2.1. Subjects

One hundred control samples (59% female, 41% male with an age-range of 19(cid:3)45 years old) were chosen at / random from the archival DNA bank of normal healthy Caucasian individuals from the Northern Ireland popu- lation stored at the Northern Ireland Histocompatibility and Immunogenetics Laboratory of Belfast City Hospi- tal. Ninety-three aged samples (70% female, 30% male with an age-range of 80(cid:3)97 years old) from the Belfast / Elderly Longitudinal Free-living Ageing Study were also included in the study (Rea et al., 2000). All DNA samples were from healthy aged Caucasian individuals from the Northern Ireland population displaying no signs of age-related disorders, still living independently and approximating SENIEUR status (Ligthart et al., 1984, 1990). There was no duplication with respect to the samples used for the previous study (Rea and Middleton, 1994).

Stringent wash temperatures and concentrations for each of the probes were empirically determined and are given above.

The aged and control subjects were HLA-A, -B and - DR typed by sequence specific oligonucleotide probes (SSOP) as previously reported (Middleton et al., 2000).

Purified PCR products (Qiagen QIAquick PCR purification kit, Qiagen Ltd, Sussex, UK) of a number of samples displaying homozygote profiles for each of the TNF polymorphisms analysed were sequenced on a 373A automated DNA sequencer (Curran et al., 1996), using the amplification primers at a concentration of 3.2 picomoles, to confirm the specificity of the amplifica- tion, and the exact location and sequence of the polymorphic nucleotide encountered.

The oligonucleotide primers employed for the ampli- fication of the 107 bp region of the TNF-a and the 772 bp region of the TNF-b genes were as reported (adapted from Barber et al., 1999):

The results of this study did not reveal any statistically significant haplotype frequency differences between the aged cohort of individuals in comparison to the younger controls, or when analysed with respect to gender. When the haplotype analysis was performed the two poly- morphic markers in the TNF genes were observed to be in tight linkage disequilibrium with the HLA haplo- types. 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%) in comparison to the controls (12.8%). When the data was analysed with respect to gender, a slight increase was observed in the aged males (7.1%) compared to the younger control male (6.1%). In contrast, 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 ageing.

Using the acquired allele data, HLA-A, -B, -DR, TNF-a and TNF-b, five loci haplotypes were estimated by the expectation-maximisation algorithm using the software package (Schneider et al., 2000). To ARLEQUIN test for departures from Hardy(cid:3)Weinberg a Markov- / chain random walk algorithm was used (Guo and Thompson, 1992) also implemented in . ARLEQUIN

2.7. Statistical analysis

Fisher’s exact probability tests were performed to assess whether the frequency differences observed be- tween the aged and control groups for haplotypes and the TNF polymorphisms were statistically significant using the computer package ‘ ’. The males as MICROSTAT a group and females as a group within each experi- mental age category were also assessed for statistically significant frequency differences using the same ap- proach. Multiple comparisons were taken into account by employing Bonferroni’s correction, a(cid:5)1(cid:1)0.951/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 the increased spontaneous release of TNF-a, and this haplotype is also associated with a high in vivo and in vitro production of TNF-a (Lio et al., 1997, 2001). On this basis it was decided to include two polymorphic nucleotide markers in the TNF cluster, also associated with increased TNF-a produc- tion, in the haplotype analysis (Hajeer and Hutchinson, 2001). The individual frequencies of the two genetic variants were also 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 G(cid:1)308A TNF- / a or G(cid:2)252A TNF-b polymorphic markers for either / the two age groups as a whole or when the data was analysed with respect to gender.

3. Results and discussion

The dysregulation of the immune function that accompanies ageing may be directed by polymorphism in the genes of the immune system. The highly poly- morphic HLA is of crucial importance for immunosur- veillance 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, were analysed using the software package ARLEQUIN (Schneider et al., 2000). There was no evidence of significant departure from Hardy(cid:3)Weinberg equili- / brium 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.