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

Expression of a truncated mRNA and protein by the mutant IRF9 allele Because the c.991G>A mutation occurs at an essential splice site at the terminal nucleotide of exon 7, we hypothesized that it would produce an aberrantly spliced transcript. We thus transiently transfected a segment of P's allele encompassing introns 5 through 8, as well as a segment from an identical region of a WT allele, into IRF9-deficient U2A fibrosarcoma cells, a gift from S. Pellegrini (Institut Pasteur, Cytokine Signaling Unit, INS ERM, Paris, France; Fig. S2 ; John et al., 1991) . Sequencing of the resulting cDNAs revealed two transcripts produced from P's IRF9 allele ( Fig. 1 E) . The dominant transcript exhibited absence of exon 7 with normal exons 6 and 8. The second, minor transcript also lacked exon 7 but used an alternative splice acceptor site in exon 8. The segment from a WT allele, however, produced four different transcripts. One transcript demonstrating correct splicing at exons 6/7 and 7/8 junctions and a second transcript lacking exon 7 but correctly using the splice sites in exons 6 and 8 were the dominant products. Two rare transcripts were also observed: one was a product of an alternative splice acceptor site in intron 5, whereas another resulted from an alternative splice acceptor site in exon 7 ( Fig. 1 E) . Only correctly spliced transcripts were observed in healthy control B-LCLs (EBV-transformed B lymphoblastoid cell lines) and SV40 large T antigen immortalized fibroblasts (F-SV40 cells), suggesting that the two alternative transcripts observed in the overexpression setting are not present endogenously. Moreover, cDNA sequencing of P's B-LCLs and F-SV40 cells did not reveal any transcript that would result in a D331N variant protein, as all transcripts lacked exon 7 ( Fig. 1 F) . This mutant transcript (and the corresponding allele) will thus hereafter be referred to as IRF9-Δex7, as the deletion is in-frame and does not introduce a premature stop codon. Next, we evaluated IRF9 expression in P's peripheral blood mononuclear cells (PBMCs) and F-SV40 cells. Consistent with the results of our cDNA sequencing, only IRF9 transcripts lacking exon 7 were detected in P's PBMCs, and a reduced number of full-length IRF9 transcripts were detected in both individuals harboring the c.991G>A mutation in proportion to the dosage of this allele (Fig. 2 A) . By Western blotting (WB), a polyclonal antibody detected a truncated IRF9 species in both P's cells and those of her mother, whereas full-length IRF9 was not detected in P's cells (Fig. 2 B) . The molecular weight of this truncated protein is roughly 12 kD lower than WT IRF9, consistent with a lack of exon 7 (142 amino acids), which forms a substantial portion of firefly luciferase reporter gene activity to constitutively expressed renilla luciferase activity (RLU, relative luciferase ratio). Representative results of three independent experiments are shown. (G) EMSA analysis of ISRE and GAS binding by IFN-stimulated B-LCLs from three healthy controls (C1, C2, and C3), the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), a STAT1-deficient patient (STAT1 −/− ), a STAT2-deficient patient (STAT2 −/− ), and an IRF7-deficient patient (IRF7 −/− ). Representative results of three independent experiments are shown. the IRF association domain (IAD) where STAT proteins interact with IRF9 ( Fig. 1 D) . Overall, these findings showed that the patient had AR IRF9 deficiency.