I.Maeve Rea

Mitochondrial DNA polymorphism: its role in longevity of the Irish population.

The mtDNA genome has been implicated as playing a pivotal role in determining the longevity and success of the human lifespan. A PCR-RFLP methodology was used to identify polymorphic restriction enzyme sites within a 2643 bp region of the mtDNA genome and a table of genetic haplotypes for a healthy aged and a younger control cohort of patients was constructed. Forty-six different mtDNA haplotypes and 11 groups of related haplotypes were identified across the two age groups but statistical analysis failed to show any significant associations. The European J haplogroup, previously reported to be associated with longevity, was not found at an increased frequency within the Irish aged population (P=0.36). However, the haplotypes comprising the J haplogroup could be differentiated into two distinct branches by the presence or absence of the two polymorphic restriction sites, 16,389g and 16,000g. The branch of haplotypes defined by 16,389g displayed a significant increased frequency in the aged samples (8%) compared to the controls (1%), P=0.015. Inversely, the branch of haplotypes defined by 16,000g displayed a significant decreased frequency in the aged samples (4%) compared to the controls (13%), P=0.011. The polymorphism (mt5178A) associated with longevity in the Japanese was not found in the Irish population, while the polymorphism (mt9055A) associated with successful ageing in the French centenarians was found at an increased frequency in the Irish aged population (9%) compared to the younger control group (5%), but failed to reach a level of statistical significance, P=0.164.

mt4216C variant in linkage with the mtDNA TJ cluster may confer a susceptibility to mitochondrial dysfunction resulting in an increased risk of Parkinson's disease in the Irish

Polymorphism of the mtDNA genome has been implicated as playing a role in the development and pathogenesis of Parkinsons disease (PD). A PCR-RFLP methodology was employed to generate genetic haplotypes for a cohort of 90 PD sufferers. No association was observed between the various mtDNA haplotypes observed and PD in comparison to healthy aged controls. The longevity-associated European J haplogroup and T haplogroup were identified and were both found to be in tight linkage with the mt4216C polymorphism. The mt4216C variant was observed at a significantly increased frequency in the PD cases (28%) in comparison to the healthy aged controls (15%; p=0.014). However, when the frequency of the mt4216C variant was examined in a cohort of 200 young controls (18-45 years) a similar frequency to the PD cases (25%) was observed. The frequencies obtained for the two branches of the J haplogroup (J1 and J2) and the T haplogroup in the cohort of PD subjects also reflected those observed for the young controls used in the previous longevity study. These findings lead one to postulate that the mt4216C variant, in linkage with the mtDNA TJ cluster, may influence mitochondrial dysfunction, resulting in an increased risk of PD.

Study of age-association with cytokine gene polymorphisms in an aged Irish population

The release of cytokines is of crucial importance in the regulation of the type and magnitude of the immune response in the elderly. A number of studies have shown different levels of cytokine production in the elderly. In the present study, a range of polymorphisms were chosen within the genes of cytokines (IL-2, IL-6, IL-8, IL-10, IL-12 and IFN-gamma) that have been observed at different levels within the elderly and analysed for age-association. No association was observed for the polymorphic cytokine markers and the healthy aged Irish population (or with respect to gender) examined in this study. These findings would suggest that polymorphism of cytokine genes may not play as crucial a role in healthy ageing as previously believed.

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

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-308A TNF-alpha and G+252A TNF-beta), associated with increased TNF-alpha 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.

Increased frequency of the 2437T allele of the heat shock protein 70-Hom gene in an aged Irish population

The frequency of the functional polymorphism, T2437C transversion (Met-->Thr), in the HSP 70-Hom gene was investigated within a healthy aged Irish population using oligonucleotide probes. The 2437T polymorphic nucleotide was observed to increase in the elderly, although not attaining statistical significance. The TT genotype was observed to be significantly increased within the Irish aged population (p=0.03), while conversely the TC genotype was significantly decreased in the aged subjects (p=0.01). These findings would support the theory that the change from a Met (non-polar and hydrophobic) residue to a Thr (polar and neutral) residue may disrupt the peptide-binding specificity of HSP 70-Hom and have an effect on its functional efficiency. One postulates that the highly significant p-value obtained for the TC genotype may infer that the presence of both the T and the C allele (heterozygosity) resulting in the generation of two different HSP 70-Hom protein species may negatively influence longevity.

Investigation of KIR diversity in immunosenecence and longevity within the Irish population

Natural killer (NK) cells play a pivotal role in the innate immune response. During the ageing process, variations occur in NK cell number and function. The cytolytic activity of NK cells is controlled by an array of activating and inhibitory cell surface receptors, including the killer cell immunoglobulin-like receptors (KIRs). In the present study, genetic diversity of the KIR loci was analysed with respect to successful ageing in the Irish population. A PCR-SSOP KIR gene identification system was employed to determine the frequency of the named KIR genes/pseudogenes and KIR genotypes within a healthy aged cohort and young control group. Although, two KIR genes (2DS3, 2DL5) displayed an initial increased frequency in the aged group, the significance of this association was lost when repeated in a second cohort. In view of the lack of studies to date, investigating the role of the KIR gene system in healthy ageing, further analysis of KIR diversity is required to fully elucidate its role in respect to age-related disease and longevity.

A novel polymorphic triplet repeat in intron five of the α-synuclein gene: no evidence of expansion or allelic association with idiopathic Parkinson's disease in the Irish

The recent discovery of two mutations associated with autosomal dominant Parkinsons disease (PD) has led to the hypothesis that the &agr;-synuclein gene plays a role in the pathogenesis of PD. Here we report a novel triplet CAA repeat within the unusually large intron 5 sequence of the &agr;-synuclein gene. Microsatellite analysis revealed a high degree of polymorphism within the Irish population with seven alleles detected, ranging from eight to 17 CAA repeats. Analysis of the allele/genotype frequency differences observed between an Irish idiopathic PD cohort (n = 98) and a healthy aged control group (n = 92) revealed no strong association with either group. All PD subjects displaying homozygous profiles were examined for expansion of the trinucleotide repeat, but no expansion was observed. These results would suggest that polymorphism of the &agr;-synuclein gene may not play as significant a role in the pathogenesis of idiopathic PD as previously hypothesised.

Hypothetical soluble KIR2DS4 natural killer cell receptor molecule does not associate with successful ageing in the Irish

The identification of immunogenetic longevity markers is a major area of molecular gerontological research. A number of genetic loci have been examined, e.g. the HLA and cytokine networks. This study investigated a genetic marker within the highly polymorphic KIR gene system with successful ageing in the Irish population. A 22 bp deletion was identified in the KIR2DS4 gene that predicts a truncated soluble KIR molecule with one intact Ig-like domain. The frequency of this variant was determined using a specific-primer PCR methodology. There was no observed association between this common polymorphic variation within this activatory KIR gene and the aged Irish population. This is the first study of KIR polymorphism in ageing and although no association was identified, the importance of the KIR network in the immune response and its polymorphic nature warrants more detailed analysis to ascertain its role in immunosenescence.