Sarah K. Westbury

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.

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.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

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.

Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

Patients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca(2+) mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1,ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin α(IIb)β(3) activation.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

High haematocrit in cyanotic congenital heart disease affects how fibrinogen activity is determined by rotational thromboelastometry

INTRODUCTION Viscoelastometry enables rapid evaluation of coagulopathy in settings such as cardiac surgery but may be influenced by red cell concentration. METHODS In order to study the effects of supra-physiological red cell concentrations on viscoelastometry, we compared ROTEM® viscoelastometry and plasma coagulation assay results in high haematocrit (HCT; 0.55-0.76 L/L) blood from patients with cyanotic congenital heart disease (CCHD), and in model high HCT blood (HCT 0.45-0.70 L/L). RESULTS High HCT blood from CCHD patients (median HCT 0.66 L/L) displayed prolonged clot initiation in the EXTEM® test compared to controls and reduced maximum clot firmness (MCF) in the EXTEM (median 51 mm vs 64 mm in controls) and FIBTEM® (7 mm vs 14 mm) tests. The plasma fibrinogen (Clauss; CF) was similar in CCHD blood to controls (median 2.94 g/L vs 2.49) but the whole blood fibrinogen concentration (WBFC) was reduced (1.27 g/L vs 1.58). The FIBTEM MCF correlated linearly with the CF (r(2)=0.68; p<0.0001) and WBFC (r(2)=0.65; p<0.0001) in control blood but this relationship was maintained only with WBFC in CCHD blood. Model high HCT blood showed abnormal ROTEM test results that were similar to CCHD blood, including reduced FIBTEM MCF (14 mm with HCT 0.32-0.44 vs 6mm with HCT 0.63-0.70). The ROTEM results were HCT dependent but independent of plasma clotting times and fibrinogen concentration. CONCLUSION Supra-physiologic HCT causes abnormal ROTEM test results consistent with increased dilution of fibrinogen and coagulation factors in whole blood by red cells. High HCT should be considered during interpretation of ROTEM test results in clinical settings.

A comprehensive high-throughput sequencing test for the diagnosis of inherited bleeding, thrombotic and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking

Objective—Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. Approach and Results—We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to &agr;-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual &agr;-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. Conclusions—Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists.