3.4. Analysis of the three cohorts combined

When studying the three cohorts combined, genotype distributions were: ε2/ε2 = 0.4%; ε2/ε3 = 13.9%; ε2/ε4 = 0.4%; ε3/ε3 = 76.9%; ε3/ε4 = 8.0%; and ε4/ε4 = 0.3% (cases) and ε2/ε2 = 0.4%; ε2/ε3 = 8.5%; ε2/ε4 = 0.9%; ε3/ε3 = 74.2%; ε3/ε4 = 14.9%; and ε4/ε4 = 1.1% (controls). The frequency of ε3/ε4 and ε4/ε4 genotypes was lower in cases than in controls (P b 0.001 and P = 0.032, respectively) whereas the ε2/ε3 genotype was more frequent in the former (P b 0.001). Allele distributions were: ε2 = 7.6%: ε3 = 87.9%; and ε4 = 4.5% (cases) and ε2 = 5.1%: ε3 = 85.9%; and ε4 = 9.0% (controls). The ε2 andε 3-allele were more frequent in centenarians than in controls (P = 0.003 and P = 0.037, respectively), whereas the ε4-allele was less frequent in b

3.3. Japanese cohort

Failure rate of genotyping was 1.75% in cases and 0.20% in controls. The distribution of ApoE genotypes was consistent with the HWE in both groups (cases, P = 0.111; controls, P = 0.293). The results of geno- type/allele frequency distributions as well as of binary logistic regression are shown in Table 5 and summarized below. The frequency of ε2/ε3 and ε3/ε3 genotypes was higher in cases than in controls (P b 0.01), whereas the frequency of ε3/ε4 and ε4/ε4 genotypes was lower in the former

Table 3

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

Cases Controls Cases vs. controls Fisher's test P-value Cases vs. controls Binary logistic (centenarians) regression n % n % OR (95%CI) P-value Genotype ε2/ε2 2 2.5 4 0.3 0.148 1.68 (0.12, 29.52) 0.64 ε2/ε3 12 15.2 50 9.5 0.060 1.94 (1.01, 3.77) 0.05 ε2/ε4 1 1.2 7 0.8 1.000 0.88 (0.09, 7.27 0.91 ε3/ε3 60 76.0 460 75.9 0.887 0.96 (0.49, 1.71) 0.84 ε3/ε4 4 5.1 70 13.0 0.084 0.41 (0.17, 1.17) 0.09 ε4/ε4 0 0.0 6 0.5 1.000 0.59 (0.04, 10.25) 0.73 Allele ε2 17 10.7 65 5.4 0.013 2.14 (1.18, 3.45) 0.01 ε3 136 86.1 1040 87.1 0.706 0.94 (0.51, 1.33) 0.68 ε4 5 3.2 89 7.5 0.046 0.41 (0.18, 0.99) 0.05

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

Table 4

Subgroup comparisons of genotype/allele distributions of the apolipoprotein E (ApoE) gene and results of binary logistic regression in Italian centenarians.

Oldest centenarians Rest of centenarians Oldest vs. the rest Oldest vs. the rest Binary logistic (oldest quartile) Fisher's test P-value regression n % n % OR (95%CI) P-value Genotype ε2/ε2 1 5.2 1 1.6 0.175 3.22 (0.18, 54.12) 0.43 ε2/ε3 8 42.1 4 6.7 0.001 10.99 (2.39, 36.74) 0.001 ε2/ε4 0 0 1 1.6 1.000 1.05 (0.04, 25.21) 0.94 ε3/ε3 9 47.5 51 85 0.002 0.17 (0.08, 0.51) 0.009 ε3/ε4 1 5.2 3 5.1 1.000 1.09 (0.17, 9.77) 0.98 ε4/ε4 0 0 0 0 – 1.00 (0.02, 255.12) 1.00 Allele ε2 10 26.3 7 5.8 0.001 5.64 (2.04, 16.27) 0.003 ε3 27 71.1 109 90.8 0.005 0.24 (0.11, 0.71) 0.008 ε4 1 2.6 4 3.4 1.000 0.79 (0.12, 7.24) 0.88

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

4. Discussion

ε3 = 88.1%; ε4 = 4.6%) are comparable to those previously reported in centenarians of the same country, e.g., ε2 = 4.5%; ε3 = 90.9%; ε4 = 4.5% (Asada et al. 1996).

Ourresultsindicate that theε4-allele wasnegatively associatedwith the likelihood of becoming a centenarian in the three cohorts. A novel finding of our study was that the ε2-allele seems to specifically favor EL in the Italian and Japanese population, although this result was not corroborated in the Spanish cohort. Of note, the ε2-allele might also specifically favor successful or healthy EL in the Italian cohort we studied. This allele was indeed more frequent in Italian centenar- ians, who were free of dementia or any other major age-related condi- tioncompared with their young controls, and was also more frequent in the oldest Italian centenarians than in the rest of this group. More research is however needed in other cohorts of disease-free cente- narians to corroborate the potential benefit of the ε2-allele on healthy EL.

Our results indicate that the ε4-allele is negatively associated with EL, a finding which was corroborated in our three independent cohorts. Some debate exists, with some authors traditionally viewing ApoE more as a ‘frailty’ or ‘disease-risk’ gene than as a longevity gene (Christensen et al., 2006; Fried et al., 2001; Gerdes et al., 2000), with its effects on death theoretically decreasing at very old ages (Christensen et al., 2006).Thisviewisinlinewiththe‘heterogeneityhypothesis’,according to which mortality of individuals carrying the ‘frail’ genotype (e.g., one or more ε4-alleles) in a population will approach that of non-carriers with advancing age because of a selection pressure on carriers; that is, surviving carriers might possess other factors compensating for the ‘frail’genotype,andthusheterogeneitybecomesamoreimportantfactor at old ages than carriage of a given risk-allele (Christensen et al., 2006). However, our results seem to be in support of previous data on Scandi- navian twins showing an increasing mortality risk associated with the ApoE ε4-allele with advancing age (Hjelmborg et al., 2006) or of those showing lower frequency of this allele in centenarians compared with younger controls of French (Blanche et al., 2001; Schachter et al., 1994), Finnish (Louhija et al., 1994), Southern Italian (Panza et al., 1999) or Chinese origin (Feng et al., 2011). Thus, our data seem in agreement with an another hypothesis, the so-called ‘multifactorial threshold model’, according to which many single factors (e.g., carriage of a frail genotype such as one or more ε4-alleles) each with small yet significant effect add up and contribute to a disorder/condition (or death) to happen with age (Jacobsen et al., 2010).