NT en fkSke ale pene ee een Re eR eee dee oko athe Rae ae620 40 860 68.0 000 620 640 080 680 700 720 740 760 780 900 820 940 800

Figure 1. Melting peak analysis of three samples on the LightCycler: The fluorescent probe complementary to the Allele carrying adnine (A) melts at 58(cid:2)C and that carrying (C) melts at 63 (cid:2)C, respectively. The samples homozygous for allele A or C show one peak corresponding to 58 or 63 (cid:2)C, respectively. Whereas the sample heterozygous for A and C shows two peaks corresponding to both the temperatures.

Table 1. List of the primers and oligonucleotide probes used for genotyping using LightCycler system.

Primers HSP70-1-forw 5¢-AGAAGACTCTGGAGAGTTC-3¢ HSP70-1-rev 5¢-AAAGGTAGTGGACTGTCGC-3¢ Probes HSP70-1-se1 5¢-AATATTCCAGGGGTTTCGCCTC-X HSP70-1-an1 5¢-LCRED640-CGTCCTGCCCCCCAGCCTT-p

X – Fluorescent dye LCRED640; p – Phosphorylated.

Results

Table 2. Genotypic and gene frequencies (g.f.) at )110 in the promoter region HSP70-1 gene in elderly Danish twins.

The genotype frequencies of the individuals homozygous for allele A (AA), heterozygous for alleles A and C (AC) and homozygous for allele C (CC) were 35% (n ¼ 142), 42.7% (n ¼ 189) and 17.2% (n ¼ 69), respectively (Table 2). The allele frequencies for allele A and allele C were 59.1% and 40.9%, respectively (Table 2) and the genotype frequency was found to be in Hardy–Weinberg equilibrium (P-value 0.59).

Genotype n=400 n (%) AA 142 (35.5) AC 189 (47.2) CC 69 (17.2) Alleles n (g.f. %) A 473 (59.1) C 327 (40.9)

The relationship of aging phenotypes with HSP70-1 genotype is shown in Tables 3–6. Of all the age-related parameters studied, only self-rated health and relative self-rated health (compared to others of the same age), showed an association (P- values of 0.0046 and 0.018, respectively) with ) polymorphism at 110 (Tables 5 and 6). As evi- dent from Tables 5 and 6, it is the heterozygous individuals who are more inclined to rate their heath as ‘poor’, ‘very poor’ or ‘worse’.

Discussion

Aging and longevity are highly complex traits influenced by genes, environment and chance (Rattan 2003). Heritability of human longevity has been estimated to be 0.26 for males and 0.23 for females from Danish twin studies (Herskind et al. 1996). However, the molecular basis of inherited components in aging is far from clear. For study-

Table 3. Genotype distribution in cases (with disease) and controls (without disease) and P-values calculated for various diseases using

Table 3. Genotype distribution in cases (with disease) and controls (without disease) and P-values calculated for various diseases using Pearson’s v2 test.

Diseases Genotypes N (cases) N (controls) P (v2) AA AC CC AA AC CC Hyperthyroidism 8 19 8 134 170 61 0.243 Hypertension 38 49 16 104 140 53 0.854 Heart failure 10 23 5 132 166 64 0.226 AMI/angina 16 20 9 126 169 60 0.858 Stroke 9 3 3 136 186 66 0.292 Cancer (except skin cancer) 7 17 6 135 172 63 0.349 Pulm disorder 10 27 7 132 162 62 0.110 Osteoporosis 10 12 2 132 177 67 0.474 Arthritis 5 9 4 137 179 65 0.683 Osteoarthrosis 41 61 27 101 128 42 0.327 Diabetes 8 14 3 134 175 66 0.622

Table 4. Mean and standard deviation (SD) of performance scores for each genotype, and P-value calculated using one way ANOVA.

Parameter Mean±SD (N) AA Mean±SD (N) AC Mean±SD (N) CC P (ANOVA) Grip strength )0.19±1.02 (131) 0.05±0.88 (183) 0.09±0.97 (65) 0.298 ADL strength )0.16±0.86 (142) )0.22±0.74 (187) )0.25±0.71 (69) 0.331 ADL endurance )0.16±0.95 (138) )0.17±0.89 (185) )0.19±0.88 (67) 0.670 MMSE 0.06±0.88 (141) 0.03±1.01 (189) 0.12±0.72 (69) 0.180 Cognitive composite )0.03±1.06 (138) 0.19±0.98 (188) 0.19±1.05 (67) 0.450 Depression )0.42±4.61 (139) )0.41±4.61 (188) )1.32±2.69 (69) 0.474

Table 5. Genotypic distribution of the self-rated health response.

Genotype Very good Good Fair Poor/very poor Total AA 55 (39%) 55 (39%) 27 (19%) 4 (3%) 141 AC 47 (25%) 87 (46%) 36 (19%) 18 (10%) 188 CC 25 (36%) 27 (39%) 17 (24%) 0 69 Total 127 169 80 22 398

P=0.0046; Pearson’s v2=18.749.

6. Genotypic distribution of the relative self-rated health response.

Genotype Self-rated health compared to others of the same age Total Better The same Worse AA 86 (61%) 49 (35%) 6 (4%) 141 AC 95 (50%) 69 (37%) 24 (13%) 188 CC 41 (59%) 26 (38%) 2 (3%) 69 Total 222 144 32 398

P=0.018; Pearson’s v2=11.919.

allele frequency might not relate directly to age but to confounding factors, as the mortality for genotypes may depend on the time of birth of the cohort because of the changes in the incidence and prevalence of the number of diseases (De Bene- dictis et al. 2001). Another weakness of cross-sec- tional studies is what epidemiologists call ‘population stratification’, that is that the ethnic compositions of various cohorts are not identical and therefore can create spurious correlations.

ing the genetic basis of human longevity, the gene- longevity association studies, which look for association between polymorphisms at candidate loci and lifespan, have been a popular and useful approach. Although a number of genes have been studied for their association with human longevity, the overall picture still remains unclear (Caruso et al. 2001). This is due to the various confounding factors which affect the longevity association studies, such as heterogeneity, which may affect the Hardy–Weinberg equilibrium of the popula- tion on which such studies are to be carried out. Special problems occur when doing cross-sectional studies, where allele frequencies a group of young controls are compared against the allele frequen- cies in the oldest-old. In this design a difference in

The association of HSP70-1 with human lon- gevity has been reported for the first time in a re- cent case/control study on samples from Calabria, a south Italian region (Altomare et al. 2003). The study showed that the presence of allele A, in the (cid:2)110 position of the promoter region of HSP70-1,