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Diagnosing mitochondrial disorders remains challenging. This is partly because the clinical phenotypes of patients overlap with those of other sporadic and inherited disorders. Although the widespread availability of genetic testing has increased the rate of diagnosis, the combination of phenotypic and genetic heterogeneity still makes it difficult to reach a timely molecular diagnosis with confidence. An objective, systematic method for describing the phenotypic spectra for each variant provides a potential solution to this problem. We curated the clinical phenotypes of 6688 published individuals with 89 pathogenic mitochondrial DNA (mtDNA) mutations, collating 26 348 human phenotype ontology (HPO) terms to establish the MitoPhen database. This enabled a hypothesis-free definition of mtDNA clinical syndromes, an overview of heteroplasmy-phenotype relationships, the identification of under-recognized phenotypes, and provides a publicly available reference dataset for objective clinical comparison with new patients using the HPO. Studying 77 patients with independently confirmed positive mtDNA diagnoses and 1083 confirmed rare disease cases with a non-mitochondrial nuclear genetic diagnosis, we show that HPO-based phenotype similarity scores can distinguish these two classes of rare disease patients with a false discovery rate

BACKGROUND: Brain monoamine vesicular transport disease is an infantile onset neurodevelopmental disorder caused by variants in SLC18A2, which codes for the vesicular monoamine transporter 2 (VMAT2) protein, involved in the transport of monoamines into synaptic vesicles and of serotonin into platelet dense granules. CASE PRESENTATION: The presented case is of a child, born of healthy consanguineous parents, who exhibited hypotonia, mental disability, epilepsy, uncontrolled movements, and gastrointestinal problems. A trial treatment with L-DOPA proved unsuccessful and the exact neurological involvement could not be discerned due to normal metabolic and brain magnetic resonance imaging results.Platelet studies and whole genome sequencing were performed. At age 4, the child's platelets showed a mild aggregation and adenosine triphosphate secretion defect that could be explained by dysmorphic dense granules observed by electron microscopy. Interestingly, the dense granules were almost completely depleted of serotonin. A novel homozygous p.P316A missense variant in VMAT2 was detected in the patient and the consanguineous parents were found to be heterozygous for this variant. Although the presence of VMAT2 on platelet dense granules has been demonstrated before, this is the first report of defective platelet dense granule function related to absent serotonin storage in a patient with VMAT2 deficiency but without obvious clinical bleeding problems. CONCLUSIONS: This study illustrates the homology between serotonin metabolism in brain and platelets, suggesting that these blood cells can be model cells for some pathways relevant for neurological diseases. The literature on VMAT2 deficiency is reviewed. ispartof: JIMD Rep vol:47 issue:1 pages:9-16 ispartof: location:United States status: Published online

Funder: Cancer Research UK, Funder: Breast Cancer Now, Funder: Addenbrookes Charitable Trust, Funder: Mark Foundation For Cancer Research; doi: http://dx.doi.org/10.13039/100014599, Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals (GB), Background: NRG1 gene fusions may be clinically actionable, since cancers carrying the fusion transcripts can be sensitive to tyrosine kinase inhibitors. The NRG1 gene encodes ligands for the HER2(ERBB2)-ERBB3 heterodimeric receptor tyrosine kinase, and the gene fusions are thought to lead to autocrine stimulation of the receptor. The NRG1 fusion expressed in the breast cancer cell line MDA-MB-175 serves as a model example of such fusions, showing the proposed autocrine loop and exceptional drug sensitivity. However, its structure has not been properly characterised, its oncogenic activity has not been fully explained, and there is limited data on such fusions in breast cancer. Methods: We analysed genomic rearrangements and transcripts of NRG1 in MDA-MB-175 and a panel of 571 breast cancers. Results: We found that the MDA-MB-175 fusion—originally reported as a DOC4(TENM4)-NRG1 fusion, lacking the cytoplasmic tail of NRG1—is in reality a double fusion, PPP6R3-TENM4-NRG1, producing multiple transcripts, some of which include the cytoplasmic tail. We hypothesise that many NRG1 fusions may be oncogenic not for lacking the cytoplasmic domain but because they do not encode NRG1’s nuclear-localised form. The fusion in MDA-MB-175 is the result of a very complex genomic rearrangement, which we partially characterised, that creates additional expressed gene fusions, RSF1-TENM4, TPCN2-RSF1, and MRPL48-GAB2. We searched for NRG1 rearrangements in 571 breast cancers subjected to genome sequencing and transcriptome sequencing and found four cases (0.7%) with fusions, WRN-NRG1, FAM91A1-NRG1, ARHGEF39-NRG1, and ZNF704-NRG1, all splicing into NRG1 at the same exon as in MDA-MB-175. However, the WRN-NRG1 and ARHGEF39-NRG1 fusions were out of frame. We identified rearrangements of NRG1 in many more (8% of) cases that seemed more likely to inactivate than to create activating fusions, or whose outcome could not be predicted because they were complex, or both. This is not surprising because NRG1 can be pro-apoptotic and is inactivated in some breast cancers. Conclusions: Our results highlight the complexity of rearrangements of NRG1 in breast cancers and confirm that some do not activate but inactivate. Careful interpretation of NRG1 rearrangements will therefore be necessary for appropriate patient management.

Supplemental Digital Content is available in the text., Background: Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH, we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. Methods: We analyzed 13 037 participants enrolled in the NBR study (NIHR BioResource—Rare Diseases), of which 1148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension, we used the Bayesian rare variant association method BeviMed. Results: Heterozygous, high impact, likely loss-of-function variants in the kinase insert domain receptor (KDR) gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (posterior probability=0.989) and older age at diagnosis (posterior probability=0.912). We also provide evidence for familial segregation of a rare nonsense KDR variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the 5 patients harboring these predicted deleterious variants in KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions: The Bayesian inference approach allowed us to independently validate KDR, which encodes for the VEGFR2 (vascular endothelial growth factor receptor 2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for KDR being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.

Transcriptional profiling of hematopoietic cell subpopulations has helped to characterize the developmental stages of the hematopoietic system and the molecular bases of malignant and non-malignant blood diseases. Previously, only the genes targeted by expression microarrays could be profiled genome-wide. High-throughput RNA sequencing, however, encompasses a broader repertoire of RNA molecules, without restriction to previously annotated genes. We analyzed the BLUEPRINT consortium RNA-sequencing data for mature hematopoietic cell types. The data comprised 90 total RNA-sequencing samples, each composed of one of 27 cell types, and 32 small RNA-sequencing samples, each composed of one of 11 cell types. We estimated gene and isoform expression levels for each cell type using existing annotations from Ensembl. We then used guided transcriptome assembly to discover unannotated transcripts. We identified hundreds of novel non-coding RNA genes and showed that the majority have cell type-dependent expression. We also characterized the expression of circular RNA and found that these are also cell type-specific. These analyses refine the active transcriptional landscape of mature hematopoietic cells, highlight abundant genes and transcriptional isoforms for each blood cell type, and provide a valuable resource for researchers of hematologic development and diseases. Finally, we made the data accessible via a web-based interface: https://blueprint.haem.cam.ac.uk/bloodatlas/. The work was funded by a grant from the European Commission 7th Framework Program (FP7/2007–2013, grant 282510, BLUEPRINT) to XE, PF, JHAM, MY, HGS and WHO. WHO is an NIHR senior investigator and receives funding from Bristol-Myers Squibb, the British Heart Foundation, the Medical Research Council and the NIHR. OGI, FJM, AF, JMM, LC and PF are funded by the Wellcome Trust (WT108749/Z/15/Z) with additional funding for specific project components such as GENCODE from the National Human Genome Research Institute of the National Institutes of Health (2U41HG007234). FP is supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisado Estado do Rio de Janeiro (FAPERJ; E-26/203.229/2016). NANJ is a recipient of a scholarship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES; Finance Code 001). MF is supported by the British Heart Foundation (FS/18/53/33863)

Several strands of evidence question the dogma that human mitochondrial DNA (mtDNA) is inherited exclusively down the maternal line, most recently in three families where several individuals harbored a ‘heteroplasmic haplotype’ consistent with biparental transmission. Here we report a similar genetic signature in 7 of 11,035 trios, with allelic fractions of 5–25%, implying biparental inheritance of mtDNA in 0.06% of offspring. However, analysing the nuclear whole genome sequence, we observe likely large rare or unique nuclear-mitochondrial DNA segments (mega-NUMTs) transmitted from the father in all 7 families. Independently detecting mega-NUMTs in 0.13% of fathers, we see autosomal transmission of the haplotype. Finally, we show the haplotype allele fraction can be explained by complex concatenated mtDNA-derived sequences rearranged within the nuclear genome. We conclude that rare cryptic mega-NUMTs can resemble paternally mtDNA heteroplasmy, but find no evidence of paternal transmission of mtDNA in humans., Recent evidence has questioned the dogma of strict maternal transmission of mitochondrial DNA (mtDNA) in humans. Wei et al. saw no evidence of paternal transmission of mtDNA in 11,035 human trios, and show that nuclear-mitochondrial segments (NUMTs) can give the impression of paternal mtDNA transmission, but are actually inherited through the nuclear genome.

Funder: Medical Research Council, BACKGROUND:The RNA helicase eIF4A1 is a key component of the translation initiation machinery and is required for the translation of many pro-oncogenic mRNAs. There is increasing interest in targeting eIF4A1 therapeutically in cancer, thus understanding how this protein leads to the selective re-programming of the translational landscape is critical. While it is known that eIF4A1-dependent mRNAs frequently have long GC-rich 5'UTRs, the details of how 5'UTR structure is resculptured by eIF4A1 to enhance the translation of specific mRNAs are unknown. RESULTS:Using Structure-seq2 and polysome profiling, we assess global mRNA structure and translational efficiency in MCF7 cells, with and without eIF4A inhibition with hippuristanol. We find that eIF4A inhibition does not lead to global increases in 5'UTR structure, but rather it leads to 5'UTR remodeling, with localized gains and losses of structure. The degree of these localized structural changes is associated with 5'UTR length, meaning that eIF4A-dependent mRNAs have greater localized gains of structure due to their increased 5'UTR length. However, it is not solely increased localized structure that causes eIF4A-dependency but the position of the structured regions, as these structured elements are located predominantly at the 3' end of the 5'UTR. CONCLUSIONS:By measuring changes in RNA structure following eIF4A inhibition, we show that eIF4A remodels local 5'UTR structures. The location of these structural elements ultimately determines the dependency on eIF4A, with increased structure just upstream of the CDS being the major limiting factor in translation, which is overcome by eIF4A activity.

The heterogeneous manifestations of MYH9‐related disorder (MYH9‐RD), characterized by macrothrombocytopenia, Döhle‐like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE‐BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9‐RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9‐RD should always be considered. A HTS‐based strategy is a reliable method to reach a conclusive diagnosis of MYH9‐RD in clinical practice., MYH9‐related disorder diagnosis is still challenging in clinical practice. We analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder and identified 50 patients with a rare variant in MYH9. In the presence of an unclassified platelet disorder with macrothrombocytes, MYH9‐RD should always be considered.

Thrombopoietin (TPO) is a lineage-specific cytokine that is synthesized predominantly in hepatocytes and is an essential regulator of self-renewal of hematopoietic stem cells and their differentiation into platelet-forming megakaryocytes. The major TPO isoform is a 353-aa peptide containing a highly glycosylated C-terminal domain and an amino-terminal receptor binding domain (RBD) that mediates interaction with the TPO receptor (c-mpl) on hematopoietic cells. TPO is encoded by the THPO gene, which maps to 3q27.1 and contains up to 6 coding exons.Increased TPO production caused by gain-of-function THPO variants results in the myeloproliferative disorder thrombocythemia-1 (Online Mendelian Inheritance in Man #187950), in which the circulating platelet count is elevated, sometimes causing bleeding or thrombosis. In contrast, biallelic loss-of-function THPO variants result in a severe reduction in the platelet count and multilineage bone marrow failure. Here, we present genetic and functional evidence that monoallelic loss-of-function THPO variants also result in reduced platelet count but do not affect other circulating blood cells.

BACKGROUND: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome. OBJECTIVE: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. METHODS: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. RESULTS: The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor-dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. CONCLUSION: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:142 issue:2 pages:630-646 ispartof: location:United States status: published

Hereditary bleeding and platelet disorders (BPDs) are characterized by marked genetic heterogeneity, far greater than previously appreciated. The list of genes involved in the regulation of megakaryopoiesis, platelet formation, platelet function and bleeding has been growing rapidly since the introduction of high-throughput sequencing (HTS) approaches in research. Thanks to the gradual adoption of HTS in diagnostic practice, these discoveries are improving the diagnostic yield for BPD patients, who may or may not present with bleeding problems and often have other clinical symptoms unrelated to the blood system. However, it was previously found that screening for all known etiologies gives a diagnostic yield of over 90% when the phenotype closely matches a known BPD but drops to 10% when the phenotype is indicative of a novel disorder. Thus, further research is needed to identify currently unknown etiologies for BPDs. Novel genes are likely to be found to be implicated in BPDs. New modes of inheritance, including digenic inheritance, are likely to play a role in some cases. Additionally, identifying and interpreting pathogenic variants outside exons is a looming challenge that can only be tackled with an improved understanding of the regulatory landscape of relevant cell types and with the transition from targeted sequencing to whole-genome sequencing in the clinic.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs ∗ 63), c.1996C>T (p.Arg666 ∗ ), c.2632G>T (p.Glu878 ∗ ), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deep phenotyping has been defined as the precise and comprehensive analysis of phenotypic abnormalities in which the individual components of the phenotype are observed and described. The three components of the Human Phenotype Ontology (HPO; www.human-phenotype-ontology.org) project are the phenotype vocabulary, disease-phenotype annotations and the algorithms that operate on these. These components are being used for computational deep phenotyping and precision medicine as well as integration of clinical data into translational research. The HPO is being increasingly adopted as a standard for phenotypic abnormalities by diverse groups such as international rare disease organizations, registries, clinical labs, biomedical resources, and clinical software tools and will thereby contribute toward nascent efforts at global data exchange for identifying disease etiologies. This update article reviews the progress of the HPO project since the debut Nucleic Acids Research database article in 2014, including specific areas of expansion such as common (complex) disease, new algorithms for phenotype driven genomic discovery and diagnostics, integration of cross-species mapping efforts with the Mammalian Phenotype Ontology, an improved quality control pipeline, and the addition of patient-friendly terminology. ispartof: Nucleic Acids Research vol:45 issue:D1 pages:D865-D876 ispartof: location:England status: published

Transcriptional profiling of hematopoietic cell subpopulations has helped characterize the developmental stages of the hematopoietic system and the molecular basis of malignant and non-malignant blood diseases for the past three decades. The introduction of high-throughput RNA sequencing has increased knowledge of the full repertoire of RNA molecules in hematopoietic cells of different types, without relying on prior gene annotation. Here, we introduce the analysis of the BLUEPRINT consortium gene expression data for mature hematopoietic cells, comprising 90 total RNA and 32 small RNA sequencing experiments, from 27 different cell types. We used these data to describe the transcriptional profile of each we used guided transcriptome assembly to extend the annotation of the transcribed genome, which led to the identification of hundreds of novel non-coding RNA genes, which display a high degree of cell type specificity. We also characterized the expression of circular RNAs and found that these are also highly cell type specific. This resource refines the active transcriptional landscape of mature hematopoietic cells, highlights abundant genes and transcriptional isoforms for each cell type, and provides valuable data and visualisation tools for the scientific community working on hematological development and diseases.

Spencer et al. report the first description of human IL-6R deficiency in two patients presenting with recurrent infections, atopy, elevated IgE, and abnormal acute-phase responses., IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R. We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

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. ispartof: HAEMATOLOGICA vol:104 issue:5 pages:1036-1045 ispartof: location:Italy status: published

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and mediating genes for more than half such disorders remain to be discovered. We implemented whole-genome sequencing (WGS) in a national healthcare system to streamline diagnosis and to discover unknown aetiological variants, in the coding and non-coding regions of the genome. In a pilot study for the 100,000 Genomes Project, we generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 patients with detailed phenotypic data. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed aetiological. Using WGS of UK Biobank1, we showed that rare alleles can explain the presence of some individuals in the tails of a quantitative red blood cell (RBC) trait. Finally, we reported 4 novel non-coding variants which cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.

Primary immunodeficiency (PID) is characterised by recurrent and often life-threatening infections, autoimmunity and cancer, and it presents major diagnostic and therapeutic challenges. Although the most severe forms present in early childhood, the majority of patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent, and up to 10% develop lymphoid malignancies1–3. Consequently, in sporadic PID genetic diagnosis is difficult and the role of genetics is not well defined. We addressed these challenges by performing whole genome sequencing (WGS) of a large PID cohort of 1,318 participants. Analysis of coding regions of 886 index cases found disease-causing mutations in known monogenic PID genes in 10.3%, while a Bayesian approach (BeviMed4) identified multiple potential new candidate genes, including IVNS1ABP. Exploration of the non-coding genome revealed deletions in regulatory regions which contribute to disease causation. Finally, a genome-wide association study (GWAS) identified PID-associated loci and uncovered evidence for co-localisation of, and interplay between, novel high penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to variable penetrance and phenotypic complexity in PID. Thus, a cohort-based WGS approach to PID diagnosis can increase diagnostic yield while deepening our understanding of the key pathways influencing human immune responsiveness.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein–coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet–platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin–mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

Primary Immune disorders affect 15,000 new patients every year in Europe. Genetic tests are usually performed on a single or very limited number of genes leaving the majority of patients without a genetic diagnosis. We designed, optimised and validated a new clinical diagnostic platform called GRID, Genomics of Rare Immune Disorders, to screen in parallel 279 genes, including 2015 IUIS genes, known to be causative of Primary Immune disorders (PID). Validation to clinical standard using more than 58,000 variants in 176 PID patients shows an excellent sensitivity, specificity. The customised and automated bioinformatics pipeline prioritises and reports pertinent Single Nucleotide Variants (SNVs), INsertions and DELetions (INDELs) as well as Copy Number Variants (CNVs). An example of the clinical utility of the GRID panel, is represented by a patient initially diagnosed with X-linked agammaglobulinemia due to a missense variant in the BTK gene with severe inflammatory bowel disease. GRID results identified two additional compound heterozygous variants in IL17RC, potentially driving the altered phenotype.

Next-generation sequencing has been invaluable in the elucidation of the genetic etiology of many subtypes of intellectual disability in recent years. Here, using exome sequencing and whole-genome sequencing, we identified three de novo truncating mutations in WAS protein family member 1 (WASF1) in five unrelated individuals with moderate to profound intellectual disability with autistic features and seizures. WASF1, also known as WAVE1, is part of the WAVE complex and acts as a mediator between Rac-GTPase and actin to induce actin polymerization. The three mutations connected by Matchmaker Exchange were c.1516C>T (p.Arg506Ter), which occurs in three unrelated individuals, c.1558C>T (p.Gln520Ter), and c.1482delinsGCCAGG (p.Ile494MetfsTer23). All three variants are predicted to partially or fully disrupt the C-terminal actin-binding WCA domain. Functional studies using fibroblast cells from two affected individuals with the c.1516C>T mutation showed a truncated WASF1 and a defect in actin remodeling. This study provides evidence that de novo heterozygous mutations in WASF1 cause a rare form of intellectual disability. ispartof: AMERICAN JOURNAL OF HUMAN GENETICS vol:103 issue:1 pages:144-153 ispartof: location:United States status: published

BACKGROUND Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant β3 integrins, displaying the biallelic HPA-1 epitopes (rHPA-1), have been shown to detect HPA-1a alloantibodies implicated in FMAIT. This report describes a multicenter validation of the beads using the results of well-characterized samples to define the optimum parameters for analysis of a large cohort of 498 clinical samples. STUDY DESIGN AND METHODS Fifty-one blinded quality assurance (QA) samples were tested by six laboratories to standardize the rHPA-1 bead assay and to develop an algorithm for sample classification. Five laboratories retrieved samples from 498 independent FMAIT cases, previously tested by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) assay, from their local archives for testing with the rHPA-1 beads. The results were evaluated using a mathematical algorithm developed to classify the samples. RESULTS The QA samples gave a mean concordance of 94% between the bead and MAIPA assays, while 97% concordance was observed with the FMAIT samples. Of the 15 discrepant samples, seven were positive by the beads but negative by MAIPA, while the contrary was observed for eight samples. Overall, the bead assay achieved 98% sensitivity for HPA-1a antibody detection in FMAIT and 98.7% specificity compared to the local MAIPA. CONCLUSION The rHPA-1 bead assay is a rapid 3-hour assay for the sensitive detection of HPA-1 antibodies. Its ease of use would enable prompt detection of maternal HPA-1a antibodies in suspected FMAIT cases, which is important supportive evidence for treatment by transfusion with HPA-1b1b PLTs.

We present a rapid and powerful inference procedure for identifying loci associated with rare hereditary disorders using Bayesian model comparison. Under a baseline model, disease risk is fixed across all individuals in a study. Under an association model, disease risk depends on a latent bipartition of rare variants into pathogenic and non-pathogenic variants, the number of pathogenic alleles that each individual carries, and the mode of inheritance. A parameter indicating presence of an association and the parameters representing the pathogenicity of each variant and the mode of inheritance can be inferred in a Bayesian framework. Variant-specific prior information derived from allele frequency databases, consequence prediction algorithms, or genomic datasets can be integrated into the inference. Association models can be fitted to different subsets of variants in a locus and compared using a model selection procedure. This procedure can improve inference if only a particular class of variants confers disease risk and can suggest particular disease etiologies related to that class. We show that our method, called BeviMed, is more powerful and informative than existing rare variant association methods in the context of dominant and recessive disorders. The high computational efficiency of our algorithm makes it feasible to test for associations in the large non-coding fraction of the genome. We have applied BeviMed to whole-genome sequencing data from 6,586 individuals with diverse rare diseases. We show that it can identify multiple loci involved in rare diseases, while correctly inferring the modes of inheritance, the likely pathogenic variants, and the variant classes responsible.

Mg2+ plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg2+]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7fl/fl-Pf4Cre) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7fl/fl-Pf4Cre MKs, which is rescued by Mg2+ supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice., Although Mg2+ is vital for platelet activation and aggregation, its regulation in these cells is still largely unknown. Here, the authors show that TRPM7, a cation channel and a protein kinase, regulates thrombopoiesis and platelet size by affecting the cytoskeleton of these cells in mice and humans.

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 (MK). 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 two unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 p.R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was associated with 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. Over-expression 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 insights into the autoregulation of DIAPH1 activity.

Rare genetic disorders, which can now be studied systematically with affordable genome sequencing, are often caused by high-penetrance rare variants. Such disorders are often heterogeneous and characterized by abnormalities spanning multiple organ systems ascertained with variable clinical precision. Existing methods for identifying genes with variants responsible for rare diseases summarize phenotypes with unstructured binary or quantitative variables. The Human Phenotype Ontology (HPO) allows composite phenotypes to be represented systematically but association methods accounting for the ontological relationship between HPO terms do not exist. We present a Bayesian method to model the association between an HPO-coded patient phenotype and genotype. Our method estimates the probability of an association together with an HPO-coded phenotype characteristic of the disease. We thus formalize a clinical approach to phenotyping that is lacking in standard regression techniques for rare disease research. We demonstrate the power of our method by uncovering a number of true associations in a large collection of genome-sequenced and HPO-coded cases with rare diseases.

Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors., The transcription factor estrogen-related receptor beta, Esrrb, regulates pluripotency genes in embryonic stem cells, but how it acts in trophoblast stem (TS) cells is unclear. Here, the authors identify Esrrb as a primary target of Fgf/Mek signaling and outline a unique TS cell-specific interactome to sustain stemness.

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. The work described in this manuscript was primarily supported by the European Commission Seventh Framework Program through the BLUEPRINT grant with code HEALTH-F5-2011-282510 (DH, FB, GC, JHAM, KD, LC, MF, SC, SF and SPG). Research in the Ouwehand laboratory is further supported by program grants from the National Institute for Health Research (NIHR, http://www.nihr.ac.uk; to AA, MK, PP, SBGJ, SN, and WHO); and the British Heart Foundation under numbers RP-PG-0310-1002 and RG/09/12/28096 (http://www.bhf.org.uk; to AR and WJA). KF and MK were supported by Marie Curie funding from the NETSIM FP7 program funded by the European Commission. The Cambridge BioResource (http://www.cambridgebioresource.org.uk), the Cell Phenotyping Hub, and the Cambridge Translational GenOmics laboratory (http://www.catgo.org.uk) are supported by an NIHR grant to the Cambridge NIHR Biomedical Research Centre (BRC). Research in the Soranzo laboratory (LV, NS and SW) is further supported by the Wellcome Trust (Grant Codes WT098051 and WT091310) and the EU FP7 EPIGENESYS initiative (Grant Code 257082). Research in the Cvejic laboratory (AC and CL) is funded by the Cancer Research UK under grant number C45041/A14953. SJS is funded by NIHR. MEF is supported by a British Heart Foundation Clinical Research Training Fellowship, number FS/12/27/29405. EBM is supported by a Wellcome Trust grant, number 084183/Z/07/Z. FAC, CL and SW are supported by MRC Clinical Training Fellowships and TB by a British Society of Haematology/NHS Blood and Transplant grant. RJR is a Principal Research Fellow of the Wellcome Trust, grant No. 082961/Z/07/Z. Research in the Flicek laboratory is also supported by the Wellcome Trust (grant number 095908) and EMBL. Research in the Bertone laboratory is supported by EMBL. KF and CvG are supported by FWO-Vlaanderen through grant G.0B17.13N

Inherited bleeding, thrombotic and platelet disorders (BPDs) affect approximately 3M people worldwide and an appreciable portion have a disorder of megakaryopoiesis and the production and function of platelets, including the formation of granules. While genetic variants in 76 genes have been implicated in BPDs, many patients remain without a molecular diagnosis (Lentaigne et al, Simeoni et al). We hypothesised that some of these disorders may be caused by compound inheritance of variants in two different genes, a mode of inheritance thus far never implicated in BPDs. For the pilot phase of the 100 000 Genomes Project we have sequenced the whole genomes of 10,000 individuals consisting of probands with molecularly unexplained rare disorders and their close relatives, with 3,000 having inherited disorders of the blood and immune system. We searched for causal variants in known BPD-related genes and employed a new statistical method for Bayesian evaluation of rare variants in Mendelian disease (BeviMed) (Greene et al) to identify novel marginal associations between rare variants and disease status. Where the identified variants could not individually explain the phenotype in full within the pedigrees, we searched for additional variants affecting other BPD-related genes or novel genes identified using BeviMed. First, we have identified a large pedigree in which certain members with mild thrombocytopenia (lower 10th percentile of the population distribution) are affected by a single variant encoding a premature stop at residue 69 in the major isoform of the tropomyosin gene TPM4 expressed in megakaryocytes (Pleines et al). Other members of the family have distinctly more severe macrothrombocytopenia (with platelet counts as low as 24x109/l) and this is due to inheritance of a second variant in the actinin gene ACTN1 leading to a Thr340Met substitution, demonstrating a compound additive effect of variants in two genes encoding cytoskeletal proteins important for actin polymerization and thereby causing inadequate platelet formation. It is noteworthy that both of these genes have been identified in a genome wide association study of the count and mean volume of platelets (Gieger et al). Second, we used BeviMed to identify genes containing variants that are marginally associated with a syndrome defined by a platelet granule defect combined with familial autism (Bijl et al). The strongest association is due to splice variants in a granule-related gene on Chr17p13 but in all four unrelated cases at least one additional variant is required to explain the observed segregation patterns. Two of these unrelated cases harbor variants in two other marginally associated genes at Chr9q32 and Chr4q31 and a third harbors a de novo copy number variant. These additional variants likely explain why the children in three of the families are affected by this syndrome while their parents are not. In conclusion, we have used whole genome sequencing, pedigree building, detailed platelet phenotyping and new association approaches to identify the first cases of digenic inheritance of BPDs. Our results illustrate the cooperative role of different cytoskeletal proteins in platelet formation and cement the role of granule biology in the function of both platelets and neurons. References: Lentaigne et al (2016) Inherited platelet disorders: towards DNA-based diagnosis. Blood127(23) 2814-2823.Simeoni et al (2016) A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders. Blood127(23) 2793-2803.Greene et al (2016) Bayesian evaluation of variant involvement in Mendelian disease. http://cran.r-project.org/web/packages/BeviMed.Pleines et al (2016) Tropomyosins regulate platelet biogenesis. (Under submission).Gieger et al (2011) New gene functions in megakaryopoiesis and platelet formation. Nature480(7376) 201-208Bijl et al (2015) Platelet studies in autism spectrum disorder patients and first-degree relatives. Molecular Autism6:57. Disclosures No relevant conflicts of interest to declare.

Background RNA Biology A common and important aim in the field of genomics is the characterization of populations of RNA molecules. Investigators within the field typically wish to uncover the sequence and concentration of each RNA in a set of samples, either as an objective in its own right or as an early step in a larger analysis pipeline. Later steps might include the identification of differentially expressed genes between treatment and control groups, the clustering of genes into sets sharing putative, common regulatory pathways, or the association of genomic polymorphisms with patterns of expression. Roughly 4% of the RNAs in a typical unprocessed RNA sample consists of messenger RNAs (mRNAs), which code for proteins. Non-coding RNAs largely comprise ribosomal and transfer RNAs, which are involved in protein synthesis but do not code for proteins themselves. The remainder of the non-coding RNAs include a set of less abundant types of molecules with diverse functions. As a result of their direct role in protein synthesis, mRNAs have been in the limelight of genomic research. Protein-coding genes are transcribed by RNA polymerase from their 5′ (upstream) end to their 3′ (downstream) end to produce pre-mRNA. As this process takes place, certain regions may be spliced out from the pre-mRNA and discarded, leaving behind only an mRNA sequence of connected exons, known as an isoform. Multiple combinations of exons may be produced, which is known as alternative splicing, and different isoforms may have different 5′ and 3′ transcript start and end sites. This allows a single gene to produce multiple distinct transcripts and contributes to the phenotypic complexity of eukaryotes. The collection of possible transcripts produced by a single gene is known as the gene model.

Gene expression levels are thought to diverge primarily via regulatory mutations in trans within species, and in cis between species. To test this hypothesis in mammals we used RNA-sequencing to measure gene expression divergence between C57BL/6J and CAST/EiJ mouse strains and allele-specific expression in their F1 progeny. We identified 535 genes with parent-of-origin specific expression patterns, although few of these showed full allelic silencing. This suggests that the number of imprinted genes in a typical mouse somatic tissue is relatively small. In the set of nonimprinted genes, 32% showed evidence of divergent expression between the two strains. Of these, 2% could be attributed purely to variants acting in trans, while 43% were attributable only to variants acting in cis. The genes with expression divergence driven by changes in trans showed significantly higher sequence constraint than genes where the divergence was explained by variants acting in cis. The remaining genes with divergent patterns of expression (55%) were regulated by a combination of variants acting in cis and variants acting in trans. Intriguingly, the changes in expression induced by the cis and trans variants were in opposite directions more frequently than expected by chance, implying that compensatory regulation to stabilize gene expression levels is widespread. We propose that expression levels of genes regulated by this mechanism are fine-tuned by cis variants that arise following regulatory changes in trans, suggesting that many cis variants are not the primary targets of natural selection.

To explore the role of p16INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16INK4a and p14ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16INK4a protein but expressing a functional 14ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16INK4a and growth arrest, EBNA3C inactivation in the p16INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs., Author Summary Epstein-Barr virus (EBV) is a causative agent of several types of B cell lymphoma. In human B cells, EBV reduces protein levels of at least two tumour suppressors that would otherwise be activated in response to over-expressed oncogenes. These proteins are BIM, which induces cell death and p16INK4a, which prevents cell proliferation. Repression of both is via epigenetic methylation of histones and is dependent on expression of both EBNA3A and EBNA3C – two EBV proteins required for the transformation of normal B cells into lymphoblastoid cell lines (LCLs). In this report we have used EBV with a conditionally active EBNA3C – active only in the presence of 4-hydroxytamoxifen – together with B cells from an individual carrying a homozygous deletion of p16INK4a to confirm that regulation of p16INK4a expression is a major function of EBNA3C and demonstrate that if B cells lack p16INK4a, then EBNA3C is no longer required for EBV-driven proliferation of LCLs. Furthermore we show that early after the infection of normal B cells, EBV induces p16INK4a accumulation that – if unchecked by EBNA3C (and EBNA3A) – prevents LCL outgrowth. Formal proof that p16INK4a is the main target of EBNA3C comes with the production of p16-null LCLs that have never expressed functional EBNA3C.

For the BRIDGE-Bleeding and Platelet Disorder Consortium. We here present a pedigree with nine cases that present with a new syndrome characterized by early onset myelofibrosis, thrombocytopenia, bleeding and bone pathologies. The syndrome is caused by a dominant gain-of-function mutation in universal tyrosine kinase SRC and it is the first example of a pathogenic germline mutation in this extensively studied kinase. The genetic discovery has been made possible by making use of our collection of 1,115 patients with inherited bleeding and platelet disorders of unknown molecular etiology enrolled by us at 25 tertiary referral centers as part of the NIHR BioResource - Rare Diseases pilot phase of the 100,000 Genomes Project. The analysis of genome sequencing data for 2 affected individuals was empowered by comparison of Human Phenotype Ontology (HPO; Westbury et al., Genome Medicine, 2015) terms of the patients with OMIM and the Mouse Phenotype Ontology terms from 7,541 strains. The selection of the SRC gene as the most likely candidate gene was also informed by the use of our Blueprint blood cell progenitor RNA-seq data (Chen et al., Science 2014). 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 COS7 cells overexpressing E527K SRC. The active form of SRC predominates in patients’ platelets, resulting in enhanced overall tyrosine phosphorylation and reduced platelet activity. Patients with myelofibrosis have a hypercellular bone marrow with trilineage dysplasia and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. Patient MKs generate platelets which are dysmorphic, low in number, highly variable in size and with a paucity of α-granules. Over-active 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 enhanced tyrosine phosphorylation levels and with reduced ploidy compared to cells transduced with wild-type SRC. Patient-derived and E527K-transduced MKs show Y419 SRC positive stained podosomes that induce a strongly altered organization of filamentous actin. Expression of mutated src in zebrafish recapitulates patients’ blood and bone phenotypes. Our findings reveal an unexpected and wide-ranging pathobiology for this universal kinase Src with podosome formation. In hematopoiesis constitutive active Src primarily affects megakaryopoiesis with ineffective formation of platelets. Its inadequate function is associated with bleeding. The kinase Src is activated frequently in human cancers hence new SFK inhibitors are undergoing clinical trials and side effects with severe bleeding are observed frequently. It remains to be determined whether this new class of SFK inhibitors inadvertently causes inadequate formation of platelets, which do not function properly. Disclosures: No relevant conflicts of interest to declare.

Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphoblastoid cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. Using Affymetrix Exon 1.0 ST microarrays analysed with the MMBGX algorithm, we have identified over 1000 genes whose regulation by EBV requires one of the EBNA3s. Remarkably, a third of the genes identified require more than one EBNA3 for their regulation, predominantly EBNA3C co-operating with either EBNA3B, EBNA3A or both. The microarray was validated by real-time PCR, while ChIP analysis of a selection of co-operatively repressed promoters indicates a role for polycomb group complexes. Targets include genes involved in apoptosis, cell migration and B-cell differentiation, and show a highly significant but subtle alteration in genes involved in mitosis. In order to assess the relevance of the BL31 system to LCLs, we analysed the transcriptome of a set of EBNA3B knockout (3BKO) LCLs. Around a third of the genes whose expression level in LCLs was altered in the absence of EBNA3B were also altered in 3BKO-BL31 cell lines. Among these are TERT and TCL1A, implying that EBV-induced changes in the expression of these genes are not required for B-cell transformation. We also identify 26 genes that require both EBNA3A and EBNA3B for their regulation in LCLs. Together, this shows the complexity of the interaction between EBV and its host, whereby multiple EBNA3 proteins co-operate to modulate the behaviour of the host cell.

Affymetrix has recently developed whole-transcript GeneChips-'Gene' and 'Exon' arrays-which interrogate exons along the length of each gene. Although each probe on these arrays is intended to hybridize perfectly to only one transcriptional target, many probes match multiple transcripts located in different parts of the genome or alternative isoforms of the same gene. Existing statistical methods for estimating expression do not take this into account and are thus prone to producing inflated estimates. We propose a method, Multi-Mapping Bayesian Gene eXpression (MMBGX), which disaggregates the signal at 'multi-match' probes. When applied to Gene arrays, MMBGX removes the upward bias of gene-level expression estimates. When applied to Exon arrays, it can further disaggregate the signal between alternative transcripts of the same gene, providing expression estimates of individual splice variants. We demonstrate the performance of MMBGX on simulated data and a tissue mixture data set. We then show that MMBGX can estimate the expression of alternative isoforms within one experimental condition, confirming our results by RT-PCR. Finally, we show that our method for detecting differential splicing has a lower error rate than standard exonlevel approaches on a previously validated colon cancer data set. © The Author(s) 2009. Published by Oxford University Press. EU (grant FP6-36495); BBSRC (grants BBG0003521 and BBC5196701). Funding for open access charge: BBSRC.

Human mRNA DeXD/H-box helicases are ubiquitous molecular motors that are required for the majority of cellular processes that involve RNA metabolism. One of the most abundant is eIF4A, which is required during the initiation phase of protein synthesis to unwind regions of highly structured mRNA that would otherwise impede the scanning ribosome. Dysregulation of protein synthesis is associated with tumorigenesis, but little is known about the detailed relationships between RNA helicase function and the malignant phenotype in solid malignancies. Therefore, immunohistochemical analysis was performed on over 3000 breast tumors to investigate the relationship among expression of eIF4A1, the helicase-modulating proteins eIF4B, eIF4E and PDCD4, and clinical outcome. We found eIF4A1, eIF4B and eIF4E to be independent predictors of poor outcome in ER-negative disease, while in contrast, the eIF4A1 inhibitor PDCD4 was related to improved outcome in ER-positive breast cancer. Consistent with these data, modulation of eIF4A1, eIF4B and PCDC4 expression in cultured MCF7 cells all restricted breast cancer cell growth and cycling. The eIF4A1-dependent translatome of MCF7 cells was defined by polysome profiling, and was shown to be highly enriched for several classes of oncogenic genes, including G-protein constituents, cyclins and protein kinases, and for mRNAs with G/C-rich 5′UTRs with potential to form G-quadruplexes and with 3′UTRs containing microRNA target sites. Overall, our data show that dysregulation of mRNA unwinding contributes to the malignant phenotype in breast cancer via preferential translation of a class of genes involved in pro-oncogenic signaling at numerous levels. Furthermore, immunohistochemical tests are promising biomarkers for tumors sensitive to anti-helicase therapies.

Authors contributed equally to this work. ~ Currently at Genomics England Ltd, London, United Kingdom Next generation sequencing is transforming our understanding of human genetic variation and is becoming a routine part of human genetic analysis. The identification of millions of new variants, which are mainly rare and assessing their implications for human health presents new challenges to researchers and clinicians. We have analyzed missense variants in the ITGB2A and ITGB3 genes obtained from whole exome and whole genome sequencing (WES & WGS) data from 5 databases: The Human Genome Mutation Database, the 1000 Genomes project, the UK10K Whole Exome Sequencing project, the UK10K Whole Genome Sequencing project, and The National Heart, Lung and Blood Institute Exome Sequencing Project. Together, these encompass variants of the platelet αIIbβ3 integrin receptor from ~32,000 alleles derived from 16,108 individuals. We identified 111 missense variants that have previously been associated with Glanzmann thrombasthenia (GT), 20 variants associated with alloimmune thrombocytopenia, and 5 variants associated with aniso/macrothrombocytopenia. None of the GT variants were found in the last four databases, indicating that they have minor allele frequencies (MAF) less than ~0.01%, attesting to both their rarity and the likelihood that they entered the population within the last ~2,500 years. We also identified 114 novel missense variants in ITGB2A affecting ~11% of the amino acids and 68 novel missense variants in ITGB3 affecting ~9% of the amino acids. 96% of the novel variants had MAF To estimate the percentage of the 114 novel identified variants that are likely to be deleterious we used 3 different algorithms, CADD, Polyphen 2-HDVI, and SIFT. The algorithms showed moderate concordance in their rankings of the likelihood that a variant is deleterious. To compare their predictive powers, we performed receiver operating characteristic (ROC) analysis based on their ability to discriminate confirmed GT missense variants (positive controls) from alloantigens (negative controls); the area under the curve (AUC) values were 0.91, 0.88, and 0.90, respectively. At cutoff values that achieved greater than 95% sensitivity for each algorithm: 1) the specificity values were 75%, 65%, and 60%, and 2) the percentages of novel αIIb+β3 missense variants predicted to be deleterious were 43%, 56%, and 58%. Polyphen 2-HDVI and SIFT identified αIIb P176H and β3 C547G as highly likely to be deleterious and αIIb P943A as much less likely to be deleterious, whereas CADD did not differentiate them in the same way. We conclude that ~1.1% of individuals in the populations studied carry at least one missense variant in αIIb or β3 and that 0.6% carry a variant that might be deleterious and therefore may result in a hemorrhagic GT-like phenotype. The rarity of almost all of the novel missense variants identified indicates that they entered the population recently. Despite having detailed knowledge of the structure and function of αIIbβ3, it is difficult to predict with certainty the impact of any single missense variant. This will pose serious challenges as more individuals undergo WES and WGS; we anticipate that linkage to health record data, as will happen for the UK 100,000 Genomes project, will aid clinical interpretation. Finally, “hypomorphic” gene variants that produce only a partial decrease in expression, such as αIIb P943A, may contribute to the wide variation in αIIbβ3 surface expression observed in the healthy population. Disclosures No relevant conflicts of interest to declare.

A genome-wide scan in nearly 70,000 individuals showed that the common SNP rs8109288 in the first intron of the human TPM4 gene exerts an effect on the volume and count of platelets (Gieger et al. Nature 2011). We isolated a mouse line with an ENU-induced missense mutation in Tpm4. Mice carrying this mutation exhibited dose-dependent macrothrombocytopenia, while other blood cell counts were normal. Bone marrow transplant experiments demonstrated that the phenotype is intrinsic to hematopoietic cells. Notably, Tpm4 insufficiency did not affect the life span or in vitro function of mutant platelets, and there was no evidence of an increased propensity to bleeding. Megakaryocyte numbers in the bone marrow were increased, although maturation as measured by ploidy appeared normal. Mutant megakaryocytes displayed altered morphology indicating fragmentation, and markedly decreased proplatelet formation in vitro. Based on Gieger et al., we examined the functional requirement for TPM4 in human megakaryocytes. We found that the localisation of TPM4 in proplatelet-forming megakaryocytes was extremely similar to the localisation in their mouse counterparts, suggesting an identical role. Furthermore, knock down of TPM4by shRNA in human megakaryocytes did not affect maturation as measured by CD41 and CD42 expression, but significantly reduced the number of proplatelet-forming cells. The occasional megakaryocyte that did form proplatelets did not exhibit the typical “beads-on-a-string” phenotype. Typically, one large bulb at the end of a protrusion or a string with no clearly distinguishable beads was observed. We therefore performed a look-up in the BRIDGE consortium database, which enrolled 542 cases with inherited bleeding and platelet disorders of unknown aetiology in the NIHR BioResource for exome sequencing. Calling of variants revealed single nucleotide variants with consequences in TPM4which were absent from ~30,000 control haplotypes, in three BRIDGE cases. Two cases with a stop codon at residue 105 and R91H variant presented with macrothrombocytopenia and mild bleeding symptoms with platelet counts of 103 and 128 x10e9/L and volumes of 15.10 and 14.00 fl, respectively. The remaining case with variant D20N (not conserved, genomic evolutionary rate profiling score of 2.68 compared to 4.62 for the other variants) had a count in the normal range (232 x10e9/L) and a reduced platelet volume of 7.20 fl. Together, these results provide compelling evidence that Tropomyosin 4 is a crucial regulator of platelet production in mice and humans, being specifically required for the terminal stages of platelet formation. Our studies demonstrate that the common intronic variant exerts a subtle effect, whilst two extremely rare variants have a more robust effect on platelet formation leading to counts and volumes at the tails of the population distribution. The lack of concordance between mice and humans with regard to bleeding may be explained by strong modifiers at loci other than TPM4. Disclosures No relevant conflicts of interest to declare.

Background: Glanzmann thrombasthenia (GT) is a moderate to severe inherited bleeding syndrome associating a lack of platelet aggregation with quantitative or qualitative deficiencies of the αIIbβ3 integrin. Rare patients with mutations in ITGA2B or ITGB3 genes show a variable GT-like phenotype associated with macrothrombocytopenia (MTP). Gain-of-function mutations that localize to the integrin cytoplasmic domains cause autosomal dominant MTP. We now report a novel heterozygous ΔLeu in ITGB3 causing a structural modification of the cytoplasmic domain of β3 in a family with moderate MTP. In silico modeling was performed to obtain structural insights into this and other known mutations affecting cytoplasmic domains of αIIb and β3 and impacting megakaryopoiesis. Aim: To help define structural changes in αIIbβ3 linked to MTP. Results: A novel in-frame heterozygous 3bp deletion (c.2230_2232delCTC) was identified by exome sequencing performed as part of the BRIDGE-BPD consortium project in a French pedigree with lifelong mucocutaneous bleeding. The consequence is the loss of β3 Leu718 (ΔL718) the second L of a LLITIHD motif of the mature protein (nomenclature used throughout). Platelets of the proband have (i) moderate expression of αIIbβ3, (ii) a major reduction in fibrinogen (Fg) and PAC-1 binding after ADP stimulation yet (iii) spontaneous Fg or PAC-1 binding to platelets was not seen. Western blotting showed a slightly slower migration of β3 and normal αIIb. The proband had a moderate MTP with platelet size heterogeneity and frequent enlarged α-granules regrouping several of normal size with Fg bound to the membrane of the α-granules as revealed by immunogold staining and electron microscopy. Platelet aggregation was much reduced with all physiologic agonists including TRAP with a reduced surface expression of P-selectin and diminished secretion. The same 3bp del in ITGB3 co-segregates with the platelet phenotype and bleeding across 3 generations in the family. We then performed molecular modeling (PyMol Molecular Graphics System version 1.3) to compare the structural effects of this mutation on αIIbβ3 with other cytoplasmic tail mutations described in the literature giving GT-like phenotypes and/or MTP. Firstly αIIb-R995Q/W or β3-D723H were confirmed to disrupt a salt bridge between the integrin subunits and induce the separation of their tails. PyMol modeling showed that the DL718 results in D723 being no longer in phase with R995 thereby disrupting the salt bridge. Significantly, introduction of a β-turn by an activating β3 L718P substitution again results in the spontaneous separation of the tails, once more through a conformational change of the mutated β3. In contrast, a previously reported ITGB3 C2268T variant resulting in Arg724*, a truncation coming after the deletion in our patient, or a β3 S752P substitution that completely blocks platelet aggregation and PAC-1 binding (and specific loss of kindlin-3 binding) despite a capacity to secrete a storage pool of Fg bound α-granule αIIbβ3 failed to give MTP despite resulting in a GT-like syndrome. Conclusions: The novel ITGB3 ΔL718 of β3 showed an intermediate phenotype with MTP, a much decreased platelet aggregation, a reduced activity of αIIbβ3 at the platelet surface but a progressive fusion of α-granules. This is despite the theoretical retention of the talin and kindlin-3 binding sites suggesting that transmembrane signaling is the principle defect. Our studies highlight how considerable phenotypic heterogeneity exists in patients with genetic variants affecting αIIb and β3 cytoplasmic domains and suggest different signaling mechanisms for surface and intracellular αIIbβ3 pools. *PN and ET; WHO and ATN made equal contributions, On behalf of the BRIDGE-Bleeding and Platelet Disorder Consortium Disclosures No relevant conflicts of interest to declare.

A fundamental challenge in genomics is to map DNA sequence variants onto changes in gene expression. Gene expression is regulated by cis-regulatory elements (CREs, i.e., enhancers, promoters, and silencers) and the trans factors (e.g., transcription factors) that act upon them. A powerful approach to dissecting cis and trans effects is to compare F1 hybrids with F0 homozygotes. Using this approach and taking advantage of the high frequency of polymorphisms in wild-derived inbred Cast/EiJ mice relative to the reference strain C57BL/6J, we conducted allele-specific mRNA-seq analysis in the adult mouse retina, a disease-relevant neural tissue. We found that cis effects account for the bulk of gene regulatory divergence in the retina. Many CREs contained functional (i.e., activating or silencing) cis-regulatory variants mapping onto altered expression of genes, including genes associated with retinal disease. By comparing our retinal data with previously published liver data, we found that most of the cis effects identified were tissue-specific. Lastly, by comparing reciprocal F1 hybrids, we identified evidence of imprinting in the retina for the first time. Our study provides a framework and resource for mapping cis-regulatory variants onto changes in gene expression, and underscores the importance of studying cis-regulatory variants in the context of retinal disease.