Overall, the FLAG study provides additional evidence for genetic linkage to regions on chromosomes 3p and 18q associated with exceptional longevity. The study also highlights the importance of careful definition of affected status when conducting genetic studies of complex traits. The limitations of the study, such as sample size and population diversity, suggest that further studies are needed to confirm and expand upon these findings. Nonetheless, the identification of genes in the linked region provides potential targets for future studies aimed at understanding the molecular mechanisms underlying exceptional longevity.Of particular interest in the 3p region is TGFBR2 because it is implicated in multiple disease etiologies [41,42]. However, of the 27 genes in the same region, most have functions that are not fully understood. Our definition of familial longevity (elevated FEL) assumes a dominant or additive model of inheritance. As a consequence, our selection criteria for affected individuals (and their familial relationship networks) are less sensitive for identifying potential recessive traits than dominant or additive traits. The familial recurrence pattern expected for a recessive trait (sib pairs scattered throughout a pedigree) would, in most cases, result in higher than expected FEL, particularly in the large sibships typical of the UPDB. Nevertheless, our study has substantially less power to identify loci with recessive effects on longevity: only 63 affected sib pairs are informative with respect to recessively inherited factors; while 607 pairs are potentially informative for dominant or additive inheritance. It is likely that variants at many loci contribute to the heritability of longevity in humans. Our independent replication of the 3p22-24 linkage peak originally reported by Boyden and Kunkel [1] should help focus the search for variants associated with longevity in this relatively small region. Advances in DNA sequencing make it practical to rapidly sequence the exons in the region, or the entire region, and hence identify the variant(s) responsible for the observed linkage signal. Other regions identified in one or both of these studies also deserve further scrutiny. We have recently described patterns of gene expression that are associated with human longevity and aging [43]. Now we can try to narrow the set of longevity-associated candidate variants at the 3p locus by searching for markers (microsatellites or SNPs) in the same 3p region that are associated with variation in gene expression patterns that are also associated with longevity. Although, in principle, this requires a complete set of GWAS data for each expression quantitative trait locus (or eQTL) of interest, several established data repositories for genome-wide eQTL studies [44,45] should greatly simplify the process.

Acknowledgments: We thank Alison Fraser, Diana Lane Reed, and Geri Mineau for valuable assistance in managing the data.

Author Contributions: Conceived and designed the experiments: RAK EO RMC KMB KRS. Performed the experiments: RAK EO RMC. Analyzed the data: RAK EO KMB. Contributed reagents/materials/analysis tools: RAK RMC KRS. Wrote the paper: RAK EO RMC.

The FLAG study differs from the HMS and other studies of exceptional longevity in several important respects. We ascertained subjects on the basis of both familial longevity and personal references.

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