An ENU-induced recessive mutation in Mpl leads to thrombocytopenia with overdominance

ObjectiveThe aim of this study was to identify and characterize the causative mutation in the thrombocytopenic mouse strain HLB219 that was generated at the Jackson Laboratory as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen.Materials and MethodsThe HLB219 mutation was identified by interval mapping of F2 mice generated from intercross breeding of HLB219 to both BALB/cByJ (BALB) and 129/SvImJ (129/Sv). Mpl was identified as a candidate gene and sequenced. The mutation was characterized in vivo in mouse hematopoietic stem/progenitor cell assays and in cell culture by expression in Ba/F3 cells.ResultsA novel mutation in the thrombopoietin (TPO) receptor Mpl in HLB219 mice caused a Cys→Arg substitution at codon 40 in the extracellular region of the receptor. Mice homozygous for the Mplhlb219 mutation had an 80% decrease in the number of platelets in comparison to the wild-type C57BL/6J strain, low numbers of bone marrow megakaryocytes, high TPO levels, and decreased competitive repopulating ability, consistent with a loss-of-function mutation in the receptor. Mice heterozygous for Mplhlb219 however, showed an overdominance effect with a significant increase in platelet number. Functional analysis in vitro demonstrated that Ba/F3 cells expressing the mutant MPLhlb219 protein failed to activate extracellular signal-regulated kinase and signal transducers and activators of transcription 5, but proliferated in the absence of TPO and required constitutive phosphorylation of RAC-alpha serine/threonine protein kinase (AKT) for cytokine-independent growth.ConclusionThrombocytopenia in HLB219 mice is caused by a recessive mutation in Mpl that abrogates mitogen-activated protein kinase–extracellular signal regulated kinase and janus kinase–signal transducers and activators of transcription signaling.