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Mechanism of KDSR-associated thrombocytopenia
tus but not from the parents, the healthy sibling, or 496 unrelated controls (Figure 3B). This finding was corrobo- rated using a LC-MS platform for the selective measure- ment of specific sphingolipids, which confirmed that KDS was detectable in both patients and absent from the plas- ma of controls (Figure 3C and Online Supplementary Figure S5). Interestingly, there was no reduction in downstream sphingolipids in the patients using either platform (Online Supplementary Tables S2 and S3A). In fact, global profiling showed levels of the KDSR product, DHS, were higher for the propositus than controls. These findings raise the hypothesis that KDSR hypofunction during de novo sphin- golipid synthesis is compensated in vivo by alternative mechanisms, for example, by the recycling of relatively abundant sphingomyelins along a pathway that normally contributes little to free DHS production.4
Depletion of kdsr in zebrafish causes thrombocytopenia
We explored the role of the enzyme on thrombocyte formation in zebrafish by MO-mediated depletion of the kdsr transcript in Tg(cd41:EGFP). As expected, this led to a reduction of the Kdsr protein level (Figure 4A and B) and resulted in curved tails, which is a typical feature for embryos with thrombocytopenia (Online Supplementary Figure S6).15 The number of thrombocytes was inversely correlated with the dose of MO injected (Figure 4C and D). Targeted sphingolipid profiling showed elevated and undetectable KDS in lysates from MO and control embryos, respectively (Figure 4E). Similar to the results obtained with the propositus’s plasma, dihydroceramides, ceramides, sphingomyelins and glycosphingolipids that are downstream of Kdsr were not significantly different between Kdsr-depleted and control fish (Online Supplementary Table S3B).
Impaired proplatelet formation in patient megakaryocytes
CFU-GEMM cultures differentiated from bone marrow HSC of the propositus showed hyperproliferation of myeloid cells (P=0.001, t-test) with a reduced myeloid/ery- throid ratio compared to the controls (Online Supplementary Figure S7). CFU-MK numbers were compa- rable to those of the control, although individual MK colonies were denser for the propositus, and liquid cul- tures showed an increased number of MK (Online Supplementary Figure S8). MK in control cultures formed proplatelets, whilst MK derived from both the propositus and the affected sister showed a strong reduction in pro- platelet formation, despite similar levels of membrane budding and a higher number of CD41 and CD42 positive cells in propositus-derived cultures when compared with control MK (Figure 5A and Online Supplementary Figures S8-S10). Patient-derived MK also showed extensive, abnormal formation of lamellipodia and reduced cell size (P=0.014, likelihood ratio test) (Figure 5B and C).
The abnormal morphological, functional, and biochemical features of the propositus’s induced pluripotent stem cells reprogrammed to megakaryocytes can be rescued
To corroborate the atypical phenotypes of MK derived from the HSC, we transduced iPSC from the propositus with lentiviral vectors containing the reference KDSR ORF (Kresc) or an inert control vector (Kev), and reprogrammed
these cells to iMK (Online Supplementary Figure S11A). Analysis of the iMK RNA-seq results showed similar KDSR gene expression but the majority of sequencing reads in the rescued iMK carried the reference allele at Chr18:61018270 G>A (p.Arg154Trp) (Online Supplementary Figure S11B-D). These findings are consis- tent with correction of the genetic defect without signifi- cant overexpression, and resulted in increased proplatelet formation compared with control iMK 4 h after seeding (P=0.047, t-test) (Figure 6A-C). The observed iMK pro- platelets were shorter and less branched than those observed following directed differentiation from stem cell cells, in keeping with published reports using this proto- col.17 Upon microscopic inspection, the rescued iMK seemed larger than the non-rescued ones, which was con- firmed to be significant by flow cytometry (Online Supplementary Figure S11E) and the increased proplatelet formation resulted by 24 h in little residual cytoplasm for the rescued versus the non-rescued iMK (Figure 6C). At the biochemical level, the rescue resulted in a significant reduction in KDS levels (P=0.02, t-test) showing the effec- tiveness of the gene therapy approach in ‘curing’ the iMK from the propositus (Figure 6D). Similar to findings in plasma and in zebrafish, there was no difference in DHS levels between the iMK with and without functional KDSR transcripts, indicating that the postulated, compen- satory mechanism is also present in iMK. We searched the iMK transcriptome landscape for possible differences in the levels of transcripts of other key enzymes that regulate sphingolipid synthesis and recycling (the enzymes exam- ined are as shown in Figure 3A). This identified only ASAH1 and CERS6 transcripts to be down- and up-regu- lated respectively (posterior probabilities 0.610 and 0.774; log-fold change -0.67 and +0.70, respectively). These two enzymes regulate the ceramide-sphingosine ratio (Figure 3A and Online Supplementary Figure S12), and in keeping with these findings, the rescued iMK showed higher sphingosine and lower ceramide levels (Figure 6D).
Discussion
Pathogenic mutations in KDSR have recently been asso- ciated with a recessively inherited syndrome of moderate- to-severe skin pathology and thrombocytopenia. We have described two novel KDSR mutations causing thrombocy- topenia in the propositus and his infant sister, expanding the phenotypic spectrum of this recently identified Mendelian disorder from severe skin pathology with no apparent hematologic involvement to profound thrombo- cytopenia and moderate anemia with spontaneous improvement across the first decade, and almost imper- ceptible dermatological abnormalities. In the propositus, BM studies also showed the novel phenotype of severe juvenile myelofibrosis; however, the sister was too young to allow confirmation of this phenotype. The biochemical sphingolipid signatures of the plasma and patient-derived iMK confirmed the predicted reduction in function with elevated levels of its substrate, KDS. This is, as expected, from the combination of a variant encoding a premature stop codon and a hypomorphic allele involving a missense variant in the catalytic domain. Unexpectedly, down- stream metabolites in the sphingolipid pathway, including DHS, ceramide, and sphingosine-1-phosphate, were not reduced in plasma, suggesting that KDSR hypofunction
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