Relative Leukocyte Telomere Length and Risk of Incident Ischemic Stroke in Men: A Prospective, Nested Case-Control Approach

Abstract

Recent data have implicated telomere-length shortening as potential risk predictor for vascular diseases, including stroke. However, to date, prospective epidemiological data are scarce in relation to ischemic stroke risk. Using leukocyte DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we examined the relationship of leukocyte telomere repeat copy number to single gene copy number (TSR), using a quantitative polymerase chain reaction protocol, amongst 259 white males who subsequently developed an ischemic stroke and amongst an equal number of age- and smoking-matched white males who remained free of reported vascular disease during follow up (controls). The observed TSRs were inversely correlated with age in the controls (p < 0.0001). However, the observed TSRs were similar between cases and controls (p = 0.92). In a multivariable adjusted analysis, no evidence was found for an association of the TSRs with ischemic stroke risk (odds ratio [OR] = 1.100, 95% confidence interval [CI], 0.506–2.392, p = 0.811). The present investigation has shown no evidence for an association of relative leukocyte telomere length with risk of incident ischemic stroke. More importantly, our present findings require replication/confirmation in future large, prospective studies.

Introduction

Vascular disorders, including ischemic stroke, are leading causes of mortality and morbidity in modern societies. The underlying pathophysiology is likely to be under the influence of both genetic and environmental factors.

Telomeres are tandem repeats of DNA sequences located at the ends of eukaryotic chromosomes with the primary function being to protect the telomeric regions from recombination and degradation, thus avoiding a DNA damage cellular response.¹ Recent evidence has suggested the relevance of telomere biology in human disorders, including vascular disease.¹ In cross-sectional and case–control studies, shortening of telomere length has recently been associated with chronic heart failure,² degenerative aortic valve stenosis,³ coronary artery diseases,^4,5 and premature myocardial infarction (MI).⁶ However, to date, prospective data examining relative telomere length and ischemic stroke are not available.

Therefore, we prospectively examined the possible association of relative leukocyte telomere length with risk of incident ischemic stroke, using a nested, matched case–control sample drawn from the prospective Physicians' Health Study (PHS) cohort.

Materials and Methods

Study design

We employed a nested case–control design within the PHS, a randomized, double-blinded, placebo-controlled trial of aspirin and β-carotene initiated in 1982 among 22,071 male, predominantly white (>94%), U.S. physicians, 40–84 years of age at study entry.⁷ Before randomization, 14,916 participants provided an ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood sample that was stored for genetic analysis. All participants were free of prior vascular disorders and cancer at study entry. Yearly follow-up self-report questionnaires provided reliable updated information on newly developed diseases and the presence or absence of other cardiovascular risk factors. History of cardiovascular risk factors, such as hypertension, diabetes, or hyperlipidemia, was defined by self-report of diagnosis at entry into the study. For all reported incident vascular events occurring after study enrollment, hospital records, death certificates, and autopsy reports were requested and reviewed by an end-points committee using standardized diagnostic criteria.

Stroke was defined by the presence of a new focal neurological deficit, with symptoms and signs persisting for >24 h and was ascertained from blinded review of medical records, autopsy results, and the judgment of a board-certified neurologist, on the basis of clinical reports, computed tomographic (CT), or magnetic resonance image (MRI) scanning.⁷

For each case, a control matched by age ± 2 years, smoking history (never, past, or current), and length of follow up since randomization was chosen among those subjects who remained free of any vascular diseases. Ischemic stroke was classified as 33% embolic, 29% thrombotic, and 38% nondifferentiable embolic–thrombotic among the ischemic stroke cases in the present study. The present association study consisted of white men only. Median length of follow up since randomization for the cases was 6.04 years (interquartile range, 3.18–8.80). The study was approved by the Brigham and Women's Hospital Institutional Review Board for Human Subjects Research.

Leukocyte telomere length determination

Genomic DNA was extracted from whole blood using the QIAmp Spin Column protocol (Qiagen, Chatsworth, CA). Telomere length was determined by a previously validated, unified quantitative polymerase chain reaction (qPCR) protocol.⁸ In brief, two master mixes of PCR reagents were prepared, one for the telomere reaction and one for the single-copy gene reaction (36B4 on chromosome 12). Telomere repeat copy number to single gene copy number ratio (TSR) was determined on an ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) in a 384-well format using the following PCR protocol: 95°C for 15 min to activate Taq polymerase, 40 cycles of denaturation at 95°C for 15 sec, and annealing–extension at 54°C for 2 min. Each 15-μL amplification reaction volume contained 1 × Qiagen Quantitect SYBR Green Master Mix (Qiagen, Chatsworth, CA) and 10 ng of template DNA. The primer sequences used have been described elsewhere.⁹ All samples for both the telomere and single-copy gene amplifications were done in duplicate on the same 384-well plate. Ct-value assignment was carried out by two independent observers, and, if necessary, a complete reamplification was performed. Duplicates of a no-template control and a reference DNA calibrator were included in each run. Melting (dissociation) curve analysis was performed on every run to verify specificity and identity of the PCR products. The Ct values generated were used to calculate the TSR for each sample using the equation: T/S = 2^−ΔCt (where ΔCt = Ct_{single-copy gene} − Ct_telomere). Results were scored blinded as to case–control status.

Statistical analysis

As previously noted, the observed TSRs had a skewed distribution and thus were log_e-transformed. The log_e-TSRs between cases and controls were compared using the paired t-test. Linear regression analysis was used to assess the relationship of age, smoking, body mass index (BMI), blood pressure, and C-reactive protein plasma concentrations with log_e-TSRs amongst the controls only. Relative risks of ischemic stroke associated with log_e-TSRs were calculated separately by conditional logistic regression analysis, adjusting for age, smoking status, and length of follow up since randomization, and further controlling for randomized treatment assignment, history of hypertension (≥140/90 mmHg or on antihypertensive medications), presence or absence of diabetes, BMI, and hyperlipidemia. Furthermore, prespecified analysis, limited to participants without baseline diabetes or hypertension (i.e., low-risk group), was performed. All analyses were carried out using SAS/Genetics 9.1 package (SAS Institute Inc., Cary, NC). A two-tailed p value of 0.05 was considered a statistically significant result.

Results

Baseline characteristics of the study participants are shown in Table 1. As expected, the case subjects had a higher prevalence of traditional vascular risk factors at baseline than did the control subjects. The observed log_e-TSRs were similar between cases and controls (p = 0.92; Table 1). As previously reported, an inverse relationship was found between the observed log_e-TSRs and age in our control group (p < 0.0001; Table 2). However, no evidence for an association of log_e-TSRs with risk of incident ischemic stroke was found in a crude or an adjusted regression analysis (adjusted odds ratio [OR] = 1.100, 95% confidence interval [CI] = 0.506–2.392, p = 0.811; Table 3). Again, similar null findings were observed in the prespecified “low-risk” analysis (Table 3). Prespecified stratified analysis by median follow-up time since randomization was also performed, and again similar null findings were observed (data not shown). The coefficients of variation of the telomere, 36B4 single gene, and TSR duplicate assays were all <2%, respectively.

Table 1.

Baseline Characteristics of Study Participants

	Controls (n = 259)	Cases (n = 259)	P
Age (years)	61.7 ± 7.9	62.1 ± 8.04	m.v.
Smoking status (%)			m.v.
Never	39.8	39.8
Past	41.7	41.7
Current	18.5	18.5
Body mass index (kg/m2)	24.8 ± 2.6	25.3 ± 2.9	0.06
Hyperlipidemia ≥240 mg/dL (%)	15.5	19.8	0.17
Hypertension (%)	31.4	51.4	<0.0001
Diabetes (%)	2.3	11.6	<0.0001
Aspirin use (%)	49.0	49.4	0.93
Family history of premature CAD <60 years (%)	8.2	9.0	0.76
Loge-TSR	3.83 ± 0.56	3.83 ± 0.55	0.92

Mean ± standard deviation (SD) unless otherwise stated. Continuous and categorical variables were tested by paired t-test and McNemar test, respectively.

m.v., Matching variable; CAD, coronary artery disease; TSR, telomere repeat copy number to single gene copy number ratio.

Table 2.

Linear Regression Analysis of Log_e-TSR with Several Baseline Variables in the Controls

TSR	Parameter estimate, t value, p
Age	−0.022, −5.20, <0.0001
Smokinga
Never	Referent
Past	0.092, 1.25, 0.214
Current	−0.108, −1.15, 0.253
Body mass indexa	−0.009, −0.74, 0.463
Systolic blood pressurea	0.000, 0.21, 0.833
Diastolic blood pressurea	0.005, 1.04, 0.297
Loge-CRPa	−0.042, −1.30, 0.194

^aAdjusted for age.

TSR, telomere repeat copy number to single gene copy number ratio; CRP, C-reactive protein concentrations.

Table 3.

Conditional Logistic Regression Analysis of Log_e-TSR

Ischemic stroke	Crude OR, 95% CI, p	Adjusted OR, 95% CI, p
All participants	1.064, 0.521–2.169, 0.866	1.100, 0.506–2.392, 0.811
Low-risk group	0.675, 0.175–2.591, 0.567	0.519, 0.127–2.119, 0.361

Crude is conditional on age, smoking status, time of follow up. Adjusted is further controlling for randomized treatment group, body mass index, hypertension, diabetes, and hyperlipidemia.

TSR, telomere repeat copy number to single gene copy number ratio; OR, odds ratio; CI, confidence interval.

Discussion

The present prospective, nested investigation is the first to examine the relationship of relative telomere length with risk of incident ischemic stroke and found no evidence for an association. Nevertheless, in concordance with prior studies, a significant inverse correlation between the observed TSRs and age in our control participants was observed. In regard to the relationship between telomere length and blood pressure (as elevated blood pressure is a well-known risk factor for stroke), our present null finding was in concordance with a recent study by Nordfjall et al., in which no association was found between telomere length and blood pressure nor pulse pressure,¹⁰ together suggesting that any association between telomere length and stroke is unlikely.

Recent animal and human studies have implicated the importance of leukocyte telomere biology in cardiovascular disease.¹ Human telomere length shortening has also been implicated in neurodegenerative conditions,^11,12 including poststroke mortality¹³ and dementia.¹⁴ A recent study by Fitzpatrick et al. that examined leukocyte telomere length and cardiovascular disease in subjects drawn from the Cardiovascular Health Study showed a significant association of leukocyte telomere length shortening with stroke. As acknowledged by the authors, their finding was based on a pilot study of 17 stroke cases aged ≤73 years, and thus larger, prospective studies are needed for further examination/confirmation.¹⁵ Because no epidemiological data of relative leukocyte telomere length on risk of incident ischemic stroke are available, no direct cross-reference comparison could be made. Furthermore, recent studies have showed an association of telomere length with severity of stroke consequences including poststroke mortality,¹³ dementia,^13,14 and cognitive decline.¹³ Because no information on these poststroke consequences is available on our study population, their potential associations with leukocyte telomere length cannot be examined in the present context.

The nature of the present investigation in which the determination of a case status was based solely on the subsequent development of disease rather than on any arbitrary selection criteria designed by the investigators, greatly reducing the possibility of bias and confounding. Nonetheless, our study population consists of white males, so our findings cannot be generalized to other ethnic groups, women, and populations with different socioeconomic backgrounds. Of note, in our study, we had the ability to detect, based on the present sample size, assuming 80% power, at an alpha of 0.05, a difference in log_e-TSR (between cases and controls) of <−0.097 or > + 0.097. Thus, the present study may have limited power to detect a true, small-to-moderate difference of telomere length between cases and controls. Because telomere biology represents a rapidly expanding research field, further thoughts on future development of the qPCR technique with comparison to the gold-standard Southern blot method is worthwhile.

In conclusion, in this prospective, nested case–control study of middle-aged white U.S. men, we found no association of relative leukocyte telomere length with risk of incident ischemic stroke. If corroborated in other large, prospective studies, our data further suggest that relative leukocyte telomere length may not be a useful predictor for risk assessment of stroke.

Acknowledgments

This research was supported by grants from the National Heart Lung and Blood Institute (CA-97193, CA-34944, CA-40360, HL-26490, HL-34595).

Authorship

Contributions: R.Y.L.Z., A.J.C., and N.S.B. designed the research, performed the research, and analyzed the data. All authors contributed to data interpretation and manuscript preparation. All authors approved the manuscript as its present form.

Author Disclosure Statement

The authors declare no competing financial interests.