Most reports indicate a lower frequency of the ApoE 4-allele in centenarians compared with younger controls in French (Blanche et al., 2001; Schachter et al., 1994), Finnish (PCR using TaqMan SNP Genotyping Assays. The Spanish cohort consisted of two groups: centenarians (175 cases, age range 100-111 years, with 144 women and 31 men), and healthy controls (1081 individuals aged 20-85 years, with 622 women and 459 men). All participants were of the same Caucasian (Spanish) descent for three generations and spoke Spanish as their mother tongue. The Japanese cohort also had two groups: centenarians (742 cases, age range 100-116 years, with 623 women and 119 men), and healthy controls (499 individuals aged 23-59 years, with 356 women and 143 men). The DNA samples of the Japanese centenarians were collected from two prospective cohorts: the Tokyo Centenarians Study (TCS) and the Semi-Supercentenarians Study in Japan (SSC-J). The Italian centenarians were recruited mainly through general practitioners in the community, representing a convenience sample that had been previously described. All Italian centenarians were free of major age-related diseases such as severe cognitive impairment, clinically evident cancer, CVD, renal insufficiency, or severe physical impairment, with only some having decreased visual or auditory acuity. Controls were in good physical health with exclusion criteria being the presence of major CVD or cerebrovascular disease, cancer, dementia, chronic autoimmune or inflammatory disorders, renal or hepatic failure, and major psychiatric disorders. Inclusion criteria for the control group were being men or women aged below 60 years, and free of diagnosed stroke, cardiac disease, and chronic renal failure. ApoE allele/genotype frequencies were compared between cases and controls in each cohort using Fisher's exact test. A P-value <0.05 was considered statistically significant.PCR was performed in the genomics laboratory of the Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan). Total DNA was extracted from venous blood using the QIAamp DNA Blood Maxi Kit (QIAGEN, Hilden, Germany). ApoE genotyping was carried out in Fall 2013 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). Each genotyping mixture consisted of 2.5 ml GTXpress master mix, 0.125 ml assay mix (40), and 1.375 ml distilled water mixed with 1 ml genomic DNA (10 ng/ml) in each reaction. One or two negative controls were included on each run. Based on the observed prevalence of the 4-allele in controls, the Japanese cohort's sample size had a 95% power to detect a relative likelihood of 1.64 for being a centenarian between 4-allele non-carriers and carriers with a significance level (alpha) of 0.05 (two-tailed). 

2.2.2 Italian cohort 

3. Results 

ApoE genotyping was performed on venous blood samples from all subjects (cases and controls) using PCR restriction fragment length polymorphism (PCR-RFLP) as previously described by Hixson and Vernier (Hixson and Vernier, 1990). In brief, a 244-bp ApoE fragment was amplified by PCR (35 cycles; 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min) with the primer pairs: 5'-GATCAAGCTTCCAATCACAGGCAGGAAG-3' and 5'-GATCCGGCCGCACACGTCCTCCATG-3'. The amplified fragment was digested using HhaI (Promega, Madison, WI), and the products were visualized on polyacrylamide gels. Each genotype gives a specific combination of HhaI fragment sizes, i.e., 2/2: 91 and 83 bp; 3/3: 91 and 48 bp; 4/4: 72 and 48 bp, and a mixed genotype; 2/3: 91, 83, and 48 bp; 3/4: 91, 72, and 48 bp; 2/4: 91, 83, 72, and 48 bp. 

3.1 Spanish cohort 

The failure rate of genotyping was 6.9% 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 binary logistic regression are shown in Table 1 and summarized below. The frequency of the 2 or 3-allele did not differ between cases and controls (P >0.05). The frequency of the 4-allele was lower in Spanish cases than in controls (P =0.027), but no difference was found for the 4/4 genotype (P =0.608). The 4-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 centenarians' group, with no significant P-value having been found. 

2.2.3 Japanese cohort 

The failure rate of genotyping was 1.75% in cases and 0.20% in controls. The distribution of ApoE genotypes met HWE in both groups (cases, P =0.513; controls, P =0.056). The results of genotype/allele frequency distributions as well as binary logistic regression are shown in Table 3 and summarized below. The frequency of the 3-allele did not differ between cases and controls (P >0.05). The frequency of the 4-allele was lower in cases than in controls (P =0.046), whereas the frequency of the 2-allele was higher in the former (P =0.013). The 4-allele was negatively associated with healthy EL [OR adjusted by sex=0.41 (95%CI:0.18-0.99), P =0.05], whereas a positive associationThe distribution of ApoE genotypes was consistent with HWE in both groups (cases, P = 0.111; controls, P = 0.293). Table 5 shows the results of genotype/allele frequency distributions as well as binary logistic regression, which are summarized below. The frequency of 2/3 and 3/3 genotypes was higher in cases than in controls (P < 0.01), whereas the frequency of 3/4 and 4/4 genotypes was lower in cases.