ACE catalyzes the conversion of angiotensin I to angio- tensin II, which is a potent vasoconstrictor and promotes vascular smooth muscle cell growth and degrades bradyki- nin, which is a vasodilator. The ACE insertion/deletion (I/D) polymorphism arises from the presence or absence of

Manuscript received December 31, 1998; revised manuscript received May 7, accepted June 23, 1999.

ACE and PAI-1 Gene Variants and Mortality

(Leiden 85-plus study [30]). The influence of the gene variants on mortality before the age of 85 years was studied in a cross-sectional design by comparing the genotype $ distributions in Leiden-born subjects 85 years old with those in young subjects whose families originated from the same geographic region (31). The relation of the gene variants to all-cause and cardiovascular mortality above the age of 85 years was investigated in a prospective study with a 10-year follow-up period using the complete elderly cohort. During follow up, the all-cause mortality rate was 89% and the cardiovascular mortality rate was 38%.

an alu repeat located in intron 16 of the ACE gene ’ (frequency of ACE D-allele in whites 0.54) (1). The ACE D-allele is associated with increased ACE activity in plasma (1,2) and tissue (3). Several studies have reported an increased frequency of the ACE D/D genotype in survivors of myocardial infarction as compared with healthy subjects (4–7). However, these results have been challenged by the negative findings from other studies (8–15). In addition to the ongoing debate on the deleteriousness of the ACE D/D genotype, a beneficial influence on survival in very old age was suggested by the increased frequency of the ACE D/D genotype in French centenarians (16).

METHODS

Subjects. The Leiden 85-plus study is a population-based study in which all inhabitants of Leiden, The Netherlands, $ aged 85 years were invited to take part (30). Of a total of 1,258 eligible subjects, 221 died before enrollment, which lasted from December 1, 1986 to March 1, 1988. Of the 1,037 remaining subjects, 977 (94%) participated and were medically interviewed at home. Diabetes was diagnosed on . the basis of the previous history or a glucose level 11.0 mmol/liter in a nonfasting blood sample, or the use of medication for treatment of diabetes. After the exclusion of 5 5 subjects with a non-Dutch (n 29) or unknown (n 69) place of birth, sufficient cell material was available from 666 (188 men and 478 women) subjects for the present genetic study. Deoxyribonucleic acid was extracted by protein pre- cipitation using potassium acetate and chloroform. The ACE genotypes were determined as previously described (32). Because the ACE I/D genotype is erroneously mistyped as ACE D/D in up to 5% of cases, the presence of the ACE D/D genotype was confirmed with an insertion- specific polymerase chain reaction (PCR) amplification (33). The PAI-1 4G/5G genotypes were determined using an allele-specific PCR amplification described by Falk et al. (34). It was not possible to determine the ACE genotype in 26 cases and the PAI-1 genotype in 22 cases for technical reasons. The ACE and PAI genotypes were independently assessed by two observers. The study was approved by the Medical Ethics Committee of the Leiden University, and informed consent was obtained from all participants.

The primary inhibitor of fibrinolysis is PAI-1. A 4/5- guanine-tract (4G/5G) polymorphism was identified in the promoter of the PAI-1 gene 675 base pairs upstream from the start of transcription (17). The PAI-1 4G allele is associated with elevated PAI-1 levels in plasma (frequency ’ of PAI-1 4G-allele in whites 0.52) (17–19). This associ- ation is especially strong among subjects with relatively high plasma levels of triglycerides, insulin or glucose (20,21). Studies in vitro indicate that the 4G-allele is unable to bind a repressor and is associated with increased transcription of the PAI-1 gene (17,18). In addition, a very low density lipoprotein responsive element was found to partly overlap the guanine tract, which may provide a molecular explana- tion for the modification effect by triglycerides (22). The elevated plasma PAI-1 level in 4G/4G homozygotes may result in an increased risk of coronary heart disease as a consequence of a diminished fibrinolytic capacity. Studies investigating this hypothesis, however, have produced con- flicting results (18,19,23–26).

If a deleterious effect of the ACE D-allele and the PAI-1 4G-allele is present, carriers of both risk alleles may be especially susceptible to disease owing to the link that exists between the renin–angiotensin system and fibrinolytic func- tion. Infusion of angiotensin II increases plasma PAI-1 activity in humans (27), and recently, two studies showed that the ACE D/D genotype was associated with elevated PAI-1 levels in plasma (28,29). Hence, the ACE and PAI-1 polymorphisms may be involved in cardiovascular disease in part by the same etiologic pathway.

Cross-sectional analysis. The ACE and PAI-1 genotype $ distributions were compared between elderly subjects 85 years old and young control subjects. To avoid false associ- ations with the ACE and PAI-1 polymorphisms due to differences in geographic origin rather than age, only those $ 5 subjects 85 years old who were born in Leiden (n 356, 55%) were compared with a control population that con- sisted of 250 (139 men and 111 women) blood donors 18 to 40 years old of Dutch descent with either two Leiden-born parents or one Leiden-born parent and the other born within a 12-km distance from Leiden. Information regard- ing the birthplace of their grandparents was obtained from a written questionnaire.

The aim of the present study was to assess whether the ACE and PAI-1 polymorphisms, either separately or in combination, are associated with mortality in the general population. This was done using two designs within a $ population-based study among subjects 85 years old