Table 2

TNF-a (rs1800629; rs361525) haplotype frequencies along the three age classes.

Sex TNF-a Groups Comparison 1 Comparison 2 v2 test of independence haplotypes (age class 2 vs. age class 1) (age class 3 vs. age class 2) between the three age classes Age class 1 Age class 2 Age class 3 O.R. 95% C.I. P O.R. 95% C.I. P value value M (cid:2)308⁄G/(cid:2)238⁄G 245 (85.1%) 429 (82.2%) 164 (84.5%) 1.000 1.000–1.000 0.293 1.000 1.000–1.000 0.458 v2 = 6.03 P = 0.197 (cid:2)308⁄G/(cid:2)238⁄A 13 (4.5%) 38 (7.3%) 17 (8.8%) 1.669 0.872–3.195 0.121 1.170 0.643–2.131 0.508 (cid:2)308⁄A/(cid:2)238⁄G 30 (10.4%) 55 (10.5%) 13 (6.7%) 1.047 0.653–1.678 0.958 0.618 0.329–1.162 0.120 D0 = 0.49, D0 = 1.00, D0 = 1.00, LD* LRS P = 0.277 LRS P = 0.416 r2 = 0.00 r2 = 0.00 r2 = 0.00 F (cid:2)308⁄G/(cid:2)238⁄G 318 (83.2%) 484 (84.9%) 159 (85.5%) 1.000 1.000–1.000 0.425 1.000 1.000–1.000 0.849 v2 = 4.50 P = 0.609 (cid:2)308⁄G/(cid:2)238⁄A 31 (8.2%) 33 (5.8%) 13 (7.0%) 0.696 0.410–1.180 0.121 1.200 0.616–2.335 0.552 (cid:2)308⁄A/(cid:2)238⁄G 32 (8.4%) 53 (9.3%) 14 (7.5%) 1.083 0.670–1.749 0.728 0.804 0.435–1.488 0.461 (cid:2)308⁄A/(cid:2)238⁄A 1 (0.2%) – – N.A. N.A. N.A. – – – D0 = 0.09, D0 = 0.84, D0 = 1.00, LD* LRS P = 0.462 LRS P = 0.656 r2 = 0.00 r2 = 0.00 r2 = 0.00

LRS, likelihood ratio test. N.A., not assessed. O.R., odds ratio. * Pairwise D0 values display no statistically significant evidence of

Table 3

Best models assessed by the GMDR for one-, two- and three-way combinations, to test ADA and TNF-a epistasis in determining human longevity. Values showing significant differences are shown in bold.

Comparison 1 (age class 1 vs. age class 2) Sex Model TBA Sign Test (P) Cross-validation consistency Odds ratios (95% C.I.) v2 (P) M ADA 22G>A 0.542 8 (0.055) 10/10 1.943 (1.063–3.511) 4.776 (0.029) ADA 22G>A/TNF-a (cid:2)238G>A 0.553 8 (0.055) 10/10 1.970 (1.195–3.248) 7.207 (0.0073) ADA 22G>A/TNF-a (cid:2)308G>A/TNF-a (cid:2)238G>A 0.554 8 (0.055) 10/10 2.054 (1.230–3.432) 7.738 (0.0054) F ADA 22G>A 0.519 6 (0.377) 9/10 1.471 (0.927–2.335) 2.705 (0.100) ADA 22G>A/TNF-a (cid:2)238G>A 0.550 9 (0.011) 10/10 1.584 (1.066–2.353) 5.216 (0.022) ADA 22G>A/TNF-a (cid:2)308G>A/TNF-a (cid:2)238G>A 0.536 8 (0.055) 10/10 1.633 (1.086–2.457) 5.589 (0.018) Comparison 2 (age class 2 vs. age class 3) M ADA 22G>A 0.562 8 (0.055) 10/10 3.122 (1.365–7.144) 7.911 (0.0049) ADA 22G>A/TNF-a (cid:2)308G>A 0.572 9 (0.011) 10/10 2.061 (1.184–3.586) 6.710 (0.0096) ADA 22G>A/TNF-a (cid:2)308G>A/TNF-a (cid:2)238G>A 0.564 9 (0.011) 10/10 2.273 (1.268–4.075) 7.853 (0.0051) F TNF-a (cid:2)308G>A 0.492 4 (0.828) 8/10 1.259 (0.662–2.395) 0.496 (0.481) ADA 22G>A/TNF-a (cid:2)308G>A 0.501 6 (0.377) 10/10 1.281 (0.744–2.206) 0.803 (0.370) ADA 22G>A/TNF-a (cid:2)308G>A/TNF-a (cid:2)238G>A 0.488 4 (0.828) 10/10 1.327 (0.758–2.234) 0.986 (0.321)

TBA, testing balanced accuracy.

>TNF-c -238G>ATNF-a -238G>AG*/G*AttG*/G*A*+ADA 22G>AG*/G*i 14,9 v1KeyA*+ ke)ADA 22G>AG*/G*Att QaTNF-o -308G>AoTNF-a -308G>AG*/G*Att TNF-& -238G>AG/G* A‘+ADA 22G>A ADA 22G>AG*/G* Att G*/G* A*+ TNF-& -238G>AG*G* A*+ADA 22G>A ADA 22G>AG*/G* A*t G*/G* A*+i eni | [I 3.0cE|_|EK 46-24

Fig. 1. Results of GMDR analyses for comparison 1 (age class 2 vs. age class 1), (A) Males best two-factor model. (B) Females best two-factor model. (C) Males three-factor model. (D) Females three-factor model, Dark gray 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 2 and age class 1, 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.

differences reported for adenosine physiological effects, several considerations can be made:

in Fig. 1, males and females seem to display opposite gene–gene interactions for comparison 1, thus suggesting the existence of a gender-specific pathway determining increased life-expectancy after reproductive age (age class 1).

a- The effect of ADA

G>A is prominent in males, which display a greater sensibility to the effect of adenosine at vascular and immunological level [

]. Also, the beneficial role of estrogen hormone may underestimate the effect of adenosine in females [

]. Furthermore, the significant dif- ference of ADA

G>A genotype frequency between males and females could be ascribed to the presence of gender- dependent transmission ratio distortion [

,

]. This hypothesis is supported by the fact that ADA

G>A SNP is located within a C

island [

] which can be epigenetically imprinted [

]. Also, there is established evidence for a cru- cial role played by ADA in human reproduction [

], possibly influencing the selection before fertilization (meiotic drive), at the time of fertilization, and after fertilization (embryonic lethality). b- Males carriers of ADA⁄

A may have a selective advantage in reaching age class

, possibly due to the protective effect of the elevated cellular concentrations of adenosine during ischemic attack, whose occurrence drastically increases in this period of life. Moreover, ADA⁄

A has been recently associated with reduced risk of Coronary Artery Disease in males [

]. In this context, it is plausible that ADA⁄

A allele is responsible for increased myocardial adenosine concentrations which exert cardio-protective effects [

]. c- The significant decrease of ADA⁄

A carriers in age class

may be due to the striking impact of ADA gene on immune functions. Adenosine triggers the release of IL-

, a disability and mortality predictor among the elderly, as well as IL-

b, a pro-inflammatory cytokine associated with poor physical performance and diminished muscle strength in older per- sons [

]. 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