The analyses were repeated while stratifying by gender since the association of SNPs with longevity has previously been suggested to differ between genders. 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.

In the German population of 1,613 oldest-old and 1,104 middle-aged individuals, the APOE and CETP SNPs were analyzed, and rs769449 (APOE) was supported at the allelic level, whereas rs9923854 (CETP) showed borderline significance at the allelic level (95% CI (OR) 0.960-1.358) and was supported at the genotype level (see Table 4). Regression analysis on survival data on the oldest-old Danes was performed to investigate whether the two SNPs also affected survival during old age. We found no effect of rs769449 (APOE), while rs9923854 (CETP) showed nominal significance in females separately, with an estimate pointing in the same direction as observed in the case-control comparison (the data are summarized in Table 4). From the regression analysis, the quantitative effect of carrying at least one rs2069827 A allele was estimated to be 0.77 and 0.74 additional years of survival for AA/AC.The corrected and processed text is:

Males and females had a mean survival time of 2.91 and 3.74 years, respectively, for rs2069827-CC males and females, indicating a considerable effect. The study evaluated the association of 102 SNPs with survival at advanced ages and estimated their quantitative effects through regression analyses of survival variables using 11 years of follow-up data from the cohort of oldest-old Danes. The Dutch prospective cohort and Danish case-control data were also used for replication studies.

Investigation of the single borderline significant IL6 SNP in the Dutch replication cohort using Cox regression supported an effect on late-life survival; HR 0.74. qPCR investigation of IL6 expression did not show any significant difference in mRNA levels between genotype groups for rs2069827 (P value 0.532).

Several candidate genes have been explored for their role in human longevity, including APOE, CETP, and MTHFR. Retrospectively inspecting these SNPs in the data shows that only rs4343 (ACE) was nominally significantly associated with longevity (P<0.05), while no effect of the other two SNPs could be noted. Thus, these results emphasize that a single-marker candidate approach might point to a specific polymorphism but does not enable conclusions on the possible relevance of variation in the gene region as such.

To facilitate the confirmation of the relevance of a number of well-known candidate genes for longevity, we extended the examination of these genes to cover the majority of the common variation in the regions by using a total of 102 tagging SNPs. Applying the case-control approach, two SNPs, rs769449 (APOE) and rs9923854 (CETP), were found to be associated with longevity. The effect of these was supported by haplotype and gene-based analyses.

The German replication population also supported these findings, pointing to the importance of genes involved in lipoprotein metabolism for longevity. The effect of APOE rs769449 most likely reflected the well-known effect of the APOE 4 haplotype defining rs429358 and was not evident in advanced age survival. Rs9923854 (CETP) did show nominal effects on survival during old age, but only in females, indicating that the effect of this SNP might become gender specific toward the most late part of life.