polymorphism is concerned. Indeed, the frequencies we found in centenarians and young donors were extremely similar. However, our results do not exclude a role of such polymorphism in the regulation of Fas expression, as we found a small difference between the two groups in the frequency of A/A genotype that was absent in centenarians. Clearly, due to the low frequency of such a genotype in young donors, a far larger number of subjects is needed to

could greatly influence the expression of Fas on the surface of the cell, and thus could play an important role in the tendency of T lymphocytes from elderly people to undergo Fas-induced apoptosis. FasL polymorphisms are located in intronic regions that are clearly not directly related to gene transcription. However, it is possible that they are in linkage disequilibrium with still unknown, polymorphic regions capable of influencing FasL expression. Studies aimed at correlating the expression of Fas and FasL with the genotypes of the subjects we have described in this paper are in course to further investigate this point, and to give new insights into the role and regulation of Fas and FasL during immunosenescence.

of DNA template were added to the reaction mixture, containing a final concentration of 200 nM of each primer, 200 mM of dNTPs, 1.5 mM MgCl , 50 mM KCl , 10 mM Tris Cl (pH 8.5) and 1 U of Taq 2 2 polymerase. The reaction was carried out in a PE 9700 Thermal Cycler for 30 cycles, with each cycle consisting of 30 s at 948C, 30 s at 568C, and 30 s at 728C. The first cycle was preceded by a single step of denaturation at 948C for 6 min, and the last one was followed by a single step of extension at 728C for 10 min. The PCR products were purified using High Pure PCR Product Purification Kit (Roche Biochemicals) and 20 ng of purified DNA were used for sequencing reaction, using BigDye DNA Sequencing Kit (Perkin Elmer). Sequencing reaction was carried out in a PE 9700 Thermal Cycler, under conditions recommended by the customer. Sequenced samples were loaded in 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.

Materials and Methods

primers (F3 and F4) with two mismatches at 3’ position were used, in couple 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 and 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 mutant allele, a product was only generated in the reaction containing the primer specific for 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 were added to the reaction mixture, containing a final concentration of 200 nM of each primer, 200 mM of dNTPs, 1.5 mM MgCl , 50 mM KCl , 10 mM Tris Cl (pH 8.5) 2 2 and1 UofTaqpolymerase(Promega).Thereactionwas carriedoutina PE 9700 Thermal Cycler for 30 cycles, with each cycle consisting of 30 s at 948C, 30 s at 628C, and 1 min at 728C. The first cycle was preceded by a single step of denaturation at 948C for 6 min, and the last one was followed by a single step of extension at 728C for 10 min.

Subjects

A group of 50 centenarians and 86 controls, all living in Northern Italy (Emilia-Romagna Region, located in the Po Valley area) was studied. The mean age of centenarians (44 females, six males) was 100.8+1.8 years (mean+S.D., range 100–106), whereas the mean age of young donors (36 females, 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 according to standardized methods by using a commercially available kit (QIA Amp DNA blood minikit, from Qiagen).

Fas polymorphism typing: analysis of MvaI

The MvaI RFLP was studied by polymerase chain reaction (PCR) amplification followed by MvaI restriction enzyme digestion, as previously described.24 The primer sequences used for PCR were MvaIDir (5’-CTACCTAAGAGCTATCTACCGTTC-3’) and MvaIRev (5’GGCTGTCCATGTTGTGGCTGC-3’). For each reaction in 25 ml, 100 ng of DNA template were added to the reaction mixture, containing a final concentration of 200 nM of each primer, 200 mM of dNTPs, 1.5 mM MgCl , 50 mM KCl , 10 mM Tris Cl (pH 8.5) and 1 U 2 2 of Taq polymerase (Promega, Madison, WI, USA). The reaction was carried out in a PE 9700 Thermal Cycler (Perkin Elmer, Boston, MA, USA) for 30 cycles, each cycle consisting of 30 s at 948C, 30 s at 628C, and 1 min at 728C. The first cycle was preceded by a single step of denaturation at 948C for 6 min, and the last one was followed by a single step of extension at 728C for 10 min. After reaction, 10 ml of reaction mixture from each sample were digested with 5 U of MvaI restriction enzyme (Roche Biochemicals, Mannheim, Germany) under recommended conditions. The product was loaded onto a 3% agarose gel and run at 90 V for 40 min. Two polymorphic alleles, allele A (233 bp) or allele G (189 bp) were produced depending on the presence of G or A at 7670 position of the Fas gene.

IVS2nt-124 FasL polymorphism typing