Compound heterozygous c-Mpl mutations in a child with congenital amegakaryocytic thrombocytopenia: functional characterization and a review of the literature

Abstract

Objective: To genetically and functionally characterize mutations of c-Mpl that lead to thrombocytopenia in a child with congenital amegakaryocytic thrombocytopenia.

Materials and methods: We identified two c-Mpl mutations in a child with clinical features of congenital amegakaryocytic thrombocytopenia, one a previously described mutation in the extracellular domain (R102P) and the other a novel mutation leading to truncation of the receptor after the box 1 homology domain (541Stop). Cell line models were created to examine the ability of the mutant receptors to signal in response to thrombopoietin and thrombopoietin-like agonists.

Results: Data from cell-line models indicate that c-Mpl R102P does not support significant signaling in response to thrombopoietin due to impaired trafficking of the mutant receptor to the cell surface. Alternative thrombopoietic agents do not circumvent this block to signaling, likely due to the inaccessibility of the receptor. In addition, previous data indicate that c-Mpl 541Stop does not support intracellular signaling due to the loss of critical intracellular domains.

Conclusions: This case demonstrates two different mechanisms by which c-Mpl mutations can impair thrombopoietin signaling, and suggests that mutations in the extracellular domain will not be rescued by c-Mpl agonists if they interfere with normal receptor expression.

Figure 1

Progression of bone marrow failure in a child with CAMT. (A) Bone marrow aspiration performed at 3 months of age shows 2 megakaryocytes in a cellular particle with background hematopoiesis including erythroid and myeloid precursors without dysplasia. (B) Bone marrow aspirate 20 months later shows a hypocellular particle with no megakaryocytes and few scattered lymphocytes and histiocytes.

Figure 2

Compound heterozygosity for mutations in exons 3 and 11 of c-Mpl. Analysis of the sequence from the proband reveals a G305>C mutation in exon 3 (confirmed to be derived from the father), which results in the amino acid substitution R102P. The second mutation, C1621>T, creates a stop codon in exon 11 and is derived from the mother.

Figure 3

BaF3 cells expressing R102P c-Mpl do not respond to TPO or a thrombopoietin mimetic. (A) Fourteen independent clones of BaF3 cells expressing R102P c-Mpl were washed of IL-3 and tested for growth in maximal IL-3, 5 ng/ml rhTPO, and 3 μM LGD4665. After 48 h, cells were lysed and an MTT assay was performed. Results are expressed relative to maximal growth in IL-3. Points were performed in triplicate. Error bars represent SEM. Growth of control cells expressing WT c-Mpl under the same conditions is shown for comparison. (B) BaF3 cells expressing WT c-Mpl or 2 representative clones of BaF3 cells expressing R102P c-Mpl were starved overnight and then stimulated with 50 ng/ml rhTPO for 15 min, lysed and assayed for phosphorylation of Akt and Erk by immunoblotting. Blots were reprobed for actin as a loading control. Blot shown is representative of 2 independent experiments.

Figure 3

BaF3 cells expressing R102P c-Mpl do not respond to TPO or a thrombopoietin mimetic. (A) Fourteen independent clones of BaF3 cells expressing R102P c-Mpl were washed of IL-3 and tested for growth in maximal IL-3, 5 ng/ml rhTPO, and 3 μM LGD4665. After 48 h, cells were lysed and an MTT assay was performed. Results are expressed relative to maximal growth in IL-3. Points were performed in triplicate. Error bars represent SEM. Growth of control cells expressing WT c-Mpl under the same conditions is shown for comparison. (B) BaF3 cells expressing WT c-Mpl or 2 representative clones of BaF3 cells expressing R102P c-Mpl were starved overnight and then stimulated with 50 ng/ml rhTPO for 15 min, lysed and assayed for phosphorylation of Akt and Erk by immunoblotting. Blots were reprobed for actin as a loading control. Blot shown is representative of 2 independent experiments.

Figure 4

R102K c-Mpl is a conservative change that does not significantly impair the function of the receptor. Three independent clones of BaF3 cells expressing R102K c-Mpl were starved of IL-3 overnight and stimulated with increasing concentrations of rhTPO and growth was analyzed by MTT at 48 hr. Results are expressed as relative to maximal growth in IL-3. Growth of cells expressing WT c-Mpl (leftmost grouping) or R102P c-Mpl (rightmost grouping) under the same conditions is shown for comparison. Points were performed in triplicate. Graph shown is representative of 2 independent experiments.

Figure 5

R102P c-Mpl does not bind TPO as efficiently as WT c-Mpl. To compare the ability of mutant and WT receptors to bind TPO, we incubated BaF3 parental cells, BaF3 cells expressing WT c-Mpl and 2 clones if BaF3 cells expressing R102P c-Mpl in medium containing fixed amounts of rhTPO and measured the ability of the cells to deplete the cytokine from the culture medium. 2×10⁷ cells were washed of IL-3 and cultured with 0.75 ng/ml rhTPO at 37°C for 30 min. TPO levels were measured in duplicate and are expressed as percentage of input TPO remaining as compared to parental BaF3 cells (which should have no specific uptake). Uptake of rhTPO by cells expressing WT c-Mpl is significantly more than that of parental cells (p=0.0012), but there is no significant difference in rhTPO uptake between parental cells and cells expressing R102P c-Mpl. Results shown are the mean of three experiments.

Figure 6

R102P c-Mpl is not normally glycosylated and is poorly expressed on the cell surface. (A) On western blotting, WT c-Mpl is usually seen as two bands: a more slowly migrating glycosylated protein that represents mature receptor (85 kD), and a more rapidly migrating, immature form (80 kD). Cells expressing R102P c-Mpl only produce the 80 kD, immature form of the receptor, whereas cells expressing R102K c-Mpl produce both forms of the receptor. (B) BaF3 cells expressing WT c-Mpl, R102P c-Mpl or R102K c-Mpl (as proven by western blotting) were incubated with an antibody that is specific for the extracellular domain of the receptor and analyzed by flow cytometry. Compared to the WT control, surface receptor is not detectable in cells expressing R102P c-Mpl but is restored in cells expressing R102K c-Mpl. Parental BaF3 cells serve as the negative control for nonspecific antibody binding.