The phenotype and disease characteristics of the Japanese centenarians are described elsewhere (Takayama et al. 2007), and the prevalence of hypertension, CAD, and dementia was of 63.6, 28.8, and 59.4 %, respectively.

Inclusion criteria for this group were man or woman aged <60 years and free of any diagnosed stroke, cardiac disease, and chronic – renal failure. During the years 2008 2012, genomic DNA was extracted from venous blood samples in 143 men and 356 women (National Institute of Health and Nutrition, Japan; see later discussion). We also used data from blood samples previously obtained in 409 men and 511 women (during year 2008) in the Department of Molecular Pathogenesis (Medical Research Institute, Tokyo Medical and Dental University, Japan), most of which have been included in another study (Hinohara et al. 2008).

Controls with CAD (n=395) Inclusion criteria for this ≤ group were (1) man or woman aged 65 years and (2) with diagnosed CAD. We used data from blood samples of 350 men and 45 women who were also previously genotyped (during the year 2008) in the Department of Molecular Pathogenesis (Medical Research Institute, Tokyo Medical and Dental University, Japan), most of which have been included in a published report on the association of rs1333049 and CAD risk (Hinohara et al. 2008).

healthy controls whose samples were genotyped in the genomics laboratory of the Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan), total DNA was isolated from venous blood by the use of QIAamp DNA Blood Maxi Kit (QIAGEN, Hilden, Germany). Genotyping was performed during Winter 2013 using custom-designed Taqman® SNP genotyping assays (assay ID C_1754666_10) (Applied Biosystems, Foster City, CA, USA). All samples were run in a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA, μ USA). A total of 5 l genotyping mixture contained 2.5 μ l GTXpressTM master mix, 0.125 μ l assay mix μ μ (40×), and 1.375 l distilled water to mix with 1 l μ genomic DNA (10 ng/ l) in each reaction. One or two negative controls were included on each plate.

Genotype assessment

In line with previous research (Emanuele et al. 2010), we focused only on the rs1333049 marker because the SNPs rs1333049, rs10757274, and rs10757278 on chromosome 9p21.3 identified as risk loci for cardiovascular disease in previous studies are in tight

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 ′ premier pairs rs1333049-F (5 -CCTTCATGCTATT ′ TTGAGGAG) and rs1333049-R (5 -GGAGATAAGT TGAGAATGTCA) (Hinohara et al. 2008).

groups are shown in Table 1. The main finding was that the frequency of the C-allele tended to be lower in P P cases than in healthy ( =0.088) or CAD controls ( = 0.078), and significant differences were found in P P 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 and genotype distributions did not differ between centenarians with CAD and/or dementia vs. those who were free of these two diseases (Table 2). Results of logistic regression are shown in Table 3.

Statistical analysis

All statistical analyses were performed using PASW (v. 18.0 for WINDOWS, Chicago, IL, USA) except for statistical power, which we calculated with StatMate software, version 2.0 (GraphPad, San Diego, CA, USA) (Emanuele et al. 2010; Minoretti et al. 2006). – We tested Hardy Weinberg equilibrium and compared the genotype/allele frequencies of cases vs. healthy/CAD controls within each cohort (Spanish and Japanese) using the χ2 test with α set at 0.05. Within each cohort of cases, we also compared allele/genotype distributions depending on disease status, i.e., centenarians with CAD and/or dementia vs. those who were free of these two conditions. We used logistic regression analysis to analyze the association between alleles and longevity within each of the two cohorts after adjusting for sex and age.

Japanese cohort Failure rate of genotyping was 1.6 % in cases and 0 % and 1.8 % in healthy and CAD controls, respectively. Thus, the final number of cases and healthy and CAD controls entered in the analyses n n was =742 (119 men, 623 women), =920 (409 men, 511 women), and n =395 (350 men, 45 women), – respectively. Genotype distributions met Hardy P Weinberg equilibrium in the three groups ( >0.05). The frequency of the risk C-allele in healthy controls (47.3 %) was comparable to that reported in another control cohort of Japanese people, i.e., 49.1 % (Hiura et al. 2008).

Allele/genotype distributions in the three Japanese groups are shown in Table 1. The main finding was that P allele ( =0.602) and genotype frequencies were P similar in cases and healthy controls ( =0.554). In contrast, significant differences were found in allele and genotype distributions between cases and CAD P “ ” 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 vs. those free of these two conditions P (Table 2). Likewise, allele ( =0.251) and genotype P 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 %). Results of logistic regression are shown in Table 3.

Results