elderly individuals. However, this approach has limitations. In this study, we investigated the association of three single nucleotide polymorphisms (SNPs) present in the HSP70 genes with human longevity using DNA samples from different population cohorts in Denmark. We found a significant association of one haplotype with male longevity and a significant difference in the survival of carriers of different genotypes in females. Additionally, we observed an age-dependent decline in the ability of peripheral blood mononuclear cells to respond to heat stress in terms of Hsp70 induction.

Human longevity is a complex trait influenced by both genetic and environmental factors. Twin studies suggest that around 25% of the observed differences in life expectancy are due to genetic variations. To identify longevity-determining genes, population-based association studies have been commonly used, but they have limitations. Heat-shock proteins (Hsps) are part of cellular safety and rescue mechanisms that respond to stress by synthesizing Hsps at the expense of other proteins, protecting cells from subsequent damage and aiding them to counteract stress. The capacity to respond rapidly to stress at the gene level determines the adaptive and survival capacity and longevity of an organism. Therefore, an association between single polymorphisms (SNPs) in Hsp genes (HSP) and longevity or parameters of aging can indicate the relationship between humans and stress response.

Of all Hsps, Hsp70 is the most prominent and best characterized family member. In humans, 11 different isoforms of HSP70 are encoded by different genes located at dispersed loci. Three isoforms are localized within the major histocompatibility complex (MHC) class III region on chromosome 6p21: intron-less HSP70A1A (HSP70-1), HSP70A1B (HSP70-2), and HSP70A1L (HSP70-Hom) genes. We studied the association of three SNPs, one in each MHC-linked HSP70 gene, with human longevity, survival, and parameters of aging. The availability of biological samples from various population cohorts in Denmark allowed us to perform novel population-based genetic association studies by mitigating many of the limiting factors.

We used DNA samples from different population cohorts in Denmark, including the Longitudinal Study of Aging Danish Twins (LSADT), which is the oldest twin registry in the world. We also collected blood samples from young and middle-aged individuals and studied age-related HSR. The gene and genotype frequency of the three SNPs was found to be in Hardy-Weinberg equilibrium. Self-rated health and relative self-rated health were associated with heterozygosity for the 110A>C polymorphism in the promoter region of HSP70-1 in LSADT.

Molecular haplotype analyses on Danish family samples revealed high LD among the three markers. On analyzing the frequency distribution of haplotypes across different age groups, we found a significant age-dependent increase in the frequency of one haplotype in males. Most genetic association studies on human longevity are performed in a case-control manner by comparing gene/genotype frequencies between young and elderly individuals. However, this approach has limitations. Our study provides new insights into the genetic basis of human longevity by using novel methods of gene association with human longevity.LLIs. This method, though widely used, has a shortcoming in that comparing the young with LLIs may give false-positive results, where the differences in the frequencies of the gene ANNALS NEW YORK ACADEMY OF SCIENCES in the study might just be owing to secular changes in population demographic and genetic parameters of the two groups and not necessarily age. To mitigate this factor, cohort studies, which focus on tracking the survival of a group of individuals born in the same time period, are appropriate alternatives. Our work on the cohort persons of Danish form in the year 1905 has provided us with the opportunity to perform such studies. We have seen a significant difference in the survival of carriers of different genotypes. Female carriers of CC, genotype of HSP70A1A, survive better than non-carriers (AA and AC) (P=0.005). It was also observed that female carriers of GG genotype in HSP70A1B survive better than non-carriers (P=0.04). (Singh et al., in preparation). We have also seen that peripheral blood mononuclear cells of young and middle-aged individuals respond differently to heat stress, confirming that in humans there is significant age-dependent attenuation in the ability to respond to stress (Singh et al., in preparation). Also, in young subjects, a positive association was found between the HSPA1L (T2Y37C) polymorphism and HSR. CC carriers had significantly lower induction than TT carriers in both monocytes (P=0.015) and lymphocytes (P=0.004). This polymorphism, which is present in the coding region of the HSPA1L gene, can affect the chaperoning function of Hsp70. Our results from the association of SNPs in three HSP70 genes with man longevity, survival, and parameters of aging have reiterated our belief that the genes involved in cellular maintenance and repair mechanisms are indeed important candidates for deciphering the genetics of human longevity. Additionally, the availability of samples from different population cohorts in Denmark has provided us with the opportunity to perform novel studies mitigating many confounding factors.

ACKNOWLEDGMENTS

We are thankful to Christian Knudsen for his technical support and to Mari Sild for her help in genotyping. We also thank the personnel of the Blood Bank, Skejby Sygehus. The studies received financial support from the Danish Center for Molecular Gerontology (DCMG), Danish Research Councils, and Nordisk Fund.