3.1. Spanish cohort

Failure rate of genotypingwas6.9%in cases and 3.9% in controls.The distribution of ApoE genotypes was consistent with the HWE in both groups (cases, P = 0.918; controls, P = 0.910). The results of genotype/ allele frequency distributions as well as of binary logistic regression are shown in Table 1 and summarized below. The frequency of the ε2 or ε3-allele did not differ between cases and controls (P N 0.05). The ε4-allele was less frequent in Spanish cases than in controls (P = 0.027), but no difference was found for the ε4/ε4 genotype (P = 0.608). The ε4-allele was negatively associated with EL [odds ratio (OR) adjusted by sex = 0.55 (95%CI: 0.33–0.94, P = 0.030]. Table 2 shows the results of the subgroup analyses within the centenar- ians' group, with no significant P-value having been found.

2.2.3. Japanese cohort

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 Fall 2013 by using custom designed Taqman® SNP genotyping assays [IDs: C___3084793_20 for Cys112Arg (rs429358) and C____904973_10 for Arg158Cys (rs7412)] (Applied Biosystems, Foster City, CA, USA). All samples were run in a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). A total of 5 ml genotyping mixture contained 2.5 ml GTXpress™ master mix, 0.125 ml assay mix (40×) and 1.375 ml distilled water to mix with 1 ml genomic DNA (10 ng/ml) in each reaction. One or two negative controls were included on each

Based on the observed prevalence of the ε4-allele in the control group, the Spanish cohort's sample size had a 95% power to detect a relative likelihood of 2.22 for being a centenarian between ε4-allele non-carriers and carriers with a significance level (alpha) of 0.05 (two-tailed).

3.2. Italian cohort

Rate failure of genotyping was 0%. The distribution of the ApoE genotypes met HWE in the two groups (cases, P = 0.513; controls, P = 0.056). The results of genotype/allele frequency distributions

Table 1

Genotype/allele distributions of the apolipoproteinE (ApoE) gene and results of binary logistic regression in the Spanish cohort.

Cases Controls Cases vs. controls Fisher's test P-value Cases vs. controls Binary logistic regression (centenarians) n % n % OR (95%CI) P-value Genotype ε2/ε2 1 0.6 4 0.4 0.518 0.31 (0.14, 12.18) 0.810 ε2/ε3 21 12.9 93 8.9 0.114 1.57 (0.94, 2.64) 0.086 ε2/ε4 1 0.6 10 1.0 1.000 0.62 (0.35, 1.09) 0.095 ε3/ε3 125 76.7 774 74.5 0.628 1.11 (0.75, 1.64) 0.617 ε3/ε4 15 9.2 150 14.4 0.086 0.55 (0.07, 4.41) 0.574 ε4/ε4 0 0.0 8 0.8 0.608 – – Allele ε2 24 7.4 111 5.3 0.153 1.42 (0.89, 2.26) 0.145 ε3 286 87.7 1791 86.2 0.487 1.15 (0.80, 1.64) 0.454 ε4 16 4.9 176 8.5 0.027 0.55 (0.33, 0.94) 0.030

Significant P-values and associations are in bold. Abbreviations; 95% CI, 95% confidence interval; OR, odds ratio (adjusted by sex).

Table 2

Subgroup comparisons of genotype/allele distributions of the apolipoprotein E (ApoE) gene in Spanish centenarians.

Oldest centenarians Rest of Oldest vs. the rest Oldest vs. the rest Binary (oldest quartile) centenarians Fisher's test P-value logistic regression n % n % OR (95%CI) P-value Genotype ε2/ε2 0 0.0 1 0.8 1.000 – – ε2/ε3 4 9.8 17 13.9 0.598 0.67 (0.21, 2.12) 0.497 ε2/ε4 0 0.0 1 0.8 1.000 – – ε3/ε3 34 82.9 91 74.6 0.393 1.65 (0.66, 4.10) 0.282 ε3/ε4 3 7.3 12 9.8 0.763 0.73 (0.19, 2.72) 0.636 ε4/ε4 0 0.0 0 0.0 1.000 – – Allele ε2 4 4.9 20 8.2 0.464 0.57 (0.19, 1.73) 0.326 ε3 75 91.5 211 86.5 0.330 1.67 (0.71, 3.94) 0.240 ε4 3 3.7 13 5.3 0.769 0.68 (0.19, 2.44) 0.551

Abbreviations: 95% CI, 95% confidence interval; OR, odds ratio (adjusted by sex).

(P b 0.05). The frequency of the ε2 andε 3-allele was higher in cases than in controls and ε4-allele frequency was lower in the former (all P b 0.01). The ε4-allele was negatively associated with healthy EL [OR adjusted by sex = 0.35 (95%CI: 0.26, 0.57), P b 0.001], whereas a positive association was found for the ε2-allele [OR adjusted by sex = 1.81 (95%CI: 1.25, 2.63), P = 0.002] and ε3-allele [OR adjusted

as well as the data of binary logistic regression are shown in Table 3 and summarized below. The frequency of the ε3-allele did not differ between cases and controls (P N 0.05). The ε4-allele was less frequent in cases than in controls (P = 0.046), whereas the ε2-allele was more frequent in the former (P = 0.013). Theε4-allele wasnegatively associated with healthy EL [OR adjusted by sex = 0.41 (95%CI: 0.18, 0.99), P = 0.05], whereas a positive association was found for the ε2-allele [OR adjusted by sex = 2.14 (95%CI: 1.18, 3.45), P = 0.01]. Table 4 shows the results of the subgroup analyses within the cente- narians' group, which are summarized below. The ε2/ε3 andε 3/ε3 genotypes were more frequent in the oldest centenarians compared with the rest of cases (P = 0.001 and P = 0.002, respectively). The frequency of the ε2- and ε3-allele was higher and lower, respec- tively, in the oldest quartile compared with the rest of centenarians (P = 0.001 and P = 0.005).

Table 6 shows the results of the subgroup analyses within the cente- narians' group, with no significant P-value having been found.

Based on the observed prevalence of the ApoE ε4 in controls, the Japanese cohort's sample size had a 95% power to detect a relative like- lihood of 1.64 for being a centenarian between ε4-allele non-carriers and carriers with a significance level (alpha) of 0.05 (two-tailed).

Based on the observed prevalence of the ε4-allele in controls, the Italian cohort's sample size had a 95% power to detect a relative likeli- hood of 3.82 for being a centenarian between ε4-allele carriers and non-carriers with α = 0.05 (two-tailed).

3.4. Analysis of the three cohorts combined