Expression of Fas on lymphocytes from elderly donors could be due to age-related changes in the susceptibility of these cells to undergo apoptosis. However, few and controversial data are present in literature. Some investigators have reported a decrease of Fas/FasL-induced apoptosis in aged animals, as well as in human CD8+ T cells reaching replicative senescence. Accordingly, we have recently reported that an inverse correlation exists between human age and the propensity of lymphocytes to undergo apoptosis induced by oxidative stress. On the other hand, other authors reported increased apoptosis in lymphocytes from elderly people following activation with anti-CD3, phytohemagglutinin, concanavalin A or activation with polyclonal mitogens plus anti-Fas treatment.

Genetic polymorphisms of apoptotic genes such as Fas and FasL may play a role in regulating the expression of a given gene. Thus, we explored whether genetic polymorphisms of apoptotic genes such as Fas and FasL exist that could help in explaining the aforementioned phenomena, taking into account that no data exist on their distribution and role in human aging and longevity. In this study, we analyzed the presence of new genetic markers, i.e. 7670 and 71377 polymorphisms in Fas promoter, and IVS2nt-124 and IVS3nt169 in FasL gene, in a group of 50 Italian centenarians and 86 young donors. No significant differences were found in the genotype and allele frequency between centenarians and controls for all polymorphisms. These data suggest that these polymorphisms likely do not confer any genetic advantage to reach the extreme limit of human life. Moreover, considering the entire group of individuals we have analyzed, our data on Fas gene are in agreement with data reported by Huang et al. for normal Caucasian population. Indeed, the frequencies of the alleles we have analyzed are very similar to those previously reported.

We found no major differences between centenarians and young donors except for the frequencies of the Fas (71377A/7670Added to the reaction mixture were a final concentration of 200 nM for each primer, 200 mM of dNTPs, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris Cl (pH 8.5), and 1 U of Taq polymerase. The reaction was conducted in a PE 9700 Thermal Cycler for 30 cycles, with each cycle consisting of 30 s at 94°C, 30 s at 56°C, and 30 s at 72°C. The first cycle was preceded by a single step of denaturation at 94°C for 6 min, and the last one was followed by a single step of extension at 72°C for 10 min. The PCR products were purified using the High Pure PCR Product Purification Kit (Roche Biochemicals), and then, 20 ng of purified DNA was used for sequencing reactions with the BigDye DNA Sequencing Kit (Perkin Elmer). The sequencing reaction was carried out in a PE 9700 Thermal Cycler under conditions recommended by the manufacturer. Sequenced samples were loaded into a PE ABI Prism 310 Genetic Analyzer (Perkin Elmer) and analyzed using ABI Prism Navigator Software. The two alleles, depending on the presence of A or G at position 71377, were observed as different fluorescence peaks in that position.

For the primers (F3 and F4) with two mismatches at position three were used in combination with a common reverse primer (F5). F3 primer is specific for allele G at position 71377, and F4 is specific for allele A. For each sample testing two PCR reactions in different tubes were performed; one reaction detected the wild-type (G) allele while the other detected the mutant (A) allele using a specific forward primer and the common reverse primer. PCR products, 393 bp fragments, from both reactions were then run on two different gels because of the exact same size of the two products. If DNA samples were homozygous for the wild-type allele, there would be only products in the reaction containing the wild-type specific primer. Conversely, if a sample was homozygous for the mutant allele, a product was only generated in the reaction containing the primer specific for the mutant allele. The presence of PCR products in both reactions indicated that the sample was heterozygous. For each reaction in 25 ml, 100 ng of DNA template was added to the reaction mixture, containing a final concentration of 200 nM of each primer, 200 mM of dNTPs, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris Cl (pH 8.5), and 1 U of Taq polymerase (Promega). The reaction was carried out in a PE 9700 Thermal Cycler for 30 cycles, with each cycle consisting of 30 s at 94°C, 30 s at 62°C, and 1 min at 72°C. The first cycle was preceded by a single step of denaturation at 94°C for 6 min, and the last one was followed by a single step of extension at 72°C for 10 min.

A group of 50 centenarians and 86 controls living in Northern Italy (Emilia-Romagna Region, located in the Po Valley area) were studied. The mean age of centenarians (44 females and six males) was 100.8±1.8 years (mean±S.D., range: 100-106), whereas the mean age of young donors (36 females and 50 males), randomly chosen among students, medical personnel, and blood donors, was 38.1±8.5 years (range: 22-54). Blood was taken from all donors after informed consent according to the Italian laws, and DNA was extracted using a commercially available kit (QIA Amp DNA blood minikit, from Qiagen).