The allele (A) in the promoter region of the HSP70-1 gene is − 110 unfavorable to longevity in women

K. , V. , D. M. S. F. S. 1 2 2 1 3 1,4 G. Rose , E. Feraco , V. Mari , G. Passarino , C. Franceschi & G. De Benedictis 1Department of Cell Biology, University of Calabria, 87030 Rende, Italy; 2Italian National Research Center on Aging (INRCA) 87100 Cosenza, Italy; 3Department of Molecular Pathology, University of Bologna, 40126 Bologna, Italy; 4Author for correspondence (e-mail: g.debenedictis@unical.it; fax: +39-0984-492911)

Received 17 December 2002; accepted in revised form 10 March 2003

Key words: aging, chaperones, genetic variation, HSP70, inflammation, longevity

Abstract

Heat shock proteins (HSPs) are crucial for maintenance of cell homeostasis and survival both during and after various stresses. The capability to cope with stress is believed to affect the chance of health and survival at organismal level. We have investigated whether the gene pool relevant to the (A/C)−110 polymorphism in the promoter region of the HSP70-1 gene changes as the population ages and survival selection occurs. A total of 591 southern Italian subjects were enrolled in the study (263 males and 328 females; age range 18–109 years), free of clinically manifest diseases and with normal haemato-chemical parameters. A significant age-related decrease of the frequency of allele (A)−110 was observed in females. The probability ratio of 0.403 (95% confidence interval [0.163, 0.910]) computed by considering female centenarians as cases and young women (18–49 years old) as controls showed that the (A)−110 allele is unfavorable to longevity in females.

Abbreviations: HSP – heat shock protein; HSE – heat shock element; HSF – heat shock factor; O.R. – odds HLA-DR – major histocompatibility complex, class II, DR alpha; dNTP – deossinucleotide tri-phosphate; PCR polymerase chain reaction

Introduction

in the search for gene factors that affect the rate and quality of human aging. In a larger survey to check the above hypothesis, we are analyzing gene markers of the HSP gene super family in population samples of healthy subjects in the age range from 18 to 100+ years. This article presents data on HSP70-1, a gene encoding for the Hsp70 protein, one of the major inducible heat shock proteins (Milner and Campbell 1990; Leppa et al. 2001). HSP70-1 is a member of the HSP70 intronless gene family which also includes HSP70-2 and HSP70-HOM genes (the latter is not heat-inducible) and maps at 6p21.3, within the class III region of the Major Histocompatibility Complex (Gene Bank reference contig NT_007592). In euka- ryotes, HSP70 gene expression is regulated essentially through the activity of heat shock transcription factors

There is increasing evidence that modulators of the rate of aging are conserved during evolution (Partridge and Gems 2002). Following hints given by studies of model organisms, the network theory of aging has proposed that human aging is also modulated by a variety of anti-stress responses (Franceschi et al. 2000a). Heat shock proteins (Hsp), due to their role in stress response as molecular chaperones (Feder and Hofmann 1999; Kregel 2002) – a role that is conserved during evolution – are likely to be closely involved in these mechanisms (Macario and Conway de Macario 2002), and HSP genes are therefore good candidates

These authors contributed equally to this work.

conditions: 5 min at 95 C, 35 cycles at 96 C for 45 s, ◦ ◦ ◦ 50 C for 30 s, 72 C for 30 s, 5 min at 72 C. The reaction was performed in a GeneAmp PCR System 9600 (PE applied Biosystems).

(HSFs) which interact with heat shock elements (HSEs) present in HSP promoters, resulting in a modulation of the transcription rates (Morimoto 1998). Interestingly, an age-related decrease in the induction of hsp70 protein levels or hsp70 synthesis has been reported in lymphocytes from rats and rhesus monkeys (Pahlavani et al. 1995) as well as in cells derived from young and adult human sub- jects (Gutsmann-Conrad et al. 1998). An A/C poly- morphism lies in the HSE region of the HSP70-1 gene at –110nt from the transcription starting site (Abravayata et al. 1991). This polymorphism is the marker chosen for the present study.

The (A/C)−110 variability was analyzed by auto- matic DNA sequencing. PCR-amplified fragments were purified by using a QIAquick PCR purification kit (Qiagen). DNA sequences were determined by a fluorescence-based automated direct sequencing tech- nique using a PE310 DNA sequencer with BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). The sequencing reaction mixture contained 4 µl of Terminator Ready Reaction mix, 150 ng of DNA template, 6.4 pmol of primer in a total volume of 20 µl. Cycle sequencing was carried out for 25 cycles at 96◦ C for 10 s, 50 ◦ C for 5 s, ◦ 60 C for 4 min in a GeneAmp PCR system 9600. The extension products were purified using Centri-SepTM template purification columns.

Materials and methods

Samples

A total of 591 unrelated subjects were analysed (263 males and 328 females, median ages 57.0 and 56.5 years respectively, age-range 18–109 years). The sample included 120 subjects older than 100 years (36 males and 84 females). All the subjects, as well as their parents and grandparents, were born in Calabria, a southern Italian region which is charac- terised by the virtual absence of immigration and a high level of endogamy (Danubio et al. 1999). Routine blood analyses were performed on all the subjects and only those having haemato-chemical parameters in the normal sex- and age-specific range were enrolled in the study. All the subjects were free of clinically overt pathologies; in particular, centenarians were in a fairly healthy condition (categories A and B of the classifi- cation reported by Franceschi et al. 2000b). Details of the recruitment criteria are reported in Garasto et al. (2003). All the subjects had given informed consent for the present study.