lack of longevity among the male Irish population. However, our study found no association between this haplotype and longevity. Two polymorphic nucleotides in the TNF cluster (G308A TNF-a and G252A TNF-b), which are associated with increased TNF-a production, were found to be in tight linkage disequilibrium with the class I and II HLA loci. Nevertheless, no age-related allele or genotype frequencies were observed for either nucleotide. The HLA complex is highly polymorphic and plays a pivotal role in immunosurveillance and the immune response. The immune system is closely linked to human longevity, and genetic determinants may reside in polymorphic loci that regulate the immune response. Dysregulation of immune function accompanying aging contributes to morbidity and mortality in the aged through an increased risk of disease, infection, tumours, and autoimmune disorders. While most studies have focused on class I and II HLA loci, there are many class III genes with the potential to modulate immune function, such as the TNF genes. The TNF locus is located within the central HLA class III region, and the multiple pro- and anti-inflammatory activities of TNF and related cytokines in the TNF cluster make them attractive targets for unraveling the molecular mechanisms underlying many diseases and aging. Polymorphism at two nucleotides (G308A TNF-a and G252A TNF-b) is associated with increased TNF-a production. Our study did not reveal any statistically significant haplotype frequency differences between the aged cohort of individuals compared to younger controls or when analyzed with respect to gender. When haplotype analysis was performed, the two polymorphic markers in the TNF genes were observed to be in tight linkage disequilibrium with the HLA haplotypes. The extended HLA-A1, -B8, -DR3, TNF-aA, TNF-bG haplotype was not observed to increase within the aged group as a whole (11.3%) compared to the controls (12.8%). When the data was analyzed with respect to gender, a slight increase was observed in the aged males (7.1%) compared to the younger control male (6.1%). However, a decrease was observed when the aged females (13.1%) were compared to the control females (17.8%). It should be noted that the numbers recruited into this study are not sufficiently large enough to provide the statistical power needed to exclude a more subtle effect on aging. Using the acquired allele data for HLA-A, -B, -DR, TNF-a and TNF-b, five loci haplotypes were estimated by the expectation-maximization algorithm using the software package. To test for departures from Hardy-Weinberg equilibrium, a Markov-chain random walk algorithm was used also implemented in ARLEQUIN. Fisher's exact probability tests were performed to assess whether the frequency differences observed between the aged and control groups for haplotypes and TNF polymorphisms were statistically significant using the computer package MICROSTAT. The males as a group and females as a group within each experimental age category were also assessed for statistically significant frequency differences using the same approach. Multiple comparisons were taken into account by employing Bonferroni's correction (α=0.05/n, where n is equal to the number of comparisons), and the level of statistical significance adjusted accordingly. One of the most characteristic features of the HLA-A1, -B8, -DR3 haplotype is increased spontaneous release of TNF-a, and this haplotype is also associated with a lack of longevity among male Irish population. However, our study found no association between this haplotype and longevity. Two polymorphic nucleotides in the TNF cluster (G308A TNF-a and G252A TNF-b), which are associated with increased TNF-a production, were found to be in tight linkage disequilibrium with the class I and II HLA loci. Nevertheless, no age-related allele or genotype frequencies were observed for either nucleotide.High in vivo and in vitro production of TNF-a (Lio et al., 1997, 2001) led to the inclusion of two polymorphic nucleotide markers in the TNF cluster, also associated with increased TNF-a production, in the haplotype analysis (Hajeer and Hutchinson, 2001). The individual frequencies of the two genetic variants were assessed for association with the Irish aged population in this study (Table 2). However, no significant frequency differences in the genotype or allele frequencies were observed for either the G308A TNF-a or G252A TNF-b polymorphic markers for either of the two age groups as a whole or when the data was analyzed with respect to gender.

3. Results and discussion

The dysregulation of immune function that accompanies ageing may be directed by polymorphism in the genes of the immune system. The highly polymorphic HLA is of crucial importance for immunosurveillance within the body and is associated with a number of autoimmune disorders and ageing. The individual HLA-A, -B, -DR antigens and two single nucleotide polymorphisms of the TNF cluster were determined, and the frequency of extended HLA haplotypes, incorporating the TNF polymorphisms, was analyzed using the software package ARLEQUIN (Schneider et al., 2000). There was no evidence of significant departure from Hardy-Weinberg equilibrium for the samples when a Markov-chain random walk algorithm was used (Guo and Thompson, 1992). As expected, a high degree of polymorphism was observed between the individuals of this study, with the most common haplotypes presented in Table 1.