FOXO3 Gene Variants and Human Aging: Coding Variants May Not Be Key Players

Timothy A. Donlon,1 J. David Curb,1,2 Qimei He,1 John S. Grove,1,3,4 Kamal H. Masaki,1,2 Beatriz Rodriguez,1,2 Ayako Elliott,1 D. Craig Willcox,5 and Bradley J. Willcox1,2

1Honolulu Heart Program, Kuakini Medical Center, Honolulu, Hawaii.

2Department of Geriatric Medicine, John A. Burns School of Medicine, University of Hawaii, Honolulu. of Public Health Sciences and Epidemiology, John A. Burns School of Medicine, University of Hawaii, 4Cancer Research Center of Hawaii, University of Hawaii, Honolulu. 5Department of Human Welfare, Okinawa International University, Ginowan, Japan.

Address correspondence to Timothy A. Donlon, Ohana Genetics, 1010 S. King Street, Suite 201, Honolulu, HI 96817. Email: donlon@hawaii.edu

FOXO3 is generally recognized as a “master” gene in aging since its association with longevity has been replicated in multiple organisms and human populations. A group of single nucleotide polymorphisms in linkage disequilibrium with a coding region has been associated with human longevity, but the actual functional variant is unidentified. Therefore, we sequenced the coding region in our long-lived Japanese American population in order to enhance resources for fine mapping this region. We demonstrate that of 38 published variants, 6 are misalignments with homologous nonallelic sequences from FOXO3B (ZNF286B), a pseudogene on a different chromosome; 2 are attributable to ZNF286B only, and the remaining 30 were unconfirmed, indicating that they are very rare and not likely involved in longevity. Furthermore, we identified a novel, unique, nonsynonymous coding variant in exon 3 (Gly566Ala; rs138174682) that is prevalent in multiple ethnic groups but appeared too rare for major longevity effects in our study populations.

Key Words: Aging—FOXO3—Genetic—Human longevity.

Received September 9, 2011; Accepted January 24, 2012

Decision Editor: Rafael de Cabo,

lations of European and Asian descent, these results point to FOXO3 playing an important role in human aging. How- ever, the “functional” SNP has yet to be identified.

enormous implications for society. Therefore, it is vitally important to understand how and why we age in order to enhance our odds of aging with better health and less disability (1). Genetics may account for 10%–50% of the variability in human life span, depending upon the population, but little is known about the effector genes (2–6). Studies of model organisms have identified several evolutionarily conserved biological pathways that have major influence on aging and age-related traits (6–8). Daf-16, an evolutionarily conserved gene in nematodes was identified among early candidates as a potential modulator of human aging (8).

SNPs are gene variants that occur on average once every 300 bp. They frequently can be used to discover nearby functional variants that are associated with a disease or other phenotype (16). Once an association between a trait and a gene variant has been discovered, it can be difficult to find which variant is actually responsible for the biologic change that produced the phenotype because several closely linked variants may be in strong linkage disequilibrium (cor- relation) with the original gene variant. There are numerous SNPs within the FOXO3 locus that are associated with lon- gevity, but they are all located in a haplotype block on the order of 100 kb in size on chromosome 6. Because there is a high degree of linkage disequilibrium, a “longevity-associated functional” variant could be anywhere in this 100+ kb region, which is centered on the second of four introns (known as “intron 2”; see Figure 2). This putative functional variant might act through any number of mechanisms. For example, it might, when translated, change an amino acid sequence. Or it could be a splice-site variant, a transcription en- hancer element, a copy-number variant, or affect some other genetic structure. The quest to identify a functional variant (typically a coding variant) in FOXO3 will undoubt- edly help us better understand the role that this gene plays in

On this basis, we tested the hypothesis that the daf-16 human homologue, FOXO3, a gene on chromosome 6 that codes for a forkhead Box O transcription factor, might be important in human aging and longevity (9; see Figure 1). We first reported on associations of three single nucleotide poly- morphisms (SNPs) of FOXO3 with healthy aging and lon- gevity in male Americans of Japanese ancestry (rs2764264 [p value = .0002], rs13217795 [p = .0006], and rs2802292 [p < .0001]), associations that were initially confirmed (with ef- fects pointing in the same direction) in German and French populations (10) and later in Italian, American (northern and western European ancestry), Chinese, and several other pop- ulations (11–15). Since the association of FOXO3 with longevity has been observed in genetically diverse popu-

Table 1. Primers Used for DNA Amplification and Sequencing

Primer Sequence Tm (°C) Length (bp) FOXO3AF: ATCATCTGGGTGCTCGGTTT 62 1,503 FOXO3AR: GACCTGCTTTGCCCACTTC 61 FOXO3A-1F: CCCAACCAGCTCCTTTAACA 60 FOXO3A-1R: TGCATAGACTGGCTGACAGG 60 FOXO3A-3F: ACCAATTCTAACGCCAGCAC 60 FOXO3A-3R: GAGTCCGAAGTGAGCAGGTC 60 FOXO3A-7F: GAATGAGGGAACTGGCAAGA 60 FOXO3A-7R: CAGGTCGTCCATGAGGTTTT 60 FOXO3A-8F: GACCTGCTCACTTCGGACTC 60 FOXO3A-8R: AAATCCAACCCATCAGCATC 60

used in BLAST queries in an attempt to explore the origins of the sequence variations. For this, a portion of the DNA adjacent to the SNP was used in the NCBI website (megablast) as well as local alignments using the Geneious software (Biomatters Ltd, Auckland, New Zealand).

Note: DNA sequencing primers used in the current study, which amplifies a 1,503 bp genomic chromosome 6–specific fragment of FOXO3 just outside the region of homology with FOXO3B.

Results

FOXO3/FOXO3B Sequence Alignment