40 20 30 40 50 60 70 80 90 100 110

Figure 1. Maximum likelihood estimates of the survival functions of allele A carriers (S (x); AA plus AG genotypes; blue curve) and noncarriers (S (x); GG A B genotype; red curve) in the sample of Italian men. The raw curves (crossed lines) are smoothed (continuous lines) by approximation to a Gompertz–Makeham mortality model (29). The synthetic survival function of the Italian male population (S(x); green curve) is also reported as a reference. The null hypothesis S (x)¼S (x)¼S(x) is A B rejected with an empirical probability level p ¼ .044.

Second, pairwise linkage disequilibrium between loci was analyzed (p , .003 in all the pairs). Third, the frequency of the four-marker haplotypes was estimated in the three age classes. The haplotype results in our sample are shown in Table 3,

where the rare haplotypes (frequency lower than 1%) are grouped with reference to allele A/G of the TNF(A/G)-308 marker. To check the difference in the haplotype frequency distribution among the three classes we applied a likelihood

Table 2. Relative Frequency (% 6 SE) of TNF(A/G)-308 Genotypes and Alleles in Participants Categorized According to Sex and Age

Men Women Age Class 1 Age Class 2 Age Class 3 Age Class 1 Age Class 2 Age Class 3 (,66 y) N ¼ 191 (66–88 y) N ¼ 99 (.88 y) N ¼ 56 (,73 y) N ¼ 155 (73–91 y) N ¼ 80 (.91 y) N ¼ 166 Genotypes Aþ 27.75 6 3.24 22.22 6 4.18 10.71 6 4.13 21.94 6 3.32 23.75 6 4.76 23.49 6 3.29 A– 72.25 6 3.24 77.78 6 4.18 89.29 6 4.13 78.06 6 3.32 76.25 6 4.76 76.51 6 3.29 p ¼ .019 NS Alleles A 15.18 6 1.84 11.11 6 2.23 6.25 6 2.29 11.94 6 1.84 11.88 6 2.56 11.75 6 1.77 G 84.82 6 1.84 88.89 6 2.23 93.75 6 2.29 88.06 6 1.84 88.12 6 2.56 88.25 6 1.77 p ¼ .028 NS

Notes: ‘‘Aþ’’¼‘‘AA’’þ‘‘AG’’ genotypes; ‘‘A–’’¼‘‘GG’’ genotype. The results of the Monte Carlo likelihood ratio chi-square test for differences in the allele/ genotype frequency distribution among age classes are shown. SE ¼ standard error; TNF ¼ tumor necrosis factor; NS ¼ not significant.

Table 3. Frequency Distribution of Four-Marker Haplotypes the TNF Region, in 346 Male Participants Categorized

the effect of which on survival may be comparable to that of alleles that affect the population’s cumulative mortality because of their association with a number of complex age- related diseases, for example, the e4 allele of the apolipo- protein E (APOE) gene (36). Interestingly, the TNF(A/G)-308 polymorphism has been suggested to play a role in the pathogenesis of cardiac ischemic damage, a major causes of death more prevalent in men; in particular, carriers of allele A are prone to more severe ischemic damages following myocardial infarction (21).

Age Class 1 Age Class 2 Age Class 3 (,66 y) (66–88 y) (.88 y) 2n ¼ 382 2n ¼ 198 2n ¼ 112 Haplotypes Freq. % 6 SE Freq. % 6 SE Freq. % 6 SE 2.1.A.3* 4.69 6 1.10 5.56 6 1.64 2.68 6 1.59 4.1.A.3 5.21 6 1.16 2.02 6 1.04 1.79 6 1.20 rare Aþ 5.21 6 1.14 3.54 6 1.31 1.79 6 1.25 1.2.G.3* 0.78 6 0.49 2.53 6 1.12 0.89 6 0.84 10.1.G.3* 19.53 6 2.14 21.72 6 2.87 23.21 6 4.00 11.1.G.3* 5.99 6 1.25 5.05 6 1.54 6.25 6 2.35 13.1.G.3* 3.39 6 0.94 10.10 6 2.13 4.46 6 1.95 2.2.G.1* 13.80 6 1.75 7.07 6 1.79 8.93 6 2.79 2.2.G.3* 4.17 6 1.10 2.53 6 1.17 5.36 6 2.15 4.1.G.3* 0.78 6 0.49 2.53 6 1.17 0.89 6 1.01 5.2.G.2 2.08 6 0.75 2.02 6 1.02 0.89 6 0.89 5.2.G.3* 2.86 6 0.90 3.03 6 1.27 5.36 6 2.25 6.1.G.3* 16.41 6 1.93 10.61 6 2.14 18.75 6 3.77 7.1.G.3* 4.43 6 1.15 8.08 6 2.02 8.93 6 2.71 7.2.G.3 4.17 6 1.10 1.51 6 1.00 0.89 6 1.01 9.2.G.3* 1.04 6 0.51 4.03 6 1.38 0.89 6 0.93 Rare Gþ 5.46 6 1.16 8.07 6 1.94 8.04 6 2.57

It is worth nothing that the TNF(A/G)-308 polymorphism is a functional polymorphism with allele-specific effects on gene expression (11,12). The finding that genotypes in- cluding the allele that increases gene expression (A allele) are detrimental for surviving up to old age is in line with the idea that inflammatory responses may contribute to devel- opment of chronic degenerative diseases (37) and that indi- viduals with a pro-inflammatory genetic asset have a higher mortality risk, as postulated by the inflammaging theory (7–9).

The detrimental effect of allele A is restricted to men. Taking into account the functional role of this poly- morphism and the sex-specificity of the physiological set- up, this finding is not unexpected. In any case, this result adds a further piece of evidence to the idea that the genetic component that modulates the probability of reaching extreme old age is gender-specific and likely more important in men than in women (38).

Notes: The haplotype nomenclature refers to alleles of the four loci, reported (from left to right) in the following order: TNFa, TNFc, TNF(A/G)-308, TNFe. Haplotypes with a frequency lower than 1% are pooled into two categories of rare haplotypes, based on the presence of allele A (rare Aþ) or G (rare Gþ) at the TNF-308 locus. Standard errors (SE) of haplotype frequencies were calculated through 1000 bootstrap resampling cycles. A likelihood ratio chi-square test for differences in the haplotype frequency distribution among age classes gave the following result: v2 ¼ 58.7, df ¼ 32, p ¼ .0027.