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Letters to the Editor
12 was reduced to 68% in immature CD33+CD15+CD11b- cells and to 39% in the more mature CD33+CD15+CD11b+ cells of the patient com- pared with control cells (Figure 2A). We also analyzed the consequences of SRP68 defect on granulocyte prolifera- tion. In the same experimental conditions, we cultured CD34+ cells purified from blood samples and observed nearly six-times less granulocytic cell proliferation in the patient compared to CD34+ cells from healthy control (Figure 2B).
These results highlight the major role of the SRP com- plex during granulocytic differentiation. This physiologi- cal process requires the production and maturation of a huge number of granule proteins.11 This context explains why granulocytic precursors and neutrophils are highly sensitive to alterations of protein synthesis, protein trans- port into ER/Golgi and ER protein misfolding. A defective co-translational targeting of nascent proteins may either affect the level of ER resident proteins as chaperones and glycosylases or lead to incorrectly translocated proteins essential for granulocytic precursor maturation as the neutrophil elastase ELANE.12 Both hypotheses will result in ER stress, activation of the unfolded protein response (UPR) and finally, to cycle cell arrest, senescence and/or death by apoptosis or necrosis of granulocytic cells. In mammals, the SRP68/SRP72 heterodimer plays an essen- tial role in the recognition of the signal peptide of nascent proteins and in their translocation through the SRP recep- tor located at the ER membrane.13 As we previously showed that SRP54 variants induce an ER stress and acti- vate the UPR pathway, we investigated several markers of UPR activation (ATF4, CHOP and spliced XBP1).3 Using in vitro sorted granulocytic cells, we found a signif- icant increase in spliced XBP1 expression level in both immature and mature granulocytic cells of the patient in comparison with control cells pointing out the specific activation of the IRE1 ER stress sensor of the UPR signal- ing (Figure 2C). 14 In contrast to SRP54, no activation of the PERK pathway, another distinct ER sensor, was observed by analyzing ATF4 and CHOP UPR-target genes.14 Under ER stress conditions, the induction of UPR leads to enhanced apoptosis reducing the number of granulocytic precursors and neutrophils. In most CN, including SRP54-related CN, apoptosis seems to be dependent on p53 pathway activation.3 We analyzed, by quantitative RT-PCR, the expression level of several P53 target genes (BAX, NOXA1, P21 and MDM2) in sorted granulocytic cells (Figure 2D). The patient displayed a higher expression level of P53 target genes than in con- trol and the activation was more pronounced in the more mature granulocytic CD33+CD15+CD11b+ cells. However, we could not definitively exclude P53-inde- pendent pathways that also lead to cell cycle arrest and apoptosis.15 Of note, P53-independent nucleolar stress has been reported in animal models of bone marrow fail- ure syndromes caused by impaired ribosomal biogenesis and function.15
Besides the profound neutropenia, this patient present- ed transient severe anemia and thrombocytopenia as also reported in patients harboring SRP54 and SRP72 defects.3,6 We could hypothesize that an aberrant SRP complex, regardless of the implicated defective SRP pro- tein, may have consequences on the targeting of proteins essential for hematopoietic stem cells or erythroid, megakaryocytic and granuloytic progenitor/precursors. Further studies are needed to characterize the mecha- nisms leading to impaired differentiation of hematopoiet- ic lineages and to identify the protein partners of the SRP complex.
In conclusion, we have identified a novel genetic entity of CN affecting another protein of the SRP complex underlying the major implication of the universally con- served co-translational targeting machinery of proteins in the pathogenesis of CN. These first observations show that loss-of-function SRP58 variants trigger an ER stress resulting in an increased P53-dependent apoptosis of granulocytic precursors and neutrophils.
Barbara Schmaltz-Panneau,1,2 Anne Pagnier,3
Séverine Clauin,4 Julien Buratti,3 Caroline Marty,1,2
Odile Fenneteau,5 Klaus Dieterich,6 Blandine Beaupain,7,8 Jean Donadieu,7,8,9 Isabelle Plo1,2
and Christine Bellanné-Chantelot1,4,8
1Gustave Roussy Cancer Center, INSERM U1287, Villejuif;
2Paris Saclay University, U1287, Villejuif; 3Department of Pediatric Hematology and Oncology, CHU Grenoble Alpes, Grenoble, GIN, Grenoble; 4AP-HP, Pitié-Salpêtrière Hospital, DMU BioGeM, Department of Genetics, Sorbonne University, Paris; 5AP-HP, Robert Debré Hospital, Laboratory of Hematology, University of Paris, Paris; 6Department of Medical Genetics, Univsersity Grenoble Alpes, INSERM U1216, CHU Grenoble Alpes, GIN, Grenoble; 7French Registry of Chronic Neutropenia, Trousseau Hospital, Paris; 8Reference Center for Chronic Neutropenia, Paris and 9AP-HP, Trousseau Hospital, Department of Pediatric Hematology and Oncology, Paris, France
Correspondence: CHRISTINE BELLANNE’-CHANTELOT christine.bellanne-chantelot@aphp.fr
doi:10.3324/haematol.2020.247825
Disclosures: no conflicts of interest to disclose
Contributions: BSP performed most of the research and analyzed functional data; AP provided samples and clinical data; SC performed and analyzed molecular experiments; JB performed bioinformatics analysis; CM performed fibroblast culture and western blot;
OF performed cytological analysis; KD provided samples and clinical data; BB collected clinical data; JD involved in the clinical part and contributed to intellectual input; IP designed the study, analyzed the data and critically reviewed the paper; CB-C designed the study, analyzed the data and wrote the paper.
Acknowledgments: the authors would like to thank the family involved in the study. The authors also thank the Cytometry Platform (PFIC) of Gustave Roussy, especially Philippe Rameau and Yann Lecluse.
Funding: the whole-exome sequencing was funded by the Foundation for rare diseases (AO9102LS) and the research was supported by grants from INCA-PLBIO 2017 (I Plo). The French Registry is supported by grants from X4 pharma, Prolong Pharma and Chugai SA to BB and JD.
References
1.Donadieu J, Beaupain B, Fenneteau O, Bellanne-Chantelot C. Congenital neutropenia in the era of genomics: classification, diagno- sis, and natural history. Br J Haematol. 2017;179 (4):557-574.
2. Carapito R, Konantz M, Paillard C, et al. Mutations in signal recog- nition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features. J Clin Invest. 2017;127 11):4090-4103.
3.Bellanne-Chantelot C, Schmaltz-Panneau B, Marty C, et al. Mutations in the SRP54 gene cause severe congenital neutropenia as well as Shwachman-Diamond-like syndrome. Blood. 2018; 132(12):1318-1331.
4. Wild K, Juaire KD, Soni K, et al. Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions. Nucleic Acids Res. 2019;47 (6):3184-3196.
5.Focia PJ, Shepotinovskaya IV, Seidler JA, Freymann DM. Heterodimeric GTPase core of the SRP targeting complex. Science. 2004;303(5656):373-377.
6. Kirwan M, Walne AJ, Plagnol V, et al. Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial apla- sia and myelodysplasia. Am J Hum Genet. 2012;90(5):888-892.
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