Results:
We analyzed the genotype and allele frequency distributions of the four polymorphisms in the three age groups. Significant changes of allele, genotype (2578/AA versus 2578/CC: OR=2.08, p=0.007; 1190/AA versus 1190/GG: OR=2.01, p=0.011) and haplotype (AAGG: 10.4% versus 14.9%, p=0group. *p and **p values were calculated using 2 or Fisher's exact tests with two degrees of freedom (for all genotypes) or with one degree of freedom (for allele comparisons). (n = 86) and subjects with 26 points (n = 80). We observed no significant difference between the two groups when genotype analysis was performed for all four polymorphisms (Supplementary data); in fact, 2578AA and 1190AA genotypes show a not significant increase in MMSE 26 subgroup; on the contrary, 1190A and 1154A alleles were significantly increased in subjects with MMSE 26 compared to MMSE > 26 subjects group (p = 0.046 and p = 0.027, respectively). Subjects ranged from 85 to 99 years of age, with a mean S.D. of 89.8 ± 2.8 years; MMSE: 25.3 ± 2.5 points; 27.3% males. As expected, the percentage of subjects carrying at least one vasde4 APOE4 allele was significantly reduced in long-lived subjects compared to younger individuals (8.1% versus 4.2%, p = 0.03). All VEGF polymorphisms were in Hardy-Weinberg equilibrium (HWE) for the entire cohort of subjects; interestingly, a statistically significant difference was observed between 2578 and 1190 HWE frequencies in young/elderly subjects compared to long-lived subjects (2578: p = 0.03; 1190: p = 0.043). 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 significantly increased in long-lived subjects (p = 0.03) compared to younger individuals. As shown in Table 2, other estimated haplotypes (i.e. CGGC, AAAG, AAAC, AGGG) were differently distributed among young/elderly and long-lived subjects in terms of allele frequencies, but they failed 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 moderately elderly individuals (Table 1). In contrast, we observed significant differences in terms of genotype and allelic distribution between the group of individuals aged more than 85 years and the two above-mentioned groups. Specifically, 2578AA and 1190AA genotypes were differently distributed 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 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 single markers, we confirmed the previously reported results with the univariate approach (2578C/A, 2, p = 0.02; 1190G/A, 2, p = 0.04).

4. Discussion

Lifespan is the result of interactions between thousands of genes and non-genetic factors. Although in the past five years we have seen the identification of several candidate genes for longevity, only the role of APOE has so far been consistently 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 analyzed.

The marked variability in the genetic background represents an important factor leading to different results in case-control studies. Genetic heterogeneity might explain the different impact of the VEGF variants on disease risks: for instance, we recently observed that C(2578)A and G(1190)A polymorphisms 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, to explain why different combinations of VEGF genotypes significantly associate with type 1 diabetes in different countries, it has been suggested a possible linkage between VEGF and other genes, such as MHC genes.

VEGF gene is highly polymorphic, with at least 25 different polymorphisms, which are organized into haplotypes, with severalPopulation (Chiappelli et al., 2006). Furthermore, the (2578A) allele was higher in APOE 4-positive MCI patients who developed AD than in those who did not. All these findings support the role of VEGF in the deterioration rate of cognitive performance in both preclinical and clinical AD. Due to the low number of long-lived subjects carrying at least one APOE 4 allele (n = 14) in the present study, we are unable to analyze the interaction between VEGF and APOE genotypes. Longitudinal studies will be required to verify how many subjects included in the present study will develop cognitive deficits, as well as MCI or AD. In addition, VEGF variability in subjects with moderate/severe dementia (having an MMSE score of 22) should be investigated.

Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.neurobiolaging.2007.05.003.