Results

FOXO3/FOXO3B Sequence Alignment

human longevity and healthy aging, but initial sequencing work is required before functional studies are undertaken.

and is composed of four exons (variant 2), of which exons 2 and 3 code for the translated protein. A variant of FOXO3 (variant 1) is missing the first exon, but this does not result in an amino acid change during translation. FOXO3 (also referred to as FOXO3A) is known to have a pseudogene on chromosome 17, referred to as FOXO3B. FOXO3 exons 2–4 appear to represent the entire FOXO3B pseudogene and support the finding that the latter is an integrated pro- cessed messenger RNA from FOXO3. Figure 2 demonstrates this homology. We observed that a portion of the FOXO3B pseudogene is annotated as being contained in exon 4 of the ZNF286B gene (Gene ID: 729288 [not shown]).

Therefore, we conducted an initial long-range sequencing effort with two purposes: (i) validate known coding variants and (ii) identify novel variants that might be responsible for the association with the longevity phenotype.

Materials and Methods

Study Populations

Ninety-five individuals of various ethnicities (Caucasians, Filipinos, Japanese, Chinese, and multiracial) living in Hawaii contributed DNA for this sequencing study, which yielded 85 intact, complete, high quality sequences for further study; an additional 282 persons with Japanese surnames living in Hawaii were used for allele frequency determination within the Japanese American population, and 675 study participants (Japanese American men) from the Kuakini Honolulu Heart Program were used for a longevity case–control study. Proce- dures were performed according to institutional guidelines and approved by the Institutional Review Board of Kuakini Medical Center.

No Validated Coding SNPs in FOXO3 in the Most Recent dbSNP Build

A recent version of the Genome Reference Consortium (GRCh37, NCBI dbSNP Build 134), released on August 8, 2011 lists FOXO3 as having 39 polymorphic variants in the coding region (later revised down to 38 polymorphic variants in the November 8 build). This included 21 synonymous, 16 nonsynonymous (including our newly found variant), and 2 frameshift variants. These variants are of great potential interest as one of them might theoretically be the actual causal variant for the longevity and healthy aging phenotypes. We will refer to “transcript variant 2” (NM_201559.2) which describes all four exons for FOXO3, but transcript variant 1 (NM_001455.3) describes two exons. Both transcript variants code for the same protein, as exons 1 and 4 are noncoding but may play roles in gene regulation.

Sequencing Strategy

Chromosome 6–specific primers (FOXO3AF and FOXO3AR) were designed using the Primer3 (17) component of Geneious and were used to amplify a 1.5 kb chromosome 6–specific genomic fragment containing exon 3, the location of the majority of the previously reported coding variants in FOXO3. Sequences were generated and screened for nucleo- tide substitutions using an ABI3100 Genetic Analyzer and Geneious software, using eight primers (shown in Table 1 and the triangles in Figure 1).

true variants but rather result from misalignment between the coding region of the FOXO3 gene with its highly homolo- gous pseudogene, FOXO3B (Gene ID = 2310). In an attempt to validate these SNPs, we designed long-range primers that were outside the region of FOXO3/FOXO3B homology that would amplify a 1,503 bp chromosome 6–specific fragment, including all of exon 3 that would serve as a template for DNA sequencing. We found a paucity of variation in this

FOXO3/FOXO3B Sequence Alignments

FOXO3 is known to have a pseudogene on chromosome 17, FOXO3B (shown in Figure 1 and 2). FOXO3 exon 3 was used in a BLAST search (18) to examine this homology in more detail. AF032887 was chosen to represent the homology

1 100 200 300 400 500 FORORAE800   1,000 1,100 1,200 1300 1,400FOXOSA-8F FOXOIA-IR FOXOSA-R, Foxosa

Figure 1. Sequencing strategy. 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.

that these SNPs are either extremely rare in our multieth- nic populations, are the result of misalignment with other highly homologous sequences, or the result of sequencing and/or assembly errors. They are clearly not responsible for the longevity association in our prior work (9) and are unlikely to be important to human longevity.

region after sequencing DNA from 95 persons in a random sample of persons living in Hawaii. These persons included primarily Caucasians, Filipinos, Japanese, and Chinese, but some were multiracial.

After completely sequencing the region of interest, we were not able to confirm the existence of any (see later) of the 38 sequence variants that were reported in the most recent version (version 134, GRCh37.p2 of November 8, 2011) of the SNP public database accessible through the NCBI website: http://www.ncbi.nlm.nih.gov/projects/

Newly Identified Coding SNP