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Mechanism of KDSR-associated thrombocytopenia
Introduction
3-keto-dihydrosphingosine reductase (KDSR) is an early, essential enzyme in the pathway of de novo sphin- golipid synthesis that catalyzes the conversion of 3-keto- dihydrosphingosine (KDS) to dihydrosphingosine (DHS) on the cytosolic leaflet of the endoplasmic reticulum.1 The canonical transcript for KDSR encodes a 332 amino acid protein. The gene is widely transcribed,1-3 consistent with the integral roles of the sphingolipid family in forming lipid rafts that facilitate membrane trafficking and in the regulation of fundamental cellular functions that include apoptosis, differentiation, and proliferation.4 The impor- tance of sphingolipid synthesis for normal cellular func- tions is illustrated by the complex multisystem pheno- types of null mice for key enzymes or receptors in the pathway, including defective platelet activation and thrombus formation.5,6 A pathway for de novo synthesis of sphingolipids in a megakaryocytic cell line has been shown, but this plays a minimal role in mature platelets, which instead acquire essential sphingolipids by incorpo- rating them from plasma or recycling plasma membrane sphingomyelins, both largely independently of KDSR.7
Consistent with these important roles of sphingolipids, compound heterozygous variants in KDSR (Figure 1) have recently been identified as causing the severe skin disorder erythrokeratodermia variabilis et progressiva 4 (EKVP4, OMIM617526), a condition characterized by neonatal onset of thick, scaly skin on the face and genitals, and milder erythematous palmo-plantar scaling.8 This observa- tion established a role for KDSR in the homeostasis of ker- atinization; however, it was unclear whether these cases had hematologic pathologies. A more recent study described four probands with EKVP4 caused by KDSR variants accompanied by severe thrombocytopenia and platelet dysfunction in infancy.9 A reduction in plasma S1P and surface-exposed ceramide in human platelets, as well
as diminished ceramide levels in affected skin, were reported. Bone marrow (BM) morphology in one patient was normal and in a second patient demonstrated increased megakaryopoiesis. For this patient, a diagnosis of immune-mediated thrombocytopenia was made with no response to corticosteroid treatment and minimal response to splenectomy. No further exploration of the molecular mechanism underlying the thrombocytopenia was undertaken.9
Here we provide evidence that in this pedigree, KDSR plays a fundamental role in megakaryopoiesis, cytoplas- mic organization, and proplatelet formation. We describe a pedigree in which compound heterozygous variants in KDSR segregate with severe thrombocytopenia and mini- mal or no skin involvement. We report novel phenotypes of progressive juvenile myelofibrosis in the propositus, who is older, and anemia in both siblings. Broad metabolic profiling complemented by targeted mass spectrometry assays confirm KDSR hypofunction and suggest activation of an alternative, compensatory pathway in vivo. Depletion of kdsr in zebrafish and studies with CD34+ stem cell- and induced pluripotent stem cell (iPSC)-derived MK show cellular and biochemical signatures in common with those observed in our patients, showing the mecha- nism by which KDSR variants mediate thrombocytope- nia.
Methods
Recruitment and sequencing
Following informed, written consent (ethical study approval ML3580), the propositus was recruited to the Bleeding, thrombot- ic and Platelet Disorders (BPD) domain of the NIHR BioResource- Rare Diseases study (UK Research Ethics Committee 13/EE/0325, https://bioresource.nihr.ac.uk). Further details are provided in the Online Supplementary Methods.
Figure 1. Reported KDSR variant genotypes and phenotypes in the context of 3-keto-dihydrosphingosine reductase (KDSR) structure and function. Protein and cDNA schematic adapted from Gupta et al.20 demonstrating location of known pathogenic KDSR variants with documented phenotypes in skin only (black), skin and platelets (red), and the novel variants reported in this manuscript in bold and underlined. Variants are linked by brackets where present in compound heterozygosity in an individual. Key structural elements of KDSR are illustrated: transmembrane anchors (blue, purple), the Rossman folds (red), and a highly conserved domain containing three putative catalytic sites (yellow). The novel p.Arg154Trp variant is within the catalytic domain.
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