FIGURE 1

Clinicopathological features of patients with biallelic loss of function variants in SH2B3. (A) Pedigree for patients 1 and 2; half‐filled squares and circles indicate carrier status for SH2B3 variants for males and females, respectively. Fully filled squares indicate homozygous SH2B3. n.t. – not tested. Double line indicates consanguinity. (B) Bone marrow aspirate from patient 1 demonstrating hypercellular marrow with megakaryocytic hyperplasia and morphological atypia (×100). (C) Bone marrow trephine biopsy from patient 2 demonstrated megakaryocytic hyperplasia and atypia (haematoxylin and eosin (H&E), ×400). (D) SH2B3 expression assessed intracellularly by flow cytometry on skin‐derived fibroblasts using a mouse anti‐human SH2B3 antibody followed by goat anti‐mouse IgG‐AlexaFluor488 staining after fixation and permeabilization using the BD Cytofix/CytoPerm buffers. Immunoreactive SH2B3 expression is present in wildtype healthy control (HC) and proband (V402M) cells. Negative control is secondary antibody (Ab) only. Numbers inside panels represent mean fluorescence intensity (MFI). (E) Western blot showing normal expression of SH2B3 in V402M skin‐derived fibroblasts; GADPH loading control. (F) Basal STAT‐5 and STAT‐3 phosphorylation was evaluated on skin‐derived fibroblasts by Phosflow assay using BD Phosflow Fix I and Perm III buffers according to manufacturer instructions (BD Biosciences). Histograms on the left show a representative example and graphs on the right the normalised mean values for four experiments. Numbers inside histograms represent mean fluorescence intensity (MFI). pSTAT3 and pSTAT5 levels were significantly elevated relative to control in the proband (V402M) samples (*indicates p < 0.05, Mann–Whitney test). (G) Increased phosphorylation of JAK2 and STAT5 in SH2B3‐V402M. Skin‐derived fibroblasts were obtained from patient 2 and from a healthy control were assessed for SH2B3, pJAK2, pSTAT3 and pSTAT5 protein expression by immunoblotting before and after 15‐min stimulation with the indicated ligands (optical density for experiment and replicates provided in histogram). Black lines next to samples in lanes 13 and 14 (pSTAT5 and GAPDH) indicate where images have been moved in the figure to allow comparison between healthy control versus patient. Graphs on the right show quantitation of multiple immunoblots with lines connecting paired samples. EPO, erythropoietin; GH, growth hormone; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; HC, healthy control; IL‐3, interleukin‐3.