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Mechanisms of Ageing and Development 128 (2007) 409–411

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of the toll-like receptor 4 polymorphism Asp299Gly in longevity and myocardial infarction in German men

a,1 a,1 a b Almut Nebel , Friederike Flachsbart , Arne Scha¨fer , Michael Nothnagel , c d a,* Susanna Nikolaus , Nour Eddine El Mokhtari , Stefan Schreiber

aInstitute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Germany bInstitute of Medical Informatics and Statistics, University Hospital Schleswig-Holstein Kiel, cHospital for General Internal Medicine, University Hospital Schleswig-Holstein Kiel, Germany dDepartment of Cardiology, University Hospital Schleswig-Holstein Kiel, Germany

Received 7 December 2006; received in revised form 30 March 2007; accepted 3 April 2007 Available online 7 April 2007

Abstract

In a previous study on Sicilian men, an association of the functional polymorphism Asp299Gly in the TLR4 gene was reported with longevity and a reduced risk of myocardial infarction (MI). Here, we sought to confirm the findings in our extensive collection of 273 long-lived men (95–107 years), 606 male early-onset MI patients and 594 appropriate controls from Germany. Despite sufficient statistical power to replicate the findings observed in the South Italians, our results rule out a noteworthy influence of the TLR4 polymorphism upon human longevity or MI in German men. Therefore, the previously described associations in Sicilians might reflect either population-specific or potentially false-positive results. # 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Toll-like receptor 4 gene; Longevity; Mycocardial infarction; Case–control association; Replication; Reproducibility; Reporting bias

Keywords: Toll-like receptor 4 gene; Longevity; Mycocardial infarction; Case–control association; Replication; Reproducibility; Reporting bias

Toll-like receptor 4 (TLR4) is involved in the activation of innate immunity, and TLR4-dependent pro-inflammatory signalling has been implicated in the pathogenesis of atherosclerosis and its clinical manifestations, such as myocardial infarction (MI; Edfeldt et al., 2002). Hence several studies have investigated the association of the functional single nucleotide polymorphism (SNP) +896 A/G (Asp299Gly) in the TLR4 gene with the risk of MI in Europeans, albeit with conflicting results. In some studies, the minor G allele was shown to be associated with a reduced risk of atherosclerosis and acute coronary events (Kiechl et al., 2002; Ameziane et al., 2003), in others it was found to predispose to MI (Edfeldt et al., 2004) or not to be associated at all with the disease (Morange et al., 2004; Zee et al., 2005; Koch et al., 2006). As MI is a leading cause of death worldwide and is tightly correlated with life expectancy, Asp299Gly has also been tested for association with longevity (Balistreri et al., 2004). In Sicilian men, the G allele was reported as an age-associated marker, being

significantly over-represented in 55 long-lived individuals (LLI) compared to 127 younger controls or 105 MI patients, respectively, reflecting a survival advantage for carriers of the SNP (Balistreri et al., 2004). In the present study, we sought to replicate the findings observed in the South Italians in our extensive collection of 544 long-lived German men and younger controls. In addition, we evaluated whether this SNP was also associated with early-onset MI in Germany using a sample of 929 male patients and healthy controls.

In this study, DNA samples of 1473 German men drawn from population-based collections were analyzed: 273 LLI (mean age: 97.9 years, range: 95–107 years) and 271 younger controls (mean age: 67.4 years; range 60–75 years; Nebel et al., 2005) as well as 606 MI patients (mean age: 54 years; range: 37–71 years, mean age of disease onset: 48 years; El Mokhtari et al., 2006) and 323 healthy controls (mean age: 62 years; range: 48–77 years; Krawczak et al., 2006) who showed no signs of any cardiac disease. SNP Asp299Gly (rs4986790) was 1 genotyped by a TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, USA). Logistic regression analysis was performed within the R statistical environment, version 2.3.1 to allow for adjustment of known MI confounding factors

* Corresponding author. Tel.: +49 431 597 2350; fax: +49 431 597 1842. E-mail address: s.schreiber@mucosa.de (S. Schreiber). 1 Both authors contributed equally.

0047-6374/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2007.04.001

A. Nebel et al./Mechanisms of Ageing and Development 128 (2007) 409–411

Table 1

Summary association statistics for the TLR4 polymorphism Asp299Gly (+896 A ! G) in German male LLI and MI patients

LLI Controls to LLI P value LLIa MI cases Controls to MI cases P value MIb P value LLI/MIa Number of males 273 271 606 323 Allele frequency G (%) 5.7 5.0 0.61 7.3 4.6 NA 0.22 Genotype frequencies (%)c 0.59 0.36 0.47 GG 0.4 0.0 0.5 0.0 AG 10.6 10.0 13.5 9.3 AA 89.0 90.0 86.0 90.7

b P value obtained bylogistic regressionanalysis froma Wald test for a non-zero regression coefficient.After adjustmentfor the fivemajor cardiac risk factors (age, BMI, smoking, diabetes type 1 and 2, hypertension). Under the dominant model (P values for the recessive and additive models were 0.99 and 0.25, respectively). c The marker Asp299Gly was in Hardy-Weinberg equilibrium (HWE) in all samples. A x2 test was used to test for deviations from HWE.

(R Development Core Team, 2006). The study was approved by the Ethics Committee of the University Hospital Schleswig- Holstein in Kiel.