A Genetic–Demographic Approach Reveals Male-Specific Association Between Survival and Tumor Necrosis Factor (A/G)-308 Polymorphism

1 1,2 1 1 Maurizio Cardelli, Luca Cavallone, Francesca Marchegiani, Fabiola Oliveri, 3 3 4 1 Serena Dato, Alberto Montesanto, Francesco Lescai, Rosamaria Lisa, 3 1,4,5,6 Giovanna De Benedictis, and Claudio Franceschi

1Italian National Research Center on Aging, Ancona, Italy. 2Department of Human Genetics, McGill University, Montreal,

4Department of Experimental Pathology and 5Centro Interdipartimentale ‘‘L. Galvani,’’ University of Bologna, Italy.

The (A/G)-308 polymorphism of the tumor necrosis factor a gene (TNF) is associated with age- related diseases, but its influence on longevity is controversial. We genotyped for this polymorphism 747 Italian volunteers (401 women and 346 men, age 19–110 years). By applying a genetic–demographic (GD) approach we found that, in men, the survival function of allele A carriers is lower than that of noncarriers at all the ages (p ¼ .044). After defining (by exploiting again demographic information) three age classes, we found that the frequency of men carrying the A allele decreases with age (p ¼ .019), thus confirming the GD analysis results. The same analyses gave negative results in women. Therefore, allele A has a detrimental effect on life expectancy, and this effect is specific to men. A haplotype analysis carried out in men by screening the TNFa, TNFc, and TNFe microsatellite polymorphisms (spanning about 20 kb) confirmed the association of the TNF region with life expectancy.

Words: TNF(A/G)-308 polymorphism—TNF demographic approach.

sion of the cytokine (11–13). The correlation between the (A/G)-308 polymorphism and age-related or autoimmune pathologies is well established (14–21). On the contrary, contrasting data have been reported as they regard the association with longevity (22–26).

the latent genetic susceptibility to two opposite traits, age-related diseases and extreme longevity, also revealing that some of the most promising genes are those encoding for inflammatory cytokines. In fact, polymorphisms of the genes involved in the regulation of the inflammatory process may influence the susceptibility to age-related diseases, and it has been shown that genotypic combinations with optimal pro-/anti-inflammatory activity can affect the probability to reach successful aging and longevity (1–6) as theorized in the context of the theory of inflammaging (7–9). In this frame, one of the most studied genes is that encoding for the tumor necrosis factor (TNF) a, a pleiotropic cytokine playing a key role in both inflammation and chronic age- related diseases. The TNF gene lies in a cluster of func- tionally related genes that also includes lymphotoxin alpha (LTA), lymphotoxin beta (LTB), and leukocyte-specific transcript-1 (LST1) and is located in the class III region of the major histocompatibility complex (MHC) on chromo- some 6. The levels of TNF-a production are influenced by inter-individual genetic variation (10). In particular, the (A/G)-308 polymorphism (rs1800629), located in the pro- moter of the TNF gene, is associated with allele-specific expression levels, with the less frequent allele (allele A, also referred as allele 2) being associated with a higher expres-

Usually, the genetic component of longevity is in- vestigated by comparing allele and/or genotype frequencies at specific candidate loci between cases and controls (gene frequency [GF] method), represented, respectively, by classes of participants of different ages, including a class of long-lived individuals. However, by dealing with cross- sectional data, the GF approach ignores cohort effects in population mortality changes. Furthermore, the definition of the age-classes is often arbitrary so that positive (or neg- ative) results may simply depend on the rather naive as- sumption that age-related allele and/or genotype frequency trajectories vary in the population always according to a monotonous function (27). The possibility of overcoming these methodological limits is offered by the addition of demographic information to genetic data (28–30). Genetic– demographic (GD) methods allow the estimation of hazard rates and survival functions in relation to candidate alleles and genotypes. In such a way it is possible to compare survival functions between individuals carrying or not carrying a candidate allele or genotype without introducing

Table 1. Polymorphisms Analyzed and Primers Used for PCR Amplification

Location Distance from TNF-308 Polymorphism Number of Alleles Primers Used for PCR Amplification Intergenic region 7.3 kb (AC)n 13 IR2*: 59-gcctctagatttcatccagccaca IR4: 59-cctctctcccctgcaacacaca Intron (LTA gene) 2.4 kb (TC)n 2 IR6: 59-ggtttctctgactgcatcttgtcc IR7*: 59-tcatggggagaacctgcagagaa (A/G)-308 Promoter (TNF gene) — G>A (rs1800629) 2 TNF1.1: 59-aggcaataggttttgagggccat TNF1.2: 59-tcctccctgctccgattccg Intron (LST1 gene) 12.8 kb (TC)n 3 IR13: 59-gtgcctggttctggagcctctc IR14**: 59-tgagacagaggataggagagacag

Notes: Loci are ordered from the most telomeric (TNFa) to the most centromeric (TNFe). *Primers labeled with 6-FAM (6-carboxyfluorescein) fluorescent dye. **Primer labeled with TET (tetrachloro-6-carboxyfluorescein) fluorescent dye. PCR ¼ polymerase chain reaction; TNF ¼ tumor necrosis factor gene; LTA ¼ lymphotoxin alpha; LST ¼ leukocyte-specific transcript-1.

recruited centenarians (35 men and 114 women) had a fairly good health status, did not suffer major pathologies or disabilities, and were classified in categories A and B according to the criteria elsewhere reported (31). All the participants were informed about the aim of the study and gave their approval according to Italian laws.