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

Timothy A. Donlon, J. David Curb, Qimei He, John S. Grove, Kamal H. Masaki, Beatriz Rodriguez, Ayako Elliott, D. Craig Willcox, and Bradley J. Willcox conducted a study on FOXO3, which is known as a master gene in aging due to its association with longevity in multiple organisms and human populations. The study aimed to enhance the resources for fine mapping the coding region in a long-lived Japanese American population since the actual functional variant is unidentified. 

The study revealed that out of 38 published variants, six are misalignments with homologous nonallelic sequences from FOXO3B (ZNF286B), a pseudogene on a different chromosome; two are attributable to ZNF286B only; and the remaining 30 were unconfirmed, indicating that they are very rare and not likely involved in longevity. A novel, unique, nonsynonymous coding variant in exon 3 (Gly566Ala; rs138174682) was identified that is prevalent in multiple ethnic groups but appeared too rare for major longevity effects in the study populations.

The study also discusses how 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. Studies of model organisms have identified several evolutionarily conserved biological pathways that have a major influence on aging and age-related traits.

SNPs are gene variants that occur on average once every 300 bp and can be used to discover nearby functional variants that are associated with a disease or other phenotype. However, it can be challenging to find which variant is responsible for the biologic change that produced the phenotype because several closely linked variants may be in strong linkage disequilibrium with the original gene variant. Numerous SNPs within the FOXO3 locus are associated with longevity, but they are all located in a haplotype block on the order of 100 kb in size on chromosome 6. A longevity-associated functional variant could be anywhere in this 100+ kb region, which is centered on the second of four introns.

The study 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. Associations of three single nucleotide polymorphisms (SNPs) of FOXO3 with healthy aging and longevity were initially reported in male Americans of Japanese ancestry and were later confirmed in German and French populations as well as Italian, American (northern and western European ancestry), Chinese, and several other populations.

The study concludes that the SNPs associated with longevity are not validated coding SNPs in FOXO3 but rather result from misalignment between the coding region of the FOXO3 gene with its highly homologous pseudogene, FOXO3B. The study 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 the study populations. The quest to identify a functional variant (typically a coding variant) in FOXO3 will undoubtedly help us better understand the role that this gene plays in human longevity and healthy aging.Paucity of variation in these 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. After sequencing DNA from 95 persons in a random sample of persons living in Hawaii, we were not able to confirm the existence of any 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/. These SNPs are either extremely rare in our multiethnic 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. In an attempt to determine the reasons for this failure 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-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 that these SNPs are either extremely rare in our multiethnic 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. One novel variant that we discovered in the current sequencing 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 substituted for a glycine at amino acid 566 (SNP ID: rs138174682).