younger controls from Germany (Nebel et al., 2011). However, whether ApoE influences exceptional longevity (EL), i.e., reaching 100+ years of age, is more controversial.

cohort of the Vallecas Project. The latter is a longitudinal study of older subjects with either no or mild cognitive impairment aimed at the early detection of AD. The history of past and current disease was collected by personal interview with the participants. Exclusion criteria were having stroke or traumatic brain injury with severe physical or cognitive impair- ment, clinically evident cancer, major psychiatric disorders or severe sys- temic diseases (e.g., chronic renal or liver failure). None of the subjects hadcriteriaofcognitiveimpairmentafterbeingsubmittedtoanextensive neuropsychological battery that included: Mini-mental-State Examina- tion (MMSE), Free and Cued Selective Reminding Test (FCSRT), Verbal Fluency Task (P, M & R), Rey-Osterrieth Complex Figure (ROCF), Drawing Clock Test, Global Deterioration Scale (GDS)-15 of Reisberg and Global Clinical Dementia Rating (CDR). All subjects with CDR N 0 were excluded from the present study.

The majority of reports indicate a lower frequency of the ApoE ε4-allele in centenarians compared with younger controls, i.e., in French (Blanche et al., 2001; Schachter et al., 1994), Finnish (Louhija et al., 1994), Southern Italian (Panza et al., 1999), or Chinese cohorts (Feng et al., 2011). However, others found no association of this variant with EL in Japanese (Asada et al., 1996), Finnish (Louhija et al., 2001) or Southern-Italian centenarians (Capurso et al., 2004). Concurrently, the ApoE ε2-allele has been reported to be more frequent in centenarians (Blanche et al., 2001; Louhija et al., 1994; Schachter et al., 1994; Seripa et al., 2006) than in younger people, but others found no differ- ences (Asada et al., 1996; Feng et al., 2011; Louhija et al., 2001; Panza et al., 1999). A meta-analysis reported a point estimate of 1.50 [95% confidence intervals (CI): 1.27, 1.78] and 0.49 (95%CI: 0.41, 0.58) for the frequency of ε2 and ε4-alleles, respectively, in centenarians vs. younger controls,but only studiespublished before2004 were included in the review (Lewis and Brunner, 2004).

2.1.2. Italian cohort

Two groups of Italian subjects born and living in Northern Italy were studied: (i) 79 cases (healthy centenarians, 100–104 years, 40 women, 39 men); and (ii) 597 healthy controls (27–81 years, 315 women, 282 men). The participants' ages were defined by the dates of birth as stated on identity cards. All patients and controls were Caucasian whites ascertained to be of Italian descent. The criterion of ‘Italian descent’ was met when all the parents and grandparents of an individual originated from Italy.

Our purpose was to determine if ApoE is associated with EL. To this end, we compared ApoE allele/genotype frequencies in centenarians (cases) and disease-free controls of the same ethnic and geographic origin among three independent cohorts from Spain, Italy and Japan. We also performed subgroup comparisons (i.e., oldest vs. the rest) within each centenarians' group. Based on previous research, we hypothesized that the ApoE gene may be associated with EL, with the ε2 and ε4-alleles playing favorable and unfavorable roles, respectively.

The Italian centenarians were ascertained mainly via general practi- tioners in the community. These centenarians represent a convenience sample that has been previously described (Emanuele et al., 2010). The history of past and current diseases was accurately collected, checking the centenarians' medical documentation and the current drug therapy. Accordingly, all the Italian centenarians were free of major age-related diseases, i.e., severe cognitive impairment, clinically evident cancer, CVD, renal insufficiency, or severe physical impairment. Only part of this group had decreased visual or auditory acuity. Thus, all of the Italian centenarians were in good health relative to their very ad-

2. Material and methods

2.1. Participants

Writtenconsentwasobtained fromeachparticipant.The studyproto- col was approved by the corresponding institutional ethics committees [European University of Madrid and Instituto de Salud Carlos III (Spanish cohort), University of Pavia (Italian cohort), and National Institute of Health and Nutrition, Medical Research Institute and Keio University (Japanesecohort)]andwasinaccordancewiththeDeclarationofHelsinki for Human Research of 1974 (last modified in 2008).

Controls were in apparent good physical health, with exclusion criteria being: presence of major CVD or cerebrovascular disease, cancer, dementia, chronic autoimmune or inflammatory disorders, renal or hepatic failure, and major psychiatric disorders.

2.1.1. Spanish cohort

Two groups of Spanish subjects (most born and living in the central area of Spain, Meseta Castellana) were investigated: (i) 175 cases (cente- narians, age range 100–111 years, 144 women, 31 men); and (ii) 1081 healthy controls (20–85 years, 622 women, 459 men). All the Spanish participants were of the same Caucasian (Spanish) descent for ≥3 gener- ations and all spoke Spanish as mother language.

2.1.3. Japanese cohort

Two groups of Japanese subjects [of the same Asian (Japanese) descent] were studied: (i) 742 cases (centenarians, 100–116 years, 623 women, 119 men); and (ii) 499 healthy controls (23–59 years, 356 women, 143 men). The group of cases was gathered from two prospective cohorts: the Tokyo Centenarians Study (TCS) and the Semi-Supercentenarians Study in Japan (SSC-J). A detailed description of population-based recruitments for the TCS has been previously re- ported (Gondo et al., 2006). The TCS cohort included 304 centenarians (65 men, 239 women) aged 100–108 years.