Methods

Study sample

The genotyped study sample is comprised of 1345 Original cohort (n=258) and Offspring (n=1087) participants who are members of the 330 largest FHS families. The Overview [24] provides further details of this sample. With respect to aging and longevity traits, 149 deaths occurred at a mean age at death of 83 years (range 46 to 99 years), and 713 participants achieved age 65 yearsinvolved in the regulation of DNA repair and genes in the evolutionarily conserved insulin/insulin-like growth factor signaling pathway [13,14] are emerging as holding great promise in the future elucidation of the underlying physiology controlling lifespan. Many of these genes have human homologs and thus have the potential to provide insights into human longevity [15-20]. Although numerous candidate genes have been proposed, studies in humans are limited and initial findings often fail replication [21,22]. More recently, genome-wide association studies (GWAS) have become feasible and offer a more comprehensive and untargeted approach to detect genes with modest phenotypic effects that underlie common complex conditions [23]. Both the Original Cohort and the Offspring Cohort remain under continuous surveillance, and all deaths that occurred prior to January 1, 2005, were included in this study. Deaths were identified using multiple strategies, including routine participant contact for research examinations or health history updates, surveillance at the local hospital, search of obituaries in the local newspaper, and if needed through use of the National Death Index. Death certificates were routinely obtained, and all hospital and nursing home records prior to death and autopsy reports (if performed) were requested. In addition, if there was insufficient information to determine a cause of death, the next of kin were interviewed by a senior investigator. All records pertinent to the death were reviewed by an endpoint panel comprised of three senior investigators. The date and cause of death (classified as due to coronary heart disease, stroke, other cardiovascular disease [CVD], cancer, other causes or unknown cause) were recorded. We had the opportunity to use the Framingham Heart Study (FHS) Affymetrix 100K SNP genotyping resource for a GWAS of longevity and aging-related phenotypes. The FHS offers the unique advantage of a longitudinal family-based community sample with participants who have been well-characterized throughout adulthood with respect to prospectively ascertained risk factors and diseases and continuously followed until death. We report several strategies for 100K SNP associations: 1) a simple low p-value SNP ranking strategy; 2) SNP selection due to associations with more than one related phenotype; and 3) SNP associations within candidate genes and regions. Cox proportional hazards models were used to generate martingale residuals using the PHREG procedure in SAS to perform the regression analysis of survival time from age at study entry to age at death. Models were sex-specific and adjusted for 1) birth cohort and 2) birth cohort, education, current smoking status (yes/no), obesity (body mass index 30 kg/m2), hypertension (blood pressure 140/90 mmHg or on antihypertensive treatment), elevated cholesterol (cholesterol > 239 mg/dL), diabetes (fasting blood sugar 126 mg/dL, random blood sugar of 200 mg/dL, or use of insulin or oral hypoglycemic agents), and comorbidity defined as CVD and cancer. Birth cohort was defined as a categorical variable for all regression models with the following categories based on year of birth: birth year prior to 1900, 1900 to 1909, 1910 to 1919, 1920 to 1929, 1930 to 1939, 1940 to 1949, and 1950 and later. All covariates were measured at study entry. Residuals from Original Cohort and Offspring participants were pooled.

Walking speed was measured on Original Cohort participants at examination 27 (January 2002 through December 2003, mean age of Original Cohort at exam 27: 86.7 years) and Offspring participants attending an ancillary study to examination 7 (1999 to 2004, mean age at exam: 62.0 years). Trained technicians timed participants walking at their normal pace on a four-meter course twice and subsequently asked participants to repeat the course walking at a rapid pace. The mean timed fast walk among Offspring participants in the 100K genotyping sample was 2.44 seconds (standard deviation 0.89). The timed fast walk was used for analysis. Sex-specific linear regression was used to generate residuals adjusted for age and height measured at the time of the walk.

Morbidity-free survival was defined as achieving age 65 years free of CVD, dementia, and cancer. CVD events included angina pectoris, coronary insufficiency, myocardial infarction, heart failure, stroke, transient ischemic attack (TIA), intermittent claudication, and coronary or CVD death. Suspected CVD events were reviewed by a panel of three investigators who adjudicated events using previously established criteria in place since study inception [25]. A separate panel of study neurologists determined the presence of stroke or TIA, and a team of at least onemenopause in the FBAT analysis, SNP associations ranked numbers 6 and 7 were rs1121980 (p-value 0.0002) and rs2278493 (p-value 0.00027), both are in the vicinity of the MSRA gene.