Survival Probability0.8oa°BR°io 4 6Years Follow-up

Subjects PAI-1 Genotype Elderly* Young† 5 5 All subjects n 354 n 250 5G/5G 67 (18.9%) 59 (23.6%) 4G/5G 190 (53.7%) 125 (50.0%) 4G/4G 97 (27.4%) 66 (26.4%) 5 5 Men only n 111 n 139 5G/5G 17 (15.3%) 36 (25.9%) 4G/5G 61 (55.0%) 70 (50.4%) 4G/4G 33 (29.7%) 33 (23.7%) 5 5 Women only n 243 n 111 5G/5G 50 (20.6%) 23 (20.7%) 4G/5G 129 (53.1%) 55 (49.5%) 4G/4G 64 (26.3%) 33 (29.7%)

1. Kaplan-Meier estimate of 10-year cumulative survival $ to ACE genotype for subjects 85-years-old.

whose families originated from the Leiden area (n 250) were in Hardy-Weinberg equilibrium (Table 3). The geno- type frequencies in young subjects were similar to those previously reported for white subjects (23,25). No overall significant differences were observed in PAI-1 genotype 5 distribution (p 0.37) or for men and women separately 5 5 (p 0.11 and p 0.78, respectively). The mortality risk up to the age of 85 years associated with the PAI-1 4G/4G genotype was estimated at 0.8 (95% CI 0.5 to 1.2) as compared with that associated with the PAI-1 5G/5G genotype.

*Median age 89 years (range 85 to 100). †Median age 31 years (range 18 to 40). Data are presented as the number (%) of subjects. 5 PAI-1 plasminogen activator inhibitor-1.

compared with carriers of the PAI-1 5G/5G genotype (Table 4). Similar estimates were obtained for men and women.

Among men, the PAI-1 4G/4G genotype was associated with an increased risk of fatal ischemic heart disease (Table 4). Assuming a recessive effect (i.e., combining PAI-1 4G/5G and 5G/5G as a single reference group), the RR was estimated at 5 3.1 (95% CI 1.2 to 7.6, p 0.015). None of the men carrying the PAI-1 4G/4G genotype who died of ischemic heart disease were diagnosed as having diabetes at baseline.

Figure 2 shows the 10-year survival of elderly subjects according to PAI-1 genotype. Elderly carriers of the PAI-1 4G/4G genotype were not at an increased risk for death from any cause (RR 0.9, 95% CI 0.7 to 1.1) or from cardiovascular disease (RR 0.9, 95% CI 0.6 to 1.3) as

Table 2. Ten-Year All-Cause and Cardiovascular Disease Mortality Risks According to Angiotensin-Converting Enzyme Inhibitor $ Genotype in Subjects 85 Years Old

Cardiovascular Ischemic Heart Cerebrovascular All-Cause Disease Disease Disease ACE Genotype n RR* 95% CI RR 95% CI RR 95% CI RR 95% CI 5 5 5 5 All subjects 638 n 569 n 241 n 61 n 79 I/I 144 1 1 1 1 I/D 316 1.2 (0.9–1.4) 1.3 (0.9–1.8) 1.2 (0.6–2.2) 1.1 (0.6–1.8) D/D 178 0.9 (0.7–1.1) 0.8 (0.6–1.2) 0.7 (0.3–1.4) 0.8 (0.4–1.5) 5 5 5 5 Men only 182 n 160 n 62 n 20 n 19 I/I 46 1 1 1 1 I/D 85 1.6 (1.1–2.3) 1.7 (0.9–3.3) 1.7 (0.6–4.7) 1.4 (0.4–4.6) D/D 51 1.2 (0.8–1.9) 1.2 (0.6–2.5) 0.6 (0.1–2.5) 1.8 (0.5–6.1) 5 5 5 5 Women only 456 n 409 n 179 n 41 n 60 I/I 98 1 1 1 1 I/D 231 1.0 (0.8–1.3) 1.1 (0.8–1.7) 1.0 (0.4–2.1) 0.9 (0.5–1.8) D/D 127 0.8 (0.6–1.0) 0.7 (0.5–1.1) 0.6 (0.3–1.6) 0.6 (0.3–1.2)

*Relative risk (RR) indicates the mortality risk as estimated by a Cox proportional hazard model adjusted for age at baseline. 5 CI confidence interval; other abbreviations as in Table 1.

1.5) from cardiovascular disease as compared with that of a reference group of double heterozygous subjects and sub- jects homozygous for the ACE I-allele and/or the PAI-1 5G-allele. It was not possible to test for an association with death from ischemic heart disease because the number of subjects was too small.

1.0     08 —— PAI 5G/5G2 . —— PAI 4G/5G5 —— PAI 4G/4G§ 06gaS0425ono0.20.0104 6 8Years Follow-up

DISCUSSION

ACE I/D polymorphism. No evidence was obtained for increased (cardiovascular) mortality among carriers of the ACE D/D genotype in the cross-sectional comparison of young and elderly subjects or in the 10-year follow-up study among elderly subjects. Other cross-sectional studies also showed similar ACE D/D frequencies in elderly subjects of about the same age as those in our study (11,37). A decreased prevalence of the ACE D/D genotype in hyper- $ tensive subjects 60 years old was reported, suggesting that the ACE D/D genotype may confer an increased mortality in this high risk subgroup (38). Increased mortality in the population at large has, however, not been observed in our study.

Figure 2. Kaplan-Meier estimate of 10-year cumulative survival $ according to PAI-1 genotype for subjects 85 years old.

The analyses were repeated for elderly subjects with 5 diabetes (n 69) because they can be expected to have elevated plasma triglycerides, insulin and glucose. In this subset, the all-cause mortality risks associated with the PAI-1 4G/4G and 4G/5G genotypes were 0.4 (95% CI 0.2 to 0.9) and 0.6 (95% CI 0.3 to 1.2), respectively, as compared with that associated with the 5G/5G genotype.

Although the neutral ACE I/D polymorphism explains 14% to 25% of the variance in ACE level (1,2), there is much debate about the significance of the relation between the ACE polymorphism and cardiovascular disease. The conflicting results of the studies performed thus far can be attributed to differences in clinical phenotype (such as myocardial infarction and coronary artery disease), differ- ences in the age of the subjects, representativeness of control subjects and differences in environmental and genetic back- ground. Moreover, a meta-analysis of the association be- tween the ACE I/D polymorphism and myocardial infarc-

Combined effects of ACE I/D and PAI-1 4G/5G poly- morphisms. The prevalence of homozygosity for both the ACE and the PAI-1 risk allele was similar among elderly subjects and young subjects of Leiden origin (6.3% and 7.6%, respectively; data not shown). Separate analysis of men and women did not reveal significant differences.

The 10-year mortality risk of elderly subjects carrying both the ACE D/D and PAI-1 4G/4G genotype was 0.8 (95% CI 0.6 to 1.2) from any cause and 0.9 (95% CI 0.5 to