Fig. 4 Impact of polymorphisms in LASS1 on gene expression. A luciferase reporter assay was used for determining the promoter activity in transfected HeLa cells. Error bars denote SEM for six independent experiments with at least six replicates for each of the four promoter constructs shown. The LASS1 promoter variants CCT and CGT associated with exceptional longevity were compared to an AGC variant. The control is a reporter lacking inserted promoter sequences.

Discussion

We have found a combination of variants in three genes that is associated not only with exceptional longevity but also with healthy aging. This is a novel finding. An association of HRAS1 with longevity was found before, and an interaction with the mitochondrial genome was demonstrated (Bonafe´ et al., 2002). A compensatory effect in late life of variants in one gene for the deleterious effects of variants in a second gene has also been described (Bergman et al., 2007). The combined effect described here involves three genes and extends the association

Circulating Aging Clearance lipids LASS APOE Vascular HDL damage Tissue infiltration S ur vi v HRAS r e s p oal a n d Lipotoxicity n s e sstr e s s Dysfunction LASS Apoptosis and morbidity

old age. Genetic interactions play an important role in complex traits, but they can be difficult to find unless a hypothesis-driven approach such as we use here is employed that reduces the scale of the search and affects the prior probabilities involved (Thorn- ton-Wells et al., 2004; Flint & Mackay, 2009). Unmasking the interactions of common variants mitigates the necessity of invoking the wide involvement of rare variants in complex traits.

The association with healthy aging and the profile of increas- ing frequencies of the haplotypes with age indicates that the three genes studied here are not frailty genes or haplotypes but instead genetic determinants of longevity. The foundation for the genetic interaction is laid by the haplotype in APOE. HRAS1 has a synergistic effect, and this combined effect is further enhanced by either of the two LASS1 haplotypes in a synergistic manner. Our results point to the importance of accounting for population heterogeneity in association studies (Dai et al., 2007). The African-origin subpopulation displays a very low fre- quency of the crucial APOE haplotype, as well as a reduced fre- quency of the LASS1 haplotypes. Thus, intrinsic genetic differences between the European and African subpopulations can explain the lack of observation of the three-gene interaction in the latter group. On the other hand, the associations we have discovered in the European-origin sample from Georgia are rep- licated in the corresponding Louisiana sample, though the hap- lotype structures in the two samples differ. It will be important to examine samples from additional populations for these asso- ciations to further generalize our findings and to explore poten-

Fig. 5 Hypothetical model of the role of APOE, HRAS1,and LASS1 interactions. Circulating lipids are cleared by APOE and by HDL, which is aided by LASS1-generated ceramide. Metabolic (lipid) stress induces protective survival and stress responses, which are mediated⁄modulated by HRAS1 and LASS1. Lipotoxicity enhances LASS1 production of ceramide, which signals apoptosis removing damaged cells. These mechanisms become overwhelmed with age resulting in dysfunction and morbidity.

isiana (Table 2). However, the congruence of the respective haplotypes in all three genes is relatively infrequent in the popu- lation at large, although it increases in frequency dramatically with age (Table 1 and Fig. 2). Thus, we have uncovered an individual path to exceptional longevity and healthy aging, and others remain to be found.

Experimental procedures

There are two different haplotypes in LASS1 that play a role in the three-gene combination described here. One appears to operate in females and the other in males to determine excep- tional longevity. Furthermore, it is only the latter that is associ- ated with healthy aging. Thus, similar but distinct mechanisms of aging are at play. Differences in genetic associations with exceptional longevity between the genders have been noted previously (De Benedictis et al., 2001). Interestingly, the cogni- tion and physical function ability of male centenarians is often greater than for their female counterparts, even though they are outnumbered by them (Terry et al., 2008). Survival to excep- tional old age, however, can occur due to delay or escape from morbidity, as well as survival in its presence, regardless of gender (Evert et al., 2003; Terry et al., 2008). The promoter analyses suggest that an enhanced LASS1 activity is conducive to longev- ity. They further suggest that healthy aging in nonagenarians depends on a somewhat lower enhancement of this promoter activity. This could, for example, be related to moderation in apoptosis-promoting activity coupled to sufficient enhancement of cholesterol clearance by ceramide. Our results support a model (Fig. 5) postulating that metabolic (lipid) stress induces protective responses. However, at some point, removal of dam- aged cells is needed. Confirmation of this model will require the longitudinal study of appropriate endophenotypes.

Study subjects