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

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD)

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility

Identification and analysis of actives cis-regulatory modules in the cardiac tube during embryogenesis in Drosophila melanogaster

Comprendre comment l’expression des gènes est régulée spécifiquement dans chaque tissu et de manière dynamique au cours du temps demeure une étape centrale de notre compréhension de l’organogénèse. L’identification des éléments cis-régulateurs de la transcription de manière tissu-spécifique peut permettre de comprendre les règles logiques d’organisation du réseau de gènes régulateur et aussi d’identifier de nouveaux acteurs (facteurs de transcription notamment). L’analyse de marques de chromatine (H3K27ac et H3K4me3) spécifiquement dans les cardioblastes (104 cellules) au cours de la différentiation a permis l’identification en masse de régions cis-régulatrices de la transcription. Via une approche d’apprentissage, de nouvelles régions régulatrices spécifiques des cardiomyocytes ainsi que 2 nouveaux facteurs de transcription (bagpipe, hamlet) ont été identifiées. L’alignement multiple des régions régulatrices suggère que les régions associées à H3K27ac dans les cellules cardiaques durant ces étapes de l’organogénèse partagent une séquence consensus. Ces nouveaux éléments régulateurs viennent compléter le réseau de gène régulateur au cours des étapes tardives de la cardiogénèse.Understanding how gene expression is spatio-temporally regulated remains a crucial step in our understanding of organogenesis. Identification of transciptional cis-regulatory elements in a tissu-specific manner could allow to understand logical rules leading regulatory network organisation and to identify new actors (in particular transcription factors). Analysis of chromatin marks (H3K27ac and H3K4me3) specifically in cardiac cells (104 cells) during differentiation allowed the identification of transcriptional cis-regulatory regions. Via a machine learning approach, new cardiac specific regulatory regions and two transcription factors (bagpipe and hamlet) have been identified. Multiple sequence alignment of regulatory regions suggests that regions associated to H3K27ac in cardiac cells during these steps of organogenesis share a consensus sequence. These new regulatory elements integrate and complete the gene regulatory network underlying late steps of cardiogenesis

LedPred: an R/bioconductor package to predict regulatory sequences using support vector machines

International audienceSupervised classification based on support vector machines (SVMs) has successfully been used for the prediction of cis-regulatory modules (CRMs). However, no integrated tool using such heterogeneous data as position-specific scoring matrices, ChIP-seq data or conservation scores is currently available. Here, we present LedPred, a flexible SVM workflow that predicts new regulatory sequences based on the annotation of known CRMs, which are associated to a large variety of feature types. LedPred is provided as an R/Bioconductor package connected to an online server to avoid installation of non-R software. Due to the heterogeneous CRM feature integration, LedPred excels at the prediction of regulatory sequences in Drosophila and mouse datasets compared with similar SVM-based software

Ndae1 expression and regulation in Drosophila embryos.

The construction and prediction of cell fate maps at the whole embryo level require the establishment of an accurate atlas of gene expression patterns throughout development and the identification of the corresponding cis-regulatory sequences. However, while the expression and regulation of genes encoding upstream developmental regulators such as transcription factors or signaling pathway components have been analyzed in detail, up to date the number of cis-regulatory sequences identified for downstream effector genes, like ion channels, pumps and exchangers, is very low. The control and regulation of ion homeostasis in each cell, including at blastoderm stages, are essential for normal embryonic development. In this study, we analyzed in detail the embryonic expression pattern and cis-regulatory modules of the Drosophila Na+-driven anion exchanger 1 (Ndae1) gene, involved in the regulation of pH homeostasis. We show that Ndae1 is expressed in a tight and complex spatial-temporal pattern. In particular, we report that this downstream effector gene is under the control of the canonical dorsal-ventral patterning cascade through dorsal, Toll, twist and snail at early embryogenesis. Moreover, we identify several cis-regulatory modules, some of which control discrete and non-overlapping aspects of endogenous gene expression throughout development

Identification of cis-regulatory modules encoding temporal dynamics during development

International audienceBackground: Developmental transcriptional regulatory networks are circuits of transcription factors (TFs) andcis-acting DNA elements (Cis Regulatory Modules, CRMs) that dynamically control expression of downstream genes. Comprehensive knowledge of these networks is an essential step towards our understanding of developmental processes. However, this knowledge is mostly based on genome-wide mapping of transcription factor binding sites, and therefore requires prior knowledge regarding the TFs involved in the network.Results: Focusing on how temporal control of gene expression is integrated within a developmental network, we applied an in silico approach to discover regulatory motifs and CRMs of co-expressed genes, with no prior knowledge about the involved TFs. Our aim was to identify regulatory motifs and potential trans-acting factors which regulate the temporal expression of co-expressed gene sets during a particular process of organogenesis, namely adult heart formation in Drosophila. Starting from whole genome tissue specific expression dynamics, we used an in silico method, cisTargetX, to predict TF binding motifs and CRMs. Potential Nuclear Receptor (NR) binding motifs were predicted to control the temporal expression profile of a gene set with increased expression levels during mid metamorphosis. The predicted CRMs and NR motifs were validated in vivo by reporter gene essays. In addition, we provide evidence that three NRs modulate CRM activity and behave as temporal regulators of target enhancers.Conclusions: Our approach was successful in identifying CRMs and potential TFs acting on the temporal regulation of target genes. In addition, our results suggest a modular architecture of the regulatory machinery, in which thetemporal and spatial regulation can be uncoupled and encoded by distinct CRMs