the present study. “Cases” (longevity phenotype) were defined as HHP and HAAS participants who had survived to beyond the upper 1% of the 1910 U.S. birth cohort– specific survival (minimum 95 years of age; n = 440 as of June 2011, 296 having died [age of death = 97.0 ± 2.2 SD; range: 95–106 years]) (28) and 144 still being alive (mean age = 96.3 ± 1.6 SD; range: 95–106 years). Controls consisted of 374 individuals from the HHP and HAAS cohort who had died near the mean for the 1910 U.S. birth cohort–specific survival for middle-aged men (77 years of age). To achieve a case:control ratio of approximately 1:1, the HHP and HAAS study population was sampled for controls who died up to the age of 81 years (mean age at death = 78.1 ± 1.8 SD years [range: 73–81 years]), consistent with the 3.5 years longer life expectancy of Japanese American men in Hawaii (29). Analyses showed no evidence of population stratification in the data set (5). All participants were drawn from records of study participants updated up to June 2011. Archived pheno- typic data and blood samples from Examination 4 of the HHP (1991–1993), which coincided with the commence- ment of the HAAS, were used as the baseline exam for this nested case–control study. We had phenotype data for only 439 cases because one subject did not attend Exam 4. Participants included 3,734 men aged 71–93 years at Exam 4 (mean age: 77.9 ± 4.7 years), representing approx- imately 80% of the survivors from the original HHP (30). Procedures performed were in accordance with institu-

FK506-binding protein (FKBP12), which then binds to and inhibits TORC1 in its complex with raptor (16,22). In contrast, rapamycin does not bind to TORC2. Binding of raptor to eukaryotic initiation factor 4E-binding protein-1 stimulates ribosomal protein S6 kinase (23) and riboso- mal protein S6 kinase, 90 kDa, polypeptide 1 (RPS6KA1). RPS6KA1 is a downstream effector of nutrient-responsive mTOR signaling. It regulates multiple transcription factors, increases cell growth—in part by increasing mTOR signal- ing—and links nutrient availability to aging in organisms as diverse as yeast and mice (24). Rapamycin reduces mTOR- mediated phosphorylation of RPS6KA1 (16), so increas- ing life span and resistance to age-related pathologies, as well as producing gene expression patterns similar to those seen in caloric restriction or pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to caloric restriction (25).

tional guidelines and were approved by the Institutional Review Board of Kuakini Medical Center. Written informed consent was obtained from all study participants or from family representatives, if participants could not provide consent.

Here we report the results of a genetic association study of tagging single-nucleotide polymorphisms (tagSNPs) that provided virtually complete coverage of key mTOR complex genes MToR, RPToR, and RicToR, as well as RPS6Ka1, in long-lived Americans of Japanese ancestry. The study tested for association of these genetic variants with living to more than 95 years of age and with multiple aging-related parameters in these subjects.

Genotyping

Total cellular DNA was isolated from leukocytes using the PureGene system (Gentra Systems) and quantified using PicoGreen staining (Molecular Probes, Eugene, OR). SNPs genotyped were from within the gene and from a 5- kb region of DNA flanking either end of the genes MToR, RPToR, RicToR, and RPS6Ka1. TagSNPs were selected using Haploview, a program that defines a haplotype based on high correlation between the first and last markers and all intermediate markers. Supplementary Figures S1–S4 show the linkage disequilibrium blocks captured by the tagSNPs used. We restricted our selection to SNPs having a minor allele frequency of ≥10% in the Japanese popula- tion (as indicated by the HapMap database: http://hapmap. ncbi.nlm.nih.gov/; HapMap release 27/phase II + III, Feb 2009 on NCBI B36 assembly, dbSNP b126 – http://www. ncbi.nlm.nih.gov/projects/SNP/). The minimal coefficient of determination (r2) value at which all alleles are to be captured was set to a threshold of .8 for the identification of all tagSNPs. Genotype data were managed through an

Methods

Study Population

We used American men of Japanese ancestry drawn from the Honolulu Heart Program (HHP) and Honolulu- Asia Aging Study (HAAS; HHP and HAAS) population. Recruitment, design, and procedures have been outlined in detail elsewhere (26,27). Briefly, recruitment of subjects aged 45–68 years (mean age: 54 years) began on Oahu in 1965–1968. These men have been followed with 12 regular examinations and blood work until the present, or death. A nested case–control design was utilized for

with cases demonstrating significantly lower blood pressure and clinical phenotypes in midlife and better grip strength and cognitive score in late life.

integrated database system sample management-data pro- cessing system that has been proven to be accurate in other successful studies (5). All positive controls on each geno- typing plate were evaluated for consistency. Call rates for markers exceeded 98%.

Genetic association Findings

SNPs for MToR, RPToR, and RicToR were genotyped at the University of Hawaii Cancer Center on the Illumina GoldenGate platform (high-throughput SNP genotyp- ing on universal bead arrays (31)). SNPs for RPS6Ka1 were detected by the TaqMan OpenArray System (Life Technologies). We implemented a series of quality control checks based on the Illumina metrics. For inclusion of data for a SNP, its call rate had to exceed .95 and the Hardy– Weinberg equilibrium p needed to be >.01. Of the 81 tag- SNPs genotyped, all met these criteria and were included. In addition, more than 96% concordance was observed for all SNPs for HapMap samples that were assayed for quality assurance.