Newly Identified Coding SNP

NCBI website: http://www.ncbi.nlm.nih.gov/projects/ SNP/snp_ref.cgi?geneId=2309 (see Table 2.). One variant that was previously reported in dbSNP (August update) is no longer listed. This variant is a T to C transition at position 108985893 (rs113367269), a serine at codon 619 previ- ously described by Watkins and colleagues (19), which does not result in an amino acid change (ie, a synonymous variant). In an attempt to determine the reasons for this fail- ure in confirming the remaining 38 variants, we performed BLAST searches of sequences adjacent to these published FOXO3 SNPs and found that many of them mapped to chromosome 17 with 100% homologies or were the result of misalignment with the location of the highly homologous FOXO3 pseudogene (20), known as FOXO3B (Figure 2). FOXO3B is homologous to exons 2, 3, and 4 of FOXO3, and there are a number of nonallelic nucleotides that align with published SNPs. None of the exon 3 SNPs was verified by our long-range chromosome 6–specific sequencing effort. In sum, none of the coding SNPs listed in dbSNP could be verified. Eight of the 38 previously published coding SNPs were found to result from misalignment of sequences between chromosomes 6 and 17, the remaining 30 could not be verified (not found in our multiethnic populations; Table 2). FOXO3B itself lies within another gene referred to as “ZNF286B,” a putative zinc finger protein 286B (Gene ID: 729288). Collectively, these results indicate

One novel variant that we discovered in the current se- quencing effort is shown in Table 3; it was a nonsynonymous SNP located at position 108985733 on chromosome 6 which results in a G to C transversion and an alanine being substi- tuted for a glycine at amino acid 566 (SNP ID: rs138174682).

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 qual- ity sequences. The ages of these participants ranged from 10 to 78 years. We found that of 85 individuals, 10 were hetero- zygous and 2 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 a further analysis. Of these 282, only 8 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 sam- ple (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

1 10,000 100,000 110,000 124,968—— \—___-~vA—¥_§{|{|—\— aaFOXO3A (v2) ¢FOXO3B mRNA \\Exons 1 & 2 Intron 2 Exon 3 Exon 4

Figure 2. FOXO3 Genomic and FOXO3B mRNA Alignment. Shown is an alignment of the FOXO3 gene (GenBank NM_201559.2), also known as FOXO3A, with the homologous genomic sequences on chromosome 17 (FOXO3B [NR_026718, complement]). Homologies are noted in the top row with green bars. The red arrows refer to the FOXO3 transcript, version 2. The yellow arrows denote the protein-coding exons 2 and 3, and the black boxes denote the noncoding FOXO3B transcript. It is apparent from this alignment that the FOXO3B pseudogene is derived from the FOXO3 processed transcript variant 2, including exons 2–4. The present study assessed single nucleotide polymorphisms from exons 2–3, shown by the yellow arrows. The large intron 2 has been truncated to reduce the size of the figure.

Table 2. Evaluation of Variants in FOXO3 From the dbSNP Public Database