Allele frequencies of G1697C (Gly566Ala). A collection of 85 random multiethnic (Caucasian, Filipino, Japanese, Chinese, and mixed ethnicity) genetic samples was chosen for allele frequency determination from the original 95 sequenced samples on the basis of intact, complete, high-quality sequences. The ages of these participants ranged from 10 to 78 years. We found that of 85 individuals, ten were heterozygous and two were homozygous for the G1697C variant (rs138174682; see Table 4). To determine if this variant might be useful to study longevity in our Kuakini Honolulu Heart Program cohort (American men of Japanese ancestry), we selected 282 persons with Japanese surnames for further analysis. Of these 282, only eight were heterozygous and none was homozygous for this variant. Because the allele frequency was not extremely rare, we decided to assess this SNP with our original Hawaii Lifespan Study case-control study sample (9) to assess whether it might be an important contributor to the longevity phenotype. The cases had a longevity phenotype defined as survival to at least 95 years of age.

Figure 1 shows the sequencing strategy used in this study. Chromosome 6-specific primers were used to amplify a 1.5-Kb genomic fragment of exon 3. Sequences were generated from 95 individuals of various ethnicities living in Hawaii and screened for nucleotide substitutions. Forward primers are denoted as dark green, whereas reverse primers are light green. The yellow box denotes the protein-coding exon 3, whereas the solid line denotes the noncoding genomic sequence.

Figure 2 shows an alignment of the FOXO3 gene (GenBank NM_201559.2), also known as FOXO3A, with the homologous genomic sequences onvariants, misalignments, sequencing/assembly errors, and population-specific variation. In this study, we focused on putative coding sequence variants in FOXO3 and verified only one SNP (located in exon 3) using long-range chromosome 6-specific sequencing. Eight of the 38 previously published coding SNPs resulted from misalignment of sequences between chromosomes 6 and 17, and the remaining 30 could not be verified in our populations. The newly discovered Gly566Ala variant (rs138174682), which is three bases away from a CREB-binding protein site at K569, might change the conformation of the FOXO3 protein and have functional implications. However, its allele frequency was too low in our study population of Japanese Americans to account for our previous study findings. Replication remains a major challenge for future studies, and care must be taken to reduce the problem of paralogous sequence variants in DNA sequencing studies. Only two genes, APOE and FOXO3, have consistent and widespread replication in multiple human populations for their associations with aging-related phenotypes and longevity.Variants from short shotgun sequencing efforts will no doubt add many invalid variants to publicly available datasets that may lead to wasted efforts and unrealistic expectations when mapping complex genetic traits (32). Through the use of longer-range DNA sequencing and careful annotation of the genome, primarily through efforts like the ENCODE Project (33), there should be an improvement in the resources that can help elucidate functional variants that are associated with complex phenotypes.

With regard to discovering the longevity-associated functional variant for FOXO3, this is as yet unresolved. It appears from the current study that it is unlikely (although not impossible) that a coding SNP will be identified as the functional variant responsible for human longevity. Interestingly, Banasik and colleagues (34) did find that one variant in intron 2, rs2802292, a noncoding SNP, demonstrates a small but significant difference in messenger RNA levels in functional studies. This same variant had the strongest association in our previous case-control study of healthy aging and longevity (9). Whether this SNP is the functional variant remains to be proven and more functional studies are required. For example, cell physiology experiments measuring messenger RNA levels need to be performed, keeping all genetic information the same except for this variant, as well as aforementioned model organism and human studies.

Some of the most basic groundwork in humans could begin with an assessment of the potential relation between mortality from the common diseases of aging, including coronary heart disease, stroke, and cancer, which account for the majority of human deaths, and variation in FOXO3. Already, there is growing evidence for FOXO3 as a tumor suppressor. For example, downregulation of FOXO3 activity is seen in various cancers (43,44). Indeed, Hawaii Lifespan Study men who had the longevity phenotype (and were more likely to have a protective FOXO3 genotype) had fewer cancers in their medical history (9). Ongoing studies with prospectively collected data can address whether and how the cause of death is modified by variation in the FOXO3 gene. This will provide further clues as to mechanisms for FOXO3's effects on human aging and longevity. Such information may enhance translational efforts that link model organism and human research and could have important implications for human health span (45).