Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim

Abstract

Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited disorder characterized at birth by thrombocytopenia with reduced megakaryocytes, which evolves into generalized bone marrow aplasia during childhood. Although CAMT is genetically heterogeneous, mutations of MPL, the gene encoding for the receptor of thrombopoietin (THPO), are the only known disease-causing alterations. We identified a family with three children affected with CAMT caused by a homozygous mutation (p.R119C) of the THPO gene. Functional studies showed that p.R119C affects not only ability of the cytokine to stimulate MPL but also its release, which is consistent with the relatively low serum THPO levels measured in patients. In all the three affected children, treatment with the THPO-mimetic romiplostim induced trilineage hematological responses, remission of bleeding and infections, and transfusion independence, which were maintained after up to 6.5 years of observation. Recognizing patients with THPO mutations among those with juvenile bone marrow failure is essential to provide them with appropriate substitutive therapy and prevent the use of invasive and unnecessary treatments, such as hematopoietic stem cell transplantation or immunosuppression.

Keywords: MPL; congenital amegakaryocytic thrombocytopenia; mutation; romiplostim; thrombopoietin.

Figure 1. Time course of hemoglobin level (Hb), white blood cell count (WBC), and platelet count (Plt) in the propositus before and during romiplostim administration

Romiplostim was started at the dosage of 1 μg/kg/week, and this schedule was maintained for 3 months. Then, a dose of 4 μg/kg was given in a monthly administration. The platelet count had wide oscillations that derived, at least partially, from the variability of the time elapsed between the drug administrations and the measurements of blood count.

Figure EV1. Identification of the p.R119C mutation of THPO

1. Family pedigree indicating the genotype and phenotype of the family members. “+” and “−” indicate the wild‐type and the mutant (c.355C>T; p.R119C) allele, respectively. Different symbols indicate the presence of severe thrombocytopenia, anemia, neutropenia, or infections, as shown in the figure.

2. Electropherogram showing the homozygous c.355C>T mutation in the proband.

Figure EV2. R119 is highly conserved, and the p.R119C is expected to affect THPO binding to its receptor

1. Multiple‐sequence alignment analysis of THPO orthologs from different species. Hs, Homo sapiens (NP_000451.1); Bt, Bos taurus (NP_001159512.1); Oa, Ovis aries (XP_011984347.1); Cl, Canis lupus familiaris (XP_005639837.1); Fc, Felis catus (NP_001157128.1); Mm, Mus musculus (NP_033405.1); Rn, Rattus norvegicus (NP_112395.1). Residues in bold are conserved among both THPO and EPO orthologs. Residues in gray are not conserved within THPO or EPO.

2. Cartoon representation of the structure of the receptor binding domain of THPO (in sky blue) in complex with a neutralizing antibody fragment (in magenta) (PDB 1V7M) (Feese et al, 2004). The inset shows a zoom of the interaction involving R119 (in red) with the D31 of the antibody fragment (in green).

3. Multiple‐sequence alignment analysis of EPO orthologs from different species. Hs, Homo sapiens (NP_000790.2); Bt, Bos taurus (NP_776334.1); Oa, Ovis aries (NP_001019908.1); Cl, Canis lupus familiaris (NP_001006647.1); Fc, Felis catus (NP_001009269.1); Mm, Mus musculus (NP_031968.1); Rn, Rattus norvegicus (NP_058697.1). Residues in bold are conserved among both THPO and EPO orthologs. Residues in gray are not conserved within THPO or EPO.

4. Cartoon representation of the structure of the receptor binding domain of EPO (in violet) in complex with its receptor (in green) (PDB 1EER) (Syed et al, 1998). The inset shows a zoom of the cluster salt bridge interactions involving R103 (in red), the analogous of R119 in THPO.

Figure 2. Transfection of constructs harboring the wild‐type or mutant THPO cDNAs induced efficient expression of THPO proteins in HEK293T cells

1. Representative images of immunoblotting of HEK293T cell lysates prepared 24 and 48 h after transfection with wild‐type (WT) or mutant (p.R119C and p.R38C) THPO‐expressing or empty vectors and then pooled into a ratio of 1:1 (Dasouki et al, 2013). THPO cDNAs were tagged with the FLAG epitope. β‐Actin was used as loading control.

2. Densitometric analysis of the bands obtained by immunoblotting of HEK293T cell lysates prepared 24 and 48 h after transfection and then pooled into a ratio of 1:1. Immunoblotting was performed on cells collected after n = 3 independent transfection experiments. THPO levels are expressed as FLAG/β‐actin ratio and THPO/β‐actin ratio.

3. Representative images of immunoblotting of HEK293T cell lysates prepared 24 and 48 h after transfection with wild‐type or mutant THPO‐expressing or empty vectors and then analyzed separately (24 and 48 h).

4. Densitometric analysis of the bands obtained by immunoblotting of HEK293T cell lysates prepared 24 and 48 h after transfection and then analyzed separately. Immunoblotting was performed on cells collected after n = 3 independent transfection experiments. Samples derive from the same experiment, and blots were processed in parallel.

Data information: In (B, D), data are presented as means ± SD. The asterisk (*) indicates a P‐value < 0.05 with respect to WT (two‐tailed Student's t‐test). The exact P‐values are reported in Appendix Table S1. Source data are available online for this figure.

Figure 3. The mutant THPO‐conditioned culture supernatants are markedly deficient in sustaining proliferation of UT7‐TPO cells

Cell proliferation measured in n = 3 independent MTT assays and expressed as absorbance units of UT7‐TPO cells incubated with medium containing 0.5 or 1.0 μl of THPO‐conditioned supernatants from HEK293T cells transfected with wild‐type (WT) or mutant (p.R119C and p.R38C) THPO‐expressing or empty vectors. Recombinant human THPO (rHuTHPO) at the concentration of 10 ng/ml was also used as control. Data are presented as means ± SD. The asterisk (*) indicates a P‐value < 0.01 with respect to WT (two‐tailed Student's t‐test). The exact P‐values are reported in Appendix Table S2. Source data are available online for this figure.

Figure 4. The p.R119C and p.R38C THPO proteins are stable as the wild‐type THPO

1. Representative images of immunoblotting of HEK293T cells transfected with wild‐type or mutant (p.R119C and p.R38C) THPO‐expressing vectors. Cell lysates were prepared just before treatment with cycloheximide (CHX) (time 0) and 8, 24, and 48 h after the addition of CHX in order to block de novo protein synthesis (CHX chase assay). DMSO alone was used as control in the WT sample. GAPDH was used as loading control.

2. Densitometric analysis of the bands obtained in n = 3 independent experiments. THPO amount was measured as THPO/GAPDH ratio and expressed as the percentage of the amount measured at time 0 in each condition (Relative protein, %). Of note, the expression of wild‐type THPO after CHX treatment was significantly lower compared with DMSO alone at each time point (P < 0.05, two‐tailed Student's t‐test), confirming that protein synthesis was efficiently blocked by CHX treatment. Data are presented as mean ± SD. The exact P‐values are reported in Appendix Table S3.

Source data are available online for this figure.

Figure 5. The p.R119C and p.R38C variants result in reduced functional activity of THPO protein

Cell proliferation measured in n = 3 independent MTT assays and expressed as absorbance units of UT7‐TPO cells incubated with medium containing different concentrations (60, 120, or 240 pg/ml) of THPO obtained from HEK293T cells supernatants transfected with wild‐type (WT) or mutant (p.R119C and p.R38C) THPO‐expressing or empty vectors. Data are presented as means ± SD. The asterisk (*) indicates a P‐value < 0.05, while (**) indicates a P‐value < 0.01 with respect to WT (two‐tailed Student's t‐test). The exact P‐values are reported in Appendix Table S4. Source data are available online for this figure.

Figure 6. Defective activation of signaling kinases downstream of the MPL receptor

1. Representative images of immunoblotting of UT7‐TPO cells recapitulating conditions of the cell proliferation assays shown in Fig 3 (incubation with 0.5 μl of HEK293T wild‐type or mutant supernatants) and Fig 5 (incubation with 240 pg/ml of THPO from HEK293T wild‐type or mutant supernatants). The supernatant (0.5 μl) obtained after transfection of the empty vector (EV) was used as a negative control. Commercial recombinant human THPO (rHuTHPO) at 10 ng/ml was used as the positive control condition. The analysis investigated the phosphorylated forms of STAT5, ERK, and AKT (p‐STAT5, p‐ERK, and p‐AKT) and the total form of the three kinases (STAT5, ERK, and AKT). GAPDH was used as loading control.

2. Densitometric analysis of the bands obtained in n = 3 independent experiments. Protein phosphorylation was expressed as the ratio between the phosphorylated and the total forms of each protein. Data are presented as means ± SD. The asterisk (*) indicates a P‐value < 0.05, while (**) indicates a P‐value < 0.01 with respect to WT (two‐tailed Student's t‐test). The exact P‐values are reported in Appendix Table S5.

Source data are available online for this figure.