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) polymorphisms 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 manufacturers' instructions.

The metabolic 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 carriers of the N363S polymorphism, higher concentrations 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 polymorphism, 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 parameters 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 cardiovascular pathologies between genotypes were tested using the binary logistic regression model adjusted for sex.

Mortality was first estimated using the Kaplan-Meier method, followed by the calculation of sex-adjusted mortality risks and 95% confidence intervals (CI) for all-cause and cardiovascular mortality with the Cox proportional hazard model. All analyses were performed with SPSS statistical software, version 12.0.

The prevalence of cardiovascular pathologies was assessed at baseline. No associations with the prevalence of myocardial infarction, myocardial ischemia, intermittent claudication, and stroke were found for the different polymorphisms (Table 4).

Table 1: Baseline characteristics of the Leiden 85-plus Study

Number: 552
Age: 85
Female: 366 (66%)
Independently living: (cm) Men: 168.1 (6.49); Women: 156.2 (6.24). Armspan (cm) Men: 178.1 (7.31); Women: 162.5 (7.03). Cardiovascular pathologies: Myocardial infarction: 134 (24%); Myocardial ischemia: 280 (51%); Intermittent claudication: 34 (6%); Stroke: 56 (10%). Metabolic profile and body composition values are presented as means (SD), except for C-reactive protein where geometric mean (SD) is presented. During the 4.2-year follow-up, 278 (50%) of the participants died, of which 115 (41%) of deaths were due to cardiovascular disease. The Kaplan Meier estimates of cumulative mortality indicated that carriers of the BclI polymorphism have lower all-cause mortality during the follow-up compared to non-carriers (Fig. 1). The all-cause mortality risk estimate for the BclI variant was 0.85 (0.67-1.08). The cardiovascular mortality analyses revealed no differences in survival for any of the ER22/23EK, N363S, and BclI polymorphism carriers (Fig. 1). The results remained unaltered after repeating the analyses for men and women separately.

All the above-mentioned analyses were also repeated with independently living participants (n=451), in order to implement the selection criteria used in other studies (van Rossum et al., 2003, 2004a). These analyses did not reveal additional associations but confirmed those observed in the whole cohort.

Table 3 shows body composition dependent on the ER22/23EK (G/A), N363S (A/G), and BclI (C/G) polymorphisms.

ER22/23EK N363S BclI GG (n=507) GA/AA (n=33) P-value AA (n=497) AG/GG (n=51) P-value CC (n=236) CG/GG (n=290) P-value mean (SE) mean (SE) mean (SE) mean (SE) mean (SE) mean (SE)
BMI (kg/m2) Men 26.3 (0.42) 26.1 (1.40) 0.91 26.3 (0.41) 26.0 (1.39) 0.84 25.8 (0.59) 26.6 (0.54) 0.30 Women 27.8 (0.26) 27.7 (1.15) 0.99 27.7 (0.27) 27.5 (0.80) 0.77 27.2 (0.39) 28.1 (0.35) 0.06
Weight (kg) Men 74.3 (1.22) 77.9 (4.12) 0.40 74.7 (1.19) 72.8 (4.09) 0.65 73.4 (1.72) 75.2 (1.59) 0.43 Women 67.4 (0.70) 69.6 (3.07) 0.48 67.5 (0.72) 67.2(2,11 ) .90;66,4(1,05 ) .19
Height(cm)
Men:167,8(0,50);171,9(1,71);0,02
Women:156,0(0,34);158,6(1,47);0,09
Armspan (cm)
Men:177,8(0,57);180,6(1,97);0,18
Women:162,4(0,39);164,3(1,66);0,26

Univariate general linear model. Data presented as means with standard errors (SE).

Table 4 shows the risk of cardiovascular pathologies at baseline dependent on the ER22/23EK (G/A), N363S (A/G), and BclI (C/G) polymorphisms.