The molecular haplotype analyses on Danish family samples have revealed high LD among the three markers (TABLE 3). Pair-wise LD calculated among (cid:2)= the three markers also showed a high LD (D 1) between the SNP at HSPA1A with the other two SNPs at HSP70A1B and HSP70A1L (FIG. 1). This infor- mation is very important when it comes to discovering genes that influence complex traits such as longevity.24 On analyzing the frequency distribution of

TABLE 3. Comparison of haplotype frequencies in the family samples (only parents) gen- erated by PHASE and from allele frequencies

Observed frequency Expected frequency Haplotypesa (generated by PHASE) (generated from allele frequency) A-A-T 0.41 0.3 A-A-C 0.18 0.09 G-A-T 0.03 0.2 G-A-C 0.02 0.06 A-C-T 0 0.16 G-C-T 0.34 0.1 G-C-C 0 0.03 A-C-C 0 0.05

Chi-square 171.457; df 7; P value 0.0001. aThe first position in the haplotypes is the marker at gene HSPA1B, second at HSPA1A, and third at HSPA1L.

SINGH et al.: HSP70 AND LONGEVITY IN DENMARK

D' FE |mn[TTT TSF <==ao o   0.0o20.81.9HEP7OR1LTSPPORLA4SP70418     7S 5 11.25 15FIGURE 1. Pair-wise measurement of LD among the three HSP70 gene markers.

FIGURE 1. Pair-wise measurement of LD among the three HSP70 gene figure shows two common ways of measuring LD, D(cid:2) , and r2. The distance the markers is the relative distance, in kilobases, where position 0 corresponds

haplotypes across different age groups from young individuals to centenar- ians we found a significant age-dependent increase in the frequency of one haplotype in males (Singh et al., in preparation).

As mentioned above most of the genetic association studies on hu- man longevity are performed in a case–control manner by comparing the gene/genotype frequencies between young and 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

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in 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 the carriers of different genotypes. Female carriers of CC = genotype of HSP70A1A survive better than the noncarriers (AA and AC) (P 0.005). It was also observed that female carriers of GG genotype in HSP70A1B = survive better than noncarriers (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 the 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 that the genes involved in cellular maintenance and repair mechanisms indeed important candidates for deciphering the genetics of human Additionally, the availability of samples from different population cohorts 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 for her help in genotyping. We also thank the personnel of the Blood Skejby Sygehus. The studies received financial support from the Danish for Molecular Gerontology (DCMG), Danish Research Councils, and Nordisk Fund.

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

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