Mutations in the thrombopoietin receptor, Mpl, in children with congenital amegakaryocytic thrombocytopenia

Abstract

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder of undefined aetiology. The disease presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. Furthermore, CAMT patients may develop bone marrow aplasia. To obtain more insight into the mechanism underlying CAMT, five children were analysed. All patients had increased plasma thrombopoietin (Tpo) levels, indicating a platelet production defect. Bone marrow‐derived CD34+ stem cells from three patients were cultured in an in vitro liquid culture system to study megakaryocytopoiesis. CD34+ cells from two of the three patients failed to differentiate into megakaryocytes. The lack of megakaryocyte formation could imply that a defect in the c‐mpl gene, encoding the Tpo receptor, exists. Sequencing of c‐mpl revealed mutations in four of five patients. Three patients had point mutations and/or a deletion in the coding regions of c‐mpl. All point mutations led to an amino acid substitution or to a premature stop codon. In one patient, a homozygous mutation in the last base of intron 10 was found that resulted in loss of a splice site. This study showed that mutations in c‐mpl could be the cause of thrombocytopenia in CAMT in the majority of patients. Furthermore, Tpo has been shown to have an anti‐apoptotic effect on stem cells. Therefore, mutations in c‐mpl might not only affect megakaryocyte formation but may also impair stem cell survival, which could explain the occurrence of bone marrow failure as final outcome in patients with CAMT.

Congenital amegakaryocytic thrombocytopenia—Report of a new c‐mpl gene missense mutation

“…While the maternally inherited mutation was previously reported [2,5], to our knowledge, the G to A transition at nucleotide 578 in exon 4 leading to a cysteine to tyrosine replacement, inherited from the patient's father, is a novel mutation.…”

“…The hallmark is absence of megakaryocytes [1], high serum thrombopoietin (tpo) levels, and in vitro megakaryocyte culture insensitivity to tpo stimulation [2]. CAMT is due to the germline mutations of both tpo receptor alleles, c-mpl [3][4][5]. c-mpl knock-out mice not only lack megakaryopoiesis but also have pluripotent stem cell defects [1].…”

“…The 12 c-mpl exons were amplified in separate PCR reactions using intron-specific primers [5]. PCR products were analyzed in a 2% agarose gel electrophoresis and extracted using the Quiaquick PCR purification kit (QIAGEN, Hilden, Germany) for sequence analysis.…”

Congenital amegakaryocytic thrombocytopenia: clinical and biological consequences of five novel mutations

“…1,[4][5][6][7][8][9][10][11] However, the diagnosis of five patients in two centers during the last 2 years raises the suspicion that the incidence of the disease is underestimated, presumably because the initial presentation of CAMT with isolated thrombocytopenia can be easily misdiagnosed with idiopathic thrombocytopenic purpura, while the late pancytopenic phase is indistinguishable from aplastic anemia. Thus, bone marrow examination should be part of the diagnostic work-up in all children with severe thrombocytopenia since birth and screening of the c-MPL gene should be performed when a reduced number of megakaryocytes is observed.…”

“…3 At least 28 different c-MPL mutated alleles have been so far identified from 32 unrelated CAMT families. 1,[4][5][6][7][8][9][10][11] Beside these mutations associated with CAMT, a gain-of-function mutation of c-MPL is responsible for familial essential thrombocythemia. 12 Interestingly, different types of mutations have been associated with different phenotypes, allowing patients to be subdivided into two groups.…”