Chris Van Geet

Transcriptional diversity during lineage commitment of human blood progenitors

Introduction Blood production in humans culminates in the daily release of around 1011 cells into the circulation, mainly platelets and red blood cells. All blood cells originate from a minute population of hematopoietic stem cells (HSCs) that expands and differentiates into progenitor cells with increasingly restricted lineage choice. Characterizing alternative splicing events involved in hematopoiesis is critical for interpreting the effects of mutations leading to inherited disorders and blood cancers and for the rational design of strategies to advance transplantation and regenerative medicine. Overview of methodology. RNA-sequencing reads from human blood progenitors [opaque cells in (A)] were mapped to the transcriptome to quantify gene and transcript expression. Reads were also mapped to the genome to identify novel splice junctions and characterize alternative splicing events (B). Rationale To address this, we explored the transcriptional diversity of human blood progenitors by sequencing RNA from six progenitor and two precursor populations representing the classical myeloid commitment stages of hematopoiesis and the main lymphoid stage. Data were aligned to the human reference transcriptome and genome to quantify known transcript isoforms and to identify novel splicing events, respectively. We used Bayesian polytomous model selection to classify transcripts into distinct expression patterns across the three cell types that comprise each differentiation step. Results We identified extensive transcriptional changes involving 6711 genes and 10,724 transcripts and validated a number of these. Many of the changes at the transcript isoform level did not result in significant changes at the gene expression level. Moreover, we identified transcripts unique to each of the progenitor populations, observing enrichment in non–protein-coding elements at the early stages of differentiation. We discovered 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes and often resulting in the gain or loss of functional domains. Of the alternative splice sites displaying differential usage, 73% resulted in exon-skipping events involving at least one protein domain (38.5%) or introducing a premature stop codon (26%). Enrichment analysis of RNA-binding motifs provided insights into the regulation of cell type–specific splicing events. To demonstrate the importance of specific isoforms in driving lineage fating events, we investigated the role of a transcription factor highlighted by our analyses. Our data show that nuclear factor I/B (NFIB) is highly expressed in megakaryocytes and that it is transcribed from an unannotated transcription start site preceding a novel exon. The novel NFIB isoform lacks the DNA binding/dimerization domain and therefore is unable to interact with its binding partner, NFIC. We further show that NFIB and NFIC are important in megakaryocyte differentiation. Conclusion We produced a quantitative catalog of transcriptional changes and splicing events representing the early progenitors of human blood. Our analyses unveil a previously undetected layer of regulation affecting cell fating, which involves transcriptional isoforms switching without noticeable changes at the gene level and resulting in the gain or loss of protein functions. A BLUEPRINT of immune cell development To determine the epigenetic mechanisms that direct blood cells to develop into the many components of our immune system, the BLUEPRINT consortium examined the regulation of DNA and RNA transcription to dissect the molecular traits that govern blood cell differentiation. By inducing immune responses, Saeed et al. document the epigenetic changes in the genome that underlie immune cell differentiation. Cheng et al. demonstrate that trained monocytes are highly dependent on the breakdown of sugars in the presence of oxygen, which allows cells to produce the energy needed to mount an immune response. Chen et al. examine RNA transcripts and find that specific cell lineages use RNA transcripts of different length and composition (isoforms) to form proteins. Together, the studies reveal how epigenetic effects can drive the development of blood cells involved in the immune system. Science, this issue 10.1126/science.1251086, 10.1126/science.1250684, 10.1126/science.1251033 RNA sequencing identifies how different cell fate decisions are made during blood cell differentiation. Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type–specific expression changes: 6711 genes and 10,724 transcripts, enriched in non–protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation—the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.

A Natural Dominant Negative P2X1 Receptor Due to Deletion of a Single Amino Acid Residue

The P2X1 receptor belongs to a family of oligomeric ATP-gated ion channels with intracellular N and C termini and two transmembrane segments separating a large extracellular domain. Here, we describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351–354). Confocal microscopy revealed proper plasma membrane localization of the mutant in stably transfected HEK293 cells. Nevertheless, voltage-clamped HEK293 cells expressing mutated P2X1 channels failed to develop an ATP or ADP-induced current. Furthermore, when co-expressed with the wild type receptor in Xenopus oocytes, the mutated protein exhibited a dose-dependent dominant negative effect on the normal ATP or ADP-induced P2X1 channel activity. These data indicate that deletion of a single apolar amino acid residue at the inner border of the P2X1 TM2 generates a nonfunctional channel. The inactive and dominant negative form of the P2X1 receptor may constitute a new tool for the study of the physiological role of this channel in native cells.

Human phenotype ontology annotation and cluster analysis to unravel genetic defects in 707 cases with unexplained bleeding and platelet disorders.

BackgroundHeritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases.MethodsWe report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms. Cluster analyses are then used to characterise groups of cases with similar HPO terms and variants in the same genes.ResultsWe show that 60% of index cases with heritable BPD enrolled at 10 European or US centres were annotated with HPO terms indicating abnormalities in organ systems other than blood or blood-forming tissues, particularly the nervous system. Cases within pedigrees clustered closely together on the bases of their HPO-coded phenotypes, as did cases sharing several clinically suspected syndromic disorders. Cases subsequently found to harbour variants in ACTN1 also clustered closely, even though diagnosis of this recently described disorder was not possible using only the clinical and laboratory data available to the enrolling clinician.ConclusionsThese findings validate our novel HPO-based phenotype clustering methodology for known BPD, thus providing a new discovery tool for BPD of unknown genetic basis. This approach will also be relevant for other rare diseases with significant genetic heterogeneity.

SCAMP5, NBEA and AMISYN: three candidate genes for autism involved in secretion of large dense-core vesicles

Autism is a neurodevelopmental disorder characterized by impaired social reciprocity, impaired communication and stereotypical behaviors. Despite strong evidence for a genetic basis, few susceptibility genes have been identified. Here, we describe the positional cloning of SCAMP5, CLIC4 and PPCDC as candidate genes for autism, starting from a person with idiopathic, sporadic autism carrying a de novo chromosomal translocation. One of these genes, SCAMP5 is silenced on the derivative chromosome, and encodes a brain-enriched protein involved in membrane trafficking, similar to the previously identified candidate genes NBEA and AMISYN. Gene silencing of Nbea, Amisyn and Scamp5 in mouse beta-TC3 cells resulted in a 2-fold increase in stimulated secretion of large dense-core vesicles (LDCVs), while overexpression suppressed secretion. Moreover, ultrastructural analysis of blood platelets from the patients with haploinsufficieny of one of the three candidate genes, showed morphological abnormalities of dense-core granules, which closely resemble LDCVs. Taken together, this study shows that in three independent patients with autism three different negative regulators of LDCV secretion are affected, respectively, suggesting that in at least a subgroup of patients the regulation of neuronal vesicle trafficking may be involved in the pathogenesis of autism.

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies

E527K hyperactive SRC results in megakaryocytes with increased podosome formation, thrombocytopenia, myelofibrosis, bleeding, and bone pathologies. SRC shows its stripes The nonreceptor tyrosine kinase SRC is a proto-oncogene that has been associated with cancer progression. Now, Turro et al. find a gain-of-function mutation in SRC in nine patients with myelofibrosis, bleeding, and bone disorders. This mutation prevented SRC from inhibiting itself, and the overactive SRC resulted in enhanced tyrosine phosphorylation in a zebrafish model as well as in patient-derived cells. In patients with myelofibrosis, this SRC mutation was associated with increased outgrowth of myeloid and megakaryocyte colonies, with abnormal platelet production, which could be rescued by SRC kinase inhibition. These findings may be important for understanding the severe bleeding in cancer patients treated with Src family kinase inhibitors. The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC’s self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients’ platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC–positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients’ blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.

Frequency of Severe Malaria and Invasive Bacterial Infections among Children Admitted to a Rural Hospital in Burkina Faso

Background Although severe malaria is an important cause of mortality among children in Burkina Faso, data on community-acquired invasive bacterial infections (IBI, bacteremia and meningitis) are lacking, as well as data on the involved pathogens and their antibiotic resistance rates. Methods The present study was conducted in a rural hospital and health center in Burkina Faso, in a seasonal malaria transmission area. Hospitalized children (<15 years) presenting with T≥38.0°C and/or signs of severe illness were enrolled upon admission. Malaria diagnosis and blood culture were performed for all participants, lumbar puncture when clinically indicated. We assessed the frequency of severe malaria (microscopically confirmed, according to World Health Organization definitions) and IBI, and the species distribution and antibiotic resistance of the bacterial pathogens causing IBI. Results From July 2012 to July 2013, a total of 711 patients were included. Severe malaria was diagnosed in 292 (41.1%) children, including 8 (2.7%) with IBI co-infection. IBI was demonstrated in 67 (9.7%) children (bacteremia, n = 63; meningitis, n = 6), 8 (11.8%) were co-infected with malaria. Non-Typhoid Salmonella spp. (NTS) was the predominant isolate from blood culture (32.8%), followed by Salmonella Typhi (18.8%), Streptococcus pneumoniae (18.8%) and Escherichia coli (12.5%). High antibiotic resistance rates to first line antibiotics were observed, particularly among Gram-negative pathogens. In addition, decreased ciprofloxacin susceptibility and extended-spectrum beta lactamase (ESBL) production was reported for one NTS isolate each. ESBL production was observed in 3/8 E. coli isolates. In-hospital mortality was 8.2% and case-fatality rates for IBI (23.4%) were significantly higher compared to severe malaria (6.8%, p<0.001). Conclusions Although severe malaria was the main cause of illness, IBI were not uncommon and had higher case-fatality rates. The high frequency, antibiotic resistance rates and mortality rates of community acquired IBI require improvement in hygiene, better diagnostic methods and revision of current treatment guidelines.

GNAS defects identified by stimulatory G protein alpha-subunit signalling studies in platelets.

CONTEXT GNAS is an imprinted region that gives rise to several transcripts, antisense transcripts, and noncoding RNAs, including transcription of RNA encoding the alpha-subunit of the stimulatory G protein (Gsalpha). The complexity of the GNAS cluster results in ubiquitous genomic imprints, tissue-specific Gsalpha expression, and multiple genotype-phenotype relationships. Phenotypes resulting from genetic and epigenetic abnormalities of the GNAS region include Albrights hereditary osteodystrophy, pseudohypoparathyroidism types Ia (PHPIa) and Ib (PHPIb), and pseudopseudohypoparathyroidism (PPHP). OBJECTIVE The aim was to study the complex GNAS pathology by a functional test as an alternative to the generally used but labor-intensive erythrocyte complementation assay. DESIGN AND PATIENTS We report the first platelet-based diagnostic test for Gsalpha hypofunction, supported by clinical, biochemical, and molecular data for six patients with PHPIa or PPHP and nine patients with PHPIb. The platelet test is based on the inhibition of platelet aggregation by cAMP, produced after Gsalpha stimulation. RESULTS Platelets are easily accessible, and platelet aggregation responses were found to reflect Gsalpha signaling defects in patients, in concordance with the patients phenotype and genotype. Gsalpha hypofunction in PHPIa and PPHP patients with GNAS mutations was clearly detected by this method. Mildly decreased or normal Gsalpha function was detected in patients with PHPIb with either an overall or exon 1A-only epigenetic defect, respectively. Platelet Gsalpha expression was reduced in both PHPIb patient groups, whereas XLalphas was up-regulated only in PHPIb patients with the broad epigenetic defect. CONCLUSION The platelet-based test is a novel tool for establishing the diagnosis of Gsalpha defects, which may otherwise be quite challenging.

An integrated proteomics and genomics analysis to unravel a heterogeneous platelet secretion defect.

Eight patients with clinical bleeding problems have evidence for platelet storage pool disease as they present with impaired platelet aggregation and secretion with low concentrations of ADP and collagen and an absence of second phase aggregation with epinephrine. Electron microscopy analysis further showed a reduced but not absent amount of platelet dense granules, and CD63 staining was decreased compared to healthy controls. The presence of alpha granules and CD62P expression after platelet activation was normal. This work aimed at identifying differentially expressed proteins in the platelet releasate and its remaining pellet after activation with A23187 and TRAP in patients and controls using DIGE-based proteomic technology. We identified 44 differentially expressed proteins in patients and the altered expression for some of them was confirmed by immunoblot analysis. Most of these proteins belong to the class of cytoskeleton-related proteins. In addition, 29 cytoskeleton-related genes showed an altered expression in platelet mRNA from patients using a real-time PCR array. In conclusion, our study shows that the dense granule secretion defect in patients with platelet storage pool disease is highly heterogeneous with evidence of an underlying cytoskeleton defect.

Compound Heterozygous Mutations in the GNAS Gene of a Boy with Morbid Obesity, Thyroid-Stimulating Hormone Resistance, Pseudohypoparathyroidism, and a Prothrombotic State

CONTEXT Pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism are characterized by Albrights hereditary osteodystrophy (AHO), respectively, with and without hormone resistance. Both clinical conditions result from decreased expression or function of the alpha-subunit of the stimulatory G protein (Gsalpha) of adenylyl cyclase due to heterozygous inactivating mutations in GNAS. Homozygous GNAS defects have not been described. OBJECTIVE A genetic and functional GNAS study was undertaken in a boy with morbid obesity (body mass index Z-score of 5 at the age of 3 yr, with a body fat fraction of 40%, which is more than twice normal), TSH resistance, pseudohypoparathyroidism, and a prothrombotic state. RESULTS The boy was found to be a first case with a compound heterozygous GNAS defect: a de novo R231C mutation on the paternal allele and on the other allele a maternally inherited unique combination of three C to T nucleotide substitutions in exon 7 (I185I), intron 7 (IVS7 + 31), and exon 13 (N371N) leading to aberrant splicing of GNAS. Platelets of this boy displayed a pronounced Gsalpha hypofunction and were spontaneously hyperreactive resulting in a prothrombotic state due to extremely low cAMP levels. CONCLUSION This report expands the human GNAS genotype-phenotype spectrum to include compound heterozygosity and a prothrombotic state.