Controls (%) Centenarians (%) –1082 IL-10 AA 19 (16) 5 (7) AG 64 (56) 34 (47) GG 32 (28) 33 (46) –308 TNF-(cid:225) AA 1 (1) 1 (1) AG 31 (27) 15 (21) GG 83 (72) 56 (78)

As far as cytokine production is concerned, the IL-10 and TNF-a polymorphisms considered are functionally important. Thus, centenarians and controls were classified into four groups,as shown in table 2.We compared the frequency of the genotype “low TNF-a and high IL-10” producer, “anti- inflammatory genotype”14 with the other genotypes in cente- narian and control men. There was a significant increase of this “anti-inflammatory genotype” in centenarians over controls (36% v 21%; p=0.038 by c 2 test). In women, there were no significant differences in the distribution of the “functional” genotypes between centenarians and younger subjects (32% v 21, p=NS by c 2 test).

All groups were in Hardy Weinberg equilibrium (p>0.05). A significantly different distribution of IL-10genotypes was observed between controls and centenarians (p=0.019 by c 2 test). Similarly, the frequency of SNP –1082G was significantly increased in centenarians over controls (0.69 v0.56, p = 0.01 by c 2 test). It is noteworthy that this significant difference was observed both in the previous sample of 25 centenarians (0.76 v0.56, p = 0.012 by c 2 test) and in the new sample of 47 centenarians (0.66 v0.56, p = 0.035 by c 2 test). No significant different distribution of TNF-(cid:225) -308 genotypes was observed between controls and centenarians by c 2 test and the frequency of SNPs –308GfiA was not significantly different between centenarians and controls by c 2 test.

DISCUSSION

The main result of this study was that the IL-10 gene SNP -1082G fi A had significant influence on the attainment of longevity in men, both separately and in association with the TNF-a gene SNP –3082Afi G. It is noteworthy that the IL-10 frequency was analysed in a large cohort of male centenar- ians, thus validating our preliminary results on a smaller sam- ple of centenarian men.7 This result is remarkable, taking into account that centenarian men are relatively rare, being many fewer than centenarian women.8

and the presence of only three different allele combinations, -1082G, -819C, and -592C GCC, ACC, and ATA, are character- istic of the IL-10 polymorphisms in white populations.15 In the previous study, only –1082 SNP was shown to be associated with longevity, so in the present study we limited ourselves to the analysis of the –1082 Gfi A SNP. This biallelic polymor- phism (GeneBank accession number: 790448) was identified using ARMS-PCR as previously described.7 The biallelic poly- morphisms at -308 (G fi A) (GeneBank Accession number CR941560) of the TNF-a gene was identified also using the ARMS-PCR method as previously described.16 Allele typing was confirmed according to Wilson et al.17

IL-10 and TNF-a are cytokines, which have complex and predominantly opposing roles in the inflammatory responses.10 11 In fact, the principal routine function of IL-10 is to limit and ultimately terminate inflammatory responses,10 whereas TNF-a determines strength, effectiveness, and dura- tion of local and systemic inflammatory reactions.11 Stimula- tion of human blood samples with bacterial lipopolysaccha- ride showed large interperson variations of IL-10 and TNF-a production, suggesting a genetic component of approximately 75% and 60%, respectively.18 Interperson differences in the regulation of IL-10 and TNF-a production may be critical with respect to the final outcome of an inflammatory response,that is within physiological limits or pathological ones.18 Several polymorphisms located close to or within the IL-10 and TNF-a genes have been shown to be associated with transcription levels. The best documented of these polymorphisms are the IL-10 gene promoter polymorphisms -1082 G fi A7 16 19–23 and a Gfi A transition at position –308 in the promoter region of TNF-a .11 13 24 25 In the presence of allele -1082A, stimulation of lymphocytes with concanavalin A resulted in lower IL-10 pro- duction than in allele -1082A negative cells. 14 21 Also, the effects of this allelic promoter region difference (relative to the transcription start site) on IL-10 gene activation have been confirmed in reporter gene assays.20 Concerning TNF-a , there are also data relating the rare -308A allele to a higher transcriptional activity than the common -308G allele, 11 13 24 25 although a lack of such an association has also been reported in non-white populations, depending on the HLA ancestral haplotype carrying this SNP.12 24 However, the functional relevance of these SNPs has been shown by their involvement in determining susceptibility to immune-inflammatory dis- eases (for IL-10 -1082G fi A,14 20 26–35 for TNF-a 11–13 24 25 36–42).

and genotypic and allelic frequencies were evaluated by gene count. The data were tested for the goodness of fit between the observed and expected genotype values (c 2 test) and their fit to Hardy-Weinberg equilibrium. c 2 tests (3 · 2 tables or 2 · 2 tables) were performed to calculate significantly different genotype or allele distributions between centenarians and controls.

RESULTS

Table 1 shows the frequency of homozygous and heterozygous genotypes for –1082 Gfi A IL-10 SNP and for –308 Gfi A TNF-a in our sample of Italian centenarian men and sex matched controls. The number of centenarians homozygous for the –1082G genotype was significantly increased in this cohort of centenarian men in comparison with younger control subjects (p=0.019). Accordingly, the SNP distribution between centenarians and controls was significantly different, with the –1082G allele increased in centenarians (0.69 v 0.56, p=0.01 by c 2 test). As already reported,7 no significant differ- ences were observed in women, between centenarians and controls (–1082G, 0.62 v 0.59, p=NS by c 2 test). The genotypic frequencies of the TNF-a promoter SNP of centenarian and