Centenarians were living mainly in nursing residencies of the Spanish centralarea, where theywererecruitedduringyears 2009–2012after we hadaccesstotheages'listsofthepeoplelivingintheresidencies.Thepar- ticipants' ages were ascertained by the dates of birth as stated on identity cards. TheirDNAwas obtained fromsalivasamples (Pinos etal., inpress). This group included the oldest European individual (111 years) alive in June 2012 (http://www.grg.org/Adams/E.HTM) and ~7% of the cohort was aged ≥105 years. The most prevalent diseases were osteoarthritis (66%), hypertension (57%), dementia (51%) and cardiovascular disease (29%). Only two centenarians were free of any diagnosed disease.

The SSC-J is a nationwide longitudinal survey consisting mainly of individuals aged 105 years or older, which started in 2002 (with n = 135 SSC). After 2002, the recruitment strategy has relied on re- sponses to local governments and nursing homes in the whole country, and direct inquiries to our research team. Consequently, a total of 450 centenarians (58 men, 392 women) were enrolled in the SSC-J by the end of November 2011. The phenotype and disease characteristics of the Japanese centenarians are described elsewhere (Takayama et al., 2007), with a prevalence of hypertension, CAD and dementia of 63.6%, 28.8% and 59.4%, respectively.

The DNA of 387 younger controls years, 170 women, 217 men) was collected from saliva samples during 2008–2009 in the European University (Madrid, Spain). This was a convenient sample composed of students and staff from this institution; all of them were freeofanymajordisease(includingCVDorAD)andhadnoknownfamily history of high longevity (90+ years), as reported in a questionnaire. The DNA of the remaining part of the control group (n = 794, 68–85 years, 452 women, 242 men) was obtained between October 2011 and April 2013 in the Fundación CIEN (Madrid, Spain) from blood samples of a

Inclusion criteria for the control group, which was recruited during the years 2008–2012 by advertising were being men or women aged b60 years, and free of diagnosed stroke, cardiac disease, and chronic renal failure (as reported in a questionnaire).

plate. TaqMan® SNP Genotyping Assays for genotype calls were analyzed by using StepOne™ Software v2.1 (Applied Biosystems, Foster City, CA, USA).

2.2. Genotype assessment

2.2.1. Spanish cohort

In the cases and younger healthy controls, genomic DNA was extract- ed frombuccal cells according to standard phenol/chloroformprocedures followed by alcohol precipitation. ApoE genotyping was performed with pre-designed Life Technologies TaqMan® SNP Genotyping Assays on de- mand considering two SNPs: Cys112Arg (rs429358; ID: C_3084793_20) and Arg158Cys (rs7412; ID: C_904973_10). Individuals ε2 were carriers of 112Cys and 158Cys alleles, individuals ε3 were carriers of 112Cys and 158Arg alleles, and individuals ε4 were carriers of 112Arg and 158Arg alleles. Polymerase chain reaction (PCR) amplification was performed by using a StepOne™ Real-Time PCR System (Life Technologies, Foster City, CA) with a denaturation stage at 95 °C for 10 min, 50 cycles of dena- turation at 92 °C for 15 s, annealing/extension at 60 °C for 1 min, and a final extension stage of 30 s at 60 °C.

2.3. Statistical analysis

ApoE allele frequencies were calculated from genotypes by using the gene-counting method. Hardy–Weinberg equilibrium (HWE) was tested with the χ2 test. Genotype/allele frequencies were compared between cases and controls in each of the three cohorts by using the Fisher exact test. We compared each genotype and allele with all other genotypes and alleles. We also conducted binary logistic regression adjusted by sex to examine the likelihood of reaching EL if carrying a given allele/ genotype. The same statistical approach was used to perform specific subgroup comparisons among centenarians in each cohort, i.e., oldest quartile vs. the rest of centenarians. A P-value ≤0.05 was considered statistically significant. All statistical analyses were performed by using the PASW (v. 18.0 for WINDOWS, Chicago), except for statistical power, which we calculated with the StatMate software, version 2.0 (GraphPad, San Diego, CA, USA).

For the controls gathered from the Vallecas Project, total DNA was isolated from peripheral blood following standard procedures. Genotyping of ApoE polymorphisms (rs429358 and rs7412) was deter- mined by Real-Time PCR (Calero et al., 2009).

2.2.2. Italian cohort

3. Results

ApoE genotyping was performed from venous blood samples in all subjects (cases and controls) using PCR restriction fragment length poly- morphism (PCR-RFLP) as described previously by Hixson and Vernier (Hixson and Vernier, 1990). In brief, a 244-bp ApoE fragment was ampli- fied 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′-GATCAAGCTTCCAATCACAGGCAGGA AG-3′ and 5′ GATCCGGCCGCACACGTCCTCCATG-3′. The amplified frag- ment was digested by using HhaI (Promega, Madison, WI) and the prod- ucts were visualized on polyacrylamide gels. Each genotype gives a specific combination of HhaI fragment sizes, i.e., ε2/2: 91 and 83 bp; ε3/3: 91 and 48 bp; ε4/4: 72 and 48 bp and a mixed genotype; ε2/3: 91, 83, and 48 bp; ε3/4: 91, 72 and 48 bp; ε2/4: 91, 83, 72 and 48 bp.

3.1. Spanish cohort