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Mechanism of KDSR-associated thrombocytopenia
mocytic, normochromic anemia with normal iron and hematinic levels (Figure 2B, Online Supplementary Table S1A and Online Supplementary Figure S1). Subsequent com- plete blood counts showed on several occasions platelet counts <100x109/L accompanied by rectal and gingival bleeding, excessive ecchymosis, and recurrent epistaxis when platelet counts were <10x109/L. Possible skin involvement was limited to a slow-to-heal perianal wound following rectal manometry. Serial BM examina- tions revealed increased numbers of dysplastic MK and progressive severe myelofibrosis (Figure 2C, Online Supplementary Table S1B and Online Supplementary Figure S2); despite this we observed significant fluctuation in the propositus’s thrombocytopenia and normalization of the hemoglobin level over the course of his first decade (Figure 2B and Online Supplementary Table S1A). The mechanism of the improvement is unclear, and occurred in the absence of identifiable environmental, therapeutic, or dietary interventions. Genetic analyses of BM DNA excluded known somatic mutations causal of myelodys- plasia or primary myelofibrosis (Online Supplementary Table S1B). Light transmission platelet aggregation was normal with the exception of an attenuated response to stimulation with collagen at low dose (Online Supplementary Table S1C). The propositus’s older brother and his parents were unaffected (Figure 2A). His sister pre- sented at birth with thrombocytopenia (Figure 2A and B, and Online Supplementary Table S1A) and mild ichthyosis in her left axilla (Figure 2D), but the skin symptoms resolved spontaneously over the first month. At 5 months of age she also developed persistent, normocytic, normochromic
anemia (Figure 2B and Online Supplementary Table S1A). Transmission EM analysis showed platelets of normal size (Figure 2E). Delta (δ)-granules appeared diminished; how- ever, it was not possible to count these accurately in the absence of whole-mount EM or a specific δ-granule mark- er (CD63 also stains lysosomal structures). There were no other marked ultrastructural abnormalities.
Pathogenic variants in KDSR
The propositus and his affected sister carry a maternally
inherited nonsense variant 18:61006104 G>A (p.Arg236*) and a paternally inherited missense variant 18:61018270 G>A (p.Arg154Trp) in KDSR (Figure 2A). The variants were confirmed by Sanger sequencing18,19 (Online Supplementary Figure S3) and have minor allele frequencies in Europeans of 4.82x10-5 and 2.32x10-4, respectively. The missense variant p.Arg154Trp is localized in the catalytic domain of KDSR (Figure 1)20 and both are found in the most abundant KDSR transcripts in MK (Online Supplementary Figure S4).2 The nonsense variant is absent from two out of three major platelet transcripts, which is in keeping with minimal de novo sphingolipid synthesis in mature platelets (Online Supplementary Figure S4).7 The results of co-segregation study were concordant with an autosomal recessive mode of inheritance (Figure 2A).
Sphingolipid profiles
We reasoned that the variants would cause reduced enzymatic function, leading to a build-up of the substrate KDS (Figure 3A). Indeed, global metabolic profiling showed KDS to be detectable in plasma from the proposi-
A
C
B
Figure 5. KDSR variants are associated with reduced proplatelet formation by megakaryocytes (MK). (A) Quantification of proplatelet formation by MK at day 11 of differentiation. On the left are the results of differentiation of bone marrow (BM)-derived hematopoietic stem cells (HSC) from the propositus and control. (Right) Results of differentiation of HSC obtained from the blood of the propositus, his affected sibling, and a second control. All MK with proplatelets and membrane bud- ding were counted as positive. Values plotted are means and Standard Deviations (SD) as quantified on 20 images. Results were analyzed by the unpaired, two- tailed t-test. (B) MK at day 11 derived from BM HSC from the propositus and a control. MK are stained for the cytoskeletal marker F-actin (red) and lysosome and delta granule marker CD63 (green). MK from the propositus and affected sibling have irregular cytoskeletal structures with lamellipodia (arrows). Further images can be found in Online Supplementary Figure S10. (C) MK area was quantified by automated analysis. Modeling was performed using a linear mixed effects model and associated P-values were computed by a likelihood ratio test. MK from affected cases were smaller compared to unrelated controls (P=0.01473).
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