Taking these two crucial factors into consideration, we have performed a longitudinal study of human survival on a group of individuals born in the year 1905 in Denmark (1905 cohort) [25]. These subjects were genotyped for three single nucleotide poly- morphisms (SNPs) present in the three heat shock protein 70 genes (HSP70), and the effects of genotypes and haplotypes on human survival were studied.

The three SNPs which we studied are, -110A>C (marker rs1008438), present in the promoter region of HSPA1A; the synonymous 1267A>G (marker rs1061581), present in the coding region of HSPA1B; and the non-synonymous 2437T>C (Marker rs2227956), present in the coding region of HSPA1L. The latter

A widely used approach for identifying genes involved in human longevity has been the population based case-control

*Address correspondence to this author at the FCMB ApS, Tysklandsvej 7, Vejle 7100, Denmark; Tel: +45 3693 8856; E-mail: rs@fcmb.dk

each SNP. The protocol for the RT-PCR is described elsewhere [13], and the program can be made available on request.

SNP leads to a change in amino acid at position 493 from a non- polar hydrophobic acid methionine (Met) to a polar neutral threo- nine (Thr) and may have molecular functional significance with respect to the stability and activity of the chaperoning protein Hsp70.

The effects of genotypes and haplotypes on human survival was estimated by using the relative risk method developed by Tan et al. [21]. With this method sex-specific haplotype relative risk (HRR) and genotype relative risk (RR) on human survival was estimated for every haplotype and genotype derived from the three SNPs. For every carrier of genotype/haplotype a relative risk of more than or less than 1 was considered to be harmful or beneficial for human survival respectively. All statistical analyses were conducted by GAUSS programming package (http://www.aptech.com) [21].

MATERIALS AND METHODS

168 DNA samples (males = 40, mean age = 92.8 ± 0.4 yr; females = 128, mean age = 92.8 ± 0.4 yr), collected in 1998 from individuals born in year 1905, were used for typing the three SNPs in the three genes. These samples were collected in Denmark as part of the first ever nationwide survey of nonagenarians [26]. Various physical and cognitive parameters were also measured on these individuals.

RESULTS

Gene and genotype frequencies of the three SNPs are given in Table 1. Fig. (1) and Table 2 show that the survival duration of the female carriers of genotypes HSPA1A-AA (RR=3.864; p=0.016) and HSPA1A-AC (RR=2.577; p=0.056) was shorter than the carriers of HSPA1A-CC.

Genotyping was done using real-time (RT) PCR on the LightCycler system (Roche Applied Sciences), which helps to monitor the amplification of PCR product in real time, using fluorescent labelled oligonucleotide probes and primers specific for

1. Gene/Genotypic Frequency Distribution of the Three SNPs in 1905 Cohort Individuals

HSPA1A (- AA AC CC A C 110A>C) M F M F M F M F M F 25 41.9 65 47 10 11.1 57.5 65.3 42.5 34.6 (10) (49) (26) (55) (4) (13) (46) (153) (34) (81) HSPA1B AA AG GG A G (1267A>G) M F M F M F M F M F 25 38 62.5 47.9 12.5 14 56.2 61.9 43.7 38.1 (10) (46) (25) (58) (5) (17) (45) (150) (35) (92) HSPA1L TT TC CC T C (2437T>C) M F M F M F M F M F 62 57.7 35.1 37.9 2.7 4.3 79.7 76.7 20.2 23.2 (23) (67) (13) (44) (1) (5) (59) (178) (15) (54)

male; F= female. Data are presented in percentage with number of individuals given in parentheses.

2. Estimated Relative Risks of the Genotypes at the Three Loci in Females

Genotype Frequency Risk p-value HSPA1A AA 0.398 3.864 0.016 AC 0.484 2.577 0.056 CC 0.118 1 - HSPA1B AA 0.359 2.764 0.039 AG 0.478 1.932 0.188 GG 0.163 1 - HSPA1L* C allele carriers 0.359 1.913 0.328

analysis was not possible due to low frequency of C allele.

(1). Survival of genotypes in HSPA1A (left) and HSPA1B (right) among females.

 jeans©SoJeAlAINSwoo0.40.30.20.1Age

Fig. (1). Survival of genotypes in HSPA1A (left) and HSPA1B (right) among females.

Cumulative Survival08:06.O40200:33500 34000 34500 35000Age in days38500 36000 36500HSPAIAJS) oeSexSceCumulative Survival° ° °2 & &°50039500 34000-4500 35000-35500 3800036500Age in days4448

Fig. (2). Association of female survival with genotypes of HSPA1A and HSPA1B respectively, using Kaplan-Meier’s survival test.

Also the females carriers of genotype HSPA1B-AA (RR=2.764; p=0.039) had shorter survival duration than carriers of HSPA1B-GG [Fig. (1)]. These results on the association of genotypes with female survival were maintained when Kaplan-Meier’s test of survival was performed [Fig. (2)]. No significant results were obtained in analyzing the male subjects.

DISCUSSION

The conventional method for performing the genetics of human longevity, in the population based association studies, is the cross- sectional design. In this method the gene, genotype or haplotype frequencies are compared between a group of old ‘cases’ and young ‘controls’. This methodology, though inexpensive and straightfor- ward, suffers from a drawback that the observed differences in the frequencies in the two groups can be due to secular changes in population demographic and genetic parameters of the two groups and not necessarily age. An alternate to this approach is the longitudinal cohort design for studying the effects of genes on human survival in a cohort of long lived individuals. In this, a group of individuals are genotyped for polymorphisms in the candidate genes, and then followed up for studying their survival. This methodology mitigates the drawbacks thus encountered by the case-control design. In the present study we have applied the