An analysis of the frequency of the haplotypes in the individual genes supports the notion that there is an intrinsic difference between the European and African subpopulations in both Georgia and Louisiana (Table 2). The major difference is in the APOE haplotype frequency, with a smaller difference in LASS1. The European populations in Georgia and Louisiana also differ, as an examination of the haplotype blocks in the three genes suggests (Fig. S1).

The lack of an association with longevity in the African-American subpopulation from both Georgia and Louisiana could be attributed to the relatively lower numbers of subjects from this group. However, it is likely that this may be an intrinsic difference in the subpopulation. An analysis of the frequency of the haplotypes in the individual genes supports this notion.

To ascertain the potential role of the three-gene interaction in promoting exceptional survival, we examined the differences in various age groups' frequency of two haplotype combinations identified above in the European subpopulation. The results are shown for combinations containing LASS1 (CCT) and (CGT) in Table 1.

The association with exceptional survival in female centenarians was found solely for combined haplotypes of the three genes that included LASS1 (CCT), showing an odds ratio of 12.52. For male centenarians, it was instead the LASS1 (CGT) haplotype with an odds ratio of 12.40.

Consistent with the increase in haplotype frequencies with age, the APOE haplotype is associated with exceptional survival in the European-origin subpopulations from both Georgia and Louisiana, with odds ratios of 1.53 (CI = 1.08-2.17; Exact P = 2.12 x 10^-2; adjusted P = 2.12 x 10^-2) and 1.37 (CI = 1.02-1.83; Exact P = 4.37 x 10^-2; adjusted P = 4.37 x 10^-2), respectively. The APOEHRAS1 haplotype combination is also associated with longevity in both of these subpopulations, with odds ratios of 2.83 (CI = 1.88-4.26; Exact P = 5.68 x 10^-7; adjusted P = 1.14 x 10^-6) and 1.89 (CI = 1.35-2.65; ExactCombination containing the LASS1 (CGT) haplotype for the oldest age group. These positive associations with longevity indicate that these are longevity haplotypes. 

Gene combination associated with healthy aging

We next examined whether the genetic combination that contributes to exceptional longevity is also associated with healthy aging. Healthy aging is a concept that is difficult to define adequately. We elected to quantify healthy aging using the frequency of self-reported deficits (Rockwood & Mitnitski, 2007) in the Louisiana nonagenarians (Table S6). For this purpose, we surveyed subjects for the presence of 42 medical conditions or functional limitations and summed them for each nonagenarian. We then partitioned the subjects into tertiles based on the number of deficits reported; tertile 1 had the fewest deficits, while tertile 3 had the greatest number of deficits. A Kaplan-Meier survival analysis was performed for each of the tertiles (Fig. 3). Examining the entire sample of nonagenarians from Louisiana, the survival curves are significantly different (Wilcoxon v2 = 20.06, P < 0.0001).

We then determined the frequencies of the APOE (ATTTC) haplotype and its combination with HRAS1 (CGCGCGT) from the three-gene haplotype combination in the various age groups. Figure 2 shows that in both cases, there is a monotonic increase with age. None of the other haplotypes in the individual genes or haplotype combinations changed in frequency with age. The patterns seen indicate that HRAS1 has a synergistic (epistatic) effect with APOE, and this effect is further enhanced.

Fig. 3 Survival of nonagenarians as a function of their healthy aging profile. Kaplan-Meier survival of all nonagenarians in the Louisiana sample is plotted after partitioning individuals into tertiles of deficit accumulation. Deficit count increases from tertile 1. Mortality was assessed on follow-up after enrollment. Censoring is indicated by the symbols.

We then searched for genetic associations with healthy aging by examining the haplotypes in the three genes in the tertile with the fewest deficits vs. that with the most (Table 1). The results indicate that the combination of the same haplotypes in the three genes is associated with healthy aging but only for the combination including LASS1 (CGT). The OR for this association was 15.48 for the European subpopulation in Louisiana.

Functional impact of gene variants

The marked difference in the effects of the two LASS1 haplotypes suggests that they could have functional consequences. Variants in the APOE promoter at -491 and -219 are known to affect the expression of this gene at the transcription level (Artiga et al., 1998a,b). The combination we have found in the haplotype associated with exceptional survival elicits a moderate level of expression, approximately half of maximal. Because LASS1 only has an effect when a combination of haplotypes in APOE and HRAS1 is present, and this effect differs depending on the particular LASS1 haplotype, we examined the variants in the LASS1 gene for their potential role in transcription of the gene, using a luciferase reporter assay. The results indicate that compared to the AGC haplotype, the CCT- and CGT-containing promoters were 48% and 29% more active, respectively (Fig. 4). Thus, the variation observed in our study appears to be functional in determining mRNA expression levels. Perhaps, more moderate expression of LASS1 in males promotes healthier aging. It is noteworthy that varying expression of the LASS1 homolog in yeast, LAG1, affects the observed life extension.