Results

Failure rate of genotyping (due in all cases to insufficient amount of DNA) was 13.1 % in cases and 5.2 % and 1.01 % in healthy and CAD controls, respectively. Thus, the final number of cases and healthy and CAD controls entered in the analyses n n was =152 (24 men, 128 women), =343 (131 men, n 212 women), and =98 (66 men, 32 women), respectively. Genotype distributions of rs1333049 – met Hardy Weinberg equilibrium in the two control P ’ P groups ( >0.05), but not in the cases group ( <0.05). “ ” The frequency of the risk C-allele we found in healthy controls (52.9 %) was similar to that previously reported in other control cohorts of Southern European

Table 1 Allele and genotype distributions of the rs1333049 (G/C) SNP in the two study cohorts

Cases Healthy controls CAD controls (centenarians) Spanish cohort Sample size per group (n) 152 343 98 χ2 and P-value for allele frequency comparison χ2=2.911 P=0.088 χ2=3.099 P=0.078 (cases vs. controls) Allele frequency, n (%) C=143 (47.0 %) C=363 (52.9 %) C=108 (55.1 %) G=161 (53.0 %) G=323 (47.1 %) G=88 (44.9 %) χ2 and P-value for genotype frequency comparisons χ2=6.766 χ2=6.220 (cases vs. controls) P=0.034 P=0.045 Genotype frequency, n (%) CC=45(29.6 % CC=105(30.6 %) CC=31(31.6 %) CG=53(34.9 %) CG=153(44.6 %)* CG=46(46.9 %) GG=54(35.5 %) GG=85(24.8 %)* GG=21(21.4 %)* Japanese cohort Sample size per group (n) 742 920 395 χ2 and P-value for allele frequency comparison χ2=0.272 χ2=23.99 (cases vs. controls) P=0.602 P<0.001 Allele frequency, n (%) C=689 (46.4 %) C=871 (47.3 %) C=452 (57.2 %) G=795 (53.6 %) G=969 (52.7 %) G=338 (42.8 %) χ2 and P-value for genotype frequency comparisons χ2=1.181 χ2=24.60 (cases vs. controls) P=0.554 P<0.001 Genotype frequency, n (%) CC=158 (21.3 %) CC=193 (21.0 %) CC=133(33.7 %)* CG=373 (50.3 %) CG=485 (52.7 %) CG=186 (47.1 %) GG=211 (28.4 %) GG=242 (26.3 %) GG=76 (19.2 %)*

CAD coronary artery disease, SNP single nucleotide polymorphism *P<0.05 for the comparison of controls vs. cases for the specific genotype

a lower proportion of this allele in the group of centenarians compared with their healthy and CAD controls. We also found a higher frequency of the GG genotype in the Spanish cases than in their healthy and CAD controls as well as a lower proportion of the CG genotype compared with healthy controls. Further, the frequency of the C-allele (47.0 %) in our cohort of centenarians, most of whom were not disease-free, 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). Although our findings overall corroborate and extend pioneer data by Emanuele et al. in another Southern European (Mediterranean) cohort, it must be emphasized that (1) the putative association between rs1333049 and extreme longevity was not so strong in our cohort and (2) the findings were not replicated in a cohort of a totally different ethnic and geographic (Japanese) “ ” origin. In the latter, the risk C-allele was in fact the minor allele in both centenarians and healthy controls

Based on the observed prevalence of the minor G-allele of the rs1333049 SNP in the CAD control group, the sample size of the Spanish cohort α ensured a power of 0.80 (with =0.05) to detect an OR of 1.48 for being a centenarian between G-allele carriers and non-carriers. Based on the observed prevalence of the minor G-allele in the CAD control group, the sample size of the Japanese cohort ensured a α power of 0.80 (with =0.05) to detect an OR of 1.20 for being a centenarian between G-allele carriers and non-carriers.

Discussion

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 that we studied, with a trends towards

Table 2 Allele and genotype distributions of the rs1333049 (G/C) SNP in cases (centenarians) depending on disease status

Centenarians free of CAD Centenarians with CAD and dementia and/or dementia Spanish cohort Sample size per group (n) 54 98 χ2 and P-value for allele frequency comparison χ2=0.005 P=0.942 Allele frequency, n (%) C=50 (46.3 %) C=58 (51.0 %) G=58 (53.7 %) G=66 (49.0 %) χ2 and P-value for genotype frequency comparison χ2 =0.543 P=0.762 Genotype frequency, n (%) CC=15 (27.8 %) CC=31 (31.6 %) CG=20 (37.0 %) CG=38 (38.8 %) GG=19 (35.2 %) GG=29 (29.6 %) Japanese cohort Sample size per group (n) 259 483 χ2 and P-value for allele frequency comparison χ2=0.361 P=0.548 Allele frequency, n (%) C=246 (47.5 %) C=443 (45.9 %) G=272 (52.5 %) G=523 (54.1 %) χ2 and P-value for genotype frequency comparison χ2=2.138 P=0.343 Genotype frequency, n (%) CC=62 (23.9 %) CC=96 (19.9 %) CG=122 (47.1 %) CG=251 (52.0 %) GG=75 (29.0 %) GG=136 (28.1 %)

χ2 and P-value for genotype frequency comparison

CAD coronary artery disease, SNP single nucleotide polymorphism

Table 3 Results of the association between the rs1333049 (G/C) SNP and the status of being a centenarian in the two cohorts

Spanish cohort Japanese cohort Centenarians vs. Centenarians vs. Centenarians vs. Centenarians vs. healthy controls CAD controls healthy controls CAD controls Dominant model CC+CG vs. GG 0.625 (95 % CI 0.409, 0.549 (95 % CI 0.269, 0.932 (95 % CI 0.743, 0.654 (95 % CI 0.436, 0.955; P=0.030) 1.121; P=0.100) 1.170; P=0.544) 0.983; P=0.041) Recessive model CC vs. CG+GG 0.932 (95 % CI 0.607, 1.106 (95 % CI 0.559, 1.120 (95 % CI 0.872, 0.696 (95 % CI 0.475, 1.429; P=0.745) 2.191; P=0.772) 1.439; P=0.374) 1.022; P=0.064) Co-dominant CG vs. GG 0.587 (95 % CI 0.362, 0.413 (95 % CI 0.191, 0.892 (95 % CI 0.701, 0.708 (95 % CI 0.450, 0.950; P=0.030) 0.890; P=0.024) 1.136; P=0.354) 1.112; P=0.134) CG vs. CC 1.185 (95 % CI 0.735, 1.766 (95 % CI 0.863, 1.165 (95 % CI 0.0.895, 0.774 (95 % CI 0.515, 1.910; P=0.487) 3.615; P=0.120) 1.517; P=0.256) 1.164; P=0.219)