Introduction:
Aging is a complex phenotype influenced by multiple determinants, including a strong genetic component that impacts survival to extreme ages. The dysregulation of immune responses that occurs with age is believed to contribute to human morbidity and mortality. Conversely, some genetic determinants of successful aging might reside in polymorphisms for immune system genes that regulate immune responses. In this study, we examined the main effects of single loci and multi-locus interactions to test the hypothesis that the adenosine deaminase (ADA) and tumor necrosis factor alpha (TNF-a) genes influence human life expectancy. ADA (22G>A, rs73598374) and TNF-a ((cid:2)308G>A, rs1800629; rs361525) functional SNPs were determined for 1071 unrelated healthy individuals from Italy (18-106 years old), divided into three gender-specific age classes defined according to information and accounting for the different survivals between sexes: for men (women), the first consists of individuals <66 years old (<73 years old), the second class of individuals 66-88 years old (73-91 years old), and the third class of individuals >88 years old (>91 years old).

Results:
Single-locus analysis showed that only ADA 22G>A is significantly associated with human life expectancy in males - comparison 1 (age class 2 vs. age class 1), O.R. 1.943, P = 0.036; comparison 2 (age class 3 vs. age class 2), O.R. 0.320, P = 0.0056). Age- and gender-specific patterns of epistasis between ADA and TNF-a were studied using Generalized Multifactor Dimensionality Reduction (GMDR). In comparison 1, a significant interaction occurs in females between ADA 22G>A and TNF-a (cid:2)238G>A (Sign Test P = 0.011). In comparison 2, both two-loci and three-loci interactions are significantly associated with increased life expectancy over 88 years in males. In conclusion, we report that a combination of functional SNPs within ADA and TNF-a genes can influence life expectancy in a gender-specific manner and that males and females have different pathways to attain longevity.

Discussion:
Human longevity is a complex trait arising from the interplay of environmental and genetic factors. The study of such phenotype gives us the opportunity to uncover the biological determinants of human survival and life expectancy. In fact, most of the information regarding the genetics of human longevity has been obtained through retrospective studies under the hypothesis that unfavorable genotypes should be dropped out of the population by a sort of demographic selection [1], which finally results in an enrichment of favorable genotypes in the gene pool of long-living people.

Materials and methods:
The study population consisted of all individuals aged 105 years or more who were alive in Italy on January 1st, 2009, plus a sample of individuals aged 90-94 years, randomly selected from the Italian population from 1890 onward (computed using death counts per calendar year and per year of birth as reported for the Italian population in the Human Mortality Database, available online at www.mortality.org) [25]. The study protocol was approved by the Joint Ethical Committee (JEC) University of Camerino-Azienda ASUR Marche ZT-10 Camerino, in accordance with the Declaration of Helsinki in its revised edition and with international and local regulatory requirements.

ADA (22G>A) and TNF-a ((cid:2)308G>A; (cid:2)238G>A) SNPs were genotyped according to RFLP-PCR methods previously published [23,26]. Briefly, for ADA, PCR was performed with primers flanking the 22G>A polymorphic region: 50-GCCCGGCCCGTTAAGAAGAGC-30 as sense primer, and 50-GGTCAAGTCAGGGGCAGAAGCAGA-30 as antisense primer.SNPs tested, only the ADA 22G>A SNP showed a significant difference in genotype frequencies between age classes in males (P = 0.036). No other significant associations were found for singular SNPs or TNF-a promoter diplotype. The GMDR method did not identify any significant gene-gene interactions between ADA and TNF-a SNPs. Haplotype association tests for TNF-a promoter SNPs also did not show any significant association with human-life expectancy.

In the materials and methods section, PCR was carried out using specific primers to amplify DNA fragments for both ADA and TNF-a SNPs. The PCR products were then digested with specific endonucleases and electrophoresed in agarose gels to detect the presence or absence of certain alleles. Statistical analysis was performed using various tests, including the chi-square test, Fisher's exact test, and logistic regression models.

The study population consisted of 1,223 individuals (654 males and 569 females) aged 18-106 years old, randomly recruited from the same geographical area of Central Italy. The population was divided into three sex-specific age classes based on demographic information. Almost all subjects were recruited in blood donor centers and through family physicians, and they provided information about their health condition to ensure that no pathological conditions existed.

In conclusion, this study did not find any significant associations between singular SNPs or TNF-a promoter diplotype with human longevity/survival. No significant gene-gene interactions were identified between ADA and TNF-a SNPs. Haplotype association tests for TNF-a promoter SNPs also did not show any significant association with human-life expectancy.needs to be further investigated for its role in immune cell development and differentiation, as well as its impact on the regulation of inflammatory responses [  ]. Overall, our results suggest that the ADA 22G>A SNP may play a gender-specific role in determining human life-expectancy, possibly through its interaction with other genetic factors such as TNF-a. Further studies are needed to confirm these findings and elucidate the underlying mechanisms involved.The text contains several errors that need correction to improve readability and coherence. Here's the processed text: