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T.K. Bariana et al.
during de novo sphingolipid synthesis is compensated by an alternative pathway. One possible alternative pathway is the recycling of relatively abundant sphingomyelins; a pathway previously shown to contribute to production of downstream sphingolipids such as dihydroceramide, ceramide, and sphingosine, but considered to contribute little, if at all, to DHS production under normal conditions.21 Post-translational modifications to sphin- golipid enzymes may also explain the metabolic profile, and further work is required to explore this possibility. Importantly, the profile of increased ceramide and reduced sphingosine in propositus-derived iMK compared with rescued iMK, and the consistent and potentially explana- tory transcriptional dysregulation of enzymes ASAH1 and CERS6, is in contrast to the limited sphingolipid quantita- tion undertaken in previous studies which showed that ceramide levels were reduced in affected skin and that platelet surface exposure of ceramide was impaired in individuals with hypo-functional KDSR variants.9
Our observation that MK lacking functional KDSR are hyperproliferative is consistent with earlier reports,9 but we expand on this characterization by showing the ex vivo generated patient MK to be smaller than controls and to be less effective in proplatelet formation. Proplatelets are pseudopodial projections of megakaryocyte cyto- plasm, supported at their core by microtubular bundles that carry granules and other platelet cargo from the body of the megakaryocyte to the tip of the proplatelet.22 Aberrant size and proplatelet formation were not only observed in MK obtained by differentiation of primary HSC obtained from the two patients, but are also present in iMK generated by forward programming of iPSC derived from the propositus’s fibroblasts. Taken together, we consider the ineffective platelet formation caused by the absence of KDSR to be the primary cause of the thrombocytopenia. Increased turnover because of a reduced platelet lifespan seems to be a less likely explana- tion because the immature platelet fraction was not sig- nificantly raised in the two patients compared with healthy controls (data not shown). We hypothesize that impaired platelet formation may, in turn, be caused by cytoskeletal disorganization and further experiments are required to explore this possibility. Pathogenic mutations in several other genes (e.g. MYH9, ACTN1, FLNA, TUBB1, DIAPH1, TPM4) encoding proteins with impor- tant functional roles in cytoskeletal reorganization and actin polymerization cause dominant forms of thrombo- cytopenia.23 However, these genetic disorders are charac- terized by enlarged platelets, and the mean volumes of the platelets of our patients are within the normal ranges for males and females, respectively.
The increased level of KDS in plasma was confirmed at the cellular level in iMK derived from the propositus. This increased level was normalized upon rescue of the propositus’s iMK with a KDSR transcript carrying the ref- erence allele. The correction of the biochemical defect was mirrored by a recovery of iMK size and improvement of their capacity to form proplatelets. To further support the central importance of KDSR in thrombopoiesis, we show that KDSR knockdown in a zebrafish model is associated with impaired thrombocyte formation. Similar approach- es have identified multiple potential regulators of throm- bopoiesis,24 though in isolation zebrafish studies these are limited by inherent differences in thrombopoiesis
between mammals and fish, notably that zebrafish have nucleate thrombocytes rather than MK.
The marked, spontaneous improvement in the proposi- tus’s thrombocytopenia and anemia led to a reversal of the decision to treat the condition by HSC transplantation. The mechanism of this improvement is unclear, given the presence of progressive myelofibrosis and in the absence of clinical features to suggest significant extramedullary hemopoiesis such as splenomegaly. Recent studies have shown spontaneous improvement in blood counts in other inherited juvenile BM failure syndromes, most notably those associated with pathogenic variants in SAMD9 or SAMD9L.25 In these cases, the improvement was attributed to the acquisition of corrective somatic mutations. Further longitudinal studies of individuals affected by pathogenic KDSR variants is essential to deter- mine whether the clinical course described is representa- tive, and whether a careful watch-and-wait approach rather than early intervention may be more appropriate in this genetically-defined subgroup of cases with inherited thrombocytopenia accompanied by BM failure.
Funding
TKB is supported by the British Society of Haematology and NHS Blood and Transplant. KF and CVG are supported by the Fund for Scientific Research-Flanders (FWO Vlaanderen, Belgium; G.0B17.13N) and by the Research Council of the University of Leuven (BOF KU Leuven‚ Belgium; OT/14/098). C.V.G is holder of the Bayer and Norbert Heimburger (CSL Behring) Chairs. The structured illumination microscope was acquired through a CLME grant from Minister Lieten to the VIB BioImaging Core, Leuven. J.H is supported by the Fund for Scientific Research-Flanders (FWO Vlaanderen, Belgium) grant no.1S00816N. AK and BJ are funded by the National Institute for Health Research (NIHR) Biomedical Research Centre (RG64245). MF is supported by the British Heart Foundation (BHF) Cambridge Centre of Excellence (RE/13/6/30180). The Ouwehand laboratory receives support from the BHF, Bristol- Myers Squibb, European Commission, MRC, NHS Blood and Transplant, Rosetrees Trust, the NIHR Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust, and the University of Cambridge.
Acknowledgments
The NIHR BioResource – Rare Disease Study is a multicenter whole-genome sequencing (WGS) study of approximately 13,000 patients. The genotype and phenotype data are being incorporated in the 100,000 Genomes Project. This study makes use of data generated by the NIHR BioResource and a full list of investigators who contributed to the generation of the data is available from https://bioresource.nihr.ac.uk/rare-diseases/con- sortia-lists/. Funding for the project was provided by the NIHR (grant number RG65966). The NIHR BioResource projects were approved by Research Ethics Committees in the UK and appro- priate national ethics authorities in non-UK enrollment centers. The authors are also grateful to all the research participants who donated their samples for this study and to Professor Andrew Mumford (University of Bristol, UK), Professor Michael Laffan (Imperial College London, UK), Dr Lining Guo (Metabolon Inc., Durham, NC, USA), and Dr Sergio Rodriguez-Cuenca and Professor Antonio Vidal-Puig from the University of Cambridge (UK) for their input. The Tg(cd41:EGF)11 line was a gift from Professor Leonard Zon (Hematology Division, Brigham and Women’s Hospital, Boston, MA, USA).
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haematologica | 2019; 104(5)