Besides the rationale for postulatingthe ApoEas a candidate to ence EL, a major strength of our study was the clear definition we used for the criterion of EL (with all cases beingcentenarians).A novelty with regards to previous research on EL and ApoE stems from the fact that (i) the analyses were conducted in three independent cohorts (with one of them, the Japanese cohort, including a very large sample of centenarians) and(ii)westudiedboth‘normal’andsuperhealthy(Italian)centenarians. Nonetheless, it must be kept in mind that the cross-sectional nature of our design precludes comments on causality and we only concentrated on EL as a categorical trait (i.e., to be centenarian or not) rather than on the continuous life-span, which runs the risk of losing some information. On the other hand, some caution is needed when attempting to extrapo- late our findings to the whole Italian, Spanish or Japanese population be- cause we used mostly convenience samples, which raises the risk of bias due to population stratification. Another limitation from our study stems from the relatively high rate of genotyping failure in the Spanish cohort, particularly in cases. The main reason might lie on the fact that DNA ex- traction from saliva in frail individuals such as centenarians might result in small amounts of DNA available for analyses (we did not collect blood samples based onethicalconstraints). Thus,blood sampling is likelypref- erable in this population segment. Also, two different methodologies were used in the Spanish cohort, i.e., TaqMan® and real-time PCR. With regard to the two aforementioned limitations, we believe the fact that enotype distributions met HWE in the two Spanish groups (cases and controls) and allele/genotype frequencies were similar to those reported in other Southern European cohorts (Blanche et al., 2001; Capurso et al., 2004) supportthevalidityof our data. Finally,interpretationofcase–con- trol studies comparing allele/genotype frequencies among centenarians and younger counterparts may be flawed by differences in date of birth, e.g., 20+ year difference in our cohorts (with centenarians and controls beenbornintheearly1900sandafter1930respectively).Indeed,mortal- ity risk depends on the interaction of genotype with environmental or lifestyle risk factors and the pattern of exposure to such risk factors is re-

The explanation for our novel i.e., that the could benefit EL in the Italian and Japanese population, and specifically favor healthy attainment of EL in the Italian cohort, is less apparent and war- rants further research. The ApoE ε2-allele has previously 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 differences (Asada et al., 1996; Capurso et al., 2004; Fengetal.,2011;Louhijaetal.,2001;Panzaetal.,1999).Ameta-analysis considering only studies of centenarians published before 2004 gave a point estimate of 1.50 (95%CI, 1.27–1.78) for ε2-allele frequency in centenarians vs. younger controls (Lewis and Brunner, 2004). However, scant data are available on the disease status of the centenarians' cohorts of the above-mentioned reports. Capurso et al. (2004) studied Southern-Italian centenarians without dementia, or other chronic neurological or cerebrovascular disease, but found no differ- ences in ApoE genotype frequencies between this group and younger controls. To date, much more data are available on the deleterious role of the ε4-allele, particularly with regards to AD risk, than on a potential protective effect of the ε2-allele. Inheritance of two copies of the ε4-allele is indeed associated with a N10-fold increased risk for de- veloping AD compared to the most common ε3/ε3 genotype (Corder et al., 1993; Strittmatter et al., 1993). There is strong evidence that ApoE ε4 facilitates Aβ deposition, with senile plaque density being significantly higher in the brains of AD ε4/ε4 patients compared with those with the ε3/ε3 genotype (Gomez-Isla et al., 1996; Rebeck et al., 1993). Although Aβ deposition is the pathological hallmark of AD, oligomeric, soluble forms of Aβ have been implicated as the synaptotoxic component,inwhichhigherlevelsofAβoligomersareincreasingtheloss

Gerontology

(

)

–

Gondo, Y., Hirose, N., Arai, Y., Inagaki, H., Masui, Y., Yamamura, K., Shimizu, K., Takayama, M., Ebihara, Y., Nakazawa, S., Kitagawa, K.,

Functional status of centenarians in Tokyo, Japan: developing better phenotypes of exceptional longevity. J. Gerontol. A Biol. Sci. Med. Sci.

,

–

Hallman, D.M., Boerwinkle, E., Saha, N., Sandholzer, C., Menzel, H.J., Csazar, A., Utermann, G.,

Theapolipoprotei

polymorphism:acomparisonof allele frequenciesand effects in nine populations. Am. J. Hum. Genet.

,

–

Hashimoto, T., Serrano-Pozo, A., Hori, Y., Adams, K.W., Takeda, S., Banerji, A.O., Mitani, A., Joyner, D., Thyssen, D.H., Bacskai, B.J., Frosch, M.P., Spires-Jones, T.L., Finn, M.B., Holtzman, D.M., Hyman, B.T.,

Apolipoprotein E, especially apolipo- protein E

, increases the oligomerization of amyloid beta peptide. J. Neurosci.

,

–

Hixson, J.E., Vernier, D.T.,

Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hh

J. Lipid Res.

,

–

Hjelmborg, J.V., Iachine, I., Skytthe, A., Vaupel, J.W., M

ue, M., Koskenvuo, M., Kaprio, J., Pedersen, N.L., Christensen, K.,

Genetic influence on human lifespan and lon- gevity. Hum. Genet.

,

–

Jacobsen,R.,Martinussen,T.,Christiansen,L.,Jeune,B.,Andersen-Ranberg,K.,Vaupel,J.W., Christensen, K.,

Increased effect of the Ap

gene on survival at advanced age in healthy and long-lived Danes: two nationwide cohort studies. Aging Cell

,

–

Kolovou, G., Yiannakouris, N., Hatzivassiliou, M., Malakos, J., Daskalova, D., Hatzigeorgiou, G., Cariolou, M.A., Cokkinos, D.V.,