Biologic age by osseographic scoring system

An osseographic scoring system (OSS) was applied to hand radiographs obtained on original cohort (1967 to 1969, mean age 58.7 years) and offspring participants (1992 to 1993, mean age 51.6 years) [10]. Biologic age was then defined as the standardized residual between the OSS predicted age and the actual age. Biologic age defined by this system predicted mortality [10,29], was very herit- able (h2 = 0.57 ± 0.06), and a genome-wide linkage anal- ysis was performed with LOD scores >1.8 present on chromosomes 3q, 11p, 16q, and 21q [10]. Sex- and cohort-specific ranked residuals generated from linear regression of age on log-OSS adjusted for height, body mass index, menopause, and estrogen therapy, were used for analysis.

Logistic regression models were used to generate deviance residuals. Models were sex-specific and adjusted for 1) birth cohort and 2) birth cohort, education, current smok- ing status, obesity, hypertension, elevated cholesterol, and diabetes. Covariates were defined as above for age at death. All covariates were measured at the examination closest to the participant attaining age 65 years using a 5 year window around age 65 years. Residuals from Origi- nal Cohort and Offspring participants were pooled.

Genotyping

Age at natural menopause

Affymetrix 100K SNP GeneChip genotyping and the Marshfield STR genotyping performed by the Mammalian Genotyping Service http://research.marshfieldclinic.org/ genetics are described in the Overview paper [24].

Natural menopause occurred after a woman had ceased menstruating naturally for one year and the age at natural menopause was the self-reported age at last menstruation. Mean age at natural menopause was similar in Original Cohort and Offspring women and the distribution of nat- urally menopausal ages in women in the 330 FHS families was similar to that of women in all 1643 FHS families [26,27]. The mean age at natural menopause in women in the 100K sample was 50.2 years (range 38 to 57 years) in Original Cohort women and 49.1 years (range 29 to 60 years) in Offspring women.

Statistical analysis

The statistical methods for genome-wide linkage and association analyses are described in the Overview [24].

Association

All residual traits described above as well as the additional traits listed in Table 1 were computed using Cox propor- tional hazards with martingale residuals for survival traits, logistic regression with deviance residuals for dichoto- mous traits, and linear regression with standard residuals for quantitative traits. The full set of FHS participants with

Crude age at natural menopause and standardized residu- als from multiple linear regressions in SAS [28] that adjusted age at natural menopause for covariates of inter- est were used as traits for analysis. Covariates were

the phenotype were used to create the residuals. The resid- uals were used to test for association between the geno- typed subset of individuals and the SNPs using additive family-based association test (FBAT) and generalized esti- mating equations (GEE) models as described in the Over- view [24]. A total of 70,987 autosomal SNPs met the criteria of genotypic call rate ≥80%, minor allele fre- quency ≥10%, Hardy-Weinberg test p ≥ 0.001, and ≥10 informative families for FBAT. The number of tests with an FBAT p < 0.001, p < 0.0001, and p < 0.00001 for all phenotypes was similar to what would be expected under the assumptions that the 70,987 tested SNPs were inde- pendent and there were no true associations. The GEE tests tended to give an excess of very small p-values over what would be expected under these assumptions.

spring participants only. Details of the sample size and covariate adjustment for each trait are provided in Table 1.

For each of the five phenotypes, Table 2a and 2b provides the top five SNPs ranked in order by lowest p-value for the GEE and FBAT models (all associations can be viewed on the web http://www.ncbi.nlm.nih.gov/projects/gap/cgi- bin/study.cgi?id=phs000007). If multiple SNPs in linkage disequilibrium (LD r2 > 0.80) were included in the top 5, additional SNPs were included until a set of 5 independ- ent associations were listed. Eight SNPs on chromosome 1 were associated with age at death in the FBAT analysis; all with p-value < 10-4 and two with p-value < 10-5. The 8 SNPs consisted of two sets of SNPs (rs10493513, rs10493514, rs6689491, rs6657082, rs1405051) and (rs10493515, rs10493518, rs10493517), clustered in two regions approximately 500 kb apart. There was exception- ally high LD across this 500 kb region: the minimum r2 between pairs of the eight SNPs was 0.58. The nearest genes in this region existing in public databases were >500 kb from any of these SNPs [31,32].

SNP prioritization