Table 1 Association of haplotypes with exceptional longevity and healthy aging

Case (N; frequency) Population Haplotype Control (N; frequency) OR (CI) Exact P (adjusted P) Georgia APOEÆHRAS1ÆLASS1 (CCT) ‡ 98 years old (147; 0.073) 7.68 (2.55–23.10) 4.1 · 10)5 (2.88 · 10)4) 20–59 years old (188; 0.010) Georgia APOEÆHRAS1ÆLASS1 (CGT) ‡ 98 years old (147; 0.191) 2.56 (1.61–4.08) 7.8 · 10)5 (4.66 · 10)4) 20–59 years old (188; 0.084) Louisiana APOEÆHRAS1ÆLASS1 (CCT) ‡ 90 years old (204; 0.105) 2.93 (1.68–5.09) 9.5 · 10)5 (5.68 · 10)4) 20–59 years old (251; 0.039) Louisiana APOEÆHRAS1ÆLASS1 (CGT) ‡ 90 years old (204; 0.175) 2.97 (1.92–4.58) 6.91 · 10)7 (5 · 10)6) 20–59 years old (251; 0.067) Georgia APOEÆHRAS1ÆLASS1 (CCT) ‡ 98 -year-old female (128; 0.099) 12.52 (2.93–53.57) 1 · 10)5 (2 · 10)5) 20–59 -year-old female (115; 0.009) Georgia APOEÆHRAS1ÆLASS1 (CGT) ‡ 98 -year-old male (19; 0.251) 12.40 (3.55–43.29) 9 · 10)6 (9 · 10)6) 20–59 -year-old male (73; 0.026) Louisiana APOEÆHRAS1ÆLASS1 (CCT) ‡ 90- year-old female (125; 0.092) 3.12 (1.47–6.63) 1.97 · 10)3 (3.93 · 10)3) 20–59- year-old female (163; 0.032) Louisiana APOEÆHRAS1ÆLASS1 (CGT) ‡ 90 -year-old male (79; 0.279) 4.86 (2.50–9.45) 1 · 10)6 (2 · 10)6) 20–59- year-old male (88; 0.074) Louisiana APOEÆHRAS1ÆLASS1 (CCT) ‡ 90 -year-old healthy (68; 0.023) 0.29 (0.08–1.04) *5.11 · 10)2 (5.11 · 10)2) ‡ 90 -year-old unhealthy (68; 0.076) Louisiana APOEÆHRAS1ÆLASS1 (CGT) ‡ 90 -year-old healthy (68; 0.295) 15.48 (5.08–47.15) 1.06 · 10)9 (2.11 · 10)9) 90- year-old unhealthy (68; 0.026)

European subpopulations (0.9 assignment probability) were examined. The haplotypes are ATTTC and CGCGCGT for APOE and HRAS1, respectively. and unhealthy were classified as described in Experimental procedures. OR, odds ratio; CI, 95% confidence interval. *Not significant.

Table 2 Haplotype frequencies in the general population

haplotype (ATTTC) and the HRAS1 (CGCGCGT) haplotype in combination with the LASS1 (CCT) or (CGT) haplotypes, in which the SNP are listed starting with the furthest upstream SNP for each gene. The odds ratios (OR) of 7.68 and 2.56 were obtained for the APOE, HRAS1, and LASS1 (CCT) or (CGT) hap- lotype combinations, respectively. The stratification of the popu- lation at the 0.9 assignment probability level was sufficient for this analysis, because no significant differences were observed in the frequencies of the ethnic affiliation markers between the control and centenarian groups in the European subpopulation.

Haplotype APOE HRAS1 LASS1 (CCT) LASS1 European Georgia (N = 220) 0.237 0.634 0.289 0.504 Louisiana (N = 293) 0.237 0.664 0.253 0.561 African Georgia (N = 145) 0.041 0.633 0.200 0.333 Louisiana (N = 52) 0.08 0.60 0.15 0.28

A Structure assignment probability of 0.8 was used for this analysis to provide a conservative evaluation of any differences. The 20- to 59 -year- old age groups were used here, as representative of the general population. APOE (ATTTC) and HRAS1 (CGCGCGT) haplotypes are

To determine whether these associations are present in the Louisiana population, we carried out the same analyses indepen- dently. The results obtained in the Georgia sample replicated in the Louisiana sample (Table 1). The same haplotype combina- tions were associated with exceptional longevity in the subpopu- lation of European origin in Louisiana, stratified with an assignment probability of 0.9, and no significant association was found in the African-origin subpopulation. The OR were 2.93 and 2.97 for the APOE, HRAS1, and LASS1 (CCT) or (CGT) hap- lotype combinations, respectively. The lower OR of the LASS1 (CCT)-containing haplotype combination in the Louisiana popu- lation is likely due to the greater demographic selection for cen- tenarians than for nonagenarians in the Georgia and Louisiana samples, respectively, but this does not appear to apply to the LASS1 (CGT).

ence in the subpopulation. Analysis of the frequency of the hapl- otypes 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 Geor- gia and Louisiana also differ, as an examination of the haplotype blocks in the three genes suggests (Fig. S1).

Haplotype frequencies differ with age

To ascertain the potential role of the three-gene interaction in promoting exceptional survival, we examined the differences in various age groups in the frequency of the two haplotype com- binations identified above in the European subpopulation. The results are shown for the combinations containing the LASS1

The lack of an association with longevity in the African-Ameri- can 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 differ-

(A) (B)

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 OR of 1.53 (CI = 1.08–2.17; Exact P = 2.12 · 10)2; adjusted P = 2.12 · 10)2) and 1.37 (CI = 1.02–1.83; Exact P = 4.37 · 10)2; adjusted P = 4.37 · 10)2) respectively. The APOEÆHRAS1 haplotype combination is also associated with longevity in both of these subpopulations, with OR of 2.83 (CI = 1.88–4.26; Exact P = 5.68 · 10)7; adjusted P = 1.14 · 10)6) and 1.89 (CI = 1.35–2.65; Exact P = 2.27 · 10)4; adjusted P = 4.54 · 10)4) respectively. The greater OR for the two-gene combination supports the epistatic effect of HRAS1. None of the other haplotypes in the individual genes or their combinations were associated with longevity (Table S5).

Haplotype combinations in women and men