Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

These data suggested that K365E, although normally expressed in patient cells, could not assemble to activate downstream signals. We therefore overexpressed wild-type or mutant MDA5 to test whether they could drive expression of luciferase from several promoters. Cells transfected with wild-type MDA5 increased baseline IFNB1 promoter activity and further augmented IFNB1 promoter activity after stimulation with intracellular poly(I:C) (Fig. 3, A and B ). In con-trast, those cells transfected with the K365E mutant showed minimal activity above nontransfected cells, which did not increase upon stimulation. K365E also failed to drive luciferase activity from IFN-stimulated response element (ISRE)and NF-κB-driven promoters (Fig. 3, C and D) . Notably, the fold induction of luciferase activity of the K365E mutation after poly(I:C) stimulation, relative to that at baseline, was larger than that of wild-type, suggesting that the defect of this mutant lies in MDA5 dimerization or oligomerization upon binding or detecting RNA, although we did not test this further. Co-transfections of the mutant with wild-type MDA5 showed no dominant-negative effect (Fig. 3 , E and F). Although biallelic loss-of-function mutations in IFIH1 have not been previously identified in humans, monoallelic gain-of-function mutations have been reported in humans with Aicardi-Goutières syndrome (Oda et al., 2014; Rice et al., 2014; Rodero and Crow, 2016) , Singleton-Merton syndrome (Rutsch et al., 2015) , and systemic lupus erythemato- sus with IgA deficiency (Van Eyck et al., 2015) . Our patient had neurodevelopmental delay, but lacked the cerebral calcifications or white matter abnormalities characteristic of Aicardi-Goutières syndrome or congenital infection (unpublished data). Moreover, her K365E mutation did not increase luciferase activity, either at baseline or after stimulation, in contrast to the known gain-of-function MDA5 mutants R337G or R779H (Fig. 2 , A, C, and D; Rice et al., 2014) . Additionally, the patient's cells did not express a type I IFN transcriptional signature characteristic of gain-of-function IFIH1 mutations (unpublished data; Rice et al., 2013) . Thus, we conclude that the K365E missense mutation was loss-of-function.