Results

Figure 1A presents the online fluorescence curves of the PCR amplification of the PAI-1 promoter region cover- ing the 4G/5G polymorphic site. All samples show an exponential increase in fluorescence intensity as a mea- surement for the quantity of amplified fragments. The negative PCR control remained negative. The real-time PCR-assisted analysis of the PAI-1 4G/5G polymor- phism was carried out by the online analysis of the melt- ing curves of the hybridization probes. Figure 1B shows melting curves of three samples (fluorescence over time), and Fig. 1C shows the first differentiation of the fluorescence (F) over time (T) (–dF/dT). The melting maximum of the 5G hybridization probe is 59.5°C for the 4G/4G genotype, 59.5°C and 65°C in the case of the 4G/5G genotype, and 65°C in the case of the 5G/5G gen- otype.

Materials and methods

Subjects

Blood samples from 205 white donors (147 women, 58 men; aged 80–98 years, mean 86.1) were recruited from three different homes for the aged in the city of Bonn, Germany. All volunteer subjects provided information on their medical history. Their mean body mass index was 22.6. The series included 44 current or former smokers, 39 persons with a history of myocardial infarc- tion, 36 with a history of stroke, 87 with arterial hypertension, 30 with diabetes mellitus, and 34 with malignant disease. Whole blood was collected from an antecubital vein into vacuum contain- ers, containing EDTA. Before entering the study, each subject gave informed consent. The study protocol was approved by the Ethics Committee of the University of Bonn.

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%; Table 2).

Subgroup analysis was performed for subjects who had a history of myocardial infarction and/or a history of stroke (Table 2). Among the combined group of patients with cardiovascular disease (33.7% of the entire cohort), the allele frequencies were nearly identical to those among subjects without a history of cardiovascular dis- ease within our study group.

DNA-extraction

Genomic DNA was isolated from whole blood using the QiAmp Blood Maxi Kit (Qiagen, Hilden, Germany).

Real-time PCR

Discussion

PCR was performed utilizing a Light-Cycler (Roche, Mannheim, Germany) using hybridization probes in combination with the Light-Cycler DNA master hybridization probes kit (Roche, Mann- heim, Germany) as described by Nauck et al. [15]. The PCR prim- ers and the hybridization probes are shown in Table 1. The hybrid- ization probes were synthesized by TIB MOLBIOL (Berlin, Ger- many). The PCR conditions were 4 mM MgCl , 1 pmol of each 2

Several sequence variations in the promoter region of the PAI-1 gene, especially the 4G/5G polymorphism, have been described [16, 17]. 4G/5G polymorphism refers to a guanosine insertion/deletion polymorphism in the pro-

moter region of the PAI-1 gene, 675 bp upstream from the start of transcription. In vitro studies suggest that the 4G allele is associated with higher PAI-1 activity than the 5G allele [18]. One possible mechanism may be de- rived from the finding that while both alleles bind a tran- scriptional activator, the 5G allele contains an additional binding site for a DNA-binding protein that could be a transcriptional repressor [18]. In the absence of bound repressor the basal level of PAI-1 transcription is in- creased [4, 18]. As gene variants of PAI-1 obviously ac- count for a significant portion of the interindividual vari- ability in circulating PAI-1 antigen concentrations in the general population [19], there is increasing interest in the determination of the genotype of PAI-1. In a large cohort of men in the United States aged 40–84 years the overall allele distribution has been reported as approximately 25% 4G/4G, 25% 5G/5G, and 50% 4G/5G genotype [20].

real-time PCR||   fluorescence (F)cyclesmelting curvesfluorescence (F)-dF/dT 

erence values for the distribution of the 4G/5G alleles among cohorts in the United States, Japan, and Europe. A similar pattern of distribution as shown in these stud- ies was also demonstrated in our group of 205 elderly subjects (Table 2). Subgroup analysis comparing the fre- quency of the PAI-1 4G/5G alleles between subjects with and without a history of cardiovascular disease yielded no further information as the allele frequencies among those two groups did not differ significantly (Table 2). Thus homozygosity for the 4G allele, despite its associa- tion with impaired fibrinolysis, is compatible with reach- ing old age. This contrasts with the findings of a previ- ous study [21] which demonstrated a higher proportion of the 4G allele and the homozygous 4G/4G genotype in centenarians than in young individuals (P=0.021 and P=0.049, respectively). The latter results suggest an even better survival for individuals with the PAI-1 4G geno- type despite its association with impaired fibrinolysis. Individuals with the homozygous 4G/4G genotype did not suffer more frequently from stroke or myocardial in-

Fig. 1A–C Analysis of the PAI-1 promoter region covering the 4G/5G polymorphic site using a real-time PCR approach. A Real- time PCR of PAI-1 4G/5G polymorphism. B Melting curves of 30 samples (fluorescence over time). C First differentiation of the fluorescence (F) over time (T; –dF/dT). The melting maximum of the 5G-hybridization probe is 59.5°C in the case of the 4G/4G genotype, 59.5°C in the case of the 4G/5G genotype, and 65.0°C in the case of the 5G/5G genotype

Table 2 Genotype distribution of the PAI-1 4G/5G polymorphism among octogenarians

n % PAI-1 4G/4G PAI-1 4G/5G PAI-1 5G/5G Total population 205 – 62±30.2 96±46.8 47±22.9 Myocardial infarction 38 18.5 13±34.2 15±39.5 10±26.3 Stroke 35 17.1 11±31.4 16±45.7 8±22.8 Cardiovascular disease 69 33.7 22±31.9 32±46.4 15±21.7 No cardiovascular disease 136 66.3 41±30.2 64±47.1 31±22.8