, USA.

for publication 22 May 2010

elegans (Tedesco et al., 2008; Mehta et al., 2009; Menuz et al., 2009).

(A) All others European African (B) All others European African

The APOE e4 allele is a risk factor for early mortality, while the alleles e2 and e3 are enriched in exceptionally old individuals in some studies (Christensen et al., 2006). Apolipoprotein E (APOE) is important in chylomicron and very low density lipoprotein (VLDL) metabolism (Mahley & Rall, 2000). Thus, it is one of the factors responsible for maintenance of circulating lipid homeostasis. This balance can break down with age, and it is clearly disrupted in some individuals resulting in an increase in the risk for cardiovascular disease and other disorders (Mahley & Rall, 2000). The rise in circulating cholesterol and triglycerides results in vascular damage and can be a cause of lipotoxicity, a potential trigger of insulin resistance and diabetes (Kusminski et al., 2009). This lipotoxicity may trigger responses that are mediated by HRAS1, a small G-protein involved in signal trans- duction (Ramos, 1999). Furthermore, some of the pathways that are induced are modulated by ceramide, a product of the cera- mide synthase LASS1 (Jazwinski & Conzelmann, 2002; Ogret- men & Hannun, 2004). Ceramide synthase responds to growth and to stress signals via Tor complex TORC2 and calcineurin, respectively (Dickson, 2008). Lipotoxicity stimulates ceramide synthesis (Kusminski et al., 2009), and the resulting ceramide can signal apoptosis, resulting in the removal of damaged cells (Koybasi et al., 2004). It can also facilitate the transfer of choles- terol from cells to high density lipoprotein (HDL) (Witting et al., 2003). Therefore, we postulate that the HRAS1 and LASS1 genes interact genetically with the APOE gene to reduce age- related increase in lipotoxic events, and that a combination of variants or haplotypes in these three genes is associated with exceptional survival.

Fig. 1 Stratification of the populations by ethnic affiliation. Alu genotypes and Structure analysis were employed to assign subjects to two major strata, European and African, in the Georgia (A) and Louisiana (B) populations. The Structure analysis was carried out in two batches for Louisiana consisting of 434 and 435 samples to speed computation, the latter of which is shown, while all 650 samples from Georgia were analyzed in one batch. Dots: red (Georgia or Louisiana samples), green (African control samples),purple (European control samples), light blue (Asian control samples),orange control samples). Indian refers to the Indian subcontinent. Not all samples are evident owing to superposition of dots.

Results

Gene combination associated with exceptional longevity

Alu used here are 95–99% (Bamshad et al., 2003). After stratifi- cation the association of APOE with longevity persisted (Table S2). However, no such association was detected for HRAS1 or LASS1 here (Table S3).

We have initiated a genetic analysis of exceptional longevity in the Georgia and Louisiana populations. Our attention in this study was exclusively directed to APOE, HRAS1, and LASS1 in a candidate gene approach. As noted earlier, variants of APOE are associated with longevity. This pattern is also found in the Geor- gia and Louisiana populations, for centenarians and nonagenari- ans respectively (Table S1). Both the Georgia and Louisiana populations are admixed with the two major strata being of European and African origin. To address this problem, we strati- fied each population by ethnic affiliation using an identical-by- descent (IBD) genetic system (Fig. 1). We placed the subjects in each population into two major groups of African and European origin. Together, these two groups constituted 81% and 83% of subjects of 20–59 years old and 72% and 77% of centenari- ans and nonagenarians in the Georgia and Louisiana samples, respectively. The ability to stratify is markedly enhanced by the Alu system used here, which can trace genealogy within the pri- mate lineage. The mean correct prediction rates with the 100

above is associated with longevity, we genotyped the Georgia sample at the three relevant genomic regions. None of the 15 SNP examined showed significance for departure from Hardy– Weinberg proportions in either the African or European-origin populations in Georgia. (The same was found separately for the Louisiana population.) We searched for combinations of haplo- types in the three genes (15 SNP) in the Georgia population present at   0.01 or greater frequency, using expectation maxi- mization. We then tested them for association with exceptional longevity (Table S4). Such associations were detected in the sub- population of European origin in Georgia, stratified with an assignment probability of 0.9 (Table 1), but not in the subpopu- lation of African origin. They involve the e3 containing APOE

Table 1 Association of haplotypes with exceptional longevity and healthy aging