Table 2. Characteristics of chromosomes 3, 9, 12, and 4 linkage peaks.

Locus 3p24.2–22.3 9q31.3–34.2 12q24.31–24.33 4q21.21–28.1 Age category reported 4 8 6 3 Total max LOD (d) 4.02 (0.288) 3.89 (0.498) 1.11 (0.168)a 1.01 (0.135)c Total max hLOD dominant (a) 3.334 (0.329) 3.883 (0.792)b NA NA Total max hLOD recessive (a) 4.652 (0.271) 2.680 (0.364)b NA NA 99% (22 LOD) left confidence boundary rs2362772 rs723706 rs606443 rs726896 Build 37.1 left boundary location 24918232 112778426 120910630 81213792 Peak maximum rs28150 rs536861 rs1732462 rs1008326 Build 37.1 maximum location 29594086 128313444 127444592 110563638 99% (22 LOD) right confidence boundary rs1382554 rs1074052b rs953182 rs1586149 Build 37.1 right boundary location 35093841 136462498b 129106410 127074788 Category-specific empirical Pvalue 0.006 0.012 NA NA Overall empirical Pvalue 0.037 0.054 NA NA Overall Pvalue upper 95% confidence limit 0.048 0.067 NA NA Previous max LOD (d) 4.49 (0.464)a 3.43 (0.636) ,1 2.20 (0.298) Previous max hLOD dominant (a) 4.029 (0.534)a 3.187 (0.833) NA 1.954 (0.358) Previous max hLOD recessive (a) 4.527 (0.389)a 2.766 (0.410) NA 1.966 (0.241) New max LOD (d) 1.19 (0.220) 1.12 (0.377) 4.05 (0.504) ,1 New max hLOD dominant (a) NA NA 3.915 (0.605) NA New max hLOD recessive (a) NA NA 3.875 (0.386) NA MC max LOD (d) 1.85 (0.260) 3.62 (0.603) ,1 ,1 MC max hLOD dominant (a) NA 3.145 (0.678) NA NA MC max hLOD recessive (a) NA 3.121 (0.377) NA NA FO max LOD (d) 2.77 (0.402)a 1.33 (0.622)b ,1 ,1 FO max hLOD dominant (a) 2.499 (0.458)a NA NA NA FO max hLOD recessive (a) 3.500 (0.357)a NA NA NA

Overall maximum LOD scores were noted if greater than or equal to 1, and hLOD scores were noted for peaks with LOD scores greater than or equal to 2. The parameters a and d represent the proportion of families that are linked (parametric analyses), and the magnitude of excess allele-sharing (nonparametric analyses), respectively. Boundaries are for the Total group for chromosomes 3 and 9, the New subgroup for chromosome 12, and the Previous subgroup for chromosome 4. The boundaries of the chromosome 9 peak are a composite of Categories 8 and 9, which had overlapping but slightly offset peaks of similar magnitude. NA, not applicable (scores were not noted or P values were not determined). aCategory 5. bCategory 9. cCategory 2. doi:10.1371/journal.pone.0012432.t002

with the RecQ helicases encoded by WRN (MIM 604611) [69] and BLM (MIM 604610) [70], in which mutations cause the progeroid disorders Werner syndrome [71] and Bloom syndrome [72], respectively. DBC1 (MIM 602865) in the chromosome 9 linkage peak directly interacts with SIRT1 (MIM 604479) and inhibits its activity [73]; SIRT1 is activated by resveratrol [74] and has been implicated in several age-related phenotypes in mammals [75]. The sirtuin family members are key regulators of lifespan in yeast [76], worms [77], and flies [78] and mediate the effects of caloric restriction [79,80], the only behavior known to increase lifespan in a wide variety of organisms, including mammals [81]. In response to nutrient withdrawal, SIRT1 is stimulated by FOXO3 [82], in which variants have been reproducibly associated with human longevity [13–18]. In addition, different SNPs in or near TLR4 (MIM 603030) in the chromosome 9 linkage peak have been associated at least once, though often not reproducibly, with exceptional longevity in men [83,84], a bone-related proxy for biological age [85], and various age-related diseases [86]. Also of note, mutations in ANK2 (MIM 106410), in the previously reported chromosome 4 linkage peak, cause long-QT syndrome

[87], and common variation in ANK2 has been reported to regulate the QT interval [88]. QT interval prolongation is a risk factor for sudden cardiac death in healthy individuals [89] as well as those with ischemic heart disease [90] and chronic congestive heart failure [91], which are more common causes of death among centenarians than younger individuals [92]. Heterozygous Ank2+/2 knockout mice displayed multiple signs of premature senescence and their lifespan was significantly reduced compared to wild-type littermates [93]. Finally, a nominally significant association was reported between a SNP in ALPK1 (MIM 607347) and both age at death and morbidity-free status at age 65 [85]. Both ANK2 and ALPK1 are within the 99% confidence (-2 LOD) [94] interval for the original chromosome 4 linkage peak [24] but are outside the 85% confidence (-1 LOD) interval tested in the study that identified MTTP [28], raising the possibility that ANK2, ALPK1, or another gene besides MTTP could explain the previous linkage result. The positions of all the above genes within linkage peaks suggest they merit attention in future gene identification efforts.

The linkage scans reported here should contribute to the analysis of both linkage and association studies on exceptional

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Since cross-sectional study designs for longevity are subject to unique methodological complications [11] and longitudinal cohort designs can be prohibitively expensive, case-control studies will benefit from direction or corroboration by linkage scans. For example, the extensive multiple testing problem encountered in genomewide association studies can be partially alleviated by a weighted Bonferroni correction or Bayesian analysis that employs linkage data [96]. Alternatively, our results could help inform deep resequencing efforts to identify rare variants that influence lifespan, a particularly suitable approach for genes in which multiple variants exert individual effects too weak to be detected by association methods. Discovery and confirmation of human longevity genes will provide insight into the biology of aging and the genetic basis for resistance to age-related disease.

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Nonparametric linkage data for Previous subgroup. Found at: doi:

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