Active TGF-β1 plasma levels were assessed in a group of 143 unrelated randomly selected subjects, including 73 centenarians (24 males and 49 females) ranging from 99 to 103 years of age and 70 young controls (22 males and 48 females) ranging from 20 to 60 years of age. Significantly increased bio-active TGF-β1 plasma levels were observed in centenarian subjects both in men and in women (Fig. 1A,B). The mean plasma levels of active TGF-β1 in male centenarians were 144.61 ± 74.8 pg mL−1 and in male controls were 92.10 ± 37.88 pg mL−1. The mean plasma levels of active TGF-β1 in female centenarians were 144.44 ± 60.25 pg mL−1 and in female controls were 113.77 ± 48.08 pg mL−1. Alleles and genotypes of four polymorphic sites at the TGF-β1 gene were estimated in the group of people described above. TGF-β1 plasma levels did not show any association with genotypes of the four polymorphisms (data not shown).

χ2 = 12.939, d.f. = 8, P = 0.08 for haplotype frequency distributions. χ2 = 7.366, d.f. = 1, P = 0.007: GCCC vs. other haplotypes.

genotypes were in agreement (P > 0.05) with those expected at Hardy–Weinberg equilibrium in every sample at each locus. No significant differences were found between young and cen- tenarian groups either for genotypes or for alleles at polymor- phic sites G/A , C/T and T/C (P > 0.05). In contrast, for −800 −509 869 the polymorphism G/C the statistical difference between 915 centenarians and younger controls for genotype and allele frequency distributions was significant (Table 2, χ2 = 6.097, d.f. = 2, P = 0.034 for genotype frequencies; χ2 = 4.848, d.f. = 1, P = 0.028 for allele frequencies). By linkage-disequilibrium analysis, nine haplotype combinations (Table 3) were observed in each group and no significant difference in the overall haplo- type frequency distributions was found between young and centenarian groups (χ2 = 12.939, d.f. = 8, P = 0.08), even when sexes were analysed separately (data not shown). A significantly decreased frequency of a particular haplotype combination (G C C C ), among the less frequent, was −800 −509 869 915 observed in centenarians compared with the control group (χ2 = 7.366, d.f. = 1, P = 0.007). The P value was not significant after Bonferroni correction (P = 0.056); however, this correc- adj tion is too stringent and has the potential to ignore important observations (Perneger, 1998). In fact, there was a survival advantage for subjects who did not have the G C C C −800 −509 869 915 haplotype combination [OR 2.25 (95% CI 1.24–4.10) P = 0.005]

Discussion

The aim of the present study was to test whether the variability of the TGF-β1 gene, a pleiotropic regulator of cellular activity and the immune system (Letterio & Roberts, 1998; Blobe et al., 2000), is involved in human longevity. In this paper, we focused on four common polymorphisms of the TGF-β1 gene: two polymorphisms, G/A and C/T , located in the promoter −800 −509 region, and two missense polymorphisms, T/C and G/C , 869 915 which change (Leu > Pro) and (Arg > Pro) , respectively, 10 25 located in the signal peptide. The genotype and allele frequen- cies found in the present study for the tested polymorphisms were not different from those of other Caucasians, thus con- firming and extending, over a large group of Italians, the data on TGF-β1 genotypes and alleles in Europeans (Cambien et al., 1996; Holla et al., 2002).

Within this population genetics perspective the novel finding of this paper is that a significant difference was found between centenarians and younger control subjects for the genotype and allele frequency distributions at the TGF-β1 +915 site in the signal

NN Active TGFB1 (pg/ml) 300.00200.00 4100.00 4 n=49 n=48     0.00  

Active TGFBI (pg/ml)300.00200.00100.000.00         

Fig.

TGF-β

plasma levels in centenarians and younger subjects. TGF-β

plasma levels in male (A) and female (B) centenarians ((cid:

)) and younger subjects ((cid:

)). Data are presented as means and

% confidence interval. (A)   vs. (cid:

): F =

, d.f. =

, P =

; (B)   vs. (cid:

): F =

, d.f. =

, P =

which is probably a common driving force of most age-associated pathologies. These data are in agreement with those reported by Forsey et al. (2003) in octogenarian and nonagenarian subjects showing a significant increase of active TGF-β1, but contrast with other data referring to much younger subjects (Young et al., 1999). These findings on TGF-β1 are also in agree- ment with previous results showing an increase in plasma levels of anti-inflammatory cytokines in centenarians (Cavallone et al., 2003). In conclusion, the data presented here provide new evidence for a relevant biological role of TGF-β1, adding human longevity to the long list of phenomena affected by this cytokine.

peptide. In most of our previous studies the association between cytokine gene polymorphisms and longevity was gender- associated, leading us to deduce that men and women follow different strategies to reach the extreme limits of human life- span (Bonafè et al., 2001; Lio et al., 2002a,c). This is not the case in the present paper: we found no gender-specific difference with regard to the genotype and allele frequency distributions at the TGF-β1 analysed loci .