]. Adenosine also inhibits the activity of NK cells that it has been reported to play a crucial role in determining successful ageing and longevity [

]. Of note, ADA deficiency

For what concern comparison 2, both two-loci and three-loci interaction are significant associated in males with increased life- expectancy over 88 years (Table 3, Fig. 2), with ADA 22G>A/TNF-a (cid:2)308G>A interaction reporting the highest testing balanced accu- racy (TBA = 0.572, Sign Test P = 0.011, CVC = 10/10) and displaying an odds ratio of 2.061 (P = 0.0096). Females does not shown any significant gene–gene interaction or attribute association with in- creased life-expectancy (Table 3).

Finally, considering the combination of three SNPs studied, we have stratified the subjects studied into three groups based on ex- pected expression of TNF-a, low, normal and high expression, respectively (Table 4). Very interestingly, females show a signifi- cant higher frequency of ‘‘low TNF-a expression’’ gene combination compared to males in age class 1 (30.9% vs. 15.3%, P = 0.0031), while age classes 2 and 3 does not shown any significant gender- specific differences. Moreover, the comparison between age class 1 and age class 2 + 3 displays a significant association of ‘‘low TNF-a expression’’ with life-expectancy, with opposite gender-spe- cific effect (Males, O.R. = 1.704, P = 0.045; Females, O.R. = 0.639, P = 0.031) (Table 4).

4. Discussion

The main purpose of the present paper was to further our understanding on the role of ADA and TNF-a genes in the determi- nation of human life-expectancy. We report a significant main ef- fect of the ADA 22G>A SNP, especially in males, with a ‘‘biphasic’’ association with human life-expectancy. Given the established evi- dence for the functional effect of A⁄ variant (historically called ADA⁄2, characterized by a (cid:3)35% lower enzymatic activity com- pared to allele G⁄, or ADA⁄1), and considering the gender-specific

>WO TNF-a -308G>AAttTNF-& -308G>AAttG*IG*ADA 22G>AG*/G*A*+ ADA 22G>AG*IG*A*+ TNF-a -308G>AAttTNF-& -308G>AAttTNF-a -238G>AGG* AttADA 22G>A ADA 22G>AG*/G* Att G*/G* Att TNF-& -238G>AG*/G* AttADA 22G>A ADA 22G>AG*/G* Att G*/G* AttG*/G* 08 -1,0

Fig. 2. Results of GMDR analyses for comparison 2 (age class 3 vs. age class 2), (A) Males best two-factor model. (B) Females best two-factor model. (C) Males three-factor model. (D) Females three-factor model, Dark grey and light grey boxes correspond to the high- and low-risk genotype combinations, respectively. The black and white bars within each box correspond to age class 3 and age class 2, respectively. The top number above each bar is the sum of scores for the corresponding group of individuals. The heights of the bars are proportional to the sum of scores in each group.

Table 4

Expected TNF-a expression based on ADA and TNFa genotypes combinations (A) and related distributions along the three age classes (B). Values showing significant differences are shown in bold.

(A) Expected TNF-a expression Low expression Normal expression High expression SNP ADA G⁄/G⁄ A⁄+ A⁄+ A⁄+ A⁄+ G⁄/G⁄ G⁄/G⁄ G⁄/G⁄ 22G>A TNF-a G⁄/G⁄ G⁄/G⁄ G⁄/G⁄ A⁄+ A⁄+ A⁄+ G⁄/G⁄ A⁄+ (cid:2)308G>A TNF-a A⁄+ G⁄/G⁄ A⁄+ A⁄+ G⁄/G⁄ A⁄+ G⁄/G⁄ G⁄/G⁄ (cid:2)238G>A (B) Groups Sex TNF-a expression Age class 1 Age class 2 Age class 3 v2 test of independence between the three age classes M Low 24 (16.7%) 71 (27.2%) 20 (20.6%) v2 = 6.67 Normal 96 (66.6%) 151 (57.9%) 64 (66.0%) P = 0.154 High 24 (16.7%) 39 (14.9%) 13 (13.4%) F Low 59 (30.9%) 64 (22.5%) 20 (21.5%) v2 = 5.5 Normal 111 (58.1%) 184 (64.6%) 63 (67.7%) P = 0.240 High 21 (11.0%) 37 (12.9%) 10 (10.8%) v2 test of independence 0.0081 0.270 0.852 between Males and Females within the age class

increases thymus apoptosis [44]. Thus, ADA⁄22A carriers may experience an increased immunological damage caused by more pronounced thymus involution compared to ADA G⁄/G⁄ homozygotes. Finally, it has been demonstrated that ADA inhibition accelerates the process of replicative senescence of CD8+ T lymphocytes, causing a reduction in overall proliferative potential, reduced telomerase activity, and blunted IL-2 gene transcription [45].

the majority of studies conducted to date [8,10–15], to our knowl- edge this is the first report on the association of TNF-a 238G>A SNP with life-expectancy.

Although either single-SNP and haplotype analysis of TNF-a gene did not yield any significant result, we detected an interest- ing gene–gene interaction between ADA and TNF-a, showing age- and sex-specific effects on life-expectancy. Of note, the epistasis between functional SNPs in ADA and TNF-a genes is also sup- ported by the established biological evidence that adenosine lev- els influence TNF-a expression [18–20]. In this respect, the opposite patterns of epistasis detected here between males and females in comparison 1 (Fig. 1) suggest that TNF-a levels exert

For what concern the TNF-a gene, our study failed to demon- strate an association of TNF-a (cid:2)308G>A and (cid:2)238G>A SNPs with human longevity. While for the former our results are in line with

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