2005).

In humans, genetic polymorphisms in the three SOD genes may play an important role in the development of age-related diseases (Caple et al. 2010; Charniot et al. 2011; Soerensen et al. 2009; Wiener et al. 2007) and longevity (Budovsky et al. 2013; Soerensen et al. 2009). The most widely studied SOD single nucleotide polymorphism (SNP) rs4880 is located in the SOD2 gene and results in a T [ C substitution causing an amino acid substitution from valine to alanine. The association results for that SNP are inconsistent. It was found that individuals carrying the T allele have an increased risk for cardiomyopathy (Hiroi et al. 1999), atherosclerosis (Kakko et al. 2003), coronary heart disease (Jones et al. 2010), diabetes mellitus (Tian et al. 2011) and lung cancer (Wang et al. 2001). Other studies have shown that the C allele is more common in patients with breast cancer (Ambro- sone et al. 1999; Hubackova et al. 2012), Parkinson’s disease (Shimoda-Matsubayashi et al. 1996), Alzhei- mer’s disease (Wiener et al. 2007), gastric cancer (Yi et al. 2010) and prostate cancer (Woodson et al. 2003). However, Soerensen et al. indicated that the C allele of rs4880 is significantly associated with decreased mortality in a longitudinal study of 1,650 LLI (age at intake: 92–93 years) from the Danish 1905 cohort (Soerensen et al. 2009). A previous study showed an increased frequency of the T allele in older Ashkenazi male subjects (75 years) compared to younger con- trols (22 years) (Stessman et al. 2005). Other case– control studies did not show an association between rs4880 and longevity (De Benedictis et al. 1998; Taufer et al. 2005). As the SOD genes (i) have been shown to influence aging processes in model organ- isms, (ii) play an important role in the regulation of oxidative stress responses and (iii) are regarded as susceptibility factors for age-related diseases, these genes appear to be attractive candidates to be studied

Superoxide dismutases (SODs) are one class of molecules with antioxidant properties that remove superoxide from organisms by catalyzing the dismu- tation of the superoxide radical to hydrogen peroxide. They are a central player in the redox biology of cells and tissues and represent an important endogenous antioxidant defense and signaling system that protects cells from free radicals (Buettner 2011; Nozik-Grayck et al. 2005).

In mammalian cells, three SOD genes have been identified that all have specific functions and cellular distributions: the cytoplasmic copper-zinc SOD1 (CuZnSOD), the mitochondrial SOD2 (MnSOD) and extracellular SOD3 (EC SOD) (reviewed in Zelko et al. 2002). The role of SODs in aging and oxidative stress regulation has been widely studied and there is growing evidence that imbalances in these processes are linked to aging in several species like yeast (Longo et al. 1999; Unlu and Koc 2007; Wawryn et al. 1999), worms (Cabreiro et al. 2011), flies (Duttaroy et al. 2003; Kirby et al. 2002; Phillips et al. 1989) and mice (Elchuri et al. 2005; Hu et al. 2007). An over-

C 0.8; p [ 0.001) to ensure that most of the HWE allelic variation of the genomic regions was captured and the common haplotypes (C2 %) were represented. Most of the chosen SNPs are located in regions of interest such as exons and 50 and 30 untranslated regions. Furthermore, polymorphisms that have already been reported to be involved in aging processes and various diseases were included in the marker set.

in our extensive German longevity sample. In addi- tion, we tested the finding for the SOD2 SNP rs4880 in our extended sample set because of the previously observed inconsistent association results with old age. To this end, we performed a case–control association study using a comprehensive haplotype tagging approach for the three SOD genes (SOD1, SOD2 and SOD3) and tested a total of 19 SNPs in our collection of 1,612 LLI (centenarians and nonagenarians) and 1,104 younger controls (Supplementary Figure 1).

Statistical analysis

Allele-based single marker case–control analyses (CCA) and odds ratio (OR) statistics were calculated with v2 statistics using the open-source analysis toolset PLINK v.1.07 (http://pngu.mgh.harvard.edu/

Methods

Subjects

sidered nominally significant. All analyzed SNPs had a minor allele frequency of at least 1 % and were tested for Hardy–Weinberg equilibrium (HWE) with the software program Haploview version 4.1 (http:// www.broad.mit.edu/mpg/haploview/) (Barrett et al. 2005) before inclusion in the analyses (p [ HWE 0.001). Furthermore, Haploview was also used for selection of tagging SNPs based on the HapMap genotypes of Europeans with the pairwise tagging option (pairwise r2 C 0.8), to calculate linkage dis- equilibrium (LD) based on the measure r2 between each marker pair, for the visualization of LD plots and to conduct haplotype association analyses.

A total sample of 1,612 unrelated German study subjects of exceptional age (age range: 95–109 years, mean age: 98.8 years), including 748 centenarians (mean age: 101.3 years) was selected for the analysis. 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 (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