Figure 3 also shows results obtained by combining HMS and FLAG data, using the unweighted mean Z-score approach of Dempfle and Loesgen [35]. The combined data yield a linkage p- value of 1.00561029 (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,

doi:10.1371/journal.pone.0034746.t003



2. Comparison of singlepoint linkage results for chromosome 3 under varying definitions of affected status (nominal

enrolled HMS subjects, nor do the original HMS data show any indication of linkage anywhere on chromosome 18.

the meta- minimum p-value by meta-analysis for the HMS 34 peak is 1.2761025 (adjusted p =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 3.461024 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 7.361024, 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. [13], for their chromosome 12 peak reported in newly-

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.

—log{pval)  LAG 2011CombinedNECS 2010FE  

3. Comparison of chromosome 3 linkage results reported here to that reported by Boyden and Kunkel [13] (nominal

Table 4. Genes in 3p22-24 linked region.

Symbol Start Stop Entrez Name NEK10 27232101 27385916 152110 NIMA (never in mitosis gene a)- related kinase 10 SLC4A7 27389218 27473249 9497 solute carrier family 4, sodium bicarbonate cotransporter, member EOMES 27732890 27738789 8320 eomesodermin CMC1 28258128 28336267 152100 COX assembly mitochondrial protein homolog (S. cerevisiae) AZI2 28339090 28365579 64343 5-azacytidine induced 2 ZCWPW2 28406991 28541636 152098 zinc finger, CW type with PWWP domain 2 RBMS3 29297807 30026890 27303 RNA binding motif, single stranded interacting protein 3 TGFBR2 30622998 30710637 7048 transforming growth factor, beta receptor II (70/80 kDa) GADL1 30742696 30911157 339896 glutamate decarboxylase-like 1

Start and stop positions are given relative to the Human March 2006 (NCBI36/hg18) doi:10.1371/journal.pone.0034746.t004

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.

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 [1] or Puca, et al. [13] 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

Genes in the consensus linked region of 3p22-24 are listed in Table 4. 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, CMC1, and AZI2 because of their potential interactions with mTOR/rapamycin [36], free radical production and detoxification [37,38], and apoptosis [39,40], respectively. Anoth- er gene of particular interest in the 3p region is TGFBR2 because it is implicated in multiple disease etiologies [41,42]), but of the 27 genes in the same region, most have functions that are not fully understood.