DNA Polymorphisms and Mutations of the Tumor Necrosis Factor-α(TNF-α) Promoter in Langerhans Cell Histiocytosis (LCH)

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Abstract
Langerhans cell histiocytosis (LCH) is a clonal proliferation of dendritic histiocytes expressing elevated levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), and leukemia inhibitory factor (LIF). The cause of the increased cytokine levels is unknown, but DNA sequence changes in promoters could alter expression. The TNF-α and IFN-γ promoter DNA sequences of 12 LCH patients were studied and compared with normal individuals by dideoxy fingerprinting and DNA sequencing. Functional consequences of polymorphic or mutated sequences were assessed by cloning altered and control promoter sequences into a luciferase reporter gene vector. Electrophoretic mobility shifts (EMSA) after binding of nuclear extracts from a macrophage cell line (U-937) by mutated promoters were compared with controls. Five of 12 LCH patients had alterations in the TNF-α promoter DNA sequence. None were found in the IFN-γ gene promoter. Of the 5 with TNF-α DNA alterations, 2 were at position —308, which has been described as a G-A polymorphism associated with upregulation of TNF-α in some patients with infections or immune-mediated diseases. The polymorphism at —308 but not the other TNF-α promoter mutations casued a 3-fold to 7-fold increased production of the luciferase reporter gene. EMSA showed that the —308 mutant promoters bound fewer nuclear proteins than normals. Polymorphisms of the TNF-α promoter in LCH patients could increase the production of that cytokine.