Results

To identify variability in the functional domains of SIRT3, we conducted DNA sequence analyses of PCR fragments around the FGE conserved motif (exon 5) in a panel of 50 unrelated subjects (25 males and 25 females randomly chosen from sample 1). We observed a VNTR polymorphism having a 72-bp core in the fifth intron of the gene (12,343 nt initial position referring to www.ncbi.nlm. nih.gov/omim; MIM 604481; contig NT_035113). After verification of Mendelian inheritance in 30 parent/offspring pairs collected in another study, we analyzed the VNTR polymorphism in all of sample 1. Six length-alleles were identified spanning one to six repeats (see Table 1); four alleles were common (alleles 1–4), while two were rare (alleles 5 and 6). Since the sample under study had been already typed for the silent variant G477T of SIRT3 [15], we analyzed linkage disequilibrium between the markers

We analyzed possible enhancer effects of specific VNTR alleles. By using constructs including the alleles lacking the DeltaEF1 site (alleles 1a, 2a, 3a, 4a) we found that these alleles act as an enhancer whose activity appears to be related to the number of VNTR repeats (Fig. 2A). One-way ANOVA showed a statistically significant difference among the luciferase activities observed in the whole experiment (p V 0.001). Then LSD post hoc tests showed that: (i) the luciferase activity sustained by each of the four alleles is b significantly higher than that present in the control (p 0.05 for every allele) and (ii) the luciferase activity is significantly different between alleles that differ for two repeats at least (p = 0.04 between alleles 1a and 3a, p = 0.01 between alleles 1a and 4a, p = 0.02 between alleles 2a and 4a). But, when the alleles harboring the DeltaEF1 site in the second repeat were assayed (alleles 2b, 3b, 4b) allele 2b showed a dramatic reduction in the enhancer activity with respect to allele 2a (Fig. 2B), although the enhancer activity of alleles 3b and 4b did not differ from that of 3a and 4a, respectively (results not shown).

Table 1

Genotypic and allelic frequencies of the VNTR polymorphism in sample (20- to 80-year-old subjects)

Genotype Count Allele Count 1.1 124 (17.6 F 1.4) 1 587 (41.7 F 1.3) 1.2 73 (10.4 F 1.2) 2 166 (11.8 F 0.9) 1.3 112 (15.9 F 1.4) 3 291 (20.7 F 1.1) 1.4 149 (21.2 F 1.5) 4 351 (25.0 F 1.2) 1.5 1 (0.1 F 0.1) 5 6 (0.4 F 0.2) 1.6 4 (0.6 F 0.3) 6 5 (0.4 F 0.2) 2.2 13 (1.9 F 0.5) Total 1406 (100.0) 2 3 27 (3.8 F 0.7) 2.4 38 (5.5 F 0.9) 2.5 2 (0.3 F 0.2) 3.3 40 (5.7 F 0.9) 3.4 69 (9.8 F 1.1) 3.5 2 (0.3 F 0.2) 3.6 1 (0.1 F 0.1) 4.4 47 (6.7 F 0.9) 4.5 1 (0.1 F 0.1) Total 703 (100.0)

By the above approach we demonstrated that the VNTR acts in vitro as an enhancer on a reporter gene. To explore a possible role of this enhancer in human longevity we analyzed the VNTR polymorphism in sample 2 (90- to 106- year-old subjects) and compared the genotypic and allelic frequencies with those of sample 1 (20- to 80-year-old subjects). The analyses were carried out according to sex because of gender effects in mortality of the oldest people and sex specificity in gene/longevity associations. On the basis of data in Table 2, we rejected the hypothesis of homogeneity between the genotypic pools of samples 1 and

Relative frequencies F standard errors ((cid:1)100) are given in parentheses. Allele nomenclature refers to the repeat number. Hardy–Weinberg equilibrium check (5000 permutations): p = 0.185.

SIRT3 geneE14 E2E3 E4 ES E6 E?1 VNTR allelesG477TTGTCAGCAGGCCTGTGCAGGGAGGAGGCTGGAGGGCAGGGGAAGGGAGGGGTACAGGAG ‘CATGGGC TGTCAGCAGGCCTGTIGCAGGGAGGAGGCTGGAGGGCAGGGGAAGGGAGGGGTACAGGAGTCACET ‘CATGGGC

  A[TGTCAGCAGGCCTETECAGGGAGGAGECTGGAGEGCAGEGGAAGGGAGEGGTAGAGGAGTTialtefin4

Fig. 1. A schematic representation of the SIRT3 gene showing the positions of the G477T marker previously identified [15] and the VNTR discovered in the present study. The alleles including the GATA3 site in the second repeat (in yellow) are named 1a–4a, while the alleles including the DeltaEF1 site (in blue) are named 2b–4b. The variable sequence occurring in the second allele repeat is also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sequencing allele 2 in all the males. Of the 89 alleles found in the young group (sample 1, Table 2B) 49 were 2a and 40 were 2b, while all 5 alleles found in the oldest group (sample 2, Table 2B) were 2a. Therefore, in the entire gene pool, allele 2a decreased from 7.8 F 1.1% in sample 1 to 2.9 F 1.3% in sample 2 (p = 0.025 by Fisher exact test), while allele 2b decreased from 6.4 F 1.0% in sample 1 to 0 in sample 2 ( p = 8  10(cid:2)5 by Fisher exact test).

2 in males (p = 0.026) but not in females (p = 0.674). Likewise, allele distribution frequencies were different b between samples 1 and 2 in males (p 0.001) but not in females (p = 0.923). In particular, in males, the frequency of allele 2 decreased from sample 1 to sample 2 (Table 2B). Since length-allele 2 included the sole allele lacking enhancer activity, we checked possible age-related varia- tions of the gene pool in terms of alleles 2a and 2b by

Discussion

>Fold inductionwoFold inductionpGL3pGL3 Allelela Allele2a Allele3a Allele4a  Allele2b Alleleza

The aim of this study was to identify a putative functional variant occurring in SIRT3 that could account for the association previously observed between longevity and a silent marker of this gene [15]. Transfection experi- ments demonstrated that the VNTR we identified in intron 5 of SIRT3 is a functional polymorphism and that different VNTR alleles are able to modulate the expression of a reporter gene in an allele-specific way, according to number of repeats and occurrence of GATA3/DeltaEF1 site in the second repeat of the length-allele 2.