Methods

We collected, after obtaining informed consent from each patient, peripheral blood samples from 203 men consecutively admitted for ACS in our coronary care unit from January 2004 to October 2005. Whole population was divided into 2 groups: 150 men aged 50 to 75 years (mean, 62 ± 7) and 53 men aged >75 years (mean, 82 ± 5). This classification was made according to data available in other significant reports. We excluded women to avoid confounding factors because some investigations have revealed that telomere length is longer in women than men. All the information was collected in a clinical and epidemiological questionnaire. The study was approved by the Ethical Committee of our hospital and conforms to the Declaration of Helsinki.

Genomic DNA was extracted from leukocytes, and telomere length was measured by relative and comparative quantitative polymerase chain reaction that compares mean telomere repeat sequence copy number to a reference single copy gene. For each DNA sample, we performed 6 quantitative polymerase chain reaction runs: 3 “T” runs (for telomere repeat sequence copy number) and 3 “S” runs (for single copy gene copy number). The reference single copy gene used in this study was 36b4. The method performed was essentially described by Gil and Coetzer in 2004 using the same primers developed by Cawthon in 2002 and the 36b4 gene as the endogenous control.

The telomere-specific primers were forward, 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′ and reverse, 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′. The 36b4-specific primers were forward, 5′-CAGCAAGTGGGAAGGTGTAATCC-3′ and reverse, 5′-CCCATTCTATCATCAACGGGTACAA-3′. The DNA amount for each reaction was 10 ng, and the quantities added of each primer were 1 μl of “forward” primer (5 μM) and 2.33 μl of “reverse” primer (5 μM) for the telomere amplification reaction and 0.4 μl (5 μM) of each primer for the 36b4 gene amplification. The number of cycles for both reactions was 40, with an annealing temperature of 60°C for 30 seconds.

After setting up the technique, our polymerase chain reaction processes had an efficiency of 88.92% and a determination coefficient of 0.976 for 36b4 gene amplification and an efficiency of 76.67% and a determination coefficient of 0.974 for the telomeres. A standard curve derived from serially diluted reference DNA was generated for each of the 3 T and 3 S runs. The average of the 3 T measurements was divided by the average of the 3 S measurements to calculate the average T/S ratio.

Patients were followed up by quarterly personal clinical interviews. To study prognosis of patients with ACS, a follow-up combined event was established. It consisted of death for any cause, new onset of heart failure, need for revascularization, and recurrent angina pectoris.

Death for any cause was determined by review of medical records and interviews to patient’s relatives. New onset of heart failure was defined by hospital admission for this cause or progression of baseline clinical status established by New York Heart Association class. New revascularization was collected from medical records and clinical interviews and was defined by percutaneous or surgical coronary revascularization for any cause. Finally, recurrent angina pectoris was established by new onset of angina symptoms or hospital admission for a new ACS.

Categorical variables are expressed by a percentage of the members of each group and quantitative ones by mean ± SD. Differences in categorical variables used to define baseline patient characteristics were studied by chi-square test. Student t test was used for quantitative ones. Receiver operating characteristic curve was used to correlate telomere length and prognosis defined by follow-up combined event in the 2 different groups. A good correlation was defined by an area under the curve of ≥0.5. In addition, this analysis was used to choose the best cut-off point to classify patients according to telomere length. After calculating the cut-off point of telomere length in both groups, we used a Kaplan-Meier survival analysis to study prognostic value of telomere length. Finally, in case of finding significant results, a Cox analysis was used to confirm the independence of this relation. Significant results were deemed statically significant if p value ≤0.05. Statistical analysis was performed using SPSS Statistics for Windows software, version 20.0 (IBM, Chicago, Illinois).

Results

Baseline characteristics were similar in both groups, except that men aged 50 to 75 years were more frequently smokers and there were more diabetic patients among men >75 years ( Table 1 ). Mean stay was 10 days, and patient treatment was chosen by their habitual doctor. During the hospital admission, reperfusion treatment (fibrinolysis or coronary revascularization) was done in 99 patients (66%) in the first group and in 28 men (53%) aged >75 years (p = 0.09). During admission, 5 patients (3.33%) died in the first group and 8 (15.1%) in the second one. Follow-up was available in 137 patients (91.33%) in the first group and 42 patients (79.25%) in the second one. Patients without follow-up died during admission or were not available to personal interview. During the follow-up, 33 patients (22%) presented combined event in the first group and 9 patients (17%) in the second one (p = 0.72). Detailed results are summarized in Table 2 .

Mean telomere length (T/S) was 4.9 ± 1.94 in men aged 50 to 75 years and 3.6 ± 1.48 in men aged >75 years (p <0.001). Receiver operating characteristic curve was plotted to study correlation between telomere lengths and combined prognostic event. In the first group, area under the curve was 0.63 (p <0.05) and in the second one was 0.36 (p = 0.203). The best cut-off point of telomere length (T/S) in men aged 50 to 75 years was 5.60 (sensitivity: 82% and specificity: 42%) and in those aged >75 years was 4.64 (sensitivity: 79% and specificity: 21%). Patients were classified according to telomere length cut-off point calculated before into 2 groups: “short telomeres” and “long telomeres.” Among men aged 50 to 75 years, 93 (62%) had “short telomeres” and 57 (38%) had “long telomeres”; in case of the second group, 44 (83%) had “short telomeres” and 9 (17%) had “long telomeres.” The distribution of combined event according to telomere length groups is explained in Table 3 .

Table 3

Presentation of prognostic events according to the telomere length

Variable	Age (yrs)
	50–75 (n = 137)		>75 (n = 42)
	Long Telomeres, n (%)	Short Telomeres, n (%)	Long Telomeres, n (%)	Short Telomeres, n (%)
Death	1 (2)	4 (4.6)	0	3 (9.1)
Revascularization	4 (7.8)	17 (19.5)	1 (11.1)	2 (6.1)
Recurrent angina pectoris	4 (7.8)	20 (23)	2 (22.2)	3 (9.1)
Heart failure	1 (2)	6 (6.9)	0	3 (9.1)
Combined event	6 (12)	27 (31)	7 (21.2)	2 (22.2)

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