Genotyping was performed in the genomics laboratory of Vall d'Hebron Hospital (Barcelona, Spain) during the summer and autumn of 2012 (centenarians and healthy controls) and summer of 2013 (CAD controls) for the Spanish cohort. The two experienced researchers in charge of genotyping were totally blinded to the participants' identities. Saliva samples were tracked solely with barcoding, and personal identities were only made available to the main study researcher who was not involved in actual genotyping. We used the classical phenol-chloroform DNA extraction protocol with alcoholic precipitation. Genomic DNA was resuspended in 50 μl milli-Q HO2 and stored at -20°C. We used custom-designed Taqman SNP genotyping assays (assay ID C_1754666_10) (Applied Biosystems, Foster City, CA, USA). All samples were run in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The analysis had a pre-read and post-read step of the plate consisting of 1 min at 60°C before and after the PCR cycle. The cycle conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 92°C, and 1 min at 60°C. To ensure proper internal control, for each genotype analysis, we used positive and negative controls from different DNA aliquots which were previously genotyped by the same method.

For healthy and CAD controls gathered from the Department of Molecular Pathogenesis (Medical Research Institute, Tokyo Medical and Dental University, Japan), venous blood samples and Taqman SNP genotyping assay (Applied Biosystems, Foster City, CA, USA) were also used for rs1333049 genotyping in PCR products with the primer pairs rs1333049-F (5'-CCTTCATGCTATT  TTGAGGAG) and rs1333049-R (5'-GGAGATAAGT TGAGAATGTCA) (Hinohara et al.). The main finding was that the frequency of the C-allele tended to be lower in PP cases than in healthy (=0.088) or CAD controls (=0.078), and significant differences were found in PP genotype distributions (=0.034 and =0.045), with a higher frequency of the GG genotype in cases than in both healthy and CAD controls and a lower proportion of the CG genotype in cases compared with healthy controls. On the other hand, allele andA cohort of Japanese individuals, comprising 49.1% (Hiura et al. 2008), was studied to determine allele/genotype distributions. Table 1 presents the distributions of alleles and genotypes in the three Japanese groups. The study found that P allele (=0.602) and genotype frequencies were similar in cases and healthy controls (=0.554). However, there were significant differences in allele and genotype distributions between cases and CAD controls (both <0.001), with the high-risk C-allele and CC genotype being less frequent and the low-risk GG genotype more frequent in the former. On the other hand, allele and genotype distributions did not differ between centenarians with CAD and/or dementia compared to those without these two conditions (Table 2). Similarly, allele (=0.251) and genotype distributions (=0.188) did not differ between TCS and SSC-J centenarians subgroups (TCS: CC=25.9%, CG=47.2%, GG=26.9%; SSC-J: CC=20.0%, CG=52.5%, GG=27.5%). Table 3 shows the results of logistic regression.

The failure rate of genotyping (due to insufficient DNA) was 13.1% in cases and 5.2% and 1.01% in healthy and CAD controls, respectively. Thus, the final number of cases, healthy controls, and CAD controls entered into the analyses were n=152 (24 men, 128 women), n=343 (131 men, 212 women), and n=98 (66 men, 32 women), respectively.

Genotype distributions of rs1333049 met Hardy Weinberg equilibrium in the two control groups (>0.05), but not in the cases group (<0.05). The frequency of the risk C-allele found in healthy controls (52.9%) was similar to that previously reported in other Southern European control cohorts. In our cohort of centenarians, most of whom were not disease-free, the frequency of the C-allele (47.0%) was similar to that recently reported (46.3%) in 80 super-healthy Italian centenarians (men and women, 100-105 years) by Emanuele et al. (2010).

Our main finding was that the risk C-allele of the rs1333049 (G/C) SNP located on chromosome 9p21.3 was associated with exceptional longevity in the Spanish cohort studied, with a trend towards this association in this population segment. The major strengths of our study were the clear definition used for the criterion of exceptional longevity (with all cases being centenarians or semi-supercentenarians) and that the analyses were performed in two totally independent cohorts from both ethnic and geographic perspectives.

There is evidence indicating an association, especially in Caucasian cohorts, of the rs1333049 SNP studied here and risk of CAD and vascular dementia (besides Alzheimer's disease), with the C-allele exerting an unfavorable effect. The rs1333049 SNP has been linked with CAD-related phenotypes in non-Caucasian cohorts, especially with myocardial infarction in Japanese people, with a frequency of the risk C-allele of 52.8% in 589 individuals who had suffered myocardial infarction (Hiura et al. 2008). Further, the C-allele was recently associated with excess mortality independent of CAD risk factors in individuals aged 71 years or older.(Dutta et al., 2011). In summary, our data overall replicate and extend the original data by Emanuele et al. (2010) in suggesting that the rs1333049 (G/C) polymorphism in chromosome 9p21.3 could be among the numerous variants contributing to exceptional longevity in Mediterranean (Southern European) cohorts. However, this association was not replicated in the healthy Japanese population, and further corroboration is needed in other ethnic/geographic groups worldwide.