Concerning the haplotype analysis, the four SNPs showed − − a linkage disequilibrium for each pair of loci ( 2578/ 634 pany D value: 0.86). Inferred haplotype frequencies are shown in Table 2; interestingly, haplotype AAGG was signifi- cantly increased in long-lived subjects (p = 0.03) compared to younger individuals. As shown in Table 2, other esti- mated haplotypes (i.e. CGGC, AAAG, AAAC, AGGG) were differently distributed among young/elderly and long-lived subjects in terms of allele frequencies, but they fall to reach a statistically significant difference.

Genotype analysis failed to find different distributions − − − − of C( 2578)A, G( 1190)A, G( 1154)A and G( 634)C polymorphisms in young subjects compared to moderate elderly individuals (Table 1). In contrast, we observed significant differences in terms of genotype and allelic dis- tribution between the group of individuals aged more than 85 years and the two above mentioned groups. Specifically, − − 2578AA and 1190AA genotypes were differently dis- tributed between the groups. Subjects carrying the “at-risk” genotypes had ORs increased up to two-fold (Table 1). We observed some differences between young/elderly and long- lived individuals when subjects were stratified according to − the gender (Supplementary data); in particular, 2578AA, − − 1190AA genotypes and 1190A allele were significantly over-represented in women and not in men.

Furthermore, through the permutation approach on sin- gle markers, we confirmed the previously reported results with the univariate approach (− 2578C/A, χ2, p = 0.02; − 1190G/A, χ2, p = 0.04).

4. Discussion

Lifespan is the result of interactions between thousands of genes and non-genetic factors. Although the past 5 years

Finally, long-lived subjects were divided into two groups according to MMSE score: subjects with MMSE > 26 points

Table 2

Estimated haplotype frequencies among young/elderly individuals and long-lived subjects

Haplotypes Allele frequencies (%) p −2578 −1190 −1154 −634 Young/elderly subjects n=648 Long-lived subjects n=332 C G G C 226 (34.9) 97 (29.1) 0.06 A A A G 180 (27.8) 103 (30.9) C G G G 119 (18.4) 52 (15.7) A A G G 68 (10.4) 50 (14.9) 0.03 C A A G 13 (2.1) 6 (1.9) A G G G 7 (1.0) 7 (2.0) A G G C 7 (1.1) 6 (1.7) A A A C 4 (0.7) 6 (1.7) Others 24 (3.7) 7 (2.1)

have seen the identification of several candidate genes for longevity, only the role of APOE has so far been consis- tently confirmed (Christensen et al., 2006). The results of the present study, for the first time, support a role for specific VEGF polymorphisms and haplotypes as an advantage for lifespan extension, at least in the cohort of Italian individuals analysed.

tant factor leading to different results in case–control studies. Genetic heterogeneity might explain the different impact of the VEGF variants on the disease’s risks: for instance, we − − recently observed that C( 2578)A and G( 1190)A poly- morphisms are associated with type 1 diabetes in Italy, while − − in Finnish individuals G( 1190) and G( 1154)A variants protect from type 1 diabetes and delay its onset (Del Bo et al., 2006a). However, in order to explain why different com- binations of VEGF genotypes significantly associate to type 1 diabetes in different countries, it has been suggested a pos- sible linkage between VEGF and other genes, such as MHC genes.

a plausible candidate gene for longevity; in fact, VEGF has been implicated in neuronal survival, regeneration, growth, endothelial cell proliferation, migration and vasopermeabil- ity in many types of tissues (Sun et al., 2003). Its expression regulation is very complex being controlled by different factors including estrogens, growth factors and cytokines, many of which have been reported to be associated with longevity. It is known that VEGF is a major regulator of angiogenesis, and both angiogenesis and neurogenesis are regulated by systemic coordinations. When dysregulated, the formation of new blood vessels contributes to numerous malignant, ischemic, inflammatory, infectious and immune disorders (Carmeliet, 2003). The neuroprotective VEGF- mediatedeffectshavebeenassociatedwithregardtoischemic insults, AD, ALS, as well as some effects associated to neurogenesis (Jin et al., 2000). The main neuroprotective mechanisms include the inhibition of potassium channel activity and caspase-3 activity, the enhancement of striatal neurogenesis and maturation of newborn neurons, as detected in adult rat brains after stroke (Sun and Guo, 2005). Abnor- mal regulation of VEGF expression has been implicated in neurodegenerative disorders and the presence of regulatory mechanisms compensating for an insufficient VEGF activity has been suggested.

− The over-representation of the 2578A allele in the long-lived individuals is an apparent paradox because we previously demonstrated that Italian patients affected by Alzheimer’s disease (AD) bear the A allele more frequently (Del Bo et al., 2005). It might be explained by the possi- bility that there is an advantage from the inheritance of the − 2578A allele that might be linked to the involvement of this polymorphism, alone or in combination with other VEGF variants, in a wide range of cellular functions which would favour increased survival rates; similarly, the angiotensin I converting enzyme 1 (ACE1) D allele has been shown to be increased both in centenarians and in patients affected by coronary artery disease (Schachter et al., 1994), although the latter association was not confirmed in a larger study with about 5000 cases and 6000 controls (Keavney et al., 2000).