Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation

Abstract

Germ-line heterozygous mutations in the hematopoietic transcription factor AML1 (RUNX1) have been identified in patients with familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), which is characterized by thrombocytopenia, abnormal platelet function, and propensity to myeloid malignancies. We identified a novel mutation in the AML1 gene in an FPD/AML pedigree characterized by a single nucleotide deletion that generates a frameshift and premature chain termination (Pro218fs-Ter225). Both wild-type and mutant transcripts were expressed in affected individuals by allele-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Thrombopoietin (TPO) binds to the Mpl receptor and is the major regulator of megakaryopoiesis. To explore the mechanisms underlying thrombocytopenia, we studied the TPO/Mpl pathway in this newly identified pedigree. TPO levels were mildly to moderately elevated. On flow cytometry and immunoblotting, Mpl receptor expression was decreased and TPOinduced signaling was impaired. While no mutations were identified in the MPL gene by sequence analysis, low MPL mRNA levels were found, suggesting decreased gene expression. Of particular interest, several AML1-binding motifs are present in the MPL promoter, suggesting MPL is an AML1 target. In conclusion, we identified a C-terminal AML1 mutation that leads to a decrease in Mpl receptor expression, providing a potential explanation for thrombocytopenia in this FPD/AML pedigree. IntroductionFamilial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder characterized by moderate thrombocytopenia, a defect in platelet function, and propensity to develop myeloid malignancy. 1 Germ-line heterozygous mutations in the hematopoietic transcription factor AML1, also known as RUNX1 and CBFA2, have been identified in 12 pedigrees reported so far, including missense, frameshift, and nonsense mutations and a large intragenic deletion. [2][3][4][5][6] AML1 is the DNA-binding subunit of the core binding factor (CBF) transcription complex. Heterodimerization with the non-DNA-binding subunit, CBF␤, increases the affinity of AML1 to DNA and protects it from proteolytic degradation. 7,8 AML1 includes a runt homology domain (RHD), which mediates DNA binding and heterodimerization with CBF␤, and a carboxy (C)-terminal domain responsible for transcriptional activation. 9 The AML1/CBF␤ complex regulates the expression of genes specific to hematopoiesis, including several cytokines and their receptors, such as granulocyte macrophage colony-stimulating factor, interleukin-3, and macrophage colony-stimulating factor receptor. 7 This transcription factor is essential for the establishment of definitive hematopoiesis, as demonstrated in AML1 Ϫ/Ϫ mice, which show a complete absence of fetal liver hematopoiesis and die during embryonic development, precluding analysis of the effects of AML1 in later stages of blood-cell development. 10 The role of this transcription factor in plate...

Myeloid neoplasms with germ line RUNX1 mutation

“…Although the incidence is less common, the mutations in the C-terminal region have been reported [25]. In this case, the mutation generates a frameshift and premature chain termination, resulting in the lack of transactivation domain [25]. Intragenic deletions, which are not identified by traditional sequence analysis, have also been reported in a few families [9,26,27].…”

“…The RUNX1 mutations identified in the individuals of FPD/AML pedigrees are generally located in the RHD, leading to disruption of DNA binding ability. Although the incidence is less common, the mutations in the C-terminal region have been reported [25]. In this case, the mutation generates a frameshift and premature chain termination, resulting in the lack of transactivation domain [25].…”

“…Thus, downregulation of MYL9 due to RUNX1 haploinsufficiency could affect abnormal platelet production and function [31]. Platelet storage pool deficiency and low levels of MPL receptor expression have also been reported [25,32].…”

Megakaryocyte development and platelet production

“…Germ line mutations of RUNX1, which cause monoallelic loss of RUNX1 complexes, are found in the autosomal dominant familial platelet disorder (FDP) with multiple platelet defects, reduced c-Mpl and predisposition to AML (Heller et al, 2005). Megakaryocytic and erythroid lineages, which are derived from a common bipotential progenitor, share many early lineage-restricted TFs (Shivdasani et al, 1997).…”

A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies

“…So far, 13 pedigrees have been reported, mainly in European countries. [5][6][7][8][9][10] Clinically, the disorder is characterized by moderate thrombocytopenia from birth, impaired platelet function and a propensity to develop myeloid malignancies. Genetic and molecular studies reveal that all reported pedigrees of FPD/MM display a mutation in the RUNX1 gene or LOH at the locus.…”