84 82 80 78 76 74 72 18 16 APOE 2 APOE 3 14 APOE 4 12 10 8 6 60 61–65 66–70 71–75 76–80 81–85 86–90 >90 Age Category, years % ,ycneuqerF elellA

Other studies with smaller sample sizes have found lower rates of mortality associated with e2 relative to e3 with age (12, 30), but our data did not support these findings, with a small but nonsignificant variation in the relation of e2 with age being observed. However, with only 66 participants over age 90 years in this study, this age category was not well represented. This may explain why previous findings of an increased e2 allele frequency in the elderly were not con- firmed in this study. Furthermore, our results did not show an effect of interaction between age and AMD phenotype on APOE allele frequency prevalence, indicative that the ob- served effect occurs independently of AMD (data not shown). Note that e4 has been previously reported to exert a protective effect against AMD (4, 15, 31, 32), and as such, an inflated frequency of this allele may be represented within the AMD- free control samples used in this study, compared with the frequency actually present in the general population.

Our findings offer further support to recently reported findings implicating the APOE region in longevity (33). Although the platform used in the study by Sebastiani et al. (33) did not genotype either rs429358 or rs7412 di- rectly, an intronic SNP rs2075650, located in a gene called TOMM40, was used as a strong proxy for rs429358, as the 2 SNPs have previously been shown to be in strong linkage disequilibrium (34). The high level of linkage disequili- brium which exists between TOMM40 and APOE makes it difficult to identify the causal variant associated with the effect observed at this locus (34). Sebastiani et al. (33) also assessed the association of this region with longevity for an interaction with gender but could find no evidence to sup- port effect modification.

Figure 1. Allele frequency distribution of apolipoprotein E gene (APOE) e2, e3, and e4 isoforms by age group in 10,623 subjects with European ancestry, 1990–2009.

isoform poses in relation to coronary heart disease, athero- sclerosis, Alzheimer’s disease, and other dementias (1–3, 5– 7). Various hypotheses have been proposed surrounding the functionality of this small, multifunctional lipid transport protein, particularly through its role as a ligand for LDL cholesterol and triglycerides and its involvement in the maintenance and repair of neuronal cells. The APOE iso- forms generated from the different alleles interact differ- ently with the lipoprotein receptors, leading to altered cholesterol levels. High levels of LDL cholesterol are asso- ciated with APOE e4, low levels with e2, and intermediate levels with e3 (29). However, the exact mechanisms regard- ing the association of APOE with increased mortality, and indeed its association with a variety of disease and patho- logic processes conferring increased or decreased risk, have yet to be elucidated.

cies needs to be carefully considered in relation to associa- tion studies of age-related conditions. Failure to do so will result in serious confounding, with an impact on unadjusted association studies that is often not appreciated; many in- vestigators assume that frequencies are constant across the life span, and this is especially so when the incidence of the disease in question increases with age (35). It has been estimated from cross-sectional studies that the frequency distribution of the e4 allele halves between the ages of 60 and 85 years (35), and this is supported by the current study. A decrease in the frequency of e4 is likely to occur as a consequence of increased risk associated with this allele in relation to coronary heart disease, atherosclerosis, Alzheimer’s disease, and other dementias (1). Association studies of age-related disorders investigating the effects of genes that may be influenced by longevity may yield greater variation in allele frequencies between controls alone than between cases and controls. While adjustment for age is im- perative to limit confounding under these circumstances, cau- tion is recommended in considering associations such as that for APOE, in case-control studies that are not well-matched for age.

A study by Seripa et al. (13) suggested an association of decreased mortality with the e2 allele in males only, partic- ularly in relation to dementia and cardiovascular disease (n ¼ 1,710; 757 males). Another smaller independent study (14) suggested that the reduced mortality associated with e2 was specific to women, and thus there is some confusion with regard to the direction and magnitude of any potential gender difference. To our knowledge, our study was one of the largest to assess the relation between APOE and age to date, and we found no evidence to suggest that the association with age and APOE was different in women compared with men; therefore, our results do not suggest that differences in longevity between men and women are explained by APOE.

The magnitude of the effect measured and the associated level of significance will be limited by sample size and the allele frequencies present within the population in question, which varies significantly in the case of APOE. APOE allele frequencies measured in this investigation (data not shown) varied by study, between 6.7% and 10.0% for e2, 75.3% and