potential longevity genes (Frederiksen et al., 2002a). In this study, we aimed to investigate the possible association between the 110A > C polymorphism in the promoter region of HSP70-1 and human longevity in a cohort of aged Danish twins. The cohort included individuals aged between 70 and 91 years (mean age 75.6 years) who were categorized based on the presence or absence of various diseases and age-related parameters for which a genetic component has already been established.

Four hundred DNA samples from the cohort underwent genotyping using real-time PCR. Aging phenotypes, including diseases, physical and cognitive functioning, were compared with genotype. Among all aging phenotypes studied, self-rated health and relative self-rated health, which represent an individual's overall sense of physical well-being and predict survival at older ages more accurately than objectively measured health status, were associated with the polymorphism.

An association was found between low self-rated health and heterozygosity for the 110A > C polymorphism in the promoter region of HSP70-1. The Hsp70 family of stress proteins plays a crucial role in protecting cells from damage induced by stress and in aiding them to counteract its effects. Hsp70 acts as a chaperone or cooperates with proteolytic pathways in cells. In humans, there are eleven different isoforms of Hsp70 encoded by different genes located at dispersed loci. Three of these isoforms are mapped within the major histocompatibility complex (MHC) class III region on the short arm of chromosome 6.

Polymorphisms in these three genes have been extensively studied for their association with various autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and insulin-dependent diabetes mellitus. However, no association between polymorphisms of the MHC-linked HSP and diseases has been found that would not be explained by linkage disequilibrium with other HLA markers. Nonetheless, protective effects of Hsp70 against oxidative stress suggest that it might be valuable to study HSP polymorphism in conditions associated with oxidant/antioxidant imbalance, such as aging.

This investigation was conducted on DNA samples collected from a cohort of 200 DZ twin pairs aged 70-91 years, from the LSADT study. These samples are part of the Danish Twin Registry, which is the first nationwide twin registry in the world. The LSADT is an ongoing longitudinal study of Danish twins who are more than 70 years old. These individuals have been offered an examination at their home every two years, which includes both a traditional health-related interview and a number of objective and cognitive tests, and a collection of blood samples. The DZ twins from LSADT-1999 wave are the basis of this investigation.

Various parameters have been used as measures of aging, including scoring of physical function such as hand grip strength, activity of daily living (ADL) strength, and ADL endurance; scoring of the mental state of the individual viz. cognitive composite score and mini mental state examination (MMSE). Furthermore, self-rated health (scored both in absolute terms measured on a scale of 15 with the possible responses very good, good, fair, poor, very poor; and in relation to others of the same age) has been shown to have a substantial genetic component.

In a recent cross-sectional study on samples from Calabria, a south Italian region, Altomare et al. (2003) reported that the presence of an allele carrying adenine (allele A), in the 110A > C polymorphism present in the 5 flanking region, 3 base pairs upstream of the heat shock element (HSE) of the promoter region of HSP70-1 gene, was unfavourable for longevity in women. In another association study between polymorphism (2437T > C) in the coding region of HSP70-Hom with human aging, done on the Irish population (Ross et al., 2003), the frequency of T allele has been shown to increase in the old population (80-90 years) as compared to the young controls (19-45 years). However, as will be discussed later, there are confounding factors which occur while performing such cross-sectional studies, where allele frequencies in a group of young controls are compared against the allele frequencies in the oldest-old. Taking this into account, we report the results of our study performed on DNA samples collected from Danish dizygotic (DZ) twin cohort of 400 individuals aged 70-91 years (mean age 75.6), from the Longitudinal Study of Aging Danish Twins (LSADT).Genetic variants of importance to mid- and late-life physical functioning have a heritability of 52% (Frederiksen et al. 2002a). Once heritability is defined for all these age-related traits, the next step is to associate these traits with specific genes. Based on these scores, we have analyzed the association of parameters correlated with human longevity with the 110A > C polymorphism in the HSP70-1 gene. 

Statistical Analysis: We grouped the participants according to their HSP70-1 genotype and compared these groups with respect to various aging phenotypes. Continuous phenotypes (physical ability scores, grip strength, depression, and cognitive scores) were compared across the genotypes using the one-way ANOVA test. Discrete variables (diseases - see Table 3) were compared across the genotypes using Pearson's v2 test. Bonferroni correction on repeated testing was applied.

Genotyping: Genotyping was done using real-time PCR on the LightCycler system (Roche Applied Sciences), which helps to monitor the amplification of PCR product simultaneously using fluorescent-labeled sequence-specific oligonucleotide probes. Probes were designed such that the oligonucleotides were complementary to the allele carrying the nucleotide.