et al., 2001 and references therein) the association between SIRT3 variability and survival was restricted to males, because no genotype specific variation of the survivorship

Survival Functions4050       60 70Age (Years)— (GT)Modeleee Experiment 80 90 100

Table 1

SIRT3-(G/T) /HRAS1-30VNTR linkage disequilibrium ðDÞ analysis in a 3223 sub-sample of 145 subjects

Haplotypes ðn ¼ 290Þ Frequency ( £ 100) Standard errors ( £ 100) G-a1 43.50 3.36 G-a2 8.67 1.79 G-a3 7.93 1.74 G-a4 4.23 1.40 T-a1 16.84 2.47 T-a2 0.99 0.86 T-a3 1.38 0.99 T-a4 4.04 1.38 Others 12.42 1.90

Haplotypic frequencies are only reported for HRAS1-30VNTR common alleles (a1, a2, a3, a4) while haplotypes containing rare alleles are grouped as Others (Bonafe` et al., 2002). Haplotypic frequencies are estimated by maximum likelihood method (Arlequin 1.1 software). The hypothesis D ¼ 0 is not rejected (p ¼ 0:124 ^ 0:001).

Fig. 2. Survival functions for carriers of GT genotype among Italian males. Empirical and modelled survival function for the whole population are also shown (please note that the empirical curve almost completely overlaps with the modelled one).

G. Rose et al. / Experimental Gerontology 38 (2003) 1065–1070

Table 2

to be sound. On the other hand the possibility that the association between SIRT3 variability and survival were due to population stratification is unlikely. Indeed, the population structure of Calabria was proved to be homogeneous and with no stratification by genotyping a moderate number of unlinked genetic markers in the same population (Rose et al., 1996; and unpublished results). What is more, the SIRT3 gene is a strong candidate in longevity taking into account the findings in model organisms. Thus, both the choice of the population sample and the choice of the candidate gene fit with the criteria suggested for genetic association studies of complex traits

SIRT3-(G/T) /IGF2-AvaII-RFLP linkage disequilibrium ðDÞ analysis in 3223 a sub-sample of 130 subjects

Haplotypes ðn ¼ 260Þ Frequency ( £ 100) Standard errors ( £ G-P0 25.54 2.99 G-A0 47.16 3.28 T-P0 7.92 2.10 T-A0 19.38 2.87

IGF2-AvaII-RFLP alleles are named according to the presence/absence (P0/A0) of the AvaII restriction site (De Luca et al., 2001). Haplotypic frequencies are estimated by maximum likelihood method (Arlequin 1.1 software). The hypothesis D ¼ 0 is not rejected (p ¼ 0:499 ^ 0:002).

On the other hand, since the G477T transversion does not change the amino acid in the conserved sirtuin domain, the observed association could be due to LD either with a functional variant of the SIRT3 gene itself or of other genes occurring in this neighbourhood. As an LD map relevant to the 11p15.5 chromosomal region is not yet available, it is not known if such a region is organised in hot or cold LD blocks (Ardlie et al., 2002). In any case, the negative findings obtained in LD analysis (Tables 1–4) show that the relationship between the G477T marker of SIRT3 and survivorship is not due to the gene/longevity associations previously found in the same 11p15.5 region, thus reinforcing the hypothesis of D-linkage between the G477T variant and an unknown functional variant of SIRT3 itself. In particular, the puzzling effects of different genotypes on the survival curves (Figs. 1–3) suggest a linkage disequilibrium of G477T with a putative multi- allelic polymorphism displaying complex allelic inter- actions as previously observed for the STR alleles of the TH gene (Tan et al., 2002). Intriguingly, molecular analyses we are carrying out in the surrounding region revealed a functional multiallelic polymorphism located in the 5th intron of the SIRT3 gene; at present, a linkage disequili- brium map of the 11p15.5 region is under construction

Table 3

SIRT3-(G/T) /INS-FokI-RFLP linkage disequilibrium ðDÞ analysis in a 3223 sub-sample of 130 subjects

Haplotypes ðn ¼ 260Þ Frequency ( £ 100) Standard errors ( £ G-P 10.57 2.17 G-A 62.12 3.26 T-P 4.43 1.68 T-A 22.88 2.83

INS-FokI-RFLP alleles are named according to the presence/absence (P/A) of the FokI restriction site (De Luca et al., 2001). Haplotypic frequencies are estimated by maximum likelihood method (Arlequin 1.1 software). The hypothesis D ¼ 0 is not rejected (p ¼ 0:810 ^ 0:001).

Table 4

SIRT3-(G/T) /HUMTHO.1-STR linkage disequilibrium ðDÞ analysis in a 3223 sub-sample of 718 subjects

Haplotypes ðn ¼ 1436Þ Frequency ( £ 100) Standard errors ( £ 100) G-6 20.84 1.19 G-7 10.01 0.92 G-8 9.06 0.86 G-9 13.52 1.03 G-10* 16.35 1.08 G-10 0.62 0.25 T-6 9.80 0.96 T-7 4.89 0.73 T-8 3.26 0.61 T-9 6.26 0.82 T-10* 4.96 0.78 T-10 0.43 0.22

Anyway, whatever the functional variant may be, an association between a marker of SIRT3 and survivorship has been found. The possibility that the Sir2p human homologue has a role in ageing is in line with the idea that, although ageing is not a programmed process as mechanisms of development are, the modulators of the rate of ageing are conserved over large evolutionary distances (Partridge and Gems, 2002). On the other hand a link among rate of ageing, gene-expression modulation, and metabolic rate is suggested by an elegant study of genome-wide transcript profiles in ageing D. melanogaster which showed age-dependent changes in transcription level nearly in 23% of the genome (Pletcher et al., 2002). These findings indicate that Sir2p activity may be an evolutionary conserved check point of this complex interplay (Chang and Ming, 2002) and therefore agree with the idea that SIRT3 variability may modulate human ageing. What is more, the localisation of the SIRT3 protein to mitochondria,

HUMTHO.1-STR alleles are named according to the number of STR repeats; allele 10* is that containing the imperfect repeat (De Luca et al., 2001; Tan et al., 2002). Haplotypic frequencies are estimated by maximum likelihood method (Arlequin 1.1 software). The hypothesis D ¼ 0 is not rejected (p ¼ 0:482 ^ 0:002).

4. Discussion