Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

Hernandez, Nicholas; Melki, Isabelle; Jing, Huie; Habib, Tanwir; Huang, Susie S.Y.; Danielson, Jeffrey; Kula, Tomasz; Drutman, Scott; Belkaya, Serkan; Rattina, Vimel; Lorenzo-Diaz, Lazaro; Boulai, Anais; Rose, Yoann; Kitabayashi, Naoki; Rodero, Mathieu P.; Dumaine, Cecile; Blanche, Stéphane; Lebras, Marie-Noëlle; Leung, Man Chun; Mathew, Lisa Sara; Boisson, Bertrand; Zhang, Shen-Ying; Boisson-Dupuis, Stephanie; Giliani, Silvia; Chaussabel, Damien; Notarangelo, Luigi D.; Elledge, Stephen J.; Ciancanelli, Michael J.; Abel, Laurent; Zhang, Qian; Marr, Nico; Crow, Yanick J.; Su, Helen C.; Casanova, Jean-Laurent

IRF9 participates in the formation of the ISGF3 transcription factor by binding with STAT1 and STAT2 via its IAD. IRF9 also enables binding of the trimer to DNA, after its translocation to the nucleus (Veals et al., 1993; Darnell et al., 1994; Taniguchi et al., 2001; Stark and Darnell, 2012) . Because P's IRF9 protein deletion occurs in the IAD region ( Fig. 1 D) , we hypothesized that IRF9-Δex7 may be unable to engage with STAT1 and STAT2 to form a functional ISGF3, consistent with a recent report of this domain's function (Rengachari et al., 2018) . To test this, we first fractionated nuclear and cytoplasmic compartments of P's F-SV40s under basal conditions to determine the localization of P's IRF9 protein ( Fig. 2 E) . Consistent with the presence of a nuclear localization sequence in the N-terminus of IRF9, a small amount of IRF9 was visible in the nucleus even under basal conditions. IRF9-Δex7, although expressed at substantially lower levels than the fulllength protein, is also visible in the nucleus. Consistently, U2A cells transfected with IRF9-Δex7 exhibited abolished luciferase transcription when luciferase production was driven from an IFN-stimulated response element (ISRE) promoter, but normal transcriptional activity when it was driven by a gamma activation sequence (GAS) promoter (Fig. 2 F) . In contrast, the Q127H and WT variants were able to induce transcription of luciferase from both ISRE and GAS promoters. Both R292Q and R292C variants displayed an intermediate phenotype with roughly 50% activity compared with the WT allele, suggesting that they are hypomorphic. Taken together, these data strongly suggest that P's IRF9 variant, Δex7, is unable to encode an IRF9 protein that forms a functional ISGF3 transcription factor, while R292C is hypomorphic and Q127H is neutral. Electrophoretic mobility shift assays (EMSAs) confirmed that P's cells were not able to form a functional ISGF3 complex following IFN-α2b stimulation, as evidenced by abolished ISRE binding in vitro, although her cells were able to form gamma-activated factor complexes and bind GAS elements under the same conditions ( Fig. 2 G) . The patient is therefore most likely homozygous for a null IRF9 allele, as IRF9-Δex7 was the only transcript detected in the cells tested. However, one cannot exclude the remote possibility that the splicing defect is leaky in untested cells, where residual D331N expression might result in a severe but not complete form of IRF9 deficiency.