.001 |
Insulin (pmol/L) | 2,941 | 138.0 (291.4) | 8,979 | 81.4 (94.9) | <0.001 |
BMI (kg/m2) | 3,019 | 29.7 (6.1) | 8,980 | 26.9 (4.8) | <0.001 |
Total cholesterol, mmol/L | 2,895 | 5.6 (1.2) | 8,971 | 5.5 (1.0) | 0.503 |
LDL cholesterol, mmol/L | 2,870 | 3.6 (1.1) | 8,832 | 3.5 (1.0) | 0.281 |
HDL cholesterol, mmol/L | 2,895 | 1.4 (0.4) | 8,973 | 1.3 (0.4) | <0.001 |
Triglycerides, mmol/L | 2,896 | 1.3 (0.9) | 8,973 | 1.5 (1.0) | <0.001 |
The results of the analysis of the association between the
TERT sequence variants and incident CHD and ischemic stroke are displayed in Tables
4 and
5, respectively. SNP rs2736122 was nominally associated with incident CHD in African-Americans both in the minimally adjusted Cox regression model (HRR = 1.20, p = 0.02, 95 % confidence interval (CI) = 1.03 – 1.40) and in a second model that was further adjusted for a panel of established cardiovascular risk factors (HRR = 1.18, p = 0.04, 95 % CI = 1.01 – 1.39). Similarly, one of the genetic variants was nominally associated with incident ischemic stroke (rs2853668) in African-Americans in a model adjusted for age and gender (HRR = 1.17, p = 0.05, 95 % CI = 1.00 – 1.38), but this relationship was attenuated after BMI, current smoking, and diabetes and hypertension case status were added to the regression models. There were also 1,203 (36.2 %) and 2,875 deaths (29.3 %) among African-American and white participants, respectively, during the mean 20.5-year follow-up period. All-cause mortality was assessed but no association with any of the
TERT sequence variants was found for either racial group (all p > 0.15) (Table
6). None of the associations described above remained significant after correction for multiple comparisons.
Table 4
TERT sequence variation and incident coronary heart disease. ARIC study (1987 – 2011)
rs2736122* | | | | | | | | | | | | |
CHD | 1.20 | 1.03, 1.40 | 0.02 | 1.18 | 1.01, 1.39 | 0.04 | 0.95 | 0.86, 1.06 | 0.38 | 0.94 | 0.85, 1.05 | 0.29 |
rs4246742* | | | | | | | | | | | | |
CHD | 1.00 | 0.87, 1.15 | 0.97 | 0.98 | 0.85, 1.14 | 0.83 | 0.96 | 0.85, 1.09 | 0.56 | 0.99 | 0.87, 1.13 | 0.88 |
rs6863494 | | | | | | | | | | | | |
CHD | 0.93 | 0.67,1.29 | 0.66 | 0.95 | 0.68, 1.32 | 0.77 | -- | | | -- | | |
rs4975605 | | | | | | | | | | | | |
CHD | 1.04 | 0.91, 1.19 | 0.55 | 1.05 | 0.91, 1.20 | 0.52 | 1.02 | 0.94, 1.12 | 0.61 | 1.02 | 0.93, 1.11 | 0.74 |
rs2736100 | | | | | | | | | | | | |
CHD | 0.99 | 0.86, 1.14 | 0.89 | 1.00 | 0.86, 1.15 | 0.97 | 0.97 | 0.88, 1.06 | 0.48 | 0.98 | 0.89, 1.07 | 0.60 |
rs2853668 | | | | | | | | | | | | |
CHD | 0.93 | 0.81, 1.07 | 0.32 | 0.98 | 0.84, 1.13 | 0.73 | 1.05 | 0.95, 1.16 | 0.35 | 1.07 | 0.97, 1.19 | 0.19 |
Table 5
TERT sequence variation and incident ischemic stroke. ARIC study (1987 – 2011)
rs2736122 | | | | | | | | | | | | |
Isch. stroke | 0.89 | 0.73, 1.08 | 0.24 | 0.90 | 0.74, 1.10 | 0.32 | 1.01 | 0.87, 1.17 | 0.89 | 1.02 | 0.87, 1.18 | 0.82 |
rs4246742 | | | | | | | | | | | | |
Isch. stroke | 0.94 | 0.79, 1.11 | 0.47 | 0.96 | 0.80, 1.15 | 0.67 | 0.99 | 0.82, 1.18 | 0.89 | 1.00 | 0.83, 1.21 | 0.99 |
rs6863494 | | | | | | | | | | | | |
Isch. stroke | 0.90 | 0.61, 1.33 | 0.59 | 0.96 | 0.65, 1.42 | 0.84 | -- | | | -- | | |
rs4975605 | | | | | | | | | | | | |
Isch. stroke | 0.94 | 0.80, 1.11 | 0.48 | 0.88 | 0.74, 1.04 | 0.14 | 1.00 | 0.88, 1.14 | 0.97 | 1.00 | 0.87, 1.14 | 0.98 |
rs2736100 | | | | | | | | | | | | |
Isch. stroke | 1.07 | 0.91, 1.27 | 0.41 | 1.03 | 0.86, 1.22 | 0.75 | 0.93 | 0.82, 1.06 | 0.31 | 0.93 | 0.81, 1.06 | 0.27 |
rs2853668 | | | | | | | | | | | | |
Isch. stroke | 1.17 | 1.00, 1.38 | 0.05 | 1.08 | 0.91, 1.28 | 0.36 | 1.02 | 0.88, 1.18 | 0.76 | 1.03 | 0.89, 1.20 | 0.71 |
Table 6
TERT sequence variation and all-cause mortality. ARIC study (1987 – 2011)
rs2736122 | | | | | | | | | | | | |
Mortality | 1.05 | 0.95, 1.15 | 0.36 | 1.04 | 0.94, 1.14 | 0.47 | 1.04 | 0.98, 1.10 | 0.22 | 1.03 | 0.97, 1.10 | 0.27 |
rs4246742 | | | | | | | | | | | | |
Mortality | 0.99 | 0.91, 1.08 | 0.85 | 0.99 | 0.90, 1.08 | 0.76 | 1.01 | 0.94, 1.08 | 0.80 | 1.02 | 0.95, 1.09 | 0.63 |
rs6863494 | | | | | | | | | | | | |
Mortality | 1.00 | 0.84, 1.20 | 0.96 | 1.00 | 0.84, 1.20 | 0.96 | -- | | | -- | | |
rs4975605 | | | | | | | | | | | | |
Mortality | 0.96 | 0.89, 1.04 | 0.37 | 0.94 | 0.87, 1.02 | 0.16 | 1.00 | 0.95, 1.05 | 0.96 | 0.99 | 0.94, 1.04 | 0.74 |
rs2736100 | | | | | | | | | | | | |
Mortality | 1.00 | 0.92, 1.09 | 0.93 | 0.98 | 0.90, 1.07 | 0.73 | 1.03 | 0.98, 1.09 | 0.21 | 1.03 | 0.97, 1.08 | 0.32 |
rs2853668* | | | | | | | | | | | | |
Mortality | 1.00 | 0.92, 1.09 | 0.94 | 0.97 | 0.89, 1.05 | 0.44 | 0.99 | 0.93, 1.05 | 0.73 | 1.02 | 0.96, 1.08 | 0.62 |
Discussion
A functional role for telomerase in the maintenance of telomere length has been established both
in vitro and
in vivo, including in the heart [
36]. In an early test of the proposed causal relationship between telomere attrition and cellular senescence, retinal pigment epithelial cells and foreskin fibroblasts that do not normally express telomerase were transfected with the enzyme’s catalytic subunit. The telomerase positive clones exhibited elongated telomeres and exceeded their normal life span by more than 20 cell divisions [
37]. Similarly, restoration of telomerase activity in
Terc-deficient mice resulted in longer telomeres and absence of premature aging [
38], and alleviated the tissue degeneration and activation of DNA damage signaling that are characteristic consequences of telomere loss [
39]. Forced expression of
TERT in cardiac muscle in mice promoted cell proliferation and cardiac myocyte survival, suggesting a possible strategy for organ regeneration after injury [
40].
Leukocyte telomere length has been shown to be associated with cardiovascular disease risk in some but not all studies [
7‐
17]. In the current study, a nominal association between rs2736122 and incident CHD in the fully adjusted model (HRR = 1.18, p = 0.04, 95 % CI = 1.01– 1.39), and rs2853668 and incident ischemic stroke in a regression model adjusted only for age and gender (HRR = 1.17, p = 0.05, 95 % CI = 1.00 – 1.38) was detected in African-American ARIC study participants. These observations are in accordance with an earlier report that 5
TERT SNPs including rs2736100 and rs4975605, and 2 variants including rs2853668 were associated with risk of incident nonfatal MI and ischemic stroke, respectively, in 23,294 individuals of European ancestry enrolled in the Women’s Genome Health Study (WGHS) [
41]. However, rs2853668 was associated with a reduced susceptibility to ischemic stroke in the WGHS (HRR (stroke) = 0.81, p = 0.03, 95 % CI = 0.66 – 0.98) after adjustment for age, BMI, smoking, diabetes, hypertension, and hormone use while the same variant increased risk in the ARIC study. Although there was adequate power to detect the same HRR observed by Zee et al., none of the three polymorphisms found to be associated with cardiovascular disease in the WGHS that were also genotyped in the ARIC study (rs2736100, rs4975605, rs2853668) [
41] were associated with stroke or CHD in white study participants. Other reasons for the discordant findings could be associations that were found by chance in either or both cohorts, as well as differences in ascertainment since WGHS included only nonfatal MI cases in the analyses while the ARIC study case definition encompassed both MI and fatal CHD.
Although the observed associations between the
TERT variants and both CHD and stroke were modest in African Americans and were no longer significant after correction for multiple testing, differences in LD could contribute to the absence of an association in whites if a true causative variant was only correlated with rs2736122 or rs285366 in African-Americans. Inspection of the LD plots generated at the
TERT locus for the Utah residents with Northern and European ancestry (CEU) and African ancestry in Southwest USA (ASW) populations included in the International HapMap Project reveals that the race-specific LD patterns are not identical, with the caveat that this region has not been densely genotyped (HapMap3 Genome Browser release #2, chromosome 5: positions 1,306,287 – 1,348,162) [
27]. Similarly, variation in linkage disequilibrium structure between whites and African-Americans could also explain the reversal in the direction of association of rs2853668 with incident ischemic stroke seen in the ARIC study when compared with the WGHS. Assuming that rs2853668 may not be the causal variant in either cohort, correlation between the polymorphism and a protective allele at another locus in WGHS participants, and with a risk allele in ARIC participants could lead to the observed results [
42]. Other reasons for the discrepancy could include chance, differences in allele frequency for the rs2853668 T allele in the two racial groups, and variation in other genetic or environmental factors that may contribute to cerebrovascular disease risk in the two study populations. The
TERT rs2736100 variant was not associated with incident CHD or stroke in either racial group. In the GWA study of telomere length in which rs2736100 was identified, there was also no relationship between this variant and prevalent coronary artery disease in a meta-analysis that combined the results for 22,233 cases and 64,762 controls of European ancestry who were enrolled in the CARDIoGRAM consortium but did not include individuals of African descent [
21,
43].
The relationship between telomere length and aging and longevity has also been assessed. A negative correlation between telomere length and age has been consistently observed when examined in multiple tissues [
3,
44‐
47]. More recently, telomere length was positively correlated with increased lifespan in the Amish Family Osteoporosis Study [
18], and Fitzpatrick et al. reported that individuals in the shortest quartile of leukocyte telomere length in the Cardiovascular Health Study were more likely to die than those in the longest quartile during a 6.1-year follow-up period [
20]. In contrast, Bischoff et al. found no correlation between telomere length and survival in a sample of 812 individuals from 3 different Danish study populations [
48]. Similar results were reported in the Scottish Lothian Birth Cohort [
49], and in a study of 3,075 participants in the population-based Health ABC Study aged 70–79 years in which neither overall survival or death from cardiovascular disease was associated with telomere length [
50]. While an association between two polymorphisms in oligonucleotide/oligosaccharide-binding fold containing 1(
OBFC1), a gene related to telomere length [
51], and decreased risk of cardiovascular death was demonstrated in women in the Cardiovascular Health Study [
52], none of the
TERT sequence variants examined here had a discernible effect on the time to death in ARIC study participants.
For all of the statistical analyses described above, it is possible that, although there was only a marginal effect on the risk of developing cardiovascular disease when the
TERT polymorphisms were considered individually, the polymorphisms may play a role in combination with other loci associated with variation in telomere length as demonstrated by Codd et al. in a genetic risk score analysis for coronary artery disease [
21]. In addition, since the association between the
TERT polymorphisms and telomere length could not be evaluated in the ARIC study, a link between increased risk of cardiovascular disease and the possible functional impact of the gene could not be explored further. It should also be noted that since several risk factors for cardiovascular disease including obesity and smoking have been shown to be associated with telomere length in leukocytes [
53], differences in the distribution of these covariates between populations or racial and ethnic groups could result in inconsistencies in the reported relationship between
TERT and a given disease outcome. Further investigation of sequence variation in
TERC [
54] as well as other genes such as
OBFC1, CTS telomere maintenance complex component 1 (
CTC1), and zinc finger protein 676 (
ZNF676) that have been identified and replicated in large-scale GWA studies of telomere length [
51,
55] but were not present on the genotyping array may also prove to be informative in the ARIC cohort.
Acknowledgements
The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C), and the National Genome Research Institute contract U01-HG-004402, and National Institutes of Health contract HHSN268200625226C. The authors thank the staff and participants of the ARIC study for their important contributions. Infrastructure was partly supported by Grant Number UL1RR025005, a component of the National Institutes of Health and NIH Roadmap for Medical Research.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JB performed the statistical analysis, participated in study design, and drafted the manuscript. NF participated in study design and data interpretation, and helped draft the manuscript. EWD participated in study design and data interpretation, and helped draft the manuscript. THM participated in study design and data interpretation, and helped draft the manuscript. ARF participated in study design and data interpretation, and helped draft the manuscript. EB conceived of the study, participated in study design and data interpretation, and helped draft the manuscript. All authors read and approved the final manuscript.