Summary. To study associations between genetic variation and disease, large bio-banks need to be created in multicenter studies. Therefore, we studied the effects of storage time and temperature on DNA quality and quantity in a simulation experiment with storage up to 28 days frozen, at 4 8C and at room temperature. In the simulation experiment, the conditions did not influence the amount or quality of DNA to an unsatisfactory level. However, the amount of extracted DNA was decreased in frozen samples and in samples that were stored for >7 days at room temperature. In a sample of patients from 24 countries of the EUROPA trial obtained by mail with transport times up to 1 month DNA yield and quality were adequate. From these results we conclude that transport of non-frozen blood by ordinary mail is usable and practical for DNA isolation for polymerase chain reaction in clinical and epidemiological studies.

The common apolipoprotein E (APOE) alleles epsilon2, epsilon3, and epsilon4 are associated with the risk of dementia and cardiovascular disease. Recently, two functional variants (- 219G/T and -491A/T) were identified in the promoter of the APOE gene that enable a further characterization of the role of the APOE locus in disease. We investigated the contribution of these APOE gene variants to dementia and cardiovascular mortality in old age using a population-based cohort of 648 subjects aged 85 years and over (Leiden 85-Plus Study). Genotypes containing an APOE epsilon4 allele were associated with a 4.1-fold (95% CI, 2.2-7.7) increased risk of dementia as compared to the epsilon3/epsilon3 genotype in old subjects. Moreover, homozygosity for the -219T allele was found to be associated with a 2.4-fold (95% CI, 1.0-5.8) increased risk independently of epsilon2 and epsilon4; the -491A/T variant was not associated with dementia. Over a 10-year follow-up period, the risk of cardiovascular mortality was not increased among epsilon4 carriers (RR, 0.6; 95% CI, 0.4-1.0) or -219T homozygous subjects (RR, 1.1; 95% CI, 0.7-1.7), nor did it decrease among -491T homozygous subjects (RR, 1.4; 95% CI, 0.6-3.1). In conclusion, both the APOE epsilon2/epsilon3/epsilon4 and the -219G/T variant were identified as risk factors for dementia but not cardiovascular mortality in old age. Our results support the hypothesis that both the isoform and the amount of APOE may influence the risk of dementia. Furthermore, they emphasize that variation at the APOE locus has a higher impact on the risk of dementia than on the risk of cardiovascular disease in old age.

High-fat diets (HFD) are thought to contribute to the development of metabolism-related diseases. The long-term impact of HFD may be mediated by epigenetic mechanisms, and indeed, HFD has been reported to induce DNA methylation changes in white adipose tissue (WAT) near metabolism related genes. However, previous studies were limited to a single WAT depot, a single time-point and primarily examined the pre-pubertal period. To define dynamic DNA methylation patterns specific for WAT depots, we investigated DNA methylation of Pparg2 and Leptin in gonadal adipose tissue (GAT) and subcutaneous adipose tissue (SAT), at baseline and after 6, 12 and 24 weeks of HFD exposure in adult mice. HFD induced hypermethylation of both the Leptin promoter (max. 19.6% at week 24, P = 2.6·10−3) and the Pparg2 promoter in GAT (max. 10.5% at week 12, P = 0.001). The differential methylation was independent of immune cell infiltration upon HFD exposure. In contrast, no differential methylation in the Pparg2 and Leptin promoter was observed in SAT. Leptin and Pparg2 DNA methylation were correlated with gene expression in GAT. Our study shows that prolonged exposure to HFD in adulthood is associated with a gradually increasing DNA methylation level at the Leptin and Pparg2 promoters in a depot-specific manner.

We conclude that the E3L.CETP mouse is a promising novel translational model to investigate the effects of new drugs, alone or in combination, that affect IR, diabetic dyslipidemia and non-alcoholic fatty liver disease (NAFLD).

An elevated level of homocysteine in plasma is associated with the occurrence of cardiovascular disease. A common ala-to-val mutation in the methylenetetrahydrofolate reductase gene (MTHFR) is associated with an elevated level of plasma homocysteine. We studied the possible detrimental effects of the MTHFR mutation on mortality. Within a population-based study in the city of Leiden, the Netherlands, we first compared the MTHFR genotype distribution among 365 elderly subjects aged 85 years and over born in Leiden, and 250 young subjects aged 18 to 40 years whose families originated from the same geographical region. Second, the complete cohort of 666 subjects aged 85 years and over was followed over a period of 10 years for all-cause and cause-specific mortality and stratified according to MTHFR genotype. The frequency of the MTHFR mutation was significantly lower in the elderly than in the young (0.30 and 0.36, respectively; P = 0.03). The difference in genotype distribution was only present in men. The estimated mortality risk up to 85 years in men carrying the val/val genotype was 3.7 (95% confidence interval (CI), 1.3-10.9). Over the age of 85, mortality in men with the val/val genotype was increased 2.0-fold (95% CI, 1.1-3.9) and appeared to be attributable to cancer rather than cardiovascular causes of death. Among women aged 85 years and over, no deleterious effect of the MTHFR mutation was observed. In conclusion, the MTHFR mutation is associated with increased mortality in men in middle and old age, but not in women.