Sphingolipid dysregulation due to lack of functional KDSR impairs proplatelet formation causing thrombocytopenia.

Sphingolipids are fundamental to membrane trafficking, apoptosis, and cell differentiation and proliferation. KDSR or 3-keto-dihydrosphingosine reductase is an essential enzyme for de novo sphingolipid synthesis, and pathogenic mutations in KDSR result in the severe skin disorder erythrokeratodermia variabilis et progressiva-4 Four of the eight reported cases also had thrombocytopenia but the underlying mechanism has remained unexplored. Here we expand upon the phenotypic spectrum of KDSR deficiency with studies in two siblings with novel compound heterozygous variants associated with thrombocytopenia, anemia, and minimal skin involvement. We report a novel phenotype of progressive juvenile myelofibrosis in the propositus, with spontaneous recovery of anemia and thrombocytopenia in the first decade of life. Examination of bone marrow biopsies showed megakaryocyte hyperproliferation and dysplasia. Megakaryocytes obtained by culture of CD34+ stem cells confirmed hyperproliferation and showed reduced proplatelet formation. The effect of KDSR insufficiency on the sphingolipid profile was unknown, and was explored in vivo and in vitro by a broad metabolomics screen that indicated activation of an in vivo compensatory pathway that leads to normalization of downstream metabolites such as ceramide. Differentiation of propositus-derived induced pluripotent stem cells to megakaryocytes followed by expression of functional KDSR showed correction of the aberrant cellular and biochemical phenotypes, corroborating the critical role of KDSR in proplatelet formation. Finally, Kdsr depletion in zebrafish recapitulated the thrombocytopenia and showed biochemical changes similar to those observed in the affected siblings. These studies support an important role for sphingolipids as regulators of cytoskeletal organization during megakaryopoiesis and proplatelet formation.

Identification of rare sequence variation underlying heritable pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disorder with a poor prognosis. Deleterious variation within components of the transforming growth factor-ß pathway, particularly the bone morphogenetic protein type 2 receptor (BMPR2), underlies most heritable forms of PAH. To identify the missing heritability we perform whole-genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses reveal significant overrepresentation of rare variants in ATP13A3, AQP1 and SOX17, and provide independent validation of a critical role for GDF2 in PAH. We demonstrate familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, lead to reduced secretion from transfected cells. In addition, we identify pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these findings contribute new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention.

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.