Is this polymorphism the functional variant we were searching for? Some hints indicate that this may be the case. First, a strong linkage disequilibrium occurs between the G477T silent marker and VNTR alleles; second, both genotypic and allelic frequency distributions differ between the group selected for longevity (male sample 2) and the younger control group (male sample 1).

Fig. 2. (A) Luciferase expression of constructs including the 1a–4a alleles reported as fold induction (pGL3 negative control). (B) Luciferase expression of constructs including the allele 2b or the allele 2a reported as fold induction (pGL3 negative control). In both A and B the values reported for transfection experiments are the means F SD of triplicate experiments.

It is intriguing that the sole allele lacking an enhancer effect in vitro, allele 2b, is completely absent in the oldest

Table 2

(A) Genotypic and (B) allelic counts of VNTR polymorphism in samples of males and females of different ages

Males Females (A) Genotype Sample 1 (20–80 years) Sample 2 (90–106 years) Sample 1 (20–80 years) Sample 2 (90–105 years) 1.1 46 (14.7 F 2.0) 14 (16.3 F 4.0) 78 (19.9 F 2.0) 27 (17.3 F 3.0) 1.2 36 (11.5 F 1.8) 3 (3.5 F 2.0) 37 (9.5 F 1.5) 13 (8.3 F 2.2) 1.3 42 (13.5 F 1.9) 15 (17.4 F 4.1) 70 (17.9 F 1.9) 31 (19.9 F 3.2) 1.4 69 (22.1 F 2.3) 23 (26.7 F 4.8) 80 (20.5 F 2.0) 33 (21.2 F 3.3) 1.5 — — 1 (0.3 F 0.3) — 1.6 — — 4 (1.0 F 0.5) — 2.2 10 (3.2 F 1.0) — 3 (0.8 F 0.4) 4 (2.6F 1.3) 2.3 12 (3.9 F 1.1) — 15 (3.8 F 1.0) 6 (3.8 F 1.5) 2.4 20 (6.4 F 1.4) 2 (2.3 F 1.6) 18 (4.6 F 1.1) 5 (3.2 F 1.4) 2.5 1 (0.3 F 0.3) — 1 (0.3 F 0.3) — 3.3 18 (5.8 F 1.3) 4 (4.7 F 2.3) 22 (5.6 F 1.2) 10 (6.4 F 2.0) 3.4 37 (11.9 F 1.8) 11 (12.8 F 3.6) 32 (8.2 F 1.4) 13 (8.3 F 2.2) 3.5 1 (0.3 F 0.3) 2 (2.3 F 1.6) 1 (0.3 F 0.3) — 3.6 — — 1 (0.3 F 0.3) — 4.4 19 (6.1 F 1.4) 10 (11.6 F 3.5) 28 (7.1 F 1.3) 11 (7.1 F 2.0) 4.5 1 (0.3 F 0.3) 1 (1.2 F 1.2) — 2 (1.3 F 0.9) 4.6 — 1 (1.2 F 1.2) — 1 (0.6 F 0.6) Total 312 (100.0) 86 (100.0) 391 (100.1) 156 (100.0) (B) Allele 1 239 (38.3 F 1.9) 69 (40.1 F 3.7) 348 (44.5 F 1.8) 131 (42.0 F 2.8) 2 89 (14.3 F 1.4) 5 (2.9 F 1.3) 77 (9.9 F 1.1) 32 (10.3 F 1.7) 3 128 (20.5 F 1.6) 36 (20.9 F 3.1) 163 (20.8 F 1.5) 70 (22.4 F 2.4) 4 165 (26.4 F 1.8) 58 (33.7 F 3.6) 186 (23.8 F 1.5) 76 (24.4 F 2.4) 5 3 (0.5 F 0.3) 3 (1.8 F 1.0) 3 (0.4 F 0.2) 2 (0.6 F 0.5) 6 — 1 (0.6 F 0.6) 5 (0.6 F 0.3) 1 (0.3 F 0.3) Total 624 (100.0) 172 (100.0) 782 (100.0) 312 (100.0)

Relative frequencies SE ((cid:1)100) are given in parentheses. (A) Genotypic homogeneity tests between samples 1 and 2: p = 0.026 in males, p = 0.674 in females. (B) Allelic homogeneity tests between samples 1 and 2: p b 0.001 in males, p = 0.923 in females.

The association between VNTR alleles and longevity was restricted to males, because no difference was observed, either in the genotypic or in the allelic pool, between female samples 1 and 2 (Table 2). This finding confirms that longevity factors are sex-specific [23–25] and that males and females probably follow different trajectories to attain longevity [16].

This finding suggests that allele 2b is detrimental for male T longevity. On the other hand as the sample s age increases the frequency of allele 2a decreases as well ( p = 0.025), thus suggesting that this allele is also unfavorable to male longevity. It follows that the GATA3/DeltaEF1 variability is not the only factor affecting the age-related variation of the gene pool and that other regulatory sites present in the 72-bp core do not act independent of the number of the VNTR repeats. We are now carrying out appropriate molecular studies to verify what is the exact portion of the VNTR repeat that links nuclear proteins, what are the proteins interacting with the repeat core, and, most importantly, how do these proteins interact according to the number of repeats.

Although cross-sectional data have been used for estimating the association between VNTR alleles and longevity, the present study should be reliable and not affected by bias caused by population stratification. In fact the population under study is highly homogeneous because of historical and geographical factors; furthermore, we did not find any stratification in this sample (unpublished results) by analyzing a panel of neutral markers spanning the genome [26].

Although the effect of the VNTR alleles as enhancer sequences has been demonstrated on a reporter gene, on the whole our findings are in line with the possibility that the VNTR acts in vivo on SIRT3 and that variations in the expression of a SIR2-like gene may modulate life span in mammals [19]. Since the SIRT3 protein could deacetylate mitochondrial proteins involved directly in apoptosis [14], possibly by mimicking SIRT1 in inhibit- ing apoptosis by p53 deacetylation [20,21], the level of expression of SIRT3 may affect longevity by acting on apoptotic mitochondrial patterns [22].

In conclusion, our study has discovered a VNTR polymorphism in intron 5 of the SIRT3 gene that has an allele-specific enhancer activity on a reporter gene. The frequency distribution of VNTR genotypes (and alleles) is significantly different between a sample of older men and a sample of younger men from the same population. Of course we need to confirm by appropriate longitudinal studies the effect of the VNTR on male longevity, and these studies are going on.

Materials and methods

sequencing in a 310 DNA sequencer with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).

Population samples

VNTR polymorphism typing