G/A Centenarians Younger controls −800 Alleles G 330 (95.9 ± 1.0) 477 (96.6 ± 1.0) A 14 (4.1 ± 1.0) 17 (3.4 ± 1.0) Genotypes GG 158 (91.9 ± 2.1) 231 (93.5 ± 1.6) GA 14 (8.1 ± 2.1) 15 (6.1 ± 1.5) AA 0 (0.0) 1 (0.4 ± 0.4) χ2 = 0.225, d.f. = 1, P = 0.711 for allele frequencies. χ2 = 1.352, d.f. = 2, P = 0.668 for genotype frequencies. C/T Centenarians Younger controls −509 Alleles C 203 (59.0 ± 2.7) 297 (60.1 ± 2.2) T 141 (41.0 ± 2.7) 197 (39.9 ± 2.2) Genotypes CC 55 (32.0 ± 3.6) 87 (35.2 ± 3.0) CT 93 (54.0 ± 3.8) 123 (49.8 ± 3.2)

The potential role of in aging and longevity has been suggested by many in vitro and in vivo studies. In particular, a TGF-β1 gene over-expression has been observed in human fibroblasts that displays a senescent-like phenotype following exposure to oxidative stress (Frippiat et al., 2001, 2002). Recently, polymorphisms identified in human TGF-β1 gene and influencing the cytokine production have been linked to age- related pathologies, such as hypertension (Li et al., 1999), oste- oporosis (Yamada, 2000) and Alzheimer’s disease (Luedecking et al., 2000).

χ2 = 0.225, d.f. = 1, P = 0.711 for allele frequencies. χ2 = 1.352, d.f. = 2, P = 0.668 for genotype

C/T Centenarians Younger controls −509 Alleles C 203 (59.0 ± 2.7) 297 (60.1 ± 2.2) T 141 (41.0 ± 2.7) 197 (39.9 ± 2.2) Genotypes CC 55 (32.0 ± 3.6) 87 (35.2 ± 3.0) CT 93 (54.0 ± 3.8) 123 (49.8 ± 3.2) TT 24 (14.0 ± 2.6) 37 (15.0 ± 2.3)

To test the hypothesis that variability of the TGF-β1 gene affects successful aging and longevity, four TGF-β1 polymor- phisms have been compared between centenarians and younger subjects (20–60 years of age) recruited in Northern and Central Italy. Two polymorphisms, G/A and C/T , are located −800 −509 upstream of the expressed gene; two polymorphisms, T/C 869 and G/C , are located in the signal peptide and are missense 915 polymorphisms which change (Leu > Pro) and (Arg > Pro) , 10 25 respectively (Cambien et al., 1996; Awad et al., 1998).

χ2 = 0.104, d.f. = 1, P = 0.775 for allele frequencies. χ2 = 0.748, d.f. = 2, P = 0.699 for genotype frequencies.

All these variants may have a functional role. In fact, recent reports have provided evidence that the T allele is associated −509 with higher transcriptional activity of TGF-β1 compared with C (Luedecking et al., 2000; Schulte et al., 2001). Likewise, −509 because the G/A polymorphism occurs in a consensus CREB −800 half-site, an allele-specific affinity for the CREB family of transcrip- tion factors is hypothesized (Grainger et al., 1999; Luedecking et al., 2000). On the other hand, the two alleles encoding pro- line (C and C ) have been found to be associated with lower 869 915 cytokine synthesis both in vitro and in vivo (Awad et al., 1998;

Table 2 Absolute allelic and genotypic frequencies of T/C and G/C 869 915 polymorphisms of the signal peptide of the TGF-β1 gene in centenarians (n = 172) and younger controls (n = 247). Relative frequencies (×100) with standard errors are given in parenthesis

T/C Centenarians Younger controls 869 Alleles T 189 (54.9 ± 2.9) 258 (52.2 ± 2.3) C 155 (45.1 ± 2.9) 236 (47.8 ± 2.3) Genotypes TT 49 (28.5 ± 3.4) 65 (26.3 ± 2.8) TC 91 (52.9 ± 3.8) 128 (51.8 ± 3.2) CC 32 (18.6 ± 3.0) 54 (21.9 ± 2.6)

In the present study, we have also investigated plasma levels of the biologically active form of TGF-β1 in randomly selected subjects, including centenarians, in order to identify the possible age-related changes in the plasma levels of this cytokine.

χ2 = 0.601, d.f. = 1, P = 0.440 for allele frequencies. χ2 = 0.723, d.f. = 2, P = 0.687 for genotype frequencies.

Results

Tgf-ββββ 1 allele, genotype, haplotype frequency distributions

G/C Centenarians Younger controls 915 Alleles G 325 (94.5 ± 1.3) 446 (90.3 ± 1.4) C 19 (5.5 ± 1.3) 48 (9.7 ± 1.4) Genotypes GG 154 (89.5 ± 2.3) 200 (81.0 ± 2.5) GC 17 (9.9 ± 2.3) 46 (18.6 ± 2.5) CC 1 (0.6 ± 0.6) 1 (0.4 ± 0.4)

No significant differences were found for the polymorphisms studied in the promoter region and in the signal peptide between women and men in each age group (data not shown). Women and men were therefore pooled to increase the power of the statistical analysis. Table 1 shows allele and genotype frequencies of the TGF-β1 promoter polymorphisms in the two age-groups, and the corresponding data referring to the signal peptide polymorphisms are shown in Table 2. The observed

χ2 = 4.848, d.f. = 1, P = 0.028 for allele frequencies. χ2 = 6.097, d.f. = 2, P = 0.034 for genotype frequencies.

to become centenarians compared with subjects with this combination.

Table 3 Haplotype frequencies in centenarians (n = 172) and younger controls (n = 247) estimated by an EM (Expectation Maximization) algorithm implemented with the Arlequin package. Relative frequencies (×100) with standard errors are given in parentheses

Active TGF-ββββ 1 plasma levels in young controls and centenarians

Haplotypes Frequencies G/A −800 C/T −509 C/T 869 G/C 915 Centenarians Younger controls A C T G 14 (4.1 ± 1.1) 17 (3.4 ± 0.8) G C C C 15 (4.4 ± 1.2) 46 (9.3 ± 1.3) G C C G 5 (1.5 ± 0.7) 4 (0.8 ± 0.4) G C T G 168 (48.8 ± 2.7) 230 (46.6 ± 2.2) G T C C 3 (0.8 ± 0.6) 1 (0.2 ± 0.2) G T C G 132 (38.4 ± 2.6) 185 (37.4 ± 2.2) G T T G 6 (1.7 ± 0.7) 10 (2.0 ± 0.6) G C T C 1 (0.3 ± 0.3) 0 (0) G T T C 0 (0) 1 (0.2 ± 0.2)