Evolution of hematologic values in the 3 patients, cytological analysis of bone marrow smears, and in vitro erythrocyte differentiation.

Variation of hemoglobin (g/dL) (A), hematocrit (%) (B), RBC (C), platelets (D), and neutrophil counts (E), in P1, P2, and P3 according to age. Gray zone indicates normal ranges in healthy individuals. The RBC counts were low in patients, even after RBC transfusion. (F) Cytological findings of bone marrow smears in P1 and P2. Irregular budding nucleus (red arrow), internuclear bridges (black arrow), acidophilic erythroblasts (green arrow), and polychromatophilic erythroblasts (orange arrow) were detected. Dysgranulopoiesis was observed in P1 as several metamyelocytes did not contain any granules (blue arrow). The slides were stained with Wright-Giemsa and H&E. Original magnification, ×100; ×175 (zoomed images). Abnormal cells are zoomed-in on the right. (G) In vitro differentiation of HSPCs into erythroblasts. Top: CD235a/CD45 staining after 7 days of culture in erythroid conditions of HSPCs from 2 healthy controls and patients P1 and P2. The red rectangle indicates the gating strategy on CD235a⁺CD45^– erythroid cells. Bottom; CD36/CD71 staining of CD235a⁺CD45^– cells. The red rectangle indicates CD36⁺CD71⁺ erythroblast cells.

Identification of a homozygous OSM mutation in the patients.

(A) Family pedigree. (B) Direct Sanger sequencing of the OSM gene in family members. Red arrowheads indicate the position of the 1 bp insertion causing a frameshift (c.507_508insG; Arg170AlafsTer124). (C) Schematic of the OSM gene showing the position of the frameshift leading to the production of a neo coding sequence (purple). (D) Protein sequences of the WT and mutated forms of OSM. Sequence in purple corresponds to the neopeptide generated by the frameshift. The red box highlights the FxxK motif critical to interact with OSMR and LIFR. (E) Domain architecture of the immature and mature forms of human OSM protein and the OSM mutated protein. Orange portion highlights the part that is missing in the mutated form of OSM and replaced by the neopeptide generated by the frameshift (in purple). Site 3 FxxK corresponds to the motif that interacts with OSMR and LIFR. (F) AlphaFold2 model of the 3D structure of WT human type II OSMR complex (OSM (UniProt P13725, aa 25-252) in complex with OSMR (UniProt Q99650, aa 141–330) and gp130 (UniProt P40189, aa 124–323). Details of the AF2 modeling are given in Supplemental Figure 6. The helices of the OSM 4-helix bundle are labeled. The phenylalanine and lysine of the FxxK motif are shown in a sphere representation (red) as well as hydrophobic amino acids of helix D (orange). The orange and red parts correspond to the missing domains in the OSMfs mutant created by the c.507_508insG variant.

Functional consequence of the OSM mutation.

(A) IL-6 concentration measured by ELISA in supernatants from LPS-activated monocytes from the healthy sister and P3. (B) OSM concentration measured by ELISA in supernatants from LPS-activated monocytes from the healthy sister and P3. (C) Schematic representation of the protocol used to measure OSM production by ELISA in supernatant from EPO-activated UT7 cells and its signaling detection in HUH7. OSM induces STAT3 phosphorylation (pSTAT3) in HUH7 as detected by FACS. (D) OSM concentration measured by ELISA in supernatants from EPO-activated WT UT7 cell line, the UT7 clone carrying the frameshift in the OSM sequence (clone C7), and 2 WT UT7 clones (C3 and G8). (E) Left: Representative FACS result demonstrating that rhOSM, used as positive control, induced a shift in STAT3 phosphorylation signal in HUH7 cells. Right: Quantitative analysis of 3 independent replicates. Statistics derived from unpaired t tests with α = 0.05. *P < 0.05. (F) Representative FACS results of p-STAT3 detection in HUH7 cells in the presence of indicated supernatants. (G) p-STAT3 induction obtained with the supernatants from EPO-activated WT UT7 cell line, the UT7 clone carrying the frameshift in the OSM sequence (clone C7), and 2 WT UT7 clones (C3 and G8). Quantitative analysis of 3 independent replicates. Statistics derived from unpaired t tests with α = 0.05. ****P < 0.0001.

Osm-morphants show erythropoiesis defect.

(A) globin:GFP embryos were injected with control- or osm- morpholino (MO), and observed by fluorescence microscopy to measure their content in erythrocytes. Dashed rectangles highlight the part of zebrafish in which the cells are detected. Numbers in bottom left of images indicate the number of embryos displaying the indicated phenotype out of the total number of embryos analyzed. Scale bars: 100 μm. (B) The strong reduction of circulating erythrocytes (detected by gata1:DsRed marker) in osm-MO is rescued by overexpressing osm (noted + osm mRNA). Numbers in bottom left of images indicate the number of embryos displaying the indicated phenotype out of the total number of embryos analyzed. Scale bars: 200 μm. (C) 23 hpf globin:GFP embryos were stained for phospho-histone 3 (pH3), a marker of proliferation. Arrows indicate colocalization of pH3 and globin:GFP. Scale bar: 100 μm. (D) Fewer globin:GFP/pH3 double-positive cells are detected in osm-morphants, demonstrating a decrease in proliferating erythroid progenitors. Statistics derived from unpaired t tests with α = 0.05. **P < 0.01. (E) Quantitative PCR performed on sorted erythrocytes (GFP⁺ cells) at different stages show that they express high levels of osmr and osm transcripts. (F) Left: mpx:GFP embryos were injected with control- or osm- morpholino (MO) and observed by fluorescence microscopy to measure their content in neutrophils. Right: Fewer mpx:GFP cells are detected in osm-morphants, demonstrating a decrease in neutrophils. Statistics derived from unpaired t tests with α = 0.05. **P < 0.01.

OSM deficiency as a novel inherited bone marrow failure syndrome.

Schematic representation of the complexity of the signaling pathways of OSM and OSMRb and the diseases associated with their deficiency in humans. The OMIM number for each disease is shown.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.