This highlights the aforementioned replication issue and points to an important additional challenge for future studies. That is, a significant proportion of SNPs in public databases is, in fact, not well curated and is composed of paralogous sequence variants. Paralogous sequence variants can arise by segmental duplications of the human genome sequence as well as by homologies with pseudogenes (30). Musumeci and colleagues (31) provide evidence that as many as 8.32% of the biallelic coding SNPs listed in the NCBI’s dbSNP database are not reliable and appear to be artifacts due to the presence of highly similar genes in the human genome. This problem arises when there is extensive DNA sequence similarity between two or more genomic regions and their sequence differences masquerade as normal SNPs. Care must be taken to reduce this problem in DNA sequencing studies so that primer selection includes unique-copy regions. In the present study, we report that all 38 of the previously reported coding SNPs in FOXO3 are either invalid or too rare to be useful for phenotype–genotype association and other genetic mapping studies.

The fact that only two genes (APOE and FOXO3) have consistent, and widespread replication is puzzling. What is the reason for the general dearth of replications for most preliminary genetic findings? One potential cause that can lead to apparent lack of replication is population-specific variants. Similar phenotypes may result from completely different variants that are common in one population but simply do not exist in other populations, such as population- specific variation within the CETP gene. A relation between variation within this gene and human longevity was first reported by Barzilai and colleagues (28) in Ashkenazi Jews but was never replicated until Koropatnick and colleagues (29) tested a population-specific variant in American men of Japanese ancestry from the Honolulu Heart Program and Hawaii Lifespan Study. The particular SNPs thought to be responsible for the shared phenotypes (elevated high density lipoprotein and exceptional longevity) do not exist in the other population. Had Koropatnick and colleagues (29) focused on the specific SNPs found in the initial study of Ashkenazi Jews (and/or those in LD), this may have led to another uninformative study. Other potentially more common reasons for lack of replication include the existence of rare

The plethora of large-scale genotyping efforts, such as genome-wide association studies that utilize nonvalidated variants from short “shotgun” sequencing efforts, will no doubt add many invalid variants to public available data sets that may lead to wasted efforts and unrealistic expectations when mapping complex genetic traits (32). Through the use of longer range DNA sequencing and careful annotation of the genome, primarily through efforts like the ENCODE Project (33), there should be an improvement in the resources that can help elucidate functional variants that are associated with complex phenotypes.

With regard to discovering the longevity-associated func- tional variant for FOXO3, this is as yet unresolved. It ap- pears from the current study that it is unlikely (although not impossible) that a coding SNP will be identified as the func- tional variant responsible for human longevity. Interestingly,

Banasik and colleagues (34) did find that one variant in in- tron 2, rs2802292, a noncoding SNP, demonstrates a small but significant difference in messenger RNA levels in func- tional studies. This same variant had the strongest associa- tion in our previous case–control study of healthy aging and longevity (9). Whether this SNP is the functional variant re- mains to be proven and more functional studies are required. For example, cell physiology experiments measuring mes- senger RNA levels need to be performed, keeping all ge- netic information the same, except for this variant, as well as aforementioned model organism and human studies.

Some of the most basic groundwork in humans could begin with assessment of the potential relation between mortality from the common diseases of aging, including coronary heart disease, stroke, and cancer, which account for the majority of human deaths, and variation in FOXO3. Already, there is growing evidence for FOXO3 as a tumor suppressor. For example, downregulation of FOXO3 activity is seen in various cancers (43,44). Indeed, the Hawaii Lifespan Study men who had the longevity phenotype (and were more likely to have a protective FOXO3 genotype) had fewer cancers in their medical history (9). Ongoing studies with prospectively collected data can address whether and how cause of death is modified by variation in the FOXO3 gene. This will provide further clues as to mechanisms for FOXO3’s effects on human aging and longevity. Such in- formation may enhance translational efforts that link model organism and human research and could have important implications for human health span (45).

from http://biomedgerontology.oxfordjournals.org/ at USDSM Wegner Health Science Info. Cntr. on November 13, 2012

In conclusion, all published nonsynonymous (coding and result in amino acid change) SNPs identified in the FOXO3 gene, to date, are either invalid or too rare to be of consequence to human longevity. The novel variant that we identified through detailed sequencing appears to be too low in frequency (0.082) to be responsible for our previously reported association of FOXO3 variants with human lon- gevity (9), although it is conceivable that this novel SNP is associated with a disease phenotype at a younger age or is important in other populations. This suggests that the func- tional SNP is most likely a noncoding SNP from intron 2, where all previously reported associations have occurred. Future research should include complete sequencing of intron 2 and annotation of regulatory elements, in order to identify candidates for functional study of the FOXO3 gene and human aging.