One immediate result of the FLAG study scan is an independent replication of Boyden and Kunkel's finding of a genetic linkage between excess longevity and a locus in the 3p22-24 region in the HMS data. compares the HMS and FLAG linked regions, based on Boyden and Kunkel's Age Category 4 (upper 2.5% of the 1900 birth cohort). Superficially, the HMS linked region appears broader than the linked region we have observed. This may be a consequence of design differences between the FLAG and HMS studies: a sib pair analysis led to the HMS result, while an analysis of a wide range of kin relationships led to the FLAG result, for approximately the same total number of pairs in each study. Our sample from UPDB's large multiplex families ensures greater variation in shared chromosomal segment length, and shorter average segments, among relative pairs contributing to linkage peaks. On the other hand, the microsatellite markers used in this study are less precise markers of location because the distance between them is much greater than for the SNP panel used by Boyden and Kunkel 
. To remedy this, we are conducting fine mapping studies to more precisely define the shape and location of the linkage signal within the 3p22-24 region.
Figure 3 Comparison of chromosome 3 linkage results reported here to that reported by Boyden and Kunkel  (nominal p-values).
also shows results obtained by combining HMS and FLAG data, using the unweighted mean Z-score approach of Dempfle and Loesgen 
. The combined data yield a linkage p-value of 1.005×10−9
(corresponding to an adjusted p
0.008) at 55 cM on 3p. The combined data provide further support for Boyden and Kunkel's suggestive linkage peak at 9q31-34 for Age Category 8 (upper 0.5% of the 1900 birth cohort). We did not identify a peak in the same region from our data alone; however, the meta- minimum p-value by meta-analysis for the HMS 9q31-34 peak is 1.27×10−5
0.37) at 127.8 cM.
There are other suggested points of overlap between our results and the HMS study: their Age Category 10 (upper 0.2% of the 1900 birth cohort) suggests linkage to a region on chromosome 8q (meta p-value of 3.4×10−4
at 120 cM, corresponding to an adjusted p-value of 0.96). They found a similar suggestion of linkage to a region on 17q (meta- p-value of 7.3×10−4
, adjusted p
0.996, at 77 cM). However, our results do not indicate support for their chromosome 4 linkage peak, originally reported by Puca et al. 
, for their chromosome 12 peak reported in newly-enrolled HMS subjects, nor do the original HMS data show any indication of linkage anywhere on chromosome 18.
Human longevity is an outcome downstream of many biological processes, and it's very likely that multiple genes influence the trait. The FLAG study was designed to capture an extreme phenotype in a particular population, and our linkage results might be difficult to derive in other population settings. However, compared to most GWAS designs, the FLAG and HMS studies have better power to identify rare variants with large effects. While such variants may not explain a large portion of variation in longevity in a given population, they might very well help to identify important mechanisms that regulate onset and/or rates of aging in general. We should also consider that particular organs of the human anatomy might respond variably to mechanisms of aging, so that variation in aging among organs might suggest areas particularly amenable to pharmacological intervention.
Genes in the consensus linked region of 3p22-24 are listed in . The consensus region is the largest region for the combined data with a meta- p-value<0.001. Note that this definition narrows the region of interest more specifically to cytogenetic band 3p23-24.1. Of particular interest on 3p24.1 are EOMES
, and AZI2
because of their potential interactions with mTOR/rapamycin 
, free radical production and detoxification 
, and apoptosis 
, respectively. Another gene of particular interest in the 3p region is TGFBR2
because it is implicated in multiple disease etiologies 
), but of the 27 genes in the same region, most have functions that are not fully understood.
Genes in 3p22-24 linked region.
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.
We have recently described patterns of gene expression that are associated with human longevity and aging 
; 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 
, should greatly simplify the process.
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 longevity, which increases the probability that subjects carry a longevity-predisposing variant, but also increases the probability that any such variant is not widely distributed in the population. Our subjects were all of primarily Northern European ancestry, and hence were genetically less diverse than would be optimal for maximum generalizability of our results. We selected pairs of individuals related to varying degree, sometimes distantly related, which allowed us to identify a fairly large sample, but prevented us from using the most powerful techniques of linkage analysis. That we did not identify some linkage peaks previously reported by Boyden and Kunkel 
or Puca, et al. 
might be the result of relatively low power, allelic heterogeneity, and/or differences in study design. Our observation of a linkage peak on 18q that is clearly not present in the HMS data might similarly be the result of any of those factors. Given the substantial differences between the FLAG and HMS studies, and their limitations, it seems striking that the 3p23-24.1 region was clearly identified in both. Moreover, while our data do not quite replicate linkage peaks on 9q, 8q, and 17q, there is considerable support in both data sets for the possibility that predisposing variants are present in those regions as well.
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 
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.