Marieke De Bruyne, Levi Hoste, Delfien J. Bogaert, Lien Van den Bossche, Simon J. Tavernier, Eef Parthoens, Mélanie Migaud, Deborah Konopnicki, Jean Cyr Yombi, Bart N. Lambrecht, Sabine van Daele, Ana Karina Alves de Medeiros, Lieve Brochez, Rudi Beyaert, Elfride De Baere, Anne Puel, Jean-Laurent Casanova, Jean-Christophe Goffard, Savvas N. Savvides, Filomeen Haerynck, Jens Staal, Melissa Dullaers
Background: Inherited CARD9 deficiency constitutes a primary immunodeficiency predisposing uniquely to chronic and invasive fungal infections. Certain mutations are shown to negatively impact CARD9 protein expression and/or NF-κB activation, but the underlying biochemical mechanism remains to be fully understood. Objectives: To investigate a possible founder origin of a known CARD9 R70W mutation in five families of Turkish origin. To explore the biochemical mechanism of immunodeficiency by R70W CARD9. Methods: We performed haplotype analysis using microsatellite markers and SNPs. We designed a model system exploiting a gain-of-function (GOF) CARD9 L213LI mutant that triggers constitutive NF-κB activation, analogous to an oncogenic CARD11 mutant, to study NF-κB signaling and signalosome formation. We performed reporter assays, immunoprecipitation and confocal imaging on HEK cells overexpressing different CARD9 variants. Results: We identified a common haplotype, thus providing evidence for a common Turkish founder. CARD9 R70W failed to activate NF-κB and abrogated NF-κB activation by WT CARD9 and by GOF CARD9. Notably, R70W CARD9 also exerted negative effects on NF-κB activation by CARD10, CARD11, and CARD14. Consistent with the NF-κB results, the R70W mutation prevented GOF CARD9 to pull down the signalosome partner proteins BCL10 and MALT1. This reflected into drastic reduction of BCL10 filamentous assemblies in a cellular context. Indeed, structural analysis revealed that position R70 in CARD9 maps at the putative interface between successive CARD domains in CARD9 filaments. Conclusions: The R70W mutation in CARD9 prevents NF-κB activation by inhibiting productive interactions with downstream BCL10 and MALT1, necessary for assembly of the filamentous CARD9-BCL10-MALT1 signalosome.