Longevity and aging phenotype definitions and residual creation

A number of potential candidate genes in a variety of bio- logical pathways have been associated with longevity in model organisms. Genes involved 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 control- ling lifespan. Many of these genes have human homologs and thus have 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 pheno- typic 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 exami- nations 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) was 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 dis- eases 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, edu- cation, current smoking status (yes/no), obesity (body mass index ≥30 kg/m2), hypertension (blood pressure ≥140/90 mmHg or on antihypertensive treatment), ele- vated cholesterol (cholesterol > 239 mg/dL), diabetes

obtained at all attended examinations prior to the onset of menopause and included mean number of cigarettes smoked per day, mean body mass index, parity (0 versus 1 or more live births), and generation (Original Cohort vs. Offspring).

(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 regres- sion 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 par- ticipants were pooled.

Walking speed

Walking speed was measured on Original Cohort partici- pants at examination 27 (January 2002 through Decem- ber 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 walk- ing at their normal pace on a four meter course twice and subsequently asked participants to repeat the course walk- ing at a rapid pace. The mean timed fast walk among Off- spring 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 at age 65 years

Morbidity-free survival was defined as achieving age 65 years free of CVD, dementia, and cancer. CVD events included angina pectoris, coronary insufficiency, myocar- dial 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 incep- tion [25]. A separate panel of study neurologists deter- mined the presence of stroke or TIA and a team of at least one neurologist and one neuropsychologist determined the presence of dementia. Two independent reviewers examined records for all cancers, and the vast majority of cancer cases were microscopically confirmed with pathol- ogy reports.

Biologic age by osseographic scoring system