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HLA haplotypes and TNF polymorphism do not associate with longevity in the Irish

Owen A. Ross *, Martin D. Curran , I. Maeve Rea , Paul Hyland , Orla Duggan , b b e b Christopher R. Barnett , Kathryn Annett , Chris Patterson , Yvonne A. Barnett , a,b,c

b School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK c School of Biology and Biochemistry, Queens University of Belfast, Belfast, Northern Ireland, UK d Department of Geriatric Medicine, Queens University of Belfast, Belfast, Northern Ireland, UK e Department of Epidemiology and Public Health, Queens University of Belfast, Belfast, Northern Ireland,

Received 26 June 2002; received in revised form 10 July 2002; accepted 20 December 2002

Abstract

Polymorphism of the human leukocyte antigen has been implicated in a number of autoimmune disorders including ageing. In the course of the present study, no association of the human leukocyte antigen (HLA)-A1, B8, DR3 haplotype with a male Irish aged population, as previously reported, was observed. Two polymorphic nucleotides in the TNF cluster (G(cid:1)308A TNF-a and G(cid:2)252A / / TNF-b), associated with increased TNF-a production, were shown to be in tight linkage disequilibrium with the class I and II HLA loci, generating HLA haplotypes with extended linkage disequilibrium. However, no age-related allele or genotype frequencies were observed for either polymorphic nucleotide. # 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: HLA; TNF; Polymorphism; Immunosenescence

1. Introduction

Pawelec, 1998; Pawelec, 1999; Franceschi et al., 2000; Malaguarnera et al., 2001). The highly polymorphic human leukocyte antigen (HLA) complex is located on chromosome 6p21 and is divided into three regions containing class I, class III and class II genes, respec- tively. In spite of the pivotal role that the polymorphic HLA antigens play in immunosurveillance and the immune response, and the multitude of studies per- formed, conclusive evidence of association with success- ful longevity has remained elusive (Reviewed in Caruso

Human longevity would appear to be inextricably linked with optimal functioning of the immune system, suggesting that specific genetic determinants may reside in polymorphic loci that regulate the immune response. Dysregulation of immune function accompanying age- ing (immunosenescence) is postulated to contribute to morbidity and mortality in the aged via an increased risk of disease, infection, tumours, and autoimmune disor- ders (Franceschi et al., 1995; Pawelec and Solana, 1997; Franceschi et al., 1998; Makinodan, 1998; Solana and

The majority of studies so far have focused on the action of class I and II HLA loci, but 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

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0047-6374/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0047-6374(03)00037-X

and related cytokines in the TNF cluster, make them attractive targets along with other HLA genes for unravelling the molecular mechanisms underlying the development of many diseases and ageing. TNF genes display strong linkage disequilibrium with HLA class I and II genes and defects in TNF production may contribute to HLA-associated disorders. Polymorphism at two nucleotides (G(cid:1)308A TNF-a and G(cid:2)252A / / TNF-b) is associated with increased TNF-a production (Makhatadze, 1998; Hajeer and Hutchinson, 2001; Lio et al., 2001).

PCR amplification was performed in 50 ml reaction volumes containing 67 mM Tris(cid:3)HCl pH 8.8; 16 mM / (NH ) SO ; 1.5 mM MgCl ; 0.01% (w/v) Tween; 100 4 2 4 2 mM of each dNTP; 0.1 mM of each primer; 1 unit Taq polymerase (Bioline UK Ltd, London, UK); 0.1(cid:3)0.5 mg / DNA. The following cycling conditions were employed; samples were held at 96 8C for 5 min, followed by 35 cycles of 96 8C for 1 min, 60 8C for 1 min, 72 8C for 1 min. After the final cycle, the samples were held at 72 8C for 5 min, and then at 15 8C until removed from the PCR machine (Perkin-Elmer 9600 Thermal Cycler). Successful amplification was confirmed using gel elec- trophoresis on a 1% agarose gel stained with ethidium bromide (0.5 mg/ml).

An increase in the frequency of the HLA haplotype A1B8Cw7DR3 was previously reported by Rea and Middleton (1994) in elderly males in the northern Irish population and this haplotype has also been associated with an enhanced TNF-a production (Lio et al., 1997, 2001). The aim of the present study was to establish the presence of this longevity marker in males from the north of Ireland and to evaluate the contribution polymorphism within the TNF cluster has on longevity.

2.4. TNF polymorphism genotyping by SSOP

Amplified DNA (2 ml) was applied to a charged nylon membrane as reported (Middleton et al., 1995). Hybri- disations, stringent washes and chemiluminescence de- tection of the hybridised digoxigenin-labelled probes were carried out as recently reported (Williams et al., 1999). The DNA sequence of the probes with the polymorphic nucleotide underlined were:

2.1. Subjects