Katja Lottermoser · Rainer Düsing · Peter Ervens Brigitte Koch · Thomas Brüning · Agapios Sachinidis Hans Vetter · Yon Ko

The plasminogen activator inhibitor 1 4G/5G polymorphism is not associated with longevity: a study in octogenarians

Received: 5 July 2000 / Accepted: 15 October 2000 / Published online: 2 March 2001 © Springer-Verlag 2001

ined the effect of the PAI-1 4G/5G genotype on longevi- ty among 205 subjects aged 80 years and older. Such studies in larger cohorts have recently become available along with new methods for the rapid and easy determi- nation of gene polymorphisms. We utilized a light-cycler assisted method which is a fast and flexible method of analyzing the PAI-1 4G/5G polymorphism on the gene level. In these 205 persons the 4G/5G allele was found in 96 persons (47%), the 4G/4G variant in 62 (30%), and the 5G/5G allele in 47 (23%). These data are similar to the allele distribution described in other large cohorts not restricted to old age. Thus the results of this study are not suggestive of an important contribution of the PAI-1 genotype on total mortality.

Abstract Accumulating evidence suggests that plasma levels of the plasminogen activator inhibitor 1 (PAI-1) may modulate the risk of coronary artery disease. The regulation of PAI-1 levels underlies not only environ- mental but also genetic influences. The 4G/5G polymor- phism of the PAI-1 gene has recently gained additional relevance as a possible cardiovascular risk factor, as the 4G allele may be associated with enhanced expression of the PAI-1 gene. This retrospective cohort study exam-

KATJA LOTTERMOSER YON KO received her M.D. degree in received his M.D. degree in medicine from the University internal medicine from the of Bonn, Germany. She is University of Bonn, Germany. presently a senior resident at He is presently head of the De- the Department of Internal partment of Hematology and Medicine of the University Oncology at the University Hospital Policlinic in Bonn. Hospital Policlinic in Bonn. Her research interests include His research interests include non-blood-pressure related ef- pharmacogenomics and gene fects of the renin-angiotensin- expression studies in various aldosterone system with spe- cell systems. cial respect to the fibrinolytic system.    

Abbreviations PAI-1: Plasminogen activator inhibitor 1 PCR: Polymerase chain reaction · t-PA: Tissue-type plasminogen activator

Introduction

The primary function of the plasminogen activator inhib- itor 1 (PAI-1) in vivo is to regulate fibrinolysis. There is increasing evidence that elevated levels of PAI-1 may also play a role in the development of cardiovascular dis- ease [1, 2, 3]. In this context it is interesting to note that raised PAI-1 plasma levels may be associated with a 1-bp guanine deletion/insertion (4G/5G) polymorphism in the promoter region of the PAI-1 gene [4, 5]. Never- theless, studies on the significance of this polymorphism as a risk predictor in individuals with or without athero- thrombotic disease to date have revealed conflicting re- sults [6, 7, 8], with the majority of recent studies denying a correlation between the PAI-1 polymorphism and car- diovascular disease [9, 10, 11, 12]. Therefore it still re- mains unclear whether the PAI-1 polymorphism, as with some other single-nucleotide polymorphisms [13], is as-

K. Lottermoser · R. Düsing ( ) · P. Ervens · B. Koch T. Brüning · A. Sachinidis · H. Vetter · Y. Ko Medizinische Universitäts-Poliklinik, Wilhelmstrasse

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Bonn, Germany e-mail: duesing@uni-bonn.de Tel.: +

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Table 1 PCR primers and hybridization probes for the PAI-1 4G/5G polymorphism. The hybridization probes were constructed as pub- lished by Nauck et al. [15] (boldfaced fluorescein-labeled 5G-hybridization probe, LCR Light-Cycler red, FL fluorescein)

sociated with an increased risk in morbidity and mortali- ty. The accurate assessment of its age dependency and proportional distribution in the population could provide additional information on the (patho)physiological rele- vance of the 4G/5G polymorphism.

hybridization probe, 20 pmol of each PCR primer, 2 µl Light-Cy- cler DNA master hybridization mix (Roche), 2 µl DNA (100 pg–10 ng) in a final volume of 20 µl. After 45 s of denatur- ation at 94°C, PCR cycles were performed with 0 s denaturation at 94°C, 5 s annealing at 57°C, and 2 s extension at 72°C for 50 cy- cles. Melting curves were achieved following a denaturation peri- od of 0 s at 94°C at a start temperature of 45°C and an end tem- perature of 75°C with a temperature increase of 0.2°C/s. PCR and melting procedure were detected online with the Light-Cycler in- strument.

As PAI-1 is of biological significance in causing an elevated risk of thromboembolic disease, the determina- tion of this PAI-1 polymorphism may have important im- plications in the early detection of asymptomatic patients who have an increased risk of developing cardiovascular disease. In the present study we evaluated the 4G/5G PAI-1 polymorphism in 205 persons aged 80 years or older to evaluate whether aging is associated with the predominance of a certain PAI-1 genotype, possibly indi- cating the selection of a favorable allele.

Statistical methods

Statistical analysis of data was performed using Excel (Microsoft Office 2000). Data are expressed as a percentage of all studied in- dividuals.

Such investigations of the biological basis of human longevity have not frequently been attempted in the past because of a lack of efficient and cost-effective methods to analyze gene polymorphisms in large numbers of indi- viduals. With a real-time polymerase chain reaction (PCR) assisted approach a high throughput analysis of the PAI-1 4G/5G polymorphism has been made possible [15].

Results