The subjects included in this study were randomly se- lected participants from the four large nation-wide surveys conducted at University of Southern Denmark, Odense, Den- mark; the Danish 1905 Cohort, which includes all Danes born in 1905 (Nybo et al., 2001); the longitudinal study of ageing Danish twins (LSADT), including all Danish twins age 70 or older (Christensen et al., 2002; Skytthe et al., 2002); the study of middle-aged Danish twins (MADT), including Danish twins born between 1931–1952 (Skytthe et al., 2002; Gaist et al., 2000); the Danish Centenarian Study, including all Danes who lived to be a 100 years old in a 14 month period of 1995–1996 (Andersen-Ranberg et al., 2001). All four surveys comprise multidimensional face-to-face interviews, assessment of functional and cogni- tive abilities and DNA sampling, either from full blood or by means of finger pricks or cheek swabs. In the case of including subject from the twin studies only one twin part- ner from each pair was selected. We have previously shown that twins and singletons have similar mortality from age six and throughout life (Christensen et al., 1995). Hence, the genetic study is population based with no exclusion criteria due to health status or residence. Furthermore, the minimal immigration into Denmark in the cohorts comprised by these surveys and the high response rate in all surveys minimize population stratification as well as selection bias within the population.

PCR I conditions were: denaturation at 94 C for 5 min ◦ followed by 15 cycles of denaturation at 94 C for 30 s, ◦ ◦ annealing at 55 C for 1 min and extension at 72 C for 15 s. ◦ PCR was terminated by extension at 72 C for 7 min and ◦ cooled to 4 C.

PCR II was then performed in a total volume of 10  l using the same primers and reaction conditions except that the primer concentration was lowered to 100 nM and Taq polymerase was replaced by Pfu polymerase (Stratagene) in order to obtain blunt-end products. 2 )pl l of a 1:5 dilution of PCR I product was used as template DNA. PCR II conditions were as for PCR I except that the number of cycles was increased to 20.

Following PCR II, 6  l of the product was digested with Tsp509I (New England Biolabs) according to the manufac- turers instructions and the digested and undigested DNA mixed in a 1:1 ratio.

2.2. Genotyping

As restriction with Tsp509I leaves the products with a 3). 3 -overhang, the pooled product was denatured in order to separate the labeled strands from the unlabelled strands, and 2 Ps l was analyzed using the ABI 377 automatic sequencer (Applied Biosystems). Alleles were defined using the Genes- can and Genotyper software.

− − 572G/C, and 174G/C, were detected by allelic ination using the Taqman technology. Primers and were designed using Primer Express® software Biosystems). Primers and probes used for − 5! - of the 597G/A alleles were: 597s: 5 3/   3/ et gtaact-3 , 597as: 5 -ctccctgtgagcggctgtt-3 , 597G: 5 3 ral5/- cctggccaccctcaaatttcg-Tamra-3 and 597A: 5 3. ggccatcctcaaatttcgt-Tamra-3 . Primers and probes for −  tion of the 572G/C alleles were: 572s: 5

The sense primer used in both PCR reactions was de- signed so as to cover and eliminate an internal Tsp509I recognition site, resulting in an unrestricted PCR product that was 119–121 nucleotides depending on the length of

L. Christiansen et al./Mechanisms of Ageing and Development 125 (2004) 391–395

tion is in accordance with the predicted values assuming Hardy–Weinberg equilibrium overall as well as for all sub-groups. We found a significant, although modest, in- − crease in the proportion of 174GG homozygotes with age P = . ( 0 01), indicating that this genotype is associated trend with increased survival chance.

the (A)n(T)m run. After digestion and denaturation the Fam-labelled restriction product containing the (A)n block was 52–54 nucleotides long and the Tet-labelled restriction product containing the (T)m block was 69–71 nucleotides long. As described by Tschentscher et al. (2000) exact al- lele determinations were possible at all times, as only one combination of the fragment containing the (A)n block and the fragment containing the (T)m block would be consistent with the length of the unrestricted fragment.

− The 572G/C genotype was also assessed in all l710 individuals. The overall distribution as well as the distri- bution in subgroups was in Hardy–Weinberg equilibrium. − The 572C allele was rare in the Danish population (al- = lele frequency 0.04) and was in all cases found associ- − ated with the 174G allele. There was no difference in the − 572G/C genotype distribution between age-groups (results not shown).

2.3. Statistics

The 373(A)n(T)m polymorphism was investigated in < 568 individuals age 70 years and 579 individuals age = − 93 years. We detected 4 different 373(A)n(T)m alleles in the Danish population; (A) (T) , (A) (T) , (A) (T) 8 12 9 11 10 10 and (A) (T) . There was no overall difference in the geno- 10 11 type distribution between the younger individuals and the group of 93 years old people, however, the frequency of the (A) (T) allele decreased slightly with age from 47.8% 8 12 in the youngest group to 43.8% in the elderly (results not P > .

Due to the conflicting reports on the implication of the 174G/C polymorphism in ageing and disease it was rele- − to investigate whether the 174C allele was only dis- when embedded in a specific haplotype.

3. Results