Results

SNP genotyping

The entire sample set of 1,612 German LLI, a subset of 748 centenarians and the control group of 1,104 younger individuals were subjected to a gender- matched case–control analysis of 19 SNPs located in the SOD1, SOD2 and SOD3 genes. All SNPs were found to be in HWE in both the cases and controls. No association was observed between the tested SNPs and the longevity phenotype, neither in the whole LLI sample (1,612 cases and 1,104 controls) (Supplemen- tary Table 2) nor in the centenarian subgroup (748 cases and 1,104 controls) (Table 1) or in a gender- specific analysis (Supplementary Table 3a, b). In the female centenarian subset, one SNP (rs8031) reached nominal significance (p = 0.035) which disappeared after correction for multiple testing (Supplementary Table 3b). Hence, we did not replicate the significant

genotyped for 19 SNPs located in the SOD1, SOD2 and SOD3 gene regions by the iPLEX Mass ARRAY technology (Sequenom, San Diego, CA), the SNPlex Genotyping System (Life Technologies Corporation, Foster City, CA) and the ABI TaqMan technology (Life Technologies Corporation, Foster City, CA). Of the 19 analyzed markers, three are located in the SOD1 gene, eight in SOD2 and eight in SOD3. The selected SOD SNPs with previously described findings are listed in the Supplementary Table 1 together with an overview of the complete marker set. The set consisted of an informative panel of SNPs selected through a haplotype tagging approach (based on the HapMap genotypes of Europeans with the pairwise tagging option; pairwise

et al. 2009; Deelen et al. 2011; Flachsbart et al. 2009; Li et al. 2009; Nebel et al. 2011; Pawlikowska et al. 2009; Schachter et al. 1994; Soerensen et al. 2010; Willcox et al. 2008). Both genes were identified by candidate gene association studies. A number of investigations were already performed on a genome-wide scale to uncover, in a hypothesis-free manner, common genetic variants that are involved in human longevity (Deelen et al. 2011; Nebel et al. 2011; Newman et al. 2010; Walter et al. 2011). However, beside APOE, genome- wide association studies (GWAS) failed to identify any additional susceptibility genes with genome-wide significance for this trait. Apparently, the influence of common genetic variation on longevity is rather modest which cannot be detected by GWAS (Deelen et al. 2011). Therefore, GWAS provide only limited statistical power as multiple testing for more than a million markers has to be taken into account. This means that candidate gene studies are still powerful tools for detecting new longevity variants.

Eighteen of the 19 analyzed SNPs form four haplotype blocks, where block 1 comprises three markers in SOD1, block 2 eight markers in SOD2, and block 3 plus block 4 seven markers in SOD3 (Supple- mentary Figure 1), which in turn results in 17 common haplotypes(each present atafrequencyofatleast 2 % in the population). None of the haplotypes showed a significant association with the longevity phenotype, neither in the entire LLI sample set (Supplementary Table 4a) nor in the centenarian subgroup analysis (Supplementary Table 4b).

Discussion

So far, only the association of genetic variants in two genes (APOE and FOXO3A) with human longevity has been validated in many different populations (Anselmi

1 Summary association statistics for the SOD1, SOD2 and SOD3 polymorphisms (centenarian subgroup compared to younger

1 Summary association statistics for the SOD1, SOD2 and SOD3 polymorphisms (centenarian subgroup compared to younger

Gene Chr SNP MAFa cases MAFa controls Pb ORc 95 % CId CCA SOD1 21 rs4998557 0.120 0.127 0.520 0.933 0.755–1.152 rs2070424 0.069 0.067 0.770 1.042 0.791–1.374 rs1041740 0.306 0.306 0.998 1.000 0.860–1.163 SOD2 6 rs6917589 0.236 0.231 0.741 1.027 0.879–1.199 rs2842980 0.206 0.200 0.689 1.036 0.873–1.229 rs7855 0.045 0.0463 0.842 0.969 0.706–1.328 rs8031 0.476 0.499 0.186 0.911 0.793–1.046 rs5746141 0.059 0.055 0.601 1.079 0.813–1.432 rs5746136 0.311 0.296 0.354 1.074 0.924–1.247 rs5746133 0.010 0.008 0.466 1.295 0.645–2.602 rs4880 0.504 0.494 0.552 1.041 0.912–1.187 SOD3 4 rs13306703 0.092 0.095 0.764 0.964 0.760–1.224 rs8192287 0.064 0.063 0.955 1.008 0.770–1.167 rs699473 0.327 0.335 0.613 0.965 0.839–1.109 rs17881426 0.090 0.098 0.425 0.907 0.715–1.152 rs1007991 0.318 0.325 0.679 0.969 0.834–1.125 rs2536512 0.341 0.340 0.942 1.005 0.875–1.155 rs8192291 0.199 0.179 0.144 1.135 0.958–1.345 rs8192290 0.025 0.030 0.428 0.841 0.548–1.291

b P , p value obtained from an allele-based case–control comparison, using a v2 test with 1 degree of freedom CCA c Odds Ratio for attaining old age with the minor allele in controls as reference allele d Confidence interval; 95 % CI is for OR

because its validity and efficacy for genetic longevity research have already been shown with the identifica- tion and validation of previous association findings (Flachsbart et al. 2009; Nebel et al. 2005). With the investigation of more than 2,700 individuals using a comprehensive haplotype tagging approach we there- fore suggest that there is no considerable influence of sequence variation in the SOD genes on human longevity in Germans.