Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice

Abstract

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.

Graphical abstract

Figure 1.

cMTFM is due to mutations in G6b-B (G6b). (A) Family pedigrees. Black arrows point to the probands in each family. Double line indicates a consanguineous relationship. Ages are provided at the time of the initial evaluation of the youngest affected individual in the family. (B) Ideograms showing cMTFM linkage region, the G6b (MPIG6B) locus and patient mutations. (C) Expression of p.Gly157Arg mutant in DT40 cells leads to protein instability and (D-E) reduced expression on the surface of the cell (mean ± standard error of the mean [SEM]; n = 2). pcDNA3 vector, blue; G6b-B WT, green; G6b-B p.Gly157Arg, red. Representative blots and histograms of 2 independent experiments. MFI, median fluorescence intensity.

Figure 2.

Pathology of cMTFM. Wright-Giemsa–stained peripheral blood smears from (A) a normal control, (B) patient 3-II-4 and (C) the clinically unaffected, genotypically G6b-mutated individual 1-III-9 (original magnification ×1000). Note the large, hypogranular platelets (arrow) and RBC anisocytosis in 3-II-4 and the rare giant platelet in 1-III-9 (arrow). Serial histologic sections of a bone marrow biopsies from (D,G,J,M) a normal control individual, (E,H,K,N) 3-II-4, and (F,I,L,O) 1-III-9 stained with (D-F) H&E (original magnification ×400), (G-I) Reticulin (original magnification ×600), (J-L) the megakaryocytic marker CD61 (original magnification ×400), and (M-O) G6b-B (original magnification ×400). Atypical megakaryocytes present in stellate clusters associated with increased reticulin staining are characteristic of cMTMF. Staining for G6b-B is entirely negative in the megakaryocytes and platelets from 3-II-4 and 1-III-9.

Figure 3.

Expression of G6b isoforms in humanized mouse model. (A) WT, G6b^+/hu, G6b^hu/hu, human and G6b^−/− washed platelet lysates (4 × 10⁸/mL), were resolved by SDS-PAGE and custom antibodies used to detect mouse G6b-B (mG6b-B), human G6b-B (hG6b-B ), and hG6b-A. Expression was quantified and normalized to (i,ii) WT or (iii,iv) human lysates, to show relevant expression levels in the mouse models. (B) Quantification of glycosylated and unglycosylated human G6b-A and -B, data normalized to show percentage of total expression. (C) hG6b-B was immunoprecipitated (IP) from basal and collagen-activated G6b^hu/hu and human washed platelet lysates (4 × 10⁸/mL). Co-IP of Shp1 and Shp2 was investigated by (i) SDS-PAGE before (ii) quantification and normalization to hG6b-B levels. Quantification was completed using Licor Odyssey system. All data represented as mean ± SEM (n = 3-4). ***P < .001.

Figure 4.

Minor alterations of G6b^hu/huplatelet functional responses. (A) Averaged aggregation and ATP release traces for washed platelets (2 × 10⁸/mL) activated with indicated concentrations of CRP (i) and anti-CLEC-2 antibody (Ab) (ii). Area under the curve (AUC) quantification of (iii) platelet aggregation and (iv) ATP release (n = 5-6 per condition, **P < .01 and *P < .05). (B) Heparin-PPACK-fragmin anticoagulated whole blood was flowed over glass coverslips coated with collagen immediately following collection. (i) Brightfield images were immediately collected, followed by staining with PE-conjugated JON/A, FITC-conjugated P-selectin, and Alexa 647–conjugated Annexin V and fluorescence imaging. Analysis quantifying (ii-vi) thrombus surface area coverage and morphological scores of adhered platelets, (n = 5-6; ***P < .001; **P < .01) and (vii-ix) surface area coverage of fluorescently labeled antibodies (n = 5-6; ***P < .001; *P < .05). (C) Total blood loss to body weight ratio of mice following excision of tail tip (n = 12-16; ***P < .001; **P < .01). All data collection and analysis in panels B and C were completed blinded, mean ± SEM. Scale bar, 10 μm.

Figure 5.

Altered tyrosine phosphorylation in response to GPVI and CLEC-2 agonists in G6b^hu/huplatelets. Representative blots (n = 3) of lysates prepared from washed platelets (4 × 10⁸/mL) activated with indicated concentrations of (Ai) CRP (90 seconds, 10 μM lotrafiban, 10 μM indomethacin, and 2 U/mL apyrase) or (Bi) CLEC-2 Ab (300 seconds, 10 μM lotrafiban) and probed with the indicated antibodies. (Aii,Bii) Src family kinase (SFK) phosphotyrosine 418 (pTyr418) and (Aiii,Biii) Syk pTyr519/20 were quantified using ImageJ and normalized to total tubulin and Syk reblots, respectively. WT, blue; G6b^hu/hu, red.

Figure 6.

Further reduction in human G6b-B expression exacerbates observed phenotype. (Ai) G6b^hu/hu mice were crossed with G6b^−/− mice to produce hemizygous (G6b^hu/−) mice. (Aii) Representative blots of mouse G6b-B (mG6b-B), human G6b-B (hG6b-B) and hG6b-A and (Aiii) quantification of hG6b-B expression in the indicated genotypes (n = 3). (B) Average aggregation and ATP release traces (Bi) and quantification of AUC (Bii,iii) investigating functional response of G6b^hu/− mouse washed platelets (2 × 10⁸/mL) in response to indicated agonists (n = 5-6; ***P < .001; *P < .05). All bar graphs presented as mean ± SEM.