Methods

Participants

The LLI sample comprised 1,384 unrelated German study participants of exceptional age (age range: 95–109 years, mean: 98.8 years), including 616 centenarians (mean age: 101.3 years). The gender ratio was 73% females versus 27% males. The 945 German control participants were be- tween 60 and 75 years of age (mean age: 66.9 years) and matched the LLI by ancestry, gender, and geographical ori- gin within the country. A detailed description of the samples and the recruitment procedure is given elsewhere (26). All participants gave informed written consent prior to partici- pation. The study was approved by the Ethics Committee of the University Hospital Schleswig-Holstein in Kiel.

As CYP2C enzymes also play an important role in the generation of ROS and are regarded as susceptibility factors for age-related diseases (15), they appear to be attractive candidates to be studied in the context of human longevity. Here, we performed a comprehensive fine mapping of the four CYP2C genes by testing altogether 56 single nucleo- tide polymorphisms (SNPs) in our extensive collection of

significant association, even without consideration of multiple testing (data not shown).

SNP Genotyping

DNA samples from LLI and control participants were an- alyzed for 56 SNPs in the CYP2C gene region (Figure 1, Table 1) by using the SNPlex Genotyping System (Applied Biosystems, Foster City, CA) (27). The complete marker set consists of (i) a maximally informative panel of SNPs, selected through a haplotype tagging approach (based on the HapMap genotypes of Europeans with the pairwise tag- ging option; pairwise r2 ≥ .8; p > .001) to ensure that HWE most of the allelic variation of the genomic regions was cap- tured and the common haplotypes (≥2%) were represented; (ii) potentially functional SNPs that are located in exons, exon–intron boundaries, promoter regions, and 5′ and 3′ un- translated regions; and (iii) polymorphisms that have already been proven to be functionally relevant in the con- text of CYP2C enzyme activity (28–30). Of the 56 analyzed markers, 14 are located in the CYP2C8 gene, 20 SNPs in CYP2C9, 10 SNPs in CYP2C18, and 12 SNPS in CYP2C19 (Figure 1, Table 1).

The 56 SNPs form three haplotype blocks (Figure 1). Block 1 comprises 20 markers, Block 2 comprises 19 mark- ers, and Block 3 comprises 11 markers (Figure 1), which define eight common haplotypes (each present at a frequency of at least 2% in the population) for each block. None of the observed haplotypes differed significantly in frequency between cases and controls (data not shown).

Discussion

Cytochrome P450 enzymes, especially the CYP2C iso- forms, are involved in the generation of ROS (15). They are expressed in tissues of the cardiovascular system and are considered susceptibility factors for age-related diseases (15). Furthermore, the CYP2C-encoding genes are known to be highly polymorphic, with a number of variants leading to changes in enzyme activity.

These observations prompted us to investigate whether allelic variation in the CYP2C-encoding genes was associ- ated with human longevity. Altogether, we genotyped 56 markers in our extensive DNA collection of more than 2,300 LLI and controls. None of the tested SNPs or haplo- types showed a statistically significant association with lon- gevity, neither in the whole sample nor in the centenarian subset.

Statistical Analysis

were performed with c2 statistics and the appropriate degrees of freedom using the open-source analysis toolset PLINK v.1.07 (http://pngu.mgh.harvard.edu/~purcell/plink/). p values smaller than .05 were considered nominally statistically significant, and Bonferroni correction for 56 tests was applied to the single-point results: Of the 56 tested markers, 28 were in strong linkage disequilibrium with each other (pairwise r2 > .8; calculated with the Haploview tagger pairwise option) so that the number of markers considered for the Bonferroni correction could be reduced to 28. As two case–control analyses (whole sample and centenarians) were performed, altogether 56 tests need to be taken into account. The software programme Haploview version 4.1 (http://www.broad.mit.edu/mpg/haploview/) was used to assess all polymorphisms for significant deviation from the Hardy–Weinberg equilibrium (HWE), to calculate linkage disequilibrium (r2) between each marker pair, and to conduct haplotype association analyses in blocks (31).

Candidate gene association studies have emerged as powerful tools in longevity research (32–47). So far, two longevity relevant genes (APOE and FOXO3A) have been confirmed in many different populations (32,35–37,39,48– 55). Because APOE and FOXO3A have been identified by candidate gene association studies, it seems that this method is still relevant for human longevity research, even in the era of genome-wide association studies. Although genome- wide association studies offer the advantage of detecting new longevity genes without a priori hypothesis, the power of hypothesis-driven candidate approaches is much higher than that of genome-wide association studies where millions of SNPs are tested, and multiple comparisons have to be taken into consideration as an essential part of determining statistical significance (56). Hence, so far it has been difficult to detect new longevity variants by genome-wide studies, and apart from the APOE locus, none of the reported genome-wide association studies signals achieved con- ventional levels of statistical significance (51,57,58). Altogether, candidate gene association studies still play an important role for the identification of longevity loci.

Results