Defects in Signal Recognition Particle (SRP) Components Reveal an Essential and Non-Redundant Role for Granule Biogenesis and Differentiation of Neutrophil Granulocytes

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.216-216
• Main Authors: Mizoguchi, Yoko, Hesse, Sebastian, Linder, Monika, Zietara, Natalia, Lyszkiewicz, Marcin, Liu, Yanshan, Tatematsu, Megumi, Grabowski, Piotr, Ahomaa, Kaarin, Jeske, Tim, Hollizeck, Sebastian, Rusha, Ejona, Saito, Megumu K, Kobayashi, Masao, Alizadeh, Zahra, Pourpak, Zahra, Iurian, Sorin, Rezaei, Nima, Unal, Ekrem, Drukker, Micha, Walzog, Barbara, Hauck, Fabian, Rappsilber, Juri, Klein, Christoph
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-130083

Neutrophil granulocyte play pivotal roles in inflammatory responses, immune defence, tissue remodeling, and cancer control. Studying rare patients with defects in differentiation and/or function of neutrophil granulocytes highlights genes and pathways orchestrating these important cellular functions. A previously not appreciated role of the signal recognition particle (SRP) has emerged when monoallelic mutations in SRP54 were associated with congenital neutropenia and pancreatic insufficiency. The eukaryotic SRP is composed of six distinct polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68, SRP72) bound to an RNA molecule (the 7SL RNA). SRP and its receptor (SRPRA and SRPRB) cooperatively transport nascent proteins emerging from ribosome to the endoplasmic reticulum (ER) lumen via a protein-conducting channel. We hypothesized that other constituents of the SRP may be involved in the pathophysiology of congenital neutropenia and that disturbances of the SRP may result in defective proteostasis in neutrophil granulocytes. By virtue of our international Care-for-Rare Alliance we identified three kindred with molecularly unsolved congenital neutropenia. Exome-sequencing revealed a de novo monoallelic mutation in SRPRA in one individual in one family and a homozygous splice-site mutation in SRP19 in five individuals from two families. Clinically, all patients with mutations in SRPRA and SRP19 suffered from various degrees of neutropenia and susceptibility to bacterial infections. Pancreatic insufficiency was only seen in patients with mutation in SRPRA, whereas SRP19 deficiency was not associated with any non-hematopoietic symptoms. To functionally validate these novel genetic variants, we set up an in-vitro neutrophil differentiation system allowing us to model the genetic defects in human SRP complex subunits. We introduced patient-specific mutations (SRPRA Q464E and SRP19 c.189+5G>A) into wildtype iPS cells using CRISPR-Cas9 editing. The engineered cells were differentiated into bona fide neutrophil granulocytes. In vitro generated wildtype neutrophil granulocytes had a high degree of resemblance to primary peripheral blood neutrophil granulocytes, as evidenced by phenotypic (light microscopy, cell surface markers, gene expression) and functional (phagocytosis, bacterial killing, rolling and adhesion, chemotaxis) characteristics. In striking contrast to wildtype iPS cells, both SRPRA and SRP19 mutated iPS cells had a significantly reduced capacity to differentia