3. Results

There are 16 possible haplotypes when considering the − − − polymorphisms 572G/C, 373(A)n(T)m and 174GC, but only 10 haplotypes were present in the Danish popula- tion. Table 2 summarizes the estimated frequencies of the six most common haplotypes (constituting more than 99% of the samples) identified in four of the seven age-groups. − The 373(A) (T) allele was almost exclusively found to- 8 12 gether with the − 174C allele, whereas the other − 373 alle- les were a little more diversely distributed, although mainly found together with the − 174G allele. There was no sta- tistically significant difference in the haplotype distribution

Initially, haplotypes for the three single point substitu- tions were assessed for 365 randomly selected participants. These preliminary results showed that there was an almost complete allelic association between the − 597G allele and the − 174G allele (∆ = − 0. 95), as has also been reported previously for other populations (Georges et al., 2001; Brull et al., 2001). Consequently, the − 597G polymorphism was not studied further. Table 1 shows the genotype distribution of the − 174G/C

Table 1 shows the genotype distribution of the − 174G/C polymorphisms in the different age-groups. The distribu-

Genotype distribution of the interleukin-6 −174G/C polymorphism in different age-groups

<50 years 50–59 years 60–69 years 70–79 years 80–89 years 90–95 years 100 −174GGa 45 (24.7) 71 (24.3) 24 (25.3) 83 (27.5) 24 (29.6) 177 (30.4) 57 (32) −174GC 98 (53.9) 158 (54.1) 47 (50) 151 (50) 43 (53.1) 284 (48.9) 84 −174CC 39 (21.4) 63 (21.7) 23 (24.5) 68 (22.5) 14 (17.3) 120 (20.7) 37

Estimated frequencies of the six most common −572GC/−373(A)n(T)m/−174GC haplotypes in middle-aged subjects and 93-year-old subjects

Age G/(A) (T) /C G/(A) (T) /G G/(A) (T) /G G/(A) (T) /G C/(A) (T) /G G/(A) (T) /C 8 12 9 11 10 11 10 10 10 10 9 11 (%) (%) (%) (%) (%) (%) <70 years (N = 568) 47.0 20.3 19.6 7.1 3.8 1.3 93 years (N = 579) 43.2 21.7 22.2 6.6 3.5 2.0

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

− − − − the 597A/ 572G/ 373(A) (T) / 174C haplotype con- 8 12 struct was significantly higher than the other haplotype con- − − − − structs including the 597A/ 572G/ 373(A) (T) / 174G 8 12 haplotype, although the absolute differences were rather

between the two age-groups. The results, however, demon- strate a small decrease with age in the frequency of the − − − P = . 572G/ 373(A) (T) / 174C haplotype ( 0 07), com- 8 12 − parable with the decrease seen for both the 174C allele − and the 373(A) (T) allele. Hence, the haplotyping results 8 12 − clearly demonstrate that looking at the 174 polymorphism alone is as informative as looking at the entire haplotype in a survival analysis in this population.

− According to our results, the 174C allele, however, nearly always specifies this exact haplotype, making it rea- sonable to only asses the former, although it should be kept − in mind that the data indicates that the 174C allele only changes with age when embedded in this haplotype. Hence, a more complete haplotyping may be necessary in other populations.

4. Discussion

The relatively little immigration in Denmark and the high genetic homogeneity in the Danish population makes this population ideal for studying the influence of genetic factors on longevity. In this paper we have investigated the impact of the four common polymorphisms in the interleukin-6 pro- moter on ageing, by comparison of the frequency of each single polymorphism separately as well as the entire pro- moter haplotype in Danish subjects ranging in age from 47 to 100 years.

Previous studies focusing mainly on the role of the − 174G/C polymorphism in ageing as well as a variety of age-related diseases have lead to contradictory conclusions, ranging from demonstration of the G allele being a risk factor, over no significance of this polymorphism, to sug- gestions of the C allele as conferring disease susceptibility (e.g. Nauck et al., 2002; Humphries et al., 2001; Rauramaa et al., 2000).

of the single polymorphisms does not provide the complete explanation for these conflicting results. It cannot be ruled out that the present study as well as the previous studies implying an association between the IL-6 gene and age- ing or age-related diseases all represents chance findings. Thus, a number of the previous studies, although not all, are limited by the use of relatively small sample-sizes, which may contribute to the heterogeneity of study results. Furthermore, the use of twins as well as population strati- fication in the present study could be a concern. However, the facts that Danish twins and singletons have similar mor- tality from age six and throughout life (Christensen et al., 1995), and that the twin studies include both complete pairs and non-complete pair twins as well as the well-known minimal immigration into Denmark in these cohorts sug- gest that the population is very well suited for studies of changing gene frequencies over age. Yet another expla- − nation could be that the effect of the 174G/C genotype (or the entire promoter haplotype) on disease susceptibility is mainly manifest in specific subgroups that are in addi- tion influenced by other risk factors. The importance of defining the relevant subgroup of susceptible individuals for a specific risk gene has previously been pointed out by Lehmann et al. (2001). Ageing and the age-related patho- logical conditions that have been associated with the IL-6 − 174G/C polymorphism are complex phenotypes likely to be influenced by several interacting genes and environ- mental factors. The contribution of any single susceptibility gene will thus be difficult to detect in a population as a whole.