Autosomal Recessive Hereditary Thrombocytosis: Functional Analysis of a Novel C-Mpl Mutation Resulting in An Unsual Mode of Constitutive Receptor Activation

Thrombopoiesis is tightly regulated by the interaction between thrombopoietin (TPO) and its receptor c-Mpl. Ligand binding results in dimerization of the receptor, internalization of the complex, and activation of downstream signaling. This activation pathway also leads to the clearance of TPO from the plasma thus establishing a negative feedback loop. Gain of function mutations of TPO and c-Mpl are exceedingly rare but provide interesting models for the biological understanding of the molecular interactions that are required for a functional TPO pathway. Here, we describe a consanguineous Qatari family with hereditary thrombocytosis. Genotype analysis revealed a novel c-Mpl P106L mutation that was found homozygously in 3 affected patients with platelet counts between 700–800/nl. The TPO gene sequence was normal. Notably, platelet counts in heterozygous relatives were within the normal range. Surprisingly, TPO plasma levels were elevated to 10 to 15-fold in homozygotes but within the normal range in heterozygotes. A simple gain of function mechanism of the c-Mpl mutation could thus be excluded. Therefore, we next functionally analyzed and compared the normal and the P106L mutated c-Mpl receptor. An analysis of HeLa and BaF3 cells that were transiently or stably, respectively, transfected with c-Mpl expression vectors showed that the P106L mutation leads to abnormal subcellular receptor distribution. Whereas normal c-Mpl was detected in the Golgi-apparatus and in the plasma membrane, c-Mpl P106L was predominantly found within the ER. Furthermore, we showed that c-Mpl P106L cannot be processed to the normal, tunicamycin sensitive, strongly glycosylated form. Functional analysis of the TPO/c-Mpl signaling pathway in stably transfected BaF3 cells demonstrated an elevated phosphorylation of Stat5 with P106L when compared to normal c-Mpl. Moreover, cells transfected with the mutant but not with the normal receptor gene were proliferating independently of extracellular growth factors such as IL3. These data indicate that c-Mpl P106L activates downstream signaling and thus stimulates cell division and platelet production in a ligand-independent fashion. The recessive mode of inheritance suggests that the presence of normal receptor molecules prevents this abnormal function and promotes the maintenance of the negative feedback loop regulating TPO plasma levels by an unknown mechanism. The raised TPO plasma levels in homozygotes and the lack of detectable mutated c-Mpl in the plasma membrane indicate that the mutated receptor does not bind and internalize its normal ligand. In conclusion, we hypothesize that the novel c-Mpl P106L receptor mutation results in spontaneous dimerization and ligand independent activation even before the receptor reaches the Golgi apparatus and is fully glycosylated and transported to the plasma membrane.