Allele-based single marker case-control analyses (CCA) and odds ratio (OR) statistics were calculated with v2 statistics using the open-source analysis toolset PL(http://www.broad.mit.edu/mpg/haploview/) (Barrett et al., 2005) was used to check for any misspelled words in the text before it was included in the analysis (p [HWE 0.001). Haploview was also utilized to select tagging SNPs based on the HapMap genotypes of Europeans with pairwise tagging option (pairwise r2 C 0.8). It was also used to calculate linkage disequilibrium (LD) based on the measure r2 between each marker pair, for visualizing LD plots and for conducting haplotype association analyses. The analysis involved a total of 1,612 unrelated German study subjects who had exceptional age (age range: 95-109 years, mean age: 98.8 years), including 748 centenarians (mean age: 101.3 years). The gender ratio was 75% females versus 25% males. At the time of recruitment, 1,104 German control subjects were between 60 and 75 years old (mean age: 66.9 years). All controls matched the LLI by ancestry, gender, and geographical origin within the country. A detailed description of the samples and the recruitment procedure is reported elsewhere (Nebel et al., 2005). Written informed consent was obtained from all participants. Ethical approval was obtained by the Ethics Committee of the Medical Faculty of the Christian-Albrechts-University, Kiel.

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) (Supplementary 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 association found for rs4880 (Soerensen et al., 2009; Stessman et al., 2005).

Genotyping for 19 SNPs located in the SOD1, SOD2 and SOD3 gene regions were carried out 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 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 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 SOD1The search for replication findings represents one of the major challenges in human longevity research. Replication failures could be due to different sample sizes and age ranges used for case and control groups, which hamper validation of association results. Associations may also be population-specific, as observed for rs4880 in several populations. For example, the Jewish sample (Stessman et al. 2005) was much smaller and younger than the German study group and represented a different ethnicity. Even though the German and Danish populations are quite similar in sample size, age range, and ethnicity, the Danish study was prospective, which might explain the different results in our retrospective case-control analysis. Furthermore, varying sets of interacting genetic and environmental factors might induce population-specific effects (Caliebe et al. 2010).

Neither the entire LLI sample nor the centenarian subgroup showed a significant association between the tested SNPs or haplotypes and the longevity phenotype. By tagging an informative subset of common variants and considering haplotype differences, comprehensive information about common SNPs and haplotypes was covered. Additionally, it should be possible to obtain statistical evidence for the effects of rare variants that are captured by one of the common haplotypes (Lin et al. 2004). The possibility that our negative association finding results from population stratification in our sample is unlikely.