rows 2, 4, and 11 were not typed but proxy SNPs typed with the Affymetrix 6.0 array confirm strong associations of variants in the same region with EL. These SNPs are summarized in Table 5 and are a good proxy for the SNPs originally typed in the NECS and SICS as shown by their proximity in terms of physical distance ,10kb and linkage disequilibrium measured by D9 and r2.

elegans [31,35]. For lifespan analysis, we focused on C. elegans, which has two orthologues of ADARB1 and ADARB2 with RNA editing activity, adr-1 and adr-2 [36] (see phylogenetic tree in Figure S1). Because C. elegans has an average lifespan of approximately 20 days, the influence of candidate genes on lifespan can be readily tested. Therefore, to evaluate whether adr loss-of-function influences C. elegans lifespan, we monitored the lifespan of single and double mutants of adr-1(gv6) and adr-2(gv42). Both of these alleles are deletions that remove at least a third of the coding sequence and are presumed null alleles [36] (Figure 6a and Figure S2). Strains carrying mutations in adr-1 and adr-2 displayed a shorter lifespan than the wild-type control N2 worms (log-rank test p,1028). Remarkably, aside from the decline in lifespan, there were no other obvious defects, in contrast with gain-of- function studies that noted lethality in Drosophila [37].

Only SNP rs2805533 reached statistical significance in the JCS set, with an effect that was consistent with the observed effect in the NECS, SICS and AJCS samples (Table 6). The SNP rs1533484 was borderline significant (Bayesian significance ,0.06) and demonstrates consistent effects with the NECS and SICS results, but the allele frequencies are substantially different. The SNP rs10903420 was also significant but with an opposite effect compared to the NECS, SICS and AJCS subject sets. Note however the substantial differences in both allele frequencies and pattern of LD that may explain the different patterns of associations in this ethnically very distinct sample. Genotype data of additional SNPs in Table 7 provide further evidence for the existence of variants in the region between physical positions 1340K and 1500K of chromosome 10 that are associated with

The insulin-like growth factor (IGF) pathway is a well known lifespan regulatory axis in worms [38], flies [39,40], mice [41], and humans [42]. Inactivation of the C. elegans insulin like receptor gene daf-2 by dsRNA increased lifespan of both the wild type N2 worms, confirming previous studies. Notably, knockdown of daf-2 by dsRNA also increased lifespan in the background of adr-1 and adr-2 null mutations, resulting in a lifespan phenocopy similar to the wild type N2 worms (Figure 6a). Similar extensions of lifespan were seen with single adr-1 or adr-2 mutants, (Figure S2). These data suggest that IGF axis mediated increases in lifespan due to daf-2 remain active in the presence of adr-1 and adr-2 background genotypes, but with less potency than in a N2 wild type background. From these data, we cannot exclude the possibility that knockdown of RNA editing genes in adr-1 or adr-2 mutants results in increased RNAi activity. In this scenario, increased RNAi might target genes downstream of daf-16 (seven daf-16 gene targets have been identified in comparative analysis of daf-2 versus daf-2::daf-16 strains [43]), thereby reducing the potency of daf-2 dependent increases in lifespan.

Age Trends

For some SNPs, a clear monotonic pattern associated with increasing age was observed (Figure 5). This monotonic pattern is consistent with a genetic effect that results from alleles positively associated with EL becoming more frequent in older individuals, while alleles that are negatively associated with EL become less frequent. This pattern has also been observed for ApoE alleles [33] and is consistent with the phenomenon of demographic selection [34]. These increases in allele frequencies with age also illustrate the increasing gain of power conferred by studying centenarians and even more so, subjects age 105+ years, in genetic studies of exceptional survival.

In a previous study, mutations in both adr-1 and adr-2 resulted in increased GFP reporter transgene silencing, suggesting that declines in ADAR function are associated with an increase in RNA interference (RNAi), which would account for the GFP silencing in those experiments. When the argonaute gene rde-1, which is essential for RNA induced silencing complex (RISC) formation, was introduced into adr-1;adr-2 worms containing the transgenes, the increased GFP silencing due to ADAR knockdown was suppressed [44]. This suggests cross-regulation between RNA editors and RNA interference that is further supported by results

Functional Studies

To go beyond statistical association, we chose to investigate the possible functional role of these genes in regulating lifespan by evaluating their influence on lifespan in the nematode, C. elegans, a robust model organism for candidate lifespan gene discovery. The A-to-I RNA editing gene family and their enzymatic editing activity has been well conserved in a broad array of species including humans, mice, flies, zebrafish, xenopus and notably, C.

susrevksiR SCJA+SCIS+SCEN)slortnoc962,dlotsedlo992(SCJASCIS+SCENHPECselellatnereferPNSwoR )A(pgisseyaBRO)A(pgisseyaBRO)A(pgisseyaBRO 22.0/72.03000.0)95.1;51.1(43.1213.0GG/GAvAA7417001sr2 01.0/21.01120.0)45.1;10.1(42.1421.0CC/CAvAA949488sr4 24.0/83.07410.0)89.0;57.0(68.0244.0GGvGA/AA4843351sr01 42.0/13.01540.0)31.2;59.0(24.112.0/71.00300.0)39.0;56.0(87.0691.0GGvGA/AA91049271sr31 84.0/34.03500.0)69.0;27.0(38.074.0GGvGA/AA347414sr51 430.0/040.02663.0)92.3;44.0(91.110.0/500.07920.0)80.1;91.0(64.0900.0GG/GAvAA9088382sr61 659.0/659.01594.0)35.2;93.0(99.099.0/599.06310.0)52.8;11.1(37.21GGvGA/AA0188382sr71 430.0/530.03754.0)60.3;83.0(60.110.0/500.09030.0)40.1;81.0(84.0900.0GG/GAvAA6188382sr81