comment. Because we only studied men, it remains to be seen whether genotypes of any of the SNPs tested could be associated with longevity or other aging-related pheno- types in women. In addition, baseline examination findings suggested that the longevity case participants were healthier than controls despite the fact that cases were on average 10.8 years older. The cases possessed significantly better biological and physiological risk factor profiles, less age- related disease, and better physical and cognitive function (Table 1). This lends additional credence to the concept of a “healthy aging” phenotype, whereby certain individuals are somehow able to delay or avoid major clinical disease and disability until late in life.

Our long-lived cases also had metabolic profiles that sug- gested higher insulin sensitivity at younger ages, namely lower waist:hip ratio, lower glucose levels, lower insulin levels, and lower homeostasis model assessment values. This profile clearly has at least some genetic basis because protective variants in FoXo3 were associated with pheno- types that reflect insulin sensitivity in previous work (5,42). Although there was bias in the selection of controls because only their deaths qualified them for control status, this was unavoidable.

An important advantage of the present study is the nested case–control study design, which selects cases and controls from an ongoing cohort study, with longitudinally col- lected data. This has advantages relative to a regular case– control study in that several phenotypes of interest, such as biological and physiological phenotypes, disease preva- lence, and functional status, were obtained by direct clini- cal examination when the participants were younger. This eliminated recall bias, namely, the differential accuracy of recall between cases and controls, because the information was collected from our subjects before case–control status was determined. In contrast, findings from studies of excep- tional survivors, such as centenarians, in which evidence for

heart dysfunction (48). Furthermore, gene expression analysis in calorically restricted mice indicates a potential neuroprotective role for the mTOR signaling pathway (49). Moreover, as mentioned earlier, there are significant differ- ences in the expression of mTOR pathway genes in older ages (38).

(43–45), could have suffered from recall bias; older partici- pants may not have recalled their past medical history pre- cisely, nor their functional status when they were younger, or may have learnt of their condition before self-reporting their exposure history to researchers. In the present study, major diseases were, moreover, adjudicated by a morbid- ity and mortality committee that used performance-based measures of physical and cognitive function to supplement self-reported information.

ies, genome-wide expression studies, and next-generation sequencing, one might question the relevance of candidate gene and pathway analyses such as that adopted in the pre- sent study. Although some headway has been made using data from published genome-wide association studies for 125 diseases and risk factors relevant to mortality in order to obtain a genetic score for time to death (50), finding actual genetic variants would require very large numbers of sub- jects of extreme old age in order for such studies to be suf- ficiently powered to generate a result that is significant at the genome-wide level. Although that particular study confirmed that longevity is a complex and highly polygenic trait, it also suggested that it is not easily explainable by common genetic variants related to diseases and physiological traits. In the largest genome-wide linkage analysis for human longevity, involving 2,118 white nonagenarian sibling pairs in 15 study centers of 11 European countries, 4 regions with significant logarithm of the odds of linkage (Lod) scores were identified (2). Fine mapping and a fixed-effect meta-analysis approach showed that aPoeε4 and aPoeε2 alleles at the ToMM40, aPoe, and aPoc1 locus explained the linkage at 19q13.3– q13.32, confirming the role of APOE as a longevity gene. The authors concluded that linkage results for the other three loci were not explained by common variants, suggesting that rare variants play an important role in longevity. Thus, although case–control studies, such as ours involving much older (>95 years) subjects than these, would seem to have value because longevity genes would be more concentrated in the extremely old, a major limitation is the difficulty of studies such as ours in obtaining sufficiently high numbers of subjects older than 95 years of age to test polymorphisms other than common variants, such as those we tested.

There are several other strengths to our study. First, the candidate genes selected for analysis were chosen a priori based on hypothesis-driven criteria. That is, studies of model organisms of aging using various methods, such as knockouts, have shown that the mTOR pathway is impor- tant for aging and longevity (46). The mTOR pathway and many of its functions appear to be evolutionarily conserved. Second, not only did the study involve a nested case–con- trol design, but it had a high event rate (deaths) during a long period of follow-up, which enhances statistical power. Third, the HHP cohort is a highly homogeneous group of individuals and no population stratification was detected in our study participants (5).