Bastiaan T. Heijmans, MS,* Rudi G. J. Westendorp, MD, PD, Dick L. Knook, PD,* C H H Cornelis Kluft, PD,* P. Eline Slagboom, PD* from Leiden, The Netherlands studied the contribution of putative risk genotypes at the angiotensin-converting enzyme inhibitor (ACE D/D) and plasminogen activator inhibitor-1 (PAI-1 4G/4G) loci to all-cause and cardiovascular mortality in a population-based cohort.

BACKGROUND 

The ACE D/D and PAI-1 4G/4G genotypes have consistently been associated with elevated plasma activities of the gene products. Although their role in cardiovascular disease has been explored intensively, it is still equivocal.

METHODS 

The ACE and PAI-1 genotypes were determined in 648 subjects aged 85 years old. In a cross-sectional analysis, the genotype distributions in a subset of 356 elderly subjects who were born in Leiden, The Netherlands were compared with those in 250 young subjects whose families originated from the same geographic region. In addition, the complete cohort of elderly subjects was followed over 10 years for all-cause and cardiovascular mortality and was stratified according to genotype.

RESULTS 

In the cross-sectional analysis, the ACE and PAI-1 genotype distributions were similar in elderly and young subjects. In the prospective follow-up study, however, the age-adjusted risk of fatal ischemic heart disease was increased threefold (95% confidence interval [CI] 1.2 to 7.6) in elderly men carrying the PAI-1 4G/4G genotype. The risk of all-cause mortality was not increased among elderly subjects carrying the PAI-1 4G/4G (relative risk [RR] 0.9, 95% CI 0.7 to 1.1) or the ACE D/D genotype (RR 0.9, 95% CI 0.7 to 1.1). Nor did we observe elevated risks of death from all cardiovascular diseases combined. There was no interaction between the genotypes.

CONCLUSIONS 

The PAI-1 4G/4G genotype may be a risk factor for fatal ischemic heart disease in elderly men. The impact of moderately increased ACE and PAI-1 activities associated with the ACE D/D and PAI-1 4G/4G genotypes is too small to affect mortality in the general population. (J Am Coll Cardiol 1999;34:1176-83) by the American College of Cardiology.

Over the last years, a number of common gene variants have been identified that are associated with plasma levels of the gene products and the risk of cardiovascular disease. Most studies performed thus far have not addressed the association of these variants with fatal cardiovascular disease or have only included a small number of fatal cases. Also, very few studies have yet explored how combinations of possible risk genotypes affect disease risk. Establishing such genetic risk profiles is only feasible when relatively large populations are available, and the putative risk alleles have a high frequency in the general population.

Two highly frequent genetic variants that may be linked to cardiovascular disease risk have been identified in the genes encoding angiotensin-converting enzyme (ACE) inhibitor and plasminogen activator inhibitor-1 (PAI-1). ACE catalyzes the conversion of angiotensin I to angiotensin II, which is a potent vasoconstrictor and promotes vascular smooth muscle cell growth, and degrades bradykinin, which is a vasodilator. The ACE insertion/deletion (I/D) polymorphism arises from the presence or absence of 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 ofIn a study conducted from December 1, 1986 to March 1, 1988, there were 1,258 eligible subjects, out of which 221 had died before enrollment. Of the remaining 1,037 subjects, 977 (94%) participated and were medically interviewed at home. Diabetes was diagnosed based on previous history or a glucose level of 11.0 mmol/liter in a nonfasting blood sample or the use of medication for diabetes treatment. After excluding 55 subjects with a non-Dutch (n=29) or unknown (n=69) birthplace, cell material was available from 666 (188 men and 478 women) subjects for the genetic study. DNA was extracted using protein precipitation with potassium acetate and chloroform. The ACE genotypes were determined as previously described (32), with confirmation of the presence of the ACE D/D genotype using 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 Leiden University, and informed consent was obtained from all participants.