Gain-of-Phosphorylation Mutations in Coiled-Coil and DNA-Binding Domain of STAT1 Identified in Japanese Patients with Chronic Mucocutaneous Candidiasis

Abstract 4831

Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 lineage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. IFN-γ stimulation results in phosphorylation of STAT1 at Tyr701 (pSTAT1) to induce the homodimerization, a gamma-activated factor (GAF), through the conformational change and the nuclear import. The GAF binds to the gamma-activated sequence (GAS) to induce the transcriptional activities. STAT1 mutations identified in patients with CMCD are gain-of-phosphorylation, gain-of-GAF DNA binding and gain-of-GAS transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased pSTAT1.

Here we report six heterozygous missense mutations in CCD and DBD of STAT1, including two novel heterozygous STAT1 mutation, 1060C>A (L354M) and 986C>T (P329L), in two sporadic and four multiplex cases with CMCD in Japan. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are gain-of-function mutations against GAF mediated transcription activity.

We then studied dephosphorylation of STAT1 using peripheral blood mononuclear cells (PBMCs) from the patients. The PBMCs were incubated with Staurosporine, tyrosine kinase inhibitor, followed by IFN-γ stimulation and analyzed by flowcytometry and immunoblot. In both experiments, PBMCs from the patients showed increased pSTAT1 after IFN-γ stimulation. Furthermore, we observed persistent pSTAT1 after Staurosporine treatment. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation. The flowcytometry analysis showed no overlap in mean flow intensity of pSTAT1 between 6 patients and 11 healthy controls after Staurosporine treatment. Therefore, this method can be useful for rapid test of STAT1 function in patients with CMCD.