Journal of Human Hypertension
Expanded Methods and Data Supplement
This Data Supplement has been provided by the authors to give readers additional information about their work.
Supplement to:Olivi et al., The –665 C>T Polymorphism in the eNOS Gene Predicts Cardiovascular Mortality and Morbidity in White Europeans.
Cardiovascular Outcome and eNOS -S1-
Blood Pressure Measurement
After the participants had rested for 5 minutes in the sitting position, the study nurses obtained five consecutive blood pressure readings (phase V diastolic pressure) to the nearest 2 mm Hg, using mercury sphygmomanometers. Standard cuffs had a 12 24 cm inflatable portion, but if upper arm girth exceeded 31 cm, larger cuffs with 15 35 cm bladders were used. For analysis, the five blood pressure readings were averaged. Quality assurance sessions were organized at 6monthly intervals to reinforce the theoretical concepts of auscultatory blood pressure and to rehearse the procedure.1,2 At each session, the observers had to pass a test requiring them to read blood pressures from a videotape featuring a falling mercury column with simultaneous Korotkoff sounds (Measuring Blood Pressure, British Medical Association, London, 1990). For each session, a specific standard was computed by averaging the film readings of experienced senior clinical researchers. All readings from each observer had to be within 5 mm Hg of the standard. Observers failing to pass the test did not participate in blood pressure measurement until re-tested successfully at a later session. We checked digit and number preference at 6-month intervals.2
Baseline Characteristics by Sex
Participants included 1434 women and 1385 men. Women compared to men (P0.0001) had lower systolic (120.5vs.126.2 mm Hg) and diastolic (73.5vs. 76.4 mm Hg) blood pressures and higher heart rate (70.6vs. 68.1 beats per minute). The prevalence of previous cardiovascular disease was not significantly different (P=0.86) between the two sexes (7.0% vs. 7.2%). Women less frequently (P≤0.005) reported smoking (24.8% vs.29.5%) and intake of alcohol (15.2% vs.38.4%). Among smokers, the median number of cigarettes smoked per day was similar (P=0.28) in women (15; interquartile range [IQR], 9–20) than men (15; IQR, 9–20). Similarly, female drinkers (10; IQR, 7–16) consumed less (P<0.0001) grams alcohol per day than male drinkers (20; IQR, 10–29). Among 1434 women, 318 (22.2% used oral contraceptive pills or took hormonal replacement therapy.
Cardiovascular Outcome and eNOS -S1-
References
1. Staessen J, Bulpitt CJ, Fagard R, Joossens JV, Lijnen P, Amery A. Familial aggregation of blood pressure, anthropometric characteristics and urinary excretion of sodium and potassium. A population study in two Belgian towns. J Chron Dis. 1985;38(5):397-407.
2. Kuznetsova T, Staessen JA, Kawecka-Jaszcz K, Babeanu S, Casiglia E, Filipovský J et al on behalf of the EPOGH Investigators. Quality control of the blood pressure phenotype in the European Project on Genes in Hypertension. Blood Press Monit. 2002;7 (4):215-224.
Cardiovascular Outcome and eNOS -S1-
Table S1 Antihypertensive drug treatment by eNOS genotype
Characteristic / TT / CT / CCNumber in category / 32 / 437 / 2350
Number with characteristics (%)
Alpha blocking / 0 / 1 (0.2) / 5 (0.2)
Beta blocking / 0 / 26 (5.9) / 140 (5.9)
Calcium entry blockers / 0 / 8 (1.8) / 35 (1.5)
Angiotensin converting blockers / 0 / 7 (1.6) / 21 (0.9)
Angiotensin II receptor antagonists / 0 / 5 (1.1) / 9 (0.4)
Diuretics / 0 / 22 (5.0) / 106 (4.5)
Centrally acting drugs / 0 / 5 (1.1) / 12 (5.4)
None of the Pvalues for an overall difference between genotypes (P≥0.12) reached significance.
Cardiovascular Outcome and eNOS -S1-
Table S2 Baseline characteristics of participants by sex
Characteristic / Women / Men / AllNumber in category / 1434 / 1385 / 2819
Number with characteristics (%)
TT homozygosity / 12 (0.8) / 20 (1.4) / 32 (1.1)
Younger than 20 years / 196 (13.7) / 191 (13.8) / 387 (13.7)
Current smoker / 356(24.8) / 409(29.5)‡ / 765 (27.1)
Drinking alcohol / 218(15.2) / 532(38.4)§ / 750 (26.6)
Diabetes mellitus / 26 (1.9) / 14(1.0) / 40 (1.4)
Hypertension / 293(20.4) / 348(25.1)‡ / 641 (22.7)
Treated hypertension / 166 (11.6) / 119 (8.6)‡ / 285 (10.1)
Previous cardiovascular disease / 100 (7.0) / 99 (7.2) / 199 (7.1)
Use of female hormones / 318 (22.2) / … / 318 (11.3)
Mean of characteristic (±SD)
Age, year / 40.2±16.4 / 40.4±16.6 / 40.3±16.5
Body mass index, kg/m2 / 24.7±4.9 / 25.3±4.1‡ / 25.0±4.5
Blood pressure, mm Hg
Systolic / 120.5±16.3 / 126.2±14.9§ / 123.3±15.9
Diastolic / 73.5±9.7 / 76.4±10.7§ / 74.9±10.3
Heart rate, beats per minute / 70.6±9.3 / 68.1±9.7§ / 69.3±9.6
Total cholesterol, mmol/L / 5.4±1.2 / 5.3±1.9 / 5.4±1.2
HDL cholesterol, mmol/L / 1.5±0.4 / 1.2±0.3§ / 1.4±0.4
Total/HDL cholesterol ratio / 3.8±1.4 / 4.6±1.8§ / 4.2±1.7
Blood glucose, mmol/L / 5.0±1.3 / 5.0±1.3 / 5.0±1.3
Serum creatinine, μmol/L / 82.0±14.0 / 97.2±16.0§ / 89.4±16.7
HDL indicates high density lipoprotein. An ellipsis denotes not applicable. Hypertension was a blood pressure of ≥140 mm Hg systolic or ≥90 mm Hg diastolic or use of antihypertensive drugs. Diabetes mellitus was a fasting or random blood glucose of ≥7.0 mmol/L or ≥11.1 mmol/L, respectively, or use of antidiabetic drugs. Significance of the sex differences:‡ P≤0.01; § P<0.0001.
Cardiovascular Outcome and eNOS -S1-
Table S3 Endpoints in 32 TT homozygotes
PatientIdentification
Number / Fatal Endpoints / Fatal plus Nonfatal Events
Total / Cardiovascular / Cardiovascular / Cardiac / Coronary
81 / dissecting aortic
aneurysm / dissecting aortic
aneurysm / dissecting aortic
aneurysm / … / …
95 / … / … / coronary syndrome / coronary syndrome / coronary syndrome
716 / acute myocardial
infarction / acute myocardial
infarction / acute myocardial
infarction / acute myocardial
infarction / acute myocardial
infarction
1506 / pancreas cancer / … / stroke / … / …
1990 / … / … / coronary syndrome / coronary syndrome / coronary syndrome
2088 / pulmonary
embolism / pulmonary
embolism / pulmonary
embolism / … / …
2334 / acute myocardial
infarction / acute myocardial
infarction / acute myocardial
infarction / acute myocardial
infarction / acute myocardial
infarction
Cardiovascular Outcome and eNOS -S1-
Table S4. Hazard ratios associated with TT homozygosity with covariables added in a stepwise fashion
Covariable Addedin Successive Steps / Cardiovascular Mortality
(4 Deaths) / Cardiovascular Events
(7 Events) / Coronary Events
(4 Events)
Hazard ratio
(95% CI) / P / Hazard ratio
(95% CI) / P / Hazard ratio
(95% CI) / P
None / 4.11 (1.51–11.2) / 0.0056 / 2.75 (1.30–5.84) / 0.0083 / 3.10 (1.15–8.41) / 0.026
Family clusters / 4.11 (1.53-11.10) / 0.0052 / 2.75 (1.32-5.73) / 0.0067 / 3.10 (1.18-8.17) / 0.022
Sex / 3.98 (1.50.10.54) / 0.0055 / 2.65 (1.35-5.19) / 0.0045 / 2.84 (1.16-6.94) / 0.0221
Age / 7.06 (2.93-17.00) / 0.0001 / 2.95 (1.49-5.81) / 0.0019 / 3.07 (1.37-6.86) / 0.0063
Systolic pressure / 5.53 (2.18-14.06) / 0.0003 / 2.65 (1.31-5.34) / 0.0066 / 2.82 (1.29-6.18) / 0.0097
Body mass index / 5.56 (2.19-14.12) / 0.0003 / 2.64 (1.31-5.33) / 0.0069 / 2.81 (1.29-6.18) / 0.0094
Smoking / 6.04 (2.41-15.11) / 0.0001 / 2.64 (1.29-5.43) / 0.0082 / 2.80 (1.23-6.38) / 0.0014
Drinking / 5.90 (2.31-15.06) / 0.0002 / 2.66 (1.29-5.48) / 0.0080 / 2.84 (1.28-6.27) / 0.0100
Total cholesterol / 5.61 (2.15-14.68) / 0.0005 / 2.58 (1.25-5-35) / 0.0106 / 2.70 (1.21-6.02) / 0.0153
Antihypertensive treatment / 6.01 (2.26-16.00) / 0.0003 / 2.64 (1.27-5.46) / 0.0091 / 2.89 (1.28-6.50) / 0.010
Hazard ratios (95% confidence interval) express the risk in TT homozygotes relative to C allele carriers. Pvalues are for the hazard ratios.
Cardiovascular Outcome and eNOS -S1-
Table S5. Hazard ratios associated with TTHomozygosity in sensitivity analyses
Endpoint(number of endpoints in TT homozygotes) / Model 1:
Removing 387 Teenagers
from Analysis / Model 2:
Substituting Systolic Pressure by Pulse pressure and Mean
Arterial Pressure / Model 3:
Removing Systolic Pressure and Antihypertensive Drug Treatment as Covariables
Hazard ratio
(95% CI) / P / Hazard ratio
(95% CI) / P / Hazard ratio
(95% CI) / P
Total mortality (5) / 2.60 (1.21–5.58) / 0.0146 / 2.64 (1.21–5.69) / 0.013 / 2.84 (1.28–6.26) / 0.010
Cardiovascular mortality (4) / 6.01(2.26–15.97) / 0.0003 / 6.60 (2.42–17.9) / 0.0002 / 7.09 (2.75–18.3) / 0.0001
Cardiovascular events (7) / 2.65 (1.28–5.49) / 0.0090 / 2.72 (1.33–5.57) / 0.0063 / 2.87 (1.39–5.92) / 0.0045
Cardiac events (4) / 2.02 (0.91–4.49) / 0.084 / 2.09 (0.95–4.58) / 0.066 / 2.20 (0.96–5.02) / 0.062
Coronary events (4) / 2.90 (1.29–6.49) / 0.0098 / 2.93 (1.32–6.53) / 0.0084 / 2.99 (1.30–6.90) / 0.010
Hazard ratios (95% confidence interval) express the risk in TT homozygotes relative to C allele carriers. All models accounted for family clusters and were adjusted for baseline characteristics including sex, age, systolic blood pressure, body mass index, smoking and drinking, total cholesterol and antihypertensive drug treatment. Pvalues are for the hazard ratios. Model 1 does not include participants younger than 20 years. In Model 2, systolic blood pressure was substituted by pulse pressure (systolic pressure minus diastolic pressure) and mean arterial pressure (diastolic pressure plus one third of pulse pressure). Model 3 did not include any blood pressure component and antihypertensive treatment.
Cardiovascular Outcome and eNOS -S1-
Figure S1. Kaplan Meier cumulative incidence estimates by eNOS genotype for all-cause (A) and for cardiac events (P). Pvalues denote the significance of the log-rank test comparing TT homozygotes with C allele carriers. The differences between CT and CC genotypes were not significant (P≥0.54).