or quantitative HSP 70 expression could cause a differential sensitivity to stresses and affect longevity.

gene in an Irish aged population in comparison to a cohort of younger controls.

A duplicated locus of two intronless genes, HSP 70-1 and HSP 70-2, encoding two identical protein products and a third intronless gene, HSP 70-Hom, are located within the human leukocyte antigen (HLA) class III region (Milner and Campbell, 1990; Feige and Polla, 1994), see Fig. 1. A relatively low degree of polymorphism is reported in all three HSP 70 genes mapping to the HLA region (Milner and Campbell, 1992). The polymorphisms identified to date have been located either in the coding region (HSP 70-2 and HSP 70-Hom) or in the regulatory regions of the genes (HSP 70-1 and HSP 70-2). HSP 70-1 and HSP 70-2 are demonstrated to contain a number of polymorphic vari- ations, some of which correspond to silent mutations (Ishihara et al., 1995; Milner and Campbell, 1990). A functional polymorphism, T2437C transversion resulting in a methionine to threonine amino acid substitution, is known to occur in the HSP 70-Hom gene, which is a HSP that has been implicated to play a role in protection against oxidative stress (Favatier et al., 1997).

2. Materials and methods

2.1. Subjects

One hundred control samples (59% female, 41% male with an age-range of 19–45 years) were chosen at random from the archival DNA bank of normal healthy Caucasian individuals from the Northern Ireland population. These samples were stored within the Northern Ireland Histocom- patibility and Immunogenetics Laboratory (NIHIL) of Belfast City Hospital. The study involved 129 aged consecutive samples (70% female, 30% male with an age- range of 80–97 years) from the Belfast Elderly Longitudi- nal Free-living Ageing Study (Rea et al., 2000). All samples were from healthy Caucasian individuals from the Northern Ireland population displaying no signs of age-related disorders, still living independently and approximate SENIEUR status (Ligthart et al., 1984).

The aim of this study was to investigate the frequency of the functional polymorphism, T2437C, in the HSP70-Hom

O.A. Ross et al. / Experimental Gerontology 38 (2003) 561–565

2.2. PCR Amplification

2.4. DNA sequence analysis

Purified PCR products (Qiagen QIAquick PCR purifi- cation kit) of a number of samples displaying homozygous profiles for each of the 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 per sequencing reaction. This confirmed the specificity of the amplification, and the exact location, and sequence of the polymorphic nucleotide encountered.

The oligonucleotide primers employed for the amplifica- tion of the 264 bp region of the HSP70-Hom gene were:

PCR amplification was performed in 50 ml reaction volumes containing 67 mM Tris–HCl pH 8.8; 16 mM (NH ) SO ; 1.5 mM MgCl ; 0.01%(w/v) Tween; 100 mM 4 2 4 2 of each dNTP; 0.1 mM of each primer; 1 unit Taq polymerase (Bioline UK Ltd, London, UK); 0.1–0.5 mg DNA. The following cycling conditions were employed: samples were held at 96 8C for 5 min, followed by 30 cycles of 96 8C for 1 min, 60 8C for 30 s, 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 amplifi- cation was confirmed using gel electrophoresis on a 1% agarose gel stained with ethidium bromide (0.5 mg/ml).

2.5. Statistical analysis

Statistical analysis was performed using 2 £ 2 contin- gency tables and the chi-squared ðx2Þ test with Yate’s correction to assess whether the frequency differences observed between the aged and control group were statistically significant using the computer package ‘Micro- stat’.

3. Results

2.3. HSP 70-Hom polymorphism by SSOP

The frequency of the polymorphic nucleotide at position 2437, a T to C transversion, resulting in an amino acid substitution of methionine to threonine in the HSP 70-Hom protein, was investigated in the Irish aged population.

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

The allele and genotype frequencies obtained during the course of the study are displayed in Table 1. There was an observed increase in the frequency of the variant HSP 70- Hom T allele within the aged cohort of samples (83%) in comparison to the control group (77%). This increased allele frequency was broadly maintained when the data was divided into males (86% vs 76%) and into females (82% vs 78%).

Hom 70-1 CAC AGC CA C GGA CAA GA Hom 70-2 CAC AGC CA T GGA CAA GA

The frequency of the heterozygote TC genotype was significantly ðx2 ¼ 6: 366; p ¼ 0: 01Þ decreased within the aged group (27%) when compared to the younger control subjects (44%). When the frequency of the heterozygote TC genotype was analysed with respect to gender there was a

Stringent wash temperatures and concentrations for each of the probes were empirically determined at 54 8C for Hom 70-1 and 52 8C for Hom 70-2 and both probes were used at a concentration of 20 picomoles.

The observed allele and genotype frequencies for the functional T2437C polymorphic variant of the HSP 70-Hom gene

CONTROLS n ¼ 100 AGED n ¼ 129 Male n ¼ 41 (%) Female n ¼ 59 (%) Total (%) Male n ¼ 39 (%) Female n ¼ 90 (%) Total (%) TT 22(54) 33(56) 55*(55) 28(72) 62(69) 90*(70) TC 18(44) 26(44) 44*(44) 11(28) 24(27) 35*(27) CC 1(2) – 1(1) – 4(4) 4(3) T 62(76) 92(78) 154(77) 67(86) 148(82) 215(83) C 20(24) 26(22) 46(23) 11(14) 32(18) 43(17)