mosome 1p13, encoding lymphoid protein tyrosine phos- phatase (LYP), plays a crucial role in the negative control of T lymphocyte activation. LYP is expressed in lymphoid cells, mainly in T lymphocytes where it associates with C-terminal Src kinase (CSK) to form a complex that sup- presses the T-cell receptor signaling kinases LCK and FYN [12, 13]. Recently, a variant of PTPN22 (rs2476601, 1858C[T) has been associated with an increase in risk for the development of multiple autoimmune diseases includ- ing type 1 diabetes (T1D) [14–16], rheumatoid arthritis [17], systemic lupus erythematosus [18], and Graves’s disease [19]. This variant is a single nucleotide change at residue 1858 from C to T, which results in a single amino acid substitution from arginine to tryptophan at position 620 of the Lyp protein (Lyp 620W). Experimental evidence suggests that the disease-associated LYP variant Trp620 prevents the interaction of LYP with CSK [14]. Conse- quently, the T-cell receptor-associated kinases might be able to induce T-cell activation in an uncontrolled manner and this may increase the overall reactivity of the immune system and predispose an individual to autoimmune dis- ease. Noteworthy, it has been demonstrated that homozy- gosity for this variant results in a profound deficit in T cell responsiveness to antigen stimulation. Heterozygosity for the variant allele is associated with reduced responsiveness of CD4? memory T cells, characterized by diminished calcium mobilization, expression of CD25, IL-10 and IL-2 production upon TCR stimulation. Additionally, the pres- ence of the variant allele is associated with an increase in

PTPN22 1858C[T (R620W) genotyping

Genomic DNA extraction from whole blood was carried out by salting-out method [21] and stored at -20(cid:3)C for the gene analysis. For the determination of the PTPN22 alleles, PCR-based restriction fragment length polymorphism (RFLP) analysis was performed as described previously [14]. Briefly, a fragment of the PTPN22 gene was ampli- fied by PCR using the forward primer 50-TCACCAGCTT CCTCAACCACA-30 and the reverse primer 50-GATAAT GTTGCTTCAACGGAATTTA-30. The C ? T transition at codon 620 (NCBI refSNP ID: rs2476601) creates in the T1858 allele a restriction site for Xcm I. The polymor- phism was identified by Xcm I (New England Biolabs, Beverly, MA, USA) restriction endonuclease digestion of

Each digestion was resolved on 2.5% agarose gel, stained with ethidium bromide and visualized by UV. Repeated typing was performed in 10% of samples, with identical results in all cases.

Statistical analysis

Indeed, we hypothesized that analysis of the genetic polymorphism of the PTPN22 enzyme associated with an increased risk of autoimmunity could also prove informa- tive as to the possible role of autoimmunity in determining human life expectancy. Therefore, we performed a popu- lation-based study analyzing the PTPN22 1858C[T (R620W) genotype frequencies in 892 healthy individuals ranging from 8 to 106 years.

HWE (Hardy–Weinberg Equilibrium) was checked by v2 test. Fisher’s exact test (two-tailed) was used to evaluate the differences in genotypic distribution (under a genetic dominant model) between the groups and the strength of association was expressed as odds ratios (O.R.s) with 95% confidence intervals. Statistical significance was set at P \ 0.05. Three-way contingency table analysis was done by a log-linear model according to Sokal and Rohlf [22]. Statistical analyses were conducted with SPSS 16.0 (SPSS Inc., Chicago, IL). Power calculations were performed using Quanto ver. 1.2.4. (available at http://hydra.usc.edu/ gxe/), taking into account sample sizes and minor allele frequencies (MAF), genetic dominant model, frequency of octogenarians and nona/centenarians in Italy (available at http://demo.istat.it/pop2010/index.html), and type I error

Materials and methods

Subjects

Eight hundred and ninety-two unrelated healthy individuals randomly selected from the same geographical area of

rate (0.05). Considering these parameters, we had a cient power (0.80) to detect an association with a relative risk (or odds ratio) B0.350 and/or C2.100.

Table 2 Three-way contingency table analysis by a log-linear model

G2 d.f. P xyz interaction 7.46 7 0.383 xy independence 0.82 4 0.936 xz independence 3.64 4 0.457 yz independence 3.4 3 0.334

Results

The distribution of the PTPN22 genotype in population studied is shown in Table 1; the maximum age in males and females in our sample population was 100 and 106 respectively. Only one subject carrier of PTPN22 T*/T* genotype was found. Allelic and genotypic frequencies are in Hardy–Weinberg equilibrium, regardless of whether males and females are considered together (v2 = 0.15, P = 0.698), or separately (males: v2 = 0.56, P = 0.454; females: v2 = 0.15, P = 0.698). The same situation occurs when age groups were considered (Table 1). For both gender, the frequency of PTPN22*T carriers does not differ significantly in nona/centenarians and in octogenarians respect to young group (Table 1). On the whole, there was a trend toward higher frequencies of PTPN22*T carriers among females (10.2 vs. 7.2%, P = 0.08), but overall, the frequency of *T carriers was not significantly associated with gender in each age group studied. Allele frequencies do not differ significantly among age groups (females v2 = 0.57, P = 0.752; males v2 = 0.33, P = 0.8479). The three-way contingency table analysis by log-linear model confirmed the irrelevant effect of PTPN22 genotype in the determination of life-span expectancy in the population

x = age group; y = PTPN22 genotype (PTPN22 C*/C* PTPN22*T carriers); z = sex