www.elsevier.com/locate/mechagedev

Modest implication of interleukin-6 promoter polymorphisms in longevity

a b a b a a a L. Christiansen , L. Bathum , K. Andersen-Ranberg , B. Jeune , K. Christensen

Department of Epidemiology, Institute of Public Health, University of Southern Denmark, Odense, Denmark b Department of Clinical Biochemistry and Clinical Genetics, Odense University Hospital, Odense, Denmark

The multifunctional interleukin-6 has been suggested to contribute to a chronic low-grade inflammatory status, thereby conferring suscep- tibility to age-related pathological conditions as well as functional decline and increased mortality. Several polymorphisms have been identified in the interleukin-6 promoter, but investigation of the effect of these on interleukin-6 levels and disease susceptibility have led to contradictory results. This study investigates the significance of the three single-point polymorphisms (−597G/A, −572G/C and −174G/C) and the AT-stretch polymorphism (−373(A)n(T)m) in ageing, by comparison of the frequency of each single polymorphism separately as well as the entire promoter haplotype in a total of 1710 Danish subjects ranging in age from 47 to 100 years. We found a modest, but significant, increase in the frequency of interleukin-6 −174GG homozygotes with age suggesting that this genotype is advantageous for longevity.

Interleukin-6; Polymorphisms; Haplotype; Ageing; Longevity

1. Introduction

Ageing has been suggested to be associated with a chronic low-grade inflammatory activity, that seem to be implicated in the pathology of several age-related diseases, conse- quently leading to an increased mortality (Bruunsgaard et al., 2001; Harris et al., 1999).

Inter-individual variations in IL-6 expression may be a of base variations located in the promoter region of IL-6 gene. Hence, putative genetic variants could partly susceptibility to diseases accompanied by alterations IL-6 levels.

The multifunctional cytokine interleukin-6 (IL-6; MIM# 147620) is one of the inflammatory markers that are sus- pected to play a role in this low-grade inflammation. IL-6 is over-expressed in many of the stress conditions that are char- acteristic features of the ageing process. Furthermore, IL-6 levels are generally believed to be highly correlated with age and increase considerably during life (Ershler and Keller, 2000; Bruunsgaard et al., 1999; Cohen et al., 1997). It has been proposed that this increase is a risk factor for develop- ment of age-related diseases, such as rheumatoid arthritis, osteoporosis, Alzheimer’s disease, cardiovascular diseases, and type 2 diabetes (Pradhan et al., 2001; Ershler and Keller, 2000; Licastro et al., 2000; Bonaccorso et al., 1998; Robak

− − Three single-point polymorphisms ( 597G/A, 572G/C − − and 174G/C) and an AT-stretch polymorphism ( 373(A)n (T)m) have been identified in the IL-6 promoter (Georges et al., 2001; Terry et al., 2000; Fishman et al., 1998). The − potential significance of the 174G/C polymorphism for both the IL-6 level and disease susceptibility has been ex- tensively investigated in a large number of studies involving a variety of diseases. The results have, however, been re- markably conflicting (Nauck et al., 2002; Humphries et al., 2001; Rauramaa et al., 2000).

One possible explanation for the inconsistent findings on − the association of the 174G/C polymorphism with disease phenotypes, is the constriction of looking at the polymor- phism in isolation. In support of this, Terry et al. (2000) have demonstrated that the IL-6 gene promoter polymorphisms are not independent, and that the differences in the haplo-

0047-6374/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.03.004

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

type, rather than the 174G/C genotype, may be determi- native for the IL-6 expression level.

aatttgaggat-3 , 572as: 5 -cagtcatctgagttcttctgtgttctg-3 , 572 ‘60  G: 5 -Fam-cagtctacaacagccgctcacagggaga-Tamra-3 , 572C: we ae3). 5 - Tet-cagtctacaacagcccctcacagggaga-Tamra-3 . Primers and probes for detection of the − 174G/C alleles were: 174s: ALIA5/- Ie  MN5/- 5 -tcaatgacgacctaagctgcact-3 , 174as: 5 -ttggaaaccttattaagat- AVE ene tgtgcaat-3 , 174G: 5 -Fam-tgacgtcctttagcatcgcaagacacaacta-    Tamra-3 and 174C: 5 -Tet-tgacgtcctttagcatggcaagacacaacta- a‘3/ Tamra-3 .

genotypes as well as the IL-6 promoter haplotypes in a large sample of Danish subjects ranging in age from 47 to 100 years, in order to detect if a specific geno- type or haplotype was associated with either longevity or increased mortality. A total of 1710 subjects were geno- typed for the − 174G/C and − 572G/C polymorphisms and − 572GC/− 373(A)n(T)m/− 174GC haplotypes were fur- thermore assessed for 1147 of the subjects.

DNA was amplified in a total volume of 10 l containing 5 ul l Taqman Universal Master Mix (Applied Biosystems), 900 nM of each primer (DNA Technology), 200 nM of each ∼ probe (MWG Biotech) and 10 ng template DNA.

PCR was performed in the ABI Prism® 7700 using the conditions recommended by the manufacturer (Applied Biosystems) and analysed using the Sequence Detection System software.

2. Materials and methods

2.1. Subjects

Detection of the 373(A)n(T)m run was performed by a modification of the PCR-RFLP method previously de- scribed by Tschentscher et al. (2000) using fluorescent la- belled primers for visualization instead of 32P labeling. As the template DNA was predominantly low concentration DNA due to isolation from finger pricks, a two-PCR am- plification method was applied; In PCR I DNA was ampli- 405/- 3/ fied using the sense primer: 5 -Fam-ttaactggaacgctatattcta-3 a 3 and the antisense primer: 5 -Tet-aagtcatgcacgaagttttac-3 in a total reaction volume of 6 D ul l containing 1 × PCR buffer, 200 ) wh M of each dNTP (Roche), 167 nM of each primer (DNA technology), 0.15 U Taq DNA polymerase (Roche) and 2  l template DNA.