However, there were also advantages to our study design. The nested case-control study design allowed us to select cases and controls from an ongoing cohort study with longitudinally collected data. This eliminated recall bias because the information was collected from our subjects before case-control status was determined. Moreover, major diseases were adjudicated by a morbidity and mortality committee that used performance-based measures of physical and cognitive function to supplement self-reported information.

Although some may question the relevance of candidate gene and pathway analyses such as ours in light of advances in technologies like genome-wide association studies and next-generation sequencing, these approaches still have value. Our study involving much older (>95 years) subjects than many others may be particularly valuable because longevity genes would be more concentrated in this population. However, a major limitation is the difficulty of obtaining sufficiently high numbers of subjects older than 95 years of age to test rare variants in addition to common variants like those we tested.Several other strengths are present in our study. Firstly, 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 important for aging and longevity (46). The mTOR pathway and many of its functions appear to be evolutionarily conserved. Secondly, not only did the study involve a nested case-control design but it had a high event rate (deaths) during a long period of follow-up, which enhances statistical power. Thirdly, the HHP cohort is a highly homogeneous group of individuals and no population stratification was detected in our study participants (5).

A possible weakness was that cases and controls had an average age difference of 10.8 years, so we cannot completely exclude the possibility of confounding by birth cohort effects. There are several reasons why this is not likely: (i) There was only a 19-year difference in birth year from oldest to youngest participants; (ii) Subgroup analyses showed no differences in socioeconomic status (education and occupation) between cases and controls; and (iii) Contrary to typical cohort studies in which younger cohorts have longer survival, older participants at baseline were most likely to have lived 95 and more years. Because our study was conducted in only one population, it should be replicated in other populations. Further research should also be directed at effects of the genetic variants with aging of specific organs.

Regarding the association between the SNPs in the mTOR pathway genes examined and longevity, it remains possible that variants having a minor allele frequency less than 0.10 or variants in other genes in this pathway could have significant effect(s) on longevity or the health or rate of aging of specific organs or both. This is particularly relevant to a genetic study of the rare phenotype of attainment of extreme old age (>95 years of age in our study). As a result, it is possible that at least some of the genetic variants that contribute to this phenotype are themselves rare or were below the minor allele frequency of 10% threshold we set for selection of SNPs for testing. Further research is needed to examine the association of uncommon and rare variants of mTOR pathway genes with longevity. Indeed, disruption of mTOR signaling has been associated with sarcopenia (47), and there is evidence that rapamycin treatment can reverse age-related.

In conclusion, the present study has found no association of common genetic variation in mTOR, RPTOR, RICTOR, and RPS6KA1 with living to age 95 or older in American men of Japanese ancestry. Further studies of these and other TOR pathway genes in both sexes and in other populations may be warranted.

Supplementary Material
Supplementary material can be found at: http://biomedgerontology.

Funding
This research was supported by the National Heart, Lung, and Blood Institute (contract NO1-HC-05102), the National Institute on Aging (contract NO1-AG-4-2149; and grants U01-AG-019349, R01-AG-038707, and R01-AG-027060), and the Hawaii Community Foundation (grant 2004-0463).

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