Diverse Mpl expression pattern among pedigrees with inherited thrombocytopenia: potential diagnostic and therapeutic implications.

HELLER PAULA G; GLEMBOTSKY ANA C; GOETTE NORA P; MARTA ROSANA F; LEV PAOLA R; MOLINAS FELISA C

Journal of Thrombosis and Haemostasis

Wiley InterScience

Año: 2008 vol. 6 p. 2215 - 2217

Diverse Mpl expression pattern among pedigrees with inherited thrombocytopenia: potential diagnostic and therapeutic implications P . G. HELLER, A. C. GLEMBOTSKY, N. P . GOETTE, R. F. MARTA, P. R. LEV and F . C. MOLINAS Hematologı´a Investigacio´ n, Instituto de Investigaciones Me´dicas Alfredo Lanari, Universidad de Buenos Aires, Buenos Aires, Argentina To cite this article: Heller PG, Glembotsky AC, Goette NP, Marta RF, Lev PR, Molinas FC. Diverse Mpl expression pattern among pedigrees with inherited thrombocytopenia: potential diagnostic and therapeutic implications. J Thromb Haemost 2008; 6: 2215–7. Inherited thrombocytopenias (IT) comprise a large group of heterogeneous disorders which differ in several biologic, molecular and clinical features. Impaired platelet production underlies the majority of disorders included among IT, although in certain cases, shortened platelet survival contributes to the pathogenesis of thrombocytopenia. Most of the genetic defects identified so far involve genes coding for megakaryocyte transcription factors, cytokine receptors or cytoskeleton proteins [1]. However, although significant advances in the understanding of the molecular basis of IT have been made in recent years, the mechanisms underlying Correspondence: Paula G. Heller, Instituto de Investigaciones Me´dicas Alfredo Lanari, Universidad de Buenos Aires, Hematologı´a Investigacio´ n, Buenos Aires, Argentina. Tel.: +54 11 45238947; fax: +54 11 45238947. E-mail: paulaheller@hotmail.com DOI: 10.1111/j.1538-7836.2008.03159.x Received 1 September 2008, accepted 13 September 2008 Letters to the Editor 2215  2008 International Society on Thrombosis and Haemostasis thrombocytopenia remain in many cases unknown. Considering that IT involves platelet production disorders and that thrombopoietin (TPO) is the main cytokine stimulating megakaryopoiesis [2], we investigated whether abnormalities in the TPO/Mpl axis are implicated in the pathogenesis of thrombocytopenia in IT. Eighteen patients belonging to eight IT pedigrees, including one previously studied [3], aged 33 (7–70) years, platelet counts 73 (10–148) · 109 L)1, and 12 patients with immune thrombocytopenic purpura (ITP) were included; pedigree I, X-linked thrombocytopenia with WASP mutation (XLT); II and III, MYH9-related disorder (MYH9-RD); IV and V, autosomal dominant macrothrombocytopenia (ADMacro); VI and VII, IT with normal platelet volume, characterized by recessive and dominant inheritance, respectively; VIII, familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/ AML)[3]. Platelet surfaceMpl expression wasmeasured byflow cytometry using an anti-Mpl antibody (Kirin Brewery, Tokyo, Japan), as previously described [3].Mean fluorescence intensity ratio between Mpl and an isotype control revealed decreased Mpl in IT compared with controls, 1.35 ± 0.34 vs. 1.70 ± 0.27, P = 0.01, Students t-test. Total platelet Mpl content was analyzed by Western blot with an anti-Mpl antibody followed by reprobing with an anti-b3 integrin antibody and a ratio between them was expressed as percentage of controls, set as 100%, as previously described [3]. In accordance with low surface Mpl, total Mpl expression was decreased in IT,whereas itwas normal or increased in ITP,Mpl/ b3 ratio was 57.8 ± 38% vs. 137.4 ± 49%, P = 0.0001, Students t-test. Correlation was found between surface and total Mpl, P = 0.0004, Spearmans rank test. However, expression pattern was variable among different pedigrees, as follows, pedigrees I (XLT), II-III (MYH9-RD) and IV-V (ADMacro) showed normal Mpl levels, whereas the receptor was decreased in pedigrees VI-VII (IT with nomal platelet volume), Fig. 1A, and VIII (FPD/AML). Levels in the last three pedigrees were 30.5 ± 17%, and, overall,Mpl was lower than 50% in 44% of IT patients. Plasma TPO levels, measured by enzyme-linked immunosorbent assay (R&D, Minneapolis, MN, USA), were mildly to moderately elevated in IT and ITP compared with controls, 99.2 (0–333) pg mL)1 and 65.7 (15–264) pg mL)1 vs. 0 (0–32.1) pg mL)1, P < 0.0001, Kruskal–Wallis test. No correlation was found between TPO, Mpl and platelet counts. In order to investigate the functional effects of low Mpl levels, we evaluated the in vitro response to TPO, regarding potentiation of platelet aggregation and phosphorylation. TPO-triggered aggregation was studied in patients who displayed normal platelet aggregation tests. First, platelet-rich plasma (PRP) was incubated with 0.5–1.5 lM ADP to induce primary aggregation. Next, platelets were incubated with 100 ng mL)1 thrombopoietin (R&D) before addition of ADP and a ratio between tracings with and without TPO (TPO+ADP/ADP) was determined. Phosphorylation pattern was evaluated in samples stimulated with 100 ng mL)1 thrombopoietin by Western blot using an anti-phosphotyrosine- HRP-conjugated antibody (Amersham, Buckinghamshire, UK), as described [3]. Patients with normal Mpl displayed normal response to TPO, showing either normal TPO-induced aggregation [TPO+ADP/ADP in pedigrees III and IV vs. controls was 2.9 (2.4–3.9) vs. 2.7 (2.3–5.3)], or phosphorylation (pedigree V). Among those with decreased Mpl, three out of four showed an impaired response to TPO, which included decreased phosphorylation in pedigree VI and both reduced aggregation (TPO+ADP/ADP ratio 1.19–1.34) and phosphorylation in pedigree VII. To determine the mechanisms underlying low receptor expression, RNA was isolated from leukocyte-depleted platelets (leukocyte reduction filter; Pall Co., New York, NY, USA) and MPL mRNA levels relative to GAPDH were measured in triplicate by real-time PCR using IQTMSYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Mean CT was calculated and fold change in mRNA was expressed relative to controls using the DDCT method. Among patients with low Mpl protein, a 10.4- and 3.5-fold decrease in mRNA was found in both patients from pedigree VI, respectively, Fig. 1B, whereas mRNA was normal in pedigree VII. The coding region and intron–exon junctions of the MPL gene were normal by sequence analysis (ABI 3100/3730XL Analyzer, Applied Biosystems, Foster City, CA, USA) in pedigrees VI and VII, excluding a mutation in MPL as the cause of defective Mpl expression. Results on TPO-induced phosphorylation, MPL mRNA and gene analysis for pedigree VIII have been published elsewhere [3]. This study revealed reduced Mpl receptor levels in IT, although a heterogeneous expression pattern was found among different pedigrees. In particular, besides the reduction previ- GAPDH 2500 2000 1500 1000 500 0 –500 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 C PVI.2 FVI.1 MPL Fluorescence Cycles 3000 2500 2000 1500 1000 500 –500 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 C 1 MPL β3integrin A PVI.1 PVI.2 C 2 B Fig. 1 Mpl expression levels in pedigree VI. (A)Mpl protein relative to b3 integrin is decreased by Western blot in patients (PVI.1 and PVI.2) compared with controls (C1 and C2). (B) Analysis of MPL transcripts by realtime PCR reveals higher CT values for patients (PVI.1 and PVI.2) compared with controls (C), reflecting decreased MPL mRNA levels, whereas CT values for GAPDH are similar (inset). 2216 Letters to the Editor  2008 International Society on Thrombosis and Haemostasis ously found in FPD/AML (pedigree VIII) [3], decreased Mpl was found in two pedigrees with IT of unknown origin, whereas Mpl was normal in patients with WASP mutation, MYH9-RD and autosomal dominant macrothrombocytopenia. We have shown that reduced Mpl in FPD/AML is associated with a defective in vitro response to its ligand [3]. Likewise, in this study, low Mpl led to an overall decrease in platelet TPO-response, suggesting this abnormality is biologically relevant and could be implicated in IT pathogenesis. Assessment of megakaryocyte growth in response to TPO would have been useful to explore the influence of decreased Mpl in megakaryopoiesis. However, bone marrow cells were not available for analysis. Severely reduced or absent Mpl expression is the hallmark of congenital amegakaryocytic thrombocytopenia (CAMT) [2], and low Mpl has been described in thrombocytopenia and absent radii (TAR) [4]. The decrease in Mpl revealed in this study for IT disorders other than CAMT and TAR indicates this finding is not restricted to these congenital syndromes. Inherited thrombocytopenias are frequently misdiagnosed as ITP and differential diagnosis between both conditions is not always clear. In this study, the difference in Mpl expression found between IT and ITP could be a useful tool to differentiate between them in certain cases. However, study of a larger number of patients would be required to determine the clinical implication of this finding. The diversity in Mpl expression pattern found among different pedigrees reflects the heterogeneous nature of these disorders and could be related to differences in the underlying molecular pathogenesis. The genetic defects leading to IT may impair each of the sequential stages of platelet production, including megakaryocyte proliferation, maturation or thrombopoiesis [1]. Thrombopoietin enhances megakaryopoiesis, although it does not stimulate proplatelet formation [2]. In this line, lowMpl expression could contribute to decreased platelet production in disorders caused by abnormalmegakaryopoiesis, as shown for FPD/LMA [3] or TAR [4], whereas disorders involving defects in thrombopoiesis are expected to be unrelated to abnormalities in the TPO/Mpl axis, as revealed for MYH9-RD. Other than MPL mutation, several mechanisms may lead to decreased Mpl expression. In one pedigree with low Mpl protein, decreasedmRNAlevelswere found, suggesting reduced gene expression. We have shown that in FPD/AML, decreased MPL gene expression seems to be linked to RUNX1 mutation leading to lower transcriptional activity of the MPL promoter and suggested thatRUNX1 regulatesMPLtranscription [3]. In the other pedigree with low protein expression, mRNA levels were normal. Discrepancy between protein and mRNA in this case could be explained by post-transcriptional defects in protein translation or intracellular processing, or increased receptor internalization.Asthe genetic defects responsible for IT in these two pedigrees could not be established, themechanisms underlying the decrease in Mpl could not be further determined. Therapeutic options in IT are limited and remain mainly supportive. Considering that IT disorders are caused by impaired platelet production, stimulation of megakaryopoiesis with TPO mimetics may be of use for treating these patients, although this option has not yet been tested. It remains to be established whether low Mpl levels will limit the response to TPO mimetics in patients harboring this defect. In conclusion, this study shows reduced expression of a key megakaryocyte cytokine receptor, Mpl, in certain IT pedigrees, providing a potential explanation for thrombocytopenia. Insight into the mechanisms underlying this defect may contribute to clarify the molecular pathways involved in normal platelet production and their dysregulation in megakaryocyte disorders. Acknowledgements We thank referring physicians, J. Drachman for MPL sequencing in pedigree VII, C. Balduini and A. Pecci for MYH9-RD studies. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. References 1 Balduini CL, Savoia A. Inherited thrombocytopenias: molecular mechanisms. Semin Thromb Hemost 2004; 30: 513–23. 2 Kaushansky K, Drachman JG. The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production. Oncogene 2002; 21: 3359–67. 3 Heller PG, Glembotsky AC, Gandhi MJ, Cummings CL, Pirola CJ, Marta RF, Kornblihtt LI, Drachman JG, Molinas FC. Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation. Blood 2005; 105: 4664–70. 4 Letestu R, Vitrat N, Masse´ A, Le Couedic JP, Lazar V, Rameau P, Wendling F, Vuillier J, Boutard P, Plouvier E, Plasse M, Favier R, Vainchenker W, Debili N. Existence of a differentiation blockage at the stage of a megakaryocyte precursor in the thrombocytopenia and absent radii (TAR) syndrome. Blood 2000; 95: 1633