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

indicated in red; other mutations indicated in blue. (E) IRF9 transcripts (left panel) and relative frequencies (right panel) produced during exon trapping in U2A cells. The results are representative of two independent experiments. (F) cDNA sequencing to detect the splicing of IRF9 mRNA from F-SV40 cells. Numbers of total and abnormal clones sequenced are indicated. Results representative of two experiments. Figure 2 . Impact of IRF9 Δex7 on IFN receptor-proximal signaling. (A) qRT-PCR measuring of IRF9 mRNA levels in PBMCs from the patient, her mother, and a healthy control with two probes-one probe spanning intron 7, and a second probe spanning intron 1. Representative results of four independent experiments are shown. (B) Top: WB of endogenous IRF9 in patient F-SV40 cells; GAP DH was used as a loading control. Bottom: STAT and phospho-STAT (pSTAT) levels were also assessed following stimulation with 1,000 U/ml of either IFN-α2b or -γ for 0.5 h on F-SV40 cells from two healthy controls (C1 and C2), the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), a STAT1-deficient patient (STAT1 −/− ), a STAT2-deficient patient (STAT2 −/− ), an IFN GR2-deficient patient (IFN GR2 −/− ), and an IRF7-deficient patient (IRF7 −/− ). Representative results of five independent experiments are shown. (C) WB of IRF9 in IRF9-deficient U2A cells stably transfected with indicated variants (green: variants reported to be loss-of-function in in vitro assays, blue: variants found in-house, red: patient). GAP DH was used as loading control. Representative results of four independent experiments are shown. (D) WB of IRF9 in patient F-SV40 cells stably transfected with indicated variants. GAP DH was used as loading control. Representative results of four independent experiments are shown. (E) WB analysis of IRF9 localization in F-SV40 cells from two healthy controls (C1 and C2), the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), a STAT1-deficient patient (STAT1 −/− ), a STAT2-deficient patient (STAT2 −/− ), an IFN GR2-deficient patient (IFN GR2 −/− ), and an IRF7-deficient patient (IRF7 −/− ). GAP DH and LaminA/C were used as loading controls. Representative results of three independent experiments are shown. (F) Reporter assays of ISRE or GAS-dependent firefly luciferase tested in U2A cells stimulated with 1,000 U/ml of either IFN-α2b or -γ for 16 h after being stably transfected with indicated variants (green: variants reported to be loss-of-function in in vitro assays, blue: variants found in-house, red: patient). The specific response to IFN stimulation was calculated by the ratio of further suggesting that it is deleterious (Kircher et al., 2014; Itan et al., 2016) . Finally, P's exome showed 6.8% homozygosity, consistent with her parents being first cousins (Belkadi et al., 2016) . Consistently, homozygous nonsynonymous rare variations (minor allele frequency <0.01) were also found in 19 other genes, none of which is a known disease-causing gene. Genes encoding topoisomerase 2A (TOP2A) and 5′-3′ exoribonuclease 2 (XRN2), the only two connected to known primary immunodeficiency (PID) genes in a connectome analysis, carried variants that were missense (Itan et al., 2013) . The only three genes that carried homozygous variations predicted to be loss-of-function were unrelated to PIDs: disheveled binding antagonist of β catenin 2 (DACT2), ENTH domain containing 1 (ENT HD1), and Forssman glycolipid synthase-like protein (GBGT1). Overall, t