Haplotype combinations in women and men

bination containing LASS1 (CGT) in centenarians was puzzling. One possibility is that this reflects the lack of a role for this haplo- type in survival at that late age. However, there is an alternate explanation. With advanced age, the ratio of females to males increases. Thus, the plateau we see could be the result of gender bias in the oldest age groups. We therefore examined the haplo- type association in the Georgia sample for females and males separately, in the European subpopulation. The results suggest that such a bias indeed exists (Table 1). The association with exceptional survival in female centenarians was found solely for the combined haplotypes of the three genes that included LASS1 (CCT), showing an OR of 12.52. For male centenarians, it was instead the LASS1 (CGT) haplotype with an OR of 12.40. The results in the Georgia sample were replicated in the Louisi- ana population (Table 1). For female nonagenarians, the com- bined haplotypes that included LASS1 (CCT) were associated with exceptional longevity with an OR of 3.12. For male nonage- narians this combination included LASS1 (CGT) and an OR of 4.86.

Fig. 2 Haplotype frequencies in different age groups. The frequencies for each haplotype or haplotype combination determined for the European subpopulation (0.9 assignment probability) were normalized to the frequencies in the < 60 year-old-age group from Georgia or Louisiana, respectively, to facilitate comparison. Error bars denote the SD (100 bootstraps). The age groups labeled ‘90’ and ‘100’ are the nonagenarians centenarians in Georgia and Louisiana, respectively. For clarity, only the haplotypes or haplotype combinations that differ significantly on frequency between age groups are shown: APOE (ATTTC), HRAS1 (CGCGCGT), and LASS1 (CCT) in A and APOE (ATTTC), HRAS1 (CGCGCGT),and LASS1 (CGT) in B. Note the difference in scale of the ordinates in A and B. The APOE and APOEÆHRAS1 frequencies are identical in the two panels.

(CCT) and (CGT) haplotypes in Fig. 2A,B, respectively. The monotonic increase in the frequencies of the haplotype combi- nations of APOEÆHRAS1ÆLASS1 with age indicates a positive association with longevity, although a plateau appears for the combination containing the LASS1 (CGT) haplotype for the old- est age group. These positive associations with longevity indi- cate that these are longevity haplotypes.

Gene combination associated with healthy aging

We next examined the question whether the genetic combi- nation that contributes to exceptional longevity is also associ- ated 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 sub- jects 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

We next 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 1–3. Mortality was assessed on follow-up after enrollment. Censoring is indicated by the symbols.



curves are significantly different (Wilcoxon v2 = 20.06, ð2Þ P < 0.0001).

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 haplo- types 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 (Arti- ga et al., 1998a,b). The combination we have found in the hap- lotype associated with exceptional survival elicits a moderate level of expression, approximately one-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, the 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