TNFSF4 (also designated OX40L, gp34 and CD134L, GenBank accession no. NM_003326), the ligand of the TNFRSF4 (OX40 receptor), is a member of the tumor necrosis factor (TNF) superfamily. TNFSF4 is a T-cell activator that seems to promote the survival (and perhaps prolong the immune response) of CD4+ T-cells at sites of inflammation [1]. T-cells are indicated to have an essential role in the development of atherosclerosis [2]. Activated CD4+ and CD8+ T-cells, B cells and vascular endothelial cells have been reported to express TNFSF4 [3].

Based on a mouse atherosclerosis model [4] to positionally identify potential human candidate genes, we have provided evidence that genetic variation in the TNFSF4 gene contributes to the risk of developing myocardial infarction (MI) [5]. A single nucleotide polymorphism (SNP) in the first intron of TNFSF4 (rs3850641) and haplotypes including this SNP were found to be associated with risk of MI in women in two independent populations and with angiographically assessed severity of CAD. TNFSF4 haplotypes were also associated with variation in some risk factors for CAD; carriers of a specific haplotype had significantly higher plasma concentrations of HDL cholesterol and serum amyloid A (SAA) than did carriers of other haplotypes. Furthermore, gene targeting of Tnfsf4 in mice showed that deficient animals had significantly smaller atherosclerotic lesions and higher levels of plasma total cholesterol and HDL cholesterol than controls, while mice over-expressing Tnfsf4 had significantly larger atherosclerotic lesions when compared to controls, indicating that Tnfsf4 is a gene influencing atherosclerosis susceptibility in mice. Further support for a role of TNFSF4 in atherosclerosis was obtained in a study where blockage of the TNFSF4/TNFRSF4 interaction reduced lesion formation in low-density lipoprotein (LDL) receptor-deficient mice [6]. In contrast, a German case-control study failed to replicate the association between TNFSF4 and MI [7].

The functional polymorphism responsible for the association between TNFSF4 and risk of MI has not been defined, nor is it known whether high or low expression of TNFSF4 is associated with MI in man. Since the observed association with MI [5] involved a haplotype spanning regions of the gene located both upstream and downstream of the transcription start site, the functional variant(s) could be located anywhere along the stretch of DNA defined by this haplotype. However, due to its location, the rs3850641 polymorphism in intron 1 is unlikely to be the functional variant. Nevertheless, interaction between the rs3850641 polymorphism and a putative enhancer binding site located in intron 1 cannot be excluded, but a functional variant in linkage disequilibrium with rs3850641 and located in the promoter or in other regulatory regions is a more plausible explanation.

In an attempt to identify the functional SNP(s) or a minimal haplotype suitable for further functional studies, we performed a detailed screening of the TNFSF4 genomic region. New genetic variants were subsequently examined in one of the clinical studies previously used to establish the association between TNFSF4 and MI. A promoter polymorphism was identified which is in linkage disequilibrium with the rs3850641 SNP and associated with MI in women. Functional analyses of the transcriptional activity of these genetic variants were then performed both in vivo, using haplotype-specific chromatin immunoprecipitation of activated polymerase II (haploChIP assay) [8], and in vitro with assays of luciferase reporter gene expression; electromobility shift assay (EMSA) was employed to analyze allele-specific binding of transcription factors in vitro.


Source Institution: Karolinska University Hospital Solna
Source Author: Ria M et al.
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