ADA genotyping

Genomic DNA was extracted according to Miller et al. (1988) and stored at -20(cid:3)C prior to gene analysis. Genomic DNA was amplified by PCR with primers flanking the G22A polymorphic region: 50-GCCCG GCCCGTTAAGAAGAGC-30 as sense primer, and 50-GGTCAAGTCAGGGGCAGAAGCAGA-30 as antisense primer (GenBank accession No. M13792). These primers yielded a 397 bp PCR product in a 20 ll reaction mixture containing 1,5 ll of template DNA, 0.6 U Phusion High Fidelty-DNA polymerase (Finn- zymes, Finland), 0.6 ll of DMSO 100%, 0.5 lM of each primer and a final concentration of 200 lM dNTPs and 1 9 Phusion HF buffer (Finnzymes). The amplification reaction was performed on a Biometra T-gradient thermal-cycler with initial denaturation at 98(cid:3)C for 3 min, followed by 35 cycles of a two-step PCR protocol consisting of denaturation step at 98(cid:3)C for 15 s and one annealing/extension step at 72(cid:3)C for

15 s, with a final extension time of 5 min at 72(cid:3)C. The PCR product were digested with TaqI (Promega) for 90 min at 65(cid:3)C, and the sample were electrophoresed in a 1.8% agarose gel. The PCR product of the ADA*1 (G22) allele was cleaved into 245-bp and 152-bp fragments. The ADA*2 (A22) allele was identified by the lack of a Taq I restriction site.

Table 2 Age classes/ADA genotypes 2-Way contingency table in males

Age (years) Genotypes ADA 1*/1* ADA*2 carriers Obs. Exp. Obs. Exp. \71 115 112.33 16 18.67 80–85 111 122.62 32 20.38 86–90 58 54.88 6 9.12 [90 59 53.16 3 8.84

Statistical analysis

The ADA genotypic distribution was tested for accordance with the Hardy-Weinberg equilibrium. A chi-square test of independence was used to evaluate the differences in ADA genotypic distribu- tion between the four age groups. Three-way contin- gency table analysis was done by a log-linear model according to Sokal and Rohlf (1981). Statistical analyses were conducted with SPSS 14.0 (SPSS Inc., Chicago, IL, 1992).

v2 = 13.923, d.f. = 3, P = 0.003

Table 3 Age classes/ADA genotypes 2-Way contingency table in females

Age (years) Genotypes ADA 1*/1* ADA*2 carriers Obs. Exp. Obs. Exp. \71 109 117.11 32 29.88 80–85 120 113.79 17 23.21 86–90 82 81.4 16 16.6 [90 91 89.7 17 18.3

Results

The distribution of the ADA genotype in individuals aged\71, 80–85, 86–90 and[90 is shown in Table 1; the maximum age in males and females in our sample population was 100 and 106 respectively. Allelic and genotypic frequencies are in Hardy–Weinberg equi- librium, regardless of whether males and females are considered together (v2 = 0.649, df = 1, P = 0.420), or separately (males: v2 = 0.017, df = 1, P = 0.896; females: v2 = 0.889, df = 1, P = 0.346).

v2 = 5.45, d.f. = 3, P = 0.14166

As seen in Table 2, compared to the control group (i.e. age \ 71), there is an increased number of ADA*2 allele carriers in the population of long-lived males aged 80–85, while among the males aged[85 there is a decrease of ADA*2 allele carriers (v2 = 13.93; df = 3; P = 0.003).

The distribution of the ADA genotype in female subjects belonging to different age groups is pre- sented in Table 3. Unlike the situation observed with males, the ADA genotype distribution does not show any significant difference between the groups (v2 = 5.45; df = 3; P = 0.142). A relative decrease in the number of ADA*2 carriers in the group of females aged 80–85 was found not to be statistically significant.

Table 1 Age classes/ADA genotypes distribution and tests for conformation to Hardy–Weinberg equilibrium

Age (years) Genotypes Total ADA 1*/1* ADA 2*/1* ADA 2*/2* M F M F M F M F \71 115 109 14 31 2 1 131 141 80–85 111 120 32 16 – 1 143 137 86–90 58 82 6 16 – – 64 98 [90 59 91 3 17 – – 62 108 Total 343 402 55 80 2 2 400 484

A significant interaction among age groups, ADA genotype distribution and sex (G = 15.086; df = 3; P = 0.00177) is shown by a three way contingency table analysis by a log linear model (Table 4). More- over, this table shows an increase of male ADA*2 carriers in the 80–85 age group (males 22.4% vs females 12.4%) and a decrease of these genotypes in the 86–90 and[90 age groups (males 9.4% vs females 16.3%; males 4.8% vs females 15.7%).

HWequilibrium : v2 = 0.649, d.f. = 1, P = 0.420 total HWequilibrium : v2 = 0.017, d.f. = 1, P = 0.896 males HWequilibrium : v2 = 0.889, d.f. = 1, P = 0.346 females

Table 4 Proportion of ADA genotypes in males and females at different age and analysis of their interaction by Three-Way contingency (Log-Linear Model)

(Horva´th et al 2005). Adenosine also con- to the activation of inflammatory cells and the production of pro-inflammatory cytokines et al. 2001). Moreover, cytokines and can increase the expression of lung aden- receptors, determining a positive feedback can increase inflammation (Husza´r et al. Zhong et al. 2006). Mice lacking adenosine exhibit an asthma-like phenotype, charac- by high IgE and adenosine levels (Blackburn Sun et al. 2006). Furthermore the pro-fibrotic of adenosine is suggested by in vivo studies in ADA-deficient and in partially ADA- mice (Chunn et al 2006). These data the hypothesis that high levels of adenosine inflammation and fibrosis. Moreover, adeno- inhibits the activity of NK and of other effector protecting tumours from immune-mediated (Raskovalova et al. 2006). Furthermore, immunological damage caused by more pro- thymic involution which occurs in males of age-group may contribute to the reduced propor- of ADA 2-1 males with respect to females (Pido- et al. 2001). This phenomenon would be enhanced in the ADA 2-1 carriers who are by having a lower adenosine deaminase

Age (years) Genotypes ADA 1*/1* ADA*2 carriers M (%) F (%) M (%) F (%) \71 87.8 77.3 12.2 22.7 80–85 77.6 87.6 22.4 12.4 86–90 90.6 83.7 9.4 16.3 [90 95.2 84.2 4.8 15.7

Three-way contingency table by a Log-linear model

G d.f. P xyz interaction 15.086 3 0.00177 xy independence 19.806 6 0.00299 xz independence 16.874 4 0.00204 yz independence 27.964 6 9.544 9 10-5