In the replication study, survival analysis using genotype data from the Leiden 85-plus Study was performed in Stata 11.1 by applying a sex-adjusted – Cox proportional hazard model. Kaplan Meier plots for the Dutch and the Danish cohorts were generated in Stata 11.1.

For all analyses, a nominal significance level was set to 0.05. Correction for multiple testing was performed by the permutation approach (applying max(T) permu- tation mode set at 10,000 permutations) for analyses carried out in the Plink and R software.

LD estimates for the APOE SNPs were obtained using the Plink software, and the LD data were used in the HaploView software (http://www.broadinstitute. org/haploview/haploview; Barrett et al. 2005) for gen- eration of an APOE LD plot.

Data on IL6 expression measurements was ana- lyzed in the STATA 11.1 statistical program by Δ linear regression of C and rs2069827 genotype t data, adjusting for age at blood sampling (the latter was initially observed to be a confounder). Assumptions of normal distribution and equal var- Δ – iance were tested for Ct by Shapiro Wilk and – – Breusch Pagan/Cook Weisberg tests. A box plot Δ of mean Ct by rs2069827 genotype groups was generated in STATA 11.1.

Two survival variables were applied in the Danish longitudinal study. First, we wanted to explore the genetic contribution to surviving for a short period versus a longer period of time during the ninth decade of life. The distribution of survival times was right skewed (data not shown); hence, a cutoff with respect

Results

(Candore et al. 2006; Franceschi et al. 2000; Li et al. 2009b; Soerensen et al. 2010), the analyses were re- peated while stratifying by gender. The sex-specific analyses of rs9923854 (CETP) and rs769449 (APOE) were in complete agreement with the result of the combined group, although rs769449 (APOE) did not pass correction for multiple testing (see Supplementary Table 3, Online Resource 4).

The 102 genotyped SNPs in the 16 candidate genes are listed in Supplementary Table 1 (Online Resource 2), whereas the Danish discovery cohorts and German and Dutch replication populations are described in Table 1.

Gene-based analysis confirmed the association of variations in CETP and APOE with longevity (see Table 3). HSPA14 and SIRT6 also showed significance in the gene-based analysis, but the gene-based P value for SIRT6 reflected the association of only a single SNP, since the remaining SIRT6 SNPs were insignificant at the single-marker level (data not shown).

Comparison of allele frequencies between middle- aged and oldest-old Danes showed more significant findings than expected by chance (see Supplementary Table 2, Online Resource 3) and left two out of 102 SNPs significant after correction for multiple testing. The minor allele frequency of rs9923854 (CETP) was – enriched in the 92 93 year olds, implying that this allele is advantageous for survival from middle-age to old age (OR >1). In contrast, rs769449 (APOE) displayed a decreased prevalence of the minor allele, thus indicating a disadvantageous effect on survival (OR <1). Analysis of genotype frequencies supported – these findings. Haplotype case control comparisons identified three haplotypes significantly associated with longevity after correction for multiple testing; all three included either rs9923854 (CETP) or rs769449 (APOE) and demonstrated effects in line with the single-SNP analysis. The data are summarized in Table 2.

ε 2/3/4 polymorphism (Bathum et al. 2006; Caliebe et al. 2010; Jacobsen et al. 2010) from 1,270 of the partic- ipants, we found that rs769449 (APOE) was in moderate LD with the ApoE ε 4 defining SNP rs429358 (R20 0.55). An LD plot for the APOE SNPs genotyped in this ε study and the two ApoE SNPs rs429358 and rs7412 is shown in Supplementary Figure 1 (Online Resource 5). In order to determine whether the effect of rs769449 was ε independent of APOE , logistic regression was per- – formed for case control status and rs769449 genotype data adjusting for gender and for either rs429358 or rs7412 genotype data. Using the 1,270 individuals with available information, the initially significant association 0 (P 0.043) remained unchanged after adjusting for

As the association of SNPs with longevity has previously been suggested to differ between genders

1 Characteristics of the Danish discovery cohorts and the German and Dutch study populations

The Danish discovery cohorts MADT 1905 Number of individuals 736 1,089 Average age in years at intake (range) 50.6 (46.0–55.0) 93.2 (92.2–93.8) Males/females (%) at intake 50.4:49.6 28.7:71.3 Mean follow-up time (SD) – 3.53 (2.5) Follow-up time (years) – 11.4 German case–control study Dutch longitudinal study Study populations for replication of initial findings Controls LLI Leiden 85-plus Study Number of individuals 1,104 1,613 563 Average age at intake (SD) 67.2 (4.07) 98.4 (2.7) 85.0 (0) Males/females (%) at intake 25.6:74.3 26.8:73.2 33.4:66.6 Mean follow-up time (SD) – – 5.49 (3.0) Follow-up time (years) – – 10.4

MADT the Study of Middle Aged Danish Twins, 1905 the Danish 1905 birth cohort, LLI long-lived individuals, SD standard deviation