Aim of the present study is to explore the VEGF gene promoter variability as a genetic determinant for longevity in an Italian population.

2.3. Statistical analysis

Two-tailed Pearson’s Chi-square and Fisher’s exact tests were used to compare genotype and allele frequency distri- butions and corresponding odds ratios (ORs) and 95% con- fidence intervals (95% CIs) were calculated. For haplotype definition, we used the algorithm developed by the haploview program (http://www.broad.mit.edu/mpg/haploview), which uses a two-marker expectation-maximization (EM) to define the eventual blocks, to estimate the maximum-likelihood val- sachay ues and to calculate the D values (Barrett et al., 2005). Haplotype phase and population frequency were inferred using a standard EM algorithm with a partition-ligation approach for blocks with greater than 10 markers. We also adjusted for multiple testing through a permutation approach on single marker. For each permutation, status was shuf- fled among both young/elderly and long-lived individuals, while the genetic data for each subject was not altered. The permutations were repeated 10,000 times.

2. Materials and methods

2.1. Subjects

A total of 490 unrelated Italian individuals (40.2% males) born and resident in Northern Italy, ranging from 25 to 99 years of age, were enrolled for this study. Clinical records were obtained for each participant. The presence of moderate/severe dementia in subjects aged more than 80 years was excluded by the mini-mental scale exami- nation (MMSE) test at time of blood collection. Subjects ≤ having a MMSE score 22 points were not included. Two groups of subjects were included in the study. Three hun- dred and four individuals (ranging from 25 to 99 years of age) were volunteers recruited either at nursing homes or at the Alzheimer Unit of “IRCCS Foundation Ospedale Mag- giore Policlinico, Mangiagalli and Regina Elena”, Milan, (non-consanguineous patients’ kindreds); 105 out of 304 subjects were aged more than 65 years; all of them had a MMSE > 26, CDR = 0 and Hachinski Ischemic score < 4 at time of sampling. In addition, 186 subjects (ranging from 80 to 99 years of age) were participants in the ongo- ing Monzino 80-plus study, a prospective door to door population-based survey among all 80 years or older resi- dents in the lower Olona Valley, some 20 km to the north of Milan (Tettamanti et al., 2006). An informed con- sent, approved by the Institutional Review Board of the “IRCCS Foundation Ospedale Maggiore Policlinico, Man- giagalli and Regina Elena”, Milan, was obtained by all participants.

3. Results

We screened 490 unrelated subjects from Italy for four well-known polymorphisms localised in the promoter and Ww&5/ 5 UTR region of VEGF gene. To carry out a compara- tive analysis of the genotypes, the whole cohort was divided into three age groups: (i) 166 young subjects (ranging from ± 25 to 65 years of age; mean standard deviation (S.D.): ± 47.4 11.2 years; 45.5% males); (ii) 158 elderly subjects ± ± (ranging from 66 to 84 years of age, mean S.D.: 75.7 5.7 years; MMSE: available only in 35 subjects aged more than ± 80years:25.6 1.8points;50.4%males);(iii)166long-lived

Table 1

VEGF genotype and allelic frequencies as detected in Italian healthy subjects

SNP Young subjects Elderly subjects Long-lived subjects p* p** OR (95% CI); p*** (%) n=166 (%) n=158 (%) n=166 −2578CC 53 (31.9) 52 (32.9) 44 (26.5) 0.03 0.03 1 (ref.) CA 85 (51.2) 79 (50.0) 74 (44.6) AA 28 (16.9) 27 (17.1) 48 (28.9) 2.08 (1.19–3.63); p=0.007 C 191 (57.5) 183 (57.9) 162 (48.8) 0.02 0.02 1 (ref.) A 141 (42.5) 133 (42.1) 170 (51.2) 1.43 (1.08–1.88); p=0.008 −1190GG 51 (30.7) 48 (30.4) 43 (25.9) 0.03 0.04 1 (ref.) GA 87 (52.4) 83 (52.5) 75 (45.2) AA 28 (16.9) 27 (17.1) 48 (28.9) 2.01 (1.14–3.52); p=0.011 G 189 (56.9) 179 (56.6) 161 (48.5) 0.03 0.03 1 (ref.) A 143 (43.1) 137 (43.4) 171 (51.5) 1.39 (1.06–1.83); p=0.014 −1154GG 68 (41.0) 75 (47.5) 71 (42.8) 0.57 0.33 GA 80 (48.2) 69 (43.7) 72 (43.4) AA 18 (10.8) 14 (8.9) 23 (13.9) G 216 (65.1) 219 (69.3) 214 (64.5) 0.93 0.21 A 116 (34.9) 97 (30.7) 118 (35.5) −634GG 62 (37.3) 58 (36.7) 78 (47.0) 0.16 0.10 GC 81 (48.8) 80 (50.6) 65 (39.2) CC 23 (13.9) 20 (12.7) 23 (13.9) G 205 (61.7) 196 (62.0) 221 (66.6) 0.22 0.25 C 127 (38.3) 120 (38.0) 111 (33.4)

*p values=young group vs. long-lived group; **p values=elderly group vs. long-lived group; ***p values=young/elderly pooled groups vs. long-lived group. *p and **p values were calculated using χ2 or Fischer’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 geno- type analysis was performed for all four polymorphisms − − (Supplementary data); in fact, 2578AA and 1190AA ≤ genotypesshowanotsignificantincreaseinMMSE 26sub- − − group; on the contrary, 1190A and 1154A alleles were ≤ significantly increased in subjects with MMSE 26 com- pared to MMSE > 26 subjects group (p = 0.046 and p = 0.027, respectively).

± subjects (ranging from 85 to 99 years of age, mean S.D.: ± ± 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 APO 4 allele was significantly reduced in long-lived sub- jects compared to younger individuals (8.1% versus 4.2%, p = 0.03).

All VEGF polymorphisms were in Hardy–Weinberg equi- librium (HWE) for the entire cohort of subjects; interestingly, a statistically significant difference was observed between − − 2578 and 1190 HWE frequencies in young/elderly sub- − jects compared to long-lived subjects ( 2578: p = 0.03; − 1190: p = 0.043).