Regarding the C1857T variant (chromosome position 108985893), at codon 619, only 2 of 85 individuals in the random Hawaii sample were heterozygous—for an allele frequency of 0.012, also precluding any meaningful power for association studies.

Discussion

Although our newly discovered Gly566Ala variant (rs138174682) does not appear to be involved in any known modification sites at amino acid 566, such as acetylation, phosphorylation, or ubiquitination, it is three bases away from a cyclic-AMP responsive element binding (CREB- binding protein) site at K569 (22,23). While this is a conserva- tive amino acid substitution, it might change the conformation of the FOXO3 protein so could theoretically have functional implications. This variant and others identified from further

FOXO3 is generally recognized as a “master” gene for human aging because it represents one of only two genes with strong associations with aging-related phenotypes and longevity that have had replications in multiple human populations (9–15). The other—ApoE—is a lipid transport gene, which, while also pleiotropic, appears to act principally via cardiovascular mechanisms on several age-related diseases (15). The E4 allele is the principal risk allele (15). Conversely,

Table 3. Validated Variants in FOXO3 exon 3

Chromosome Position mRNA Position SNP ID MAF Type Allele Amino Acid Codon Amino Acid 108985733 1697 n/a 0.082 Missense G Gly [G] 2 566 C Ala [A] 2 566 108985893 1857 n/a 0.012 Synonymous C Ser [S] 3 619 T Ser [S] 3 619

Note: Chromosome position is in reference to the Genome Reference Consortium (GRCh37, NCBI dbSNP Build 132 [NT_025741]) released October 9, 2010. The mRNA position is in reference to the “A” in the AUG start codon, SNP IDs are those designated in dbSNP, and codon refers to the position in the amino acid codon at which the variant occurs. A multiethnic group of 85 participants were sequenced for a total of 170 chromosomes. MAF = minor allele frequency, mRNA = messenger RNA, and SNP = single nucleotide polymorphism.

Table 4. Allele Frequencies of G1697C in Different Populations

variants, misalignments with other highly homologous quences, sequencing/assembly errors, among other causes.

Random Multiethnic Japanese Surnames KHHP in Hawaii (n = 85) in Hawaii (n = 282) (n = 675) 14/170 alleles (0.082) 8/564 (0.014) 14/1,350 (0.0104)

Regarding the current study, rapid progress in genomic methodology has made sequencing a viable and cost-effective approach to uncover variants that can then be assessed for functional significance. SNPs found within a coding sequence have generally been of most interest because they are more likely to alter the biological function of a protein than those in noncoding sequences. Therefore, the current investiga- tion focused on all putative coding sequence variants (n = 38) that were reported in the most recent version of the SNP public database (see earlier). Surprisingly, only one of the SNPs (located in exon 3) was verified by our long-range chromosome 6–specific sequencing (a noncoding SNP). Further investigation revealed that, in fact, 8 of the 38 previ- ously published coding SNPs resulted from misalignment of sequences between chromosomes 6 and 17 (Table 2), the remaining 30 are from exon 3 and could not be verified (not found at all in our populations).

Note: Allele frequencies for the G566A variant at chromosome position 108985733 were determined in (1) a random sample of 85 participants living in Honolulu, HI, including Filipinos, Japanese, Chinese, and Caucasian (Random Hawaii); (2) 282 random samples from participants with Japanese surnames living in Honolulu, HI; and (3) 675 participants from the Kuakini Honolulu Heart Program (KHHP).

sequencing efforts will require validation in other populations. Unfortunately, the variant was too infrequent in our study population of Japanese Americans to account for our previous study findings (9), but it might theoretically contribute to longevity in other populations.

While this variant might appear promising, there is a plethora of human studies that have found one or more genetic variants associated with longevity, but the vast majority has not been replicated. This has historically been the rule rather than the exception. For example, in 1987, in the first candidate gene studies of human longevity, several HLA genes found in Okinawans (24) were not replicated until over a decade later when one of the original findings (DR alleles) were replicated in a European population (25). The second major genetic finding, the APOE gene (26) was widely replicated over the next several years (for review, see [15]) and inspired a plethora of studies of new candidate longevity genes and genome-wide scans (27). Over the next decade, multiple novel genetic findings were reported with inconsistent or no replications until the FOXO3 gene finding (9). Thereafter, multiple replications occurred in less than 2 years (for review, see [15]).