and a region on chromosome 9q31-34 with an Overall maximum LOD score of 3.89 in Category 8. No other intervals achieved a LOD score above 3.0 (Figure 1), and given the high probability that peaks below that magnitude represent false positives, they were not considered further. By averaging the simulation results across all ten age categories, we calculated the per-category empirical threshold for genomewide significance with a type I error rate of a =0.05 to be a LOD score of approximately 3.1, likely reflecting the imperfect informativity of the 10K panel [37]. Correcting for all categories, a LOD score of approximately 3.9 was required for Overall genomewide significance. For the chromosomes 3 and 9 linkage peaks, respectively, we observed 36 and 53 distinct genomic locations among 1000 sets of replicates for which a LOD score in any age category met or exceeded the observed scores, resulting in corresponding empirical P values of 0.037 [upper 95% confidence limit 0.048] and 0.054 [upper 95% confidence limit 0.067]. Only a single replicate achieved two LOD scores of at least 3.89 at distinct locations across all categories, yielding an empirical P value for the combined result of 0.002 [upper 95% confidence limit 0.006]. The stability of the chromosomes 3 and 9 linkage peaks was evaluated by dividing the marker panel into two independent subsets containing every other SNP, and both regions retained strong evidence for linkage in the halved panels (data not shown).

maximum hLOD score for the chromosome 9 peak was higher under the dominant model (3.883) than the recessive model (2.680). The dominant model on chromosome 9 also yielded positive conventional parametric LOD scores of 1.047 in Category 8 and 2.840 in Category 9, suggesting reduced locus heterogeneity in the oldest sibships. The chromosome 12 peak achieved similar maximum hLOD scores of 3.915 and 3.875 under the dominant and recessive models, respectively, which likewise produced similar results on chromosome 4. We did not assess whether differences in hLOD scores under the dominant and recessive models were significant. Overall maximum nonparametric LOD and paramet- ric hLOD scores, P values, and linkage peak locations for chromosomes 3, 9, 12, and 4 are given in Table 2.

Discussion

We performed the second genomewide linkage scan on families of exceptionally long-lived siblings, with substantial improvements in power over the first scan [24]. A filtered panel of 9751 SNPs with an average minor allele frequency of 0.27, equivalent to approximately 4000 microsatellite markers [44], provided a ten- fold effective increase in marker density over the original scan. The resulting advantage in information content and power of similar SNP arrays over microsatellite panels has been demonstrated in theory [45] and in practice [46]. Our analysis of 279 families in Total Category 1 included 129 of the 137 sibships in the previous study plus an independent cohort of 150 sibships. For a set of 300 sibpairs the power to detect linkage is nearly 100% for an allele conferring a two-fold increased risk to siblings of affected individuals [47,48]. If genes with such magnitudes of effect exist they may be the most relevant to the study of human aging, and our scan would be well-powered to detect them.

The LOD scores for all linkage peaks varied considerably by group (Figure 2). The chromosome 4q22-25 linkage peak reported previously [24] produced an Overall maximum LOD score of 1.01 in Total Category 2. In the same region in the Previous subgroup we obtained maximum LOD scores of 2.14, 1.99, and 2.20 in Categories 1 to 3, respectively, whereas in the New subgroup, there was no evidence for linkage. For chromosomes 3 and 9, the Previous subgroup provided strong evidence for linkage with LOD scores of 3.90 in Category 4 (Overall maximum 4.49 in Category 5) and 3.43 in Category 8, respectively, while in the New subgroup the corresponding maximum LOD scores were 1.19 and 1.12. Analysis of the New subgroup also revealed a third novel locus on chromosome 12q24 with an Overall maximum LOD score of 4.05 in Category 6. This peak was completely absent from the Previous subgroup, and accordingly, the original cohort [24]. The Overall maximum LOD score for the chromosome 12 peak in the Total group was 1.11 in Category 5.

Across ten categories of minimum expected age at death requirements, we identified three novel loci of interest: an Overall genomewide significant peak on chromosome 3p24-22 (LOD = 4.02, P= 0.037), a highly suggestive peak on chromosome 9q31-34 (LOD = 3.89, P=0.054), and a peak on chromosome 12q24 (LOD =4.05) in the newly recruited subset of our subjects. These linkage peaks are preliminary results that warrant replication studies, as several factors may influence their significance (Text S6). None of our scans provided substantial evidence for linkage to FOXO3 on chromosome 6, probably because of the relatively small effect sizes conferred by SNPs at that locus [13–18]. Consistent with previous studies [24,49], the APOE gene on chromosome 19 was likewise not linked in any analysis, again possibly due to the low relative mortality risks of the e2 and e4 haplotypes [50]. In addition, the e4 haplotype predisposes young individuals to elevated mortality rates and is thus depleted in the exceptionally old [11], but such negatively selected alleles do not result in significant excess allele sharing among long-lived siblings [49]. By contrast, the e2 haplotype is enriched in the exceptionally old [11], but the apparent protective effect may be mediated by a heterozygote advantage mechanism [50-52]. The e2 haplotype is then analogous to a rare dominant variant, for which the power of allele-sharing methods is also low [49,53].