By contrast, a loss of allele C of the GC heterozygous 915 and 915 genotype was observed in centenarians with respect to the young control group; consequently, an increase of allele G 915 and the GG homozygous genotype was evident. In the same 915 groups no significant differences in allele or genotype frequen- cies were observed for the polymorphisms G/A , C/T and −800 −509 T/C Moreover, a significant decreased frequency of the hap- 869. lotype combination G C C C was observed in cente- −800 −509 869 915 narians in comparison with the young control group, suggesting that G/C polymorphism is associated with long life expect- 915 ancy. As this polymorphism occurs at codon 25 within the signal peptide that is cleaved from the TGF-β1 precursor at the level of codon 29, it is possible that the (Arg > Pro) substitution 25 could play a role in the efficient production of the mature TGF- β1. Moreover, because the signal peptide allows export of the cytokine precursor across the membrane of the endoplasmic reticulum, it could be hypothesized that the polymorphism at codon 25 affects this process. Finally, Hoffmann et al. (2001) have observed that codon 25 regulates gene transcription, which therefore might be affected by nucleotide change at the +915 site. All these functional characteristics of G/C poly- 915 morphism could play a role in explaining the difference in allele and genotype frequencies between centenarians and younger controls. Previous studies showed that the G allele at codon 25 was associated with fibrotic diseases (Awad et al., 1998) and higher blood pressure (Li et al., 1999), and thus the increase of the G allele found in our centenarian sample is an apparent par- adox. However, unexpected and counter-intuitive results have often been found in association studies on longevity candidate loci (Mari et al., 1996; Rose et al., 2001; Bonafè et al., 2002; Coppola et al., 2003; Garasto et al., 2003; Rea et al., 2004). We are pursuing the hypothesis that polymorphic variants in certain genes could be related to longevity or diseases as a consequence of peculiar interactions with other individual-specific variants in the nuclear as well as in the mitochondrial DNA (Rose et al., 2001; Garasto et al., 2003; Franceschi et al., in press).

Experimental procedures

Subjects

Two sample groups recruited in Central and Northern Italy were studied. The young group comprised 247 subjects (94 males and 153 females), ranging from 20 to 60 years of age. The sample of centenarians comprised 172 subjects (50 males and 122 females), 99–103 years of age. Each group was free of clinically manifest pathologies; in particular, centenarians belonged to classes A and B, according to the centenarians’ health status classification; category A included centenarians in good health status, defined as absence of physical disabilities, absence of severe cognitive impairment, absence of severe diseases such as cancer, ictus, liver diseases, etc., category C included cente- narians in poor health status and category B included centenar- ians in an intermediate health status (Franceschi et al., 2000b). Most of the subjects included in the present study had already been analysed in previous investigations to test the possible role of other genetic markers in human longevity (Bonafè et al., 1999, 2001; Lio et al., 2002a,b; Cavallone et al., 2003). All sub- jects had been informed about the aim of the study and had given their consent, in accordance with Italian laws.

Genotyping

DNA was extracted from blood lymphocytes by phenol/chloro- form following standard procedures. Genotyping of all subjects participating in the study was performed by polymerase chain reaction (PCR) and PCR product digestion with specific restriction enzymes. Primers were designed to amplify regions containing the analysed polymorphisms (G/A and C/T : 5′- −800 −509 ACCGTGCACCTGGAGATCG-3′, 5′-CCAGGCGGAGAAGGCT- TAAT-3′; T/C and G/C : 5′-CGCTTCACCAGCTCCATGTC- 869 915 GATAG-3′, 5′-CACACCAGCCCTGTTCGCGCTCT-3′). The amplification reaction for the polymorphisms in the promoter region was performed on a ‘PCR express Hybaid’ thermal cycler with initial denaturation at 95 °C for 2 min, followed by 30 cycles, each one comprising denaturation at 94 °C for 45 min, annealing at 60 °C for 25 min and extension at 72 °C for 50 min, with a final extension time of 5 min at 72 °C. The ampli- fied PCR fragments for markers −800 and −509 were digested

Recent reports have also shown that the G allele at the +915 site in the TGF-β1 gene is associated with increased production of this anti-inflammatory cytokine both in vitro and in vivo (Awad et al., 1998; El-Gamel et al., 1998; Arkwright et al., 2000). In the present paper we have shown increased plasma levels of bio-active (naturally processed) TGF-β1 in the cente- narians both in women and in men, without any association with genotypes of the four TGF-β1 polymorphisms analysed. Moreover, because TGF-β1 is immunosuppressive, the age-related increase of the active cytokine suggests that it could counteract and counterbalance the harmful effects of inflamm-aging,

fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation

,

–

Bidwell J, Keen L, Gallagher G, Kimberly R, Huizinga T, M

ermott MF, Oksenberg J, M

icholl J, Pociot F, Hardt C, D’Alfonso S (

) Cytokine gene polymorphism in human disease: on-line databases. Genes Immun.

,

–

Bidwell J, Keen L, Gallagher G, Kimberly R, Huizinga T, M

ermott MF, Oksenberg J, M

icholl J, Pociot F, Hardt C, D’Alfonso S (

) Cytokine gene polymorphism in human disease: on-line databases (Suppl.

). Genes Immun.

,

–

Blobe GC, Schiemann WP, Lodish HF (

) Role of transforming growth factor beta in human disease. N. Engl. J. Med.

,

–