Discussion

The screening of two polymorphisms in the hu- man metallothionein gene 1a coding region from old and very old healthy female has revealed for the first time their involvement in the longevity. In particular, the polymorphism corresponding to A/C (Asp/Thr) transition at 647 nt position seems to be the more involved rather than the poly- morphism corresponding to A/G (Lys/Arg) tran- sition at 1,245 nt position. Indeed, for the first SNP, a significant increment of AA (Asp/Asp) genotype (P < 0.0001) and Asp allele (P = 0.0002, odds ratio = 2.164, CI = 1.386– 3.380) occurs in nonagenarian subjects as com- pared to old ones. For the second SNP, instead, no significant different distribution of genotypes and alleles between old and nonagenarian sub- jects is observed. These findings clearly show that these polymorphisms are differently involved in the longevity at least in women, where the pres- ence of the genotype Asp/Asp is crucial for the achievement of successful ageing. The determi- nation of the haplotypes in which the allele Asp is more present in nonagenarians with respect to other alleles is in line with this assumption. That this fact is not a mere coincidence, the determi- nation of some functional parameters linked to the inflammatory status and zinc ion availability is a further support. It has been reported that nonagenarians display a lower degree of the

Haplotype determination

Two single nucleotide polymorphisms of MT1a cluster were determined and the frequency of haplotypes were analysed using the Arlequin software package. The polymorphism of +647 MT1a was in tight linkage disequilibrium with the +1245 MT1a one (P < 0.05). Moreover, a statis- tically significant haplotype frequency difference between the old female individuals and female nonagenarians was found (Likelihood Ratio = 13.3, df = 3, P = 0.004 by Pearson Chi-Squared) (Table 5). The MT1a Arg/Asp haplotype increased in nonagenarians (7.1%) in comparison to old individuals (2.1%). Conversely, the MT1a Lys/Thr haplotype decreased in nonagenarians (8%) with respect to

Table 4 Plasma zinc, zinc ion availability, IL-6 and MT detection for +1245MT1a polymorphism

+1245 MT1a Zp1 (MFI – MFI ) D MFI IL-6 (pg/ml) Zn (lM) MT (F.U.) min Old G+ carriers 73.18 ± 16.39 17.60 ± 5.67 21.56 ± 2.40 11.53 ± 0.25 31.14 ± Old G– carriers 66.71 ± 6.28 14.28 ± 3.26 20.53 ± 1.22 11.90 ± 0.18 21.26 ± Nonagenarian G+ carriers 48.50 ± 21.70 11.00 ± 4.02 32.89 ± 8.60 10.78 ± 0.46 27.80 ± Nonagenarian G– carriers 43.75 ± 14.88 13.00 ± 6.01 37.24 ± 6.09 10.18 ± 0.34 15.44 ±

The data of Zp1, Delta and MT were obtained in n. 20 subjects/group

Table 5 Haplotype frequency of MT1a cluster in old and nonagenarian healthy female

more involved in zinc release by MT and subse- quently in longevity, might be assumed that the presence of an additional amino group (NH3) in asparagine in subjects carrying A allele of +647 MT1a polymorphism may affect the zinc affinity in the cysteine cluster, with consequent better zinc homeostasis. In this context, further study is still in progress (Zincage project). In conclusion, the +647 MT1a polymorphism may be essential to reach health longevity at least in women, further confirming the pivotal role played by MT and zinc homeostasis as biological and genetic marker for the ageing process (Mocchegiani et al. 2006), even if these data were obtained in one restricted geographic area (Central Italy). Further analyses are in progress in different European Countries (Zincage project).

Haplotype Lys/Asp Lys/Thr Arg/Asp Arg/Thr Old (N.183) 71.6% 18.7% 2.1% 7.6% Adjusted –1.6 2.7 –2.5 0.8 residual Nonagenarians 79.6% 8% 7.1% 5.3% (N.100) Adjusted +1.6 –2.7 +2.5 –0.8 residual

P = 0.004, Likelihood Ratio = 13.3, df = 3 by Pearson Chi- Squared

Data in bold represent the more significant haplotypes for longevity (Arg/Asp) or not (Lys/Thr) taking into account the adjusted residual

gp130 as well as lower values of circulating IL-6 (Mocchegiani et al. 2002; Moroni et al. 2005). Concomitantly, the gene expression of MT is also lower than old individuals associated with a sat- isfactory zinc ion availability and good immune and antioxidant performances (Mocchegiani et al. 2003; Mecocci et al. 2000). Since the zinc ion availability is fundamental for immune response and antioxidant activity (Wellinghaus and Rink 1998), the capacity of the MT in the zinc release is a crucial point for the ageing process, because of the possible inability of MT in zinc release due to the presence of chronic inflammation (Mocchegiani et al. 2000). The determination of the zinc release by MT using NO donor has revealed that old and very old subjects carrying Asp/Asp genotype for +647 MT1a polymorphism (herein called C– subjects) display more zinc release by MT (as determined by higher Delta MIF) and less inflammation (lower IL-6 plasma levels) coupled with also satisfactory plasma zinc levels (Table 3). Conversely, no differences in all these functional parameters occur for +1245 MT1a polymorphism. Therefore, the polymor- phism of +647 MT1a is fundamental to achieve a good functioning of many body homeostatic mechanisms, via zinc release by MT and lower inflammation, with subsequent healthy ageing and longevity, at least in old women.

Acknowledgements This paper was supported by Euro- pean Commission (Zincage project, Contract No. FOOD- CT-2003-506850; Scientific Coordinator Dr. Eugenio Mocchegiani).

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