Subjects and methods

Subjects

The Leiden 85-plus Study is a prospective population based study, in which all 85-year-old inhabitants of Leiden, the Netherlands were invited to take part. There were no selection criteria related to health or demographic characteristics. The study population consists of 599 subjects, all members of the 1912– 1914 birth cohort, enrolled in the month of their 85th birthday between 1997 and 1999 (Bootsma-van der Wiel et al. 2002). For the present study DNA was available for 563 people. All participants of the Leiden 85-plus Study were followed for mortality until April 1st 2004. Primary causes of death were obtained from the Dutch Central Bureau of Statistics and categorized according to the 10th International Classification of Diseases (ICD-10). The Medical Ethical Committee of the Leiden University Medical Centre approved the study and informed consent was obtained from all participants. We also genotyped 370 blood donors (median age 32, inerquartile range (27–36)) from Leiden and surrounding areas for a cross-sectional analysis of genotype frequencies (Heijmans et al. 1999).

C-reactive protein, high-density lipoprotein (HDL)- cholesterol, triglycerides and HbA1c (hemoglobin A1c) concentrations were determined in serum using fully automated analyzers (Hitachi 747 and 911; Hitachi, Ltd, Tokyo, Japan). Low-density lipoprotein (LDL)-cholesterol was estimated with the Frieden- wald equation (Friedewald et al. 1972). Cortisol was determined by a fluorescence polarization immuno- assay using the Abbott TDx (Abbott Laboratories, Abbott Park, IL, USA) according to the manufac- turer’s instructions. The within-assay coefficient of variation (CV) was below 5% at different levels. Body weight (kg) and height (cm) were measured in all participants and body mass index (BMI, kg/m2) was calculated. The measurement of height is often

In this study we assessed the impact of the ER22/ 23EK, N363S and BclI variants in the GR gene on metabolic profile, body composition, and on the prevalence of cardiovascular pathologies. Also the risks of allcause and cardiovascular mortalities dependent on the polymorphisms were calculated. The study was carried out in the population-based

Results

The prevalence and number of cardiovascular pathol- ogies were obtained from the participant’s general practitioner or nursing home physician. In addition, electrocardiograms were recorded on a Siemens Sic- card 440 and transmitted by telephone to the ECG Core Lab in Glasgow for automated Minnesota coding (Macfarlane and Latif 1996). Cardiovascular pathol- ogies were classified as: myocardial infarction, myo- cardial ichemia, intermittent claudication or stroke (van Exel et al. 2002).

DNA was available for 563 participants, of which 11 were excluded due to the use of corticosteroids. The baseline characteristics of the remaining 552 partici- pants are presented in Table 1. All study subjects were genotyped for the ER22/23EK, N363S and BclI polymorphisms with a genotyping error of lower than 5%. Therefore, the total number of analyzed partici- pants was 540 for ER22/23EK, 548 for N363S and 526 for the BclI polymorphisms. The minor allele frequencies were 0.03, 0.05 and 0.34 for the ER22/ 23EK, N363S and BclI polymorphisms, respectively. The overall genotype distributions and resulting allelic frequencies of the SNPs were in agreement with the distribution predicted by the Hardy–Wein- berg equilibrium. In this study, the calculated allele and genotype frequencies did not differ between elderly and young subjects (n=370, median age 32 years)(data not shown).

Genotyping

The ER22/23EK variant consists of two single nucleotide polymorphisms (SNPs) in codons 22 (A/ G, rs6189) and 23 (G/A, rs6190). The ER22/23EK, N363S (A/G, rs6195) and BclI (C/G) (van Rossum et al. 2003) polymorphims were genotyped using an Assay-by-Design (Applied Biosystems), consisting of PCR primers and TaqMan MGB probes on an ABI 7900 HT with real-time PCR (Applied Biosystems). Amplification reactions and parameters were based on manufacturer’s instructions.

profile dependent on the ER22/23EK, N363S and BclI variants were assessed. The analyses revealed that carriers of the ER22/23EK variant have higher HbA1c levels (6.54 (0.19) vs. 5.74 (0.05), P < 0.001) compared to the non-carriers (Table 2). For the car- riers of the N363S polymorphism, higher concentra- tions of LDL cholesterol (4.18 (0.13) vs. 3.62 (0.04), P < 0.001) and triglycerides (1.60 (1.06) vs. 1.39 (1.02), P=0.03) compared to the non-carriers were observed (Table 2). No associations between the metabolic profile and the BclI polymorphism were found. The analyses of body composition showed only differences in height for the ER22/23EK poly- morphism, where men carrying the ER22/23EK variant were taller (171.9 (1.71) vs. 167.8 (0.50), P=0.02) compared to the non-carriers. The same trend was observed for women (Table 3).

Statistical analysis

Allele frequencies were calculated and analyzed for deviation from the Hardy–Weinberg equilibrium using the v2-test. Mean differences in the parame- ters of the metabolic profile and body composition dependent on the polymorphisms were assessed with the univariate general linear model. All continuous variables were normally distributed, except for the C-reactive protein and triglyceride levels, which were ln-transformed. Differences in the prevalence of car- diovascular pathologies between genotypes were tes- ted using the binary logistic regression model adjusted for sex. Mortality was first estimated using the Kap- lan–Meier method, followed by the calculation of sex adjusted mortality risks and 95% confidence intervals (CI) for allcause and cardiovascular mortality with the Cox proportional hazard model. All analyses were performed with SPSS statistical software, version 12.0