controls, and (2) alleles in genes associated with Hsp70 explain these differences. In this cross-sectional study, we assessed serum Hsp70 levels from participants enrolled in the New England Centenarian Study (NECS) or the Longevity Genes Project (LGP). The study included 87 LGP centenarians, 93 NECS centenarian offspring, and 126 controls (43 NECS, 83 LGP). We also examined genotypic and allelic frequencies of polymorphisms in HSP70-A1A and HSP70-A1B in 347 centenarians (266 from NECS, 81 from LGP), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls; LGP: 79 spousal controls). The adjusted mean serum Hsp70 levels for the NECS centenarian offspring, LGP centenarians, LGP spousal controls, and NECS controls were 1.05, 1.13, 3.07, and 6.93 ng/mL, respectively. These results suggest that a low serum Hsp70 level is associated with longevity; however, no genetic associations were found with two SNPs within two Hsp70 genes. 

Heat shock proteins are highly conserved proteins that protect stress-exposed cells when produced intracellularly. Conversely, extracellular heat shock protein 70 (Hsp70) has been shown to have both protective and deleterious effects. In this study, we assessed the potential role of heat shock protein 70 in human longevity. Various studies have examined the relationship of extracellular Hsp70 with aging. Rea et al. demonstrated a progressive decline in serum Hsp70 levels in older age groups in a cross-sectional study of individuals aged between 20 and 96 years. Similarly, Jin et al. demonstrated a decline in serum Hsp70 at older ages in their study of healthy male donors aged between 15 and 50 years. These findings suggest that serum Hsp70 levels decline at older ages; however, they do not indicate whether levels decline over time within an individual. They also do not indicate whether there is a decline in Hsp70 function over time, although in vitro studies suggest that cell lines from centenarians, when exposed to heat stress, have similar Hsp70 synthesis, have a similar transcriptional response to the Hsp70 gene, and are less prone to heat-induced apoptosis when compared to cell lines from younger individuals.

Blood samples were collected from 87 LGP centenarian subjects, 93 NECS centenarian offspring, and 126 spousal controls (NECS controls: 43, LGP: 83). Blood samples were not obtained in the acute phase of an illness. Once collected, the blood samples were divided into aliquots and stored at -80°C. The serum was then analyzed using the classical sandwich enzyme-linked immunosorbent assay (ELISA) method previously described by Rea and co-workers with minor modifications. The interassay variability of the Hsp70 immunoassays was <10%.Controls and (2) alleles in genes associated with Hsp70 explain these differences. Few studies have examined the role of genetics in the relationship between Hsp70 and survival to very old age. Altomare et al. (2003) found that a polymorphism within the hsp70 gene promoter was associated with longevity only in females. Subsequently, Marini et al. (2004) reported that in centenarians, the A/A genotype of the (A/C)-genetic 110 polymorphism in the promoter region was associated with lower expression of Hsp70 protein after heat stress, compared to the C/C genotype. In addition to analyzing serum Hsp70, we examined two single nucleotide polymorphisms (SNPs) of hsp70 genes for their potential role in exceptional longevity.

2.3. SNP genotyping
Whole blood samples were obtained from 347 centenarians (NECS: 266, LGP: 81), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls, LGP: 79 spousal controls). Of note, although sera were not available from all NECS subjects for Hsp70 assays, whole blood was available for genetic studies. DNA was isolated from each subject's peripheral blood leukocytes using the Qiagen DNA purification system (Qiagen, Valencia, CA). Real-time PCR-based SNP genotyping assays were purchased from Applied Biosystems Assays-on-DemandTM and performed in accordance with the manufacturer's instructions. Reaction products were analyzed on an ABI Prism 7900 Sequence detection system. HSP70-A1A and HSP70-A1B are encoding gene members of the hsp70 family located within the cluster of human leukocyte antigen class III genes on chromosome 6p21 in the neighborhood of the tumor necrosis factor (TNF) locus. These genes share the characteristic of having only one linkage disequilibrium (LD) block. Thus, we genotyped one tagSNP per gene: rs1043618 (hcv 11917510) in HSP70-A1A and rs6457452 (hcv3052604) in HSP70-A1B.