The Deubiquitinase OTULIN Is an Essential Negative Regulator of Inflammation and Autoimmunity.

Methionine-1 (M1)-linked ubiquitin chains regulate the activity of NF-κB, immune homeostasis, and responses to infection. The importance of negative regulators of M1-linked chains in vivo remains poorly understood. Here, we show that the M1-specific deubiquitinase OTULIN is essential for preventing TNF-associated systemic inflammation in humans and mice. A homozygous hypomorphic mutation in human OTULIN causes a potentially fatal autoinflammatory condition termed OTULIN-related autoinflammatory syndrome (ORAS). Four independent OTULIN mouse models reveal that OTULIN deficiency in immune cells results in cell-type-specific effects, ranging from over-production of inflammatory cytokines and autoimmunity due to accumulation of M1-linked polyubiquitin and spontaneous NF-κB activation in myeloid cells to downregulation of M1-polyubiquitin signaling by degradation of LUBAC in B and T cells. Remarkably, treatment with anti-TNF neutralizing antibodies ameliorates inflammation in ORAS patients and rescues mouse phenotypes. Hence, OTULIN is critical for restraining life-threatening spontaneous inflammation and maintaining immune homeostasis.

Dual proteolytic pathways govern glycolysis and immune competence.

Proteasomes and lysosomes constitute the major cellular systems that catabolize proteins to recycle free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPPII) is a large cytosolic proteolytic complex that functions in tandem with the proteasome-ubiquitin protein degradation pathway. We found that autosomal recessive TPP2 mutations cause recurrent infections, autoimmunity, and neurodevelopmental delay in humans. We show that a major function of TPPII in mammalian cells is to maintain amino acid levels and that TPPII-deficient cells compensate by increasing lysosome number and proteolytic activity. However, the overabundant lysosomes derange cellular metabolism by consuming the key glycolytic enzyme hexokinase-2 through chaperone-mediated autophagy. This reduces glycolysis and impairs the production of effector cytokines, including IFN-γ and IL-1ß. Thus, TPPII controls the balance between intracellular amino acid availability, lysosome number, and glycolysis, which is vital for adaptive and innate immunity and neurodevelopmental health.

Inherited ADAMTS13 mutations associated with Thrombotic Thrombocytopenic Purpura: a short review and update.

ADAMTS13 is a plasma metalloprotease with the primary function of cleaving VWF to maintain hemostasis. Circulating ADAMTS13 is in the closed conformation until blood vessel injury triggers a VWF-dependant activation to the open active form of the protein. ADAMTS13 is a multi-domain protein with the domains broadly functioning to interact and cleave VWF or maintain global latency of ADAMTS13. Thrombotic Thrombocytopenic Purpura is a disease characterized by excessive thrombi formation in the microvasculature, diagnosis is made when ADAMTS13 activity is <10%. In the hereditary form, a variety of mutations are found throughout all domains of ADAMTS13, examples are given alongside details of each domain in this article. ADAMTS13 mutations can inhibit the binding and cleavage of VWF directly or indirectly through reduced secretion, leading to increased size of VWF multimers and platelet recruitment. Molecular characterization of ADAMTS13 may provide insight into the mechanisms of TTP to aid in both scientific and clinical research.

Germline TET2 loss of function causes childhood immunodeficiency and lymphoma.

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Post-translational polymodification of ß1-tubulin regulates motor protein localisation in platelet production and function.

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which ß1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Prevalence and natural history of variants in the ANKRD26 gene: a short review and update of reported cases.

ANKRD26 is a highly conserved gene located on chromosome 10p12.1 which has shown to play a role in normal megakaryocyte differentiation. ANKRD26-related thrombocytopenia, or thrombocytopenia 2, is an inherited thrombocytopenia with mild bleeding diathesis resulting from point mutations the 5'UTR of the ANKRD26 gene. Point mutations in the 5'UTR region have been shown to prevent transcription factor-mediated downregulation of ANKRD26 in normal megakaryocyte differentiation. Patients with ANKRD26-related thrombocytopenia have a predisposition to developing hematological malignancies, with acute myeloid leukemia and myelodysplastic syndrome most commonly described in the literature. We review the clinical features and biological mechanisms of ANKRD26-related thrombocytopenia and summarize known cases in the literature.

A novel RUNX1 exon 3 - 7 deletion causing a familial platelet disorder.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline RUNX1 variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3-7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.

An adaptable analysis workflow for characterization of platelet spreading and morphology.

The assessment of platelet spreading through light microscopy, and the subsequent quantification of parameters such as surface area and circularity, is a key assay for many platelet biologists. Here we present an analysis workflow which robustly segments individual platelets to facilitate the analysis of large numbers of cells while minimizing user bias. Image segmentation is performed by interactive learning and touching platelets are separated with an efficient semi-automated protocol. We also use machine learning methods to robustly automate the classification of platelets into different subtypes. These adaptable and reproducible workflows are made freely available and are implemented using the open-source software KNIME and ilastik.

A comprehensive bioinformatic analysis of 126 patients with an inherited platelet disorder to identify both sequence and copy number genetic variants.

Inherited bleeding disorders (IBDs) comprise an extremely heterogeneous group of diseases that reflect abnormalities of blood vessels, coagulation proteins, and platelets. Previously the UK-GAPP study has used whole-exome sequencing in combination with deep platelet phenotyping to identify pathogenic genetic variants in both known and novel genes in approximately 40% of the patients. To interrogate the remaining "unknown" cohort and improve this detection rate, we employed an IBD-specific gene panel of 119 genes using the Congenica Clinical Interpretation Platform to detect both single-nucleotide variants and copy number variants in 126 patients. In total, 135 different heterozygous variants in genes implicated in bleeding disorders were identified. Of which, 22 were classified pathogenic, 26 likely pathogenic, and the remaining were of uncertain significance. There were marked differences in the number of reported variants in individuals between the four patient groups: platelet count (35), platelet function (43), combined platelet count and function (59), and normal count (17). Additionally, we report three novel copy number variations (CNVs) not previously detected. We show that a combined single-nucleotide variation (SNV)/CNV analysis using the Congenica platform not only improves detection rates for IBDs, suggesting that such an approach can be applied to other genetic disorders where there is a high degree of heterogeneity.

Role of the novel endoribonuclease SLFN14 and its disease-causing mutations in ribosomal degradation.

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by a low platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants colocalize with ribosomes and mediate rRNA endonucleolytic degradation. Compared to SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients' platelets. Overall, we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

Investigation of the contribution of an underlying platelet defect in women with unexplained heavy menstrual bleeding.

Heavy menstrual bleeding (HMB) is often undiagnosed in women and can cause discomfort and distress. A haemostatic cause for excessive bleeding is often not routinely investigated and can lead to hysterectomy at an early age. A prospective cohort study was carried out to determine whether certain patients with unexplained HMB have an underlying platelet function defect (PFD). The Genotyping and Phenotyping of Platelets (GAPP) study recruited 175 women with HMB and 44 unrelated volunteers from 25 Haemophilia Centres across the UK, and a tertiary gynaecology service. Bleeding history was assessed using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT). Platelet count, platelet size, haemoglobin and mean corpuscular volume were measured in whole blood using the Sysmex XN-1000 Haematology Analyzer. Platelet function testing using lumiaggregometry and flow cytometry was performed in patients included in this study. A PFD was identified in 47% (82/175) of patients with HMB. Cutaneous bleeding was the most frequent additional bleeding symptom (89% in PFD and 83% with no PFD). Whole blood platelet count was significantly lower (P < 0.0001) between the PFD group and no PFD group. The prevalence of anaemia did not differ between patients and healthy volunteers. Clinical evaluation alone is insufficient to determine presence of an underlying PFD in patients with HMB. Platelet function tests may be considered and clinical guidelines may include them in their algorithms. An appropriate diagnosis and subsequent tailored management of HMB may prevent unnecessary surgery and help manage future haemostatic challenges.

Evaluation of the Total Thrombus-Formation System (T-TAS): application to human and mouse blood analysis.

The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of in vitro thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyze platelet (PL) or fibrin-rich thrombus formation, respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N = 22) increased with shear within PL (4:40 ± 1.11, 3:25 ± 0.43 and 3:12 ± 0.48 mins [1000, 1500 and 2000s-1]) and AR chips (3:55 ± 0.42 and 1:49 ± 0.19 [240s-1 and 600s-1]). The area under the curve (AUC) on the PL chip was also reduced at 1000s-1 compared to 1500/2000s-1 (260 ± 51.7, 317 ± 55.4 and 301 ± 66.2, respectively). In contrast, no differences in the AUC between 240s-1 and 600s-1 were observed in the AR chip (1593 ± 122 and 1591 ± 158). The intra-assay coefficient of variation (CV) (n = 10) in the PL chip (1000s-1) and AR chip (240s-1) were T1014.1%, T6016.7%, T10-6022.8% and AUC1024.4% or T10 9.03%, T808.64%, T10-8023.8% and AUC305.1%. AR chip thrombus formation was inhibited by rivaroxaban (1 µM), but not with ticagrelor (10 µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n = 30) with normal PL counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a PL function defect. The onset (T10) of thrombus formation in WT mice (N = 4) was shorter when compared to humans e.g. PL chip (1000s-1) T10 were 02:02 ± 00:23 and 03:30 ± 0:45, respectively). T-TAS measures in vitro thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected PL function defects and monitoring PL function within mice.

Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-regulation of RET expression.

Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERß expression in the MTC tumour. ERα and ERß form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERß represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis.

Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders.

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.

Inherited platelet disorders: Insight from platelet genomics using next-generation sequencing.

Inherited platelet disorders (IPDs) are a heterogeneous group of disorders associated with normal or reduced platelet counts and bleeding diatheses of varying severities. The identification of the underlying cause of IPDs is clinically challenging due to the absence of a gold-standard platelet test, and is often based on a clinical presentation and normal values in other hematology assays. As a consequence, a DNA-based approach has a potentially important role in the investigation of these patients. Next-generation sequencing (NGS) technologies are allowing the rapid analysis of genes that have been previously implicated in IPDs or that are known to have a key role in platelet regulation, as well as novel genes that have not been previously implicated in platelet dysfunction. The potential limitations of NGS arise with the interpretation of the sheer volume of genetic information obtained from whole exome sequencing (WES) or whole genome sequencing (WGS) in order to identify function-disrupting variants. Following on from bioinformatic analysis, a number of candidate genetic variants usually remain, therefore adding to the difficulty of phenotype-genotype segregation verification. Linking genetic changes to an underlying bleeding disorder is an ongoing challenge and may not always be feasible due to the multifactorial nature of IPDs. Nevertheless, NGS will play a key role in our understanding of the mechanisms of platelet function and the genetics involved.

Whole exome sequencing identifies a mutation in thrombomodulin as the genetic cause of a suspected platelet disorder in a family with normal platelet function.

Here, we describe a mother and son with a lifelong bleeding tendency and posttraumatic bleeding who were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study with a suspected platelet function disorder. However, despite a clinically significant bleeding score, both had normal platelet counts and normal platelet function. The patients' blood was analyzed by light transmission aggregometry and genotyping by whole exome sequencing, as outlined by the GAPP study. Approximately 25 000 genetic variants were found for each patient as a result of sequencing and were filtered using a specialized bioinformatics pipeline. A heterozygous variant displaying autosomal dominant inheritance (c.1611 C>A) was found in the gene THBD which encodes the glycoprotein thrombomodulin. This sequence change results in a stop codon (p.Cys537Stop) and truncation of the protein and has been previously described in two other families with bleeding events which suggests it may be a recurrent mutation. In summary, this study shows that patients with a suspected platelet disorder but who present with a normal pattern of platelet aggregation should be investigated for defects in nonplatelet genes.

Defective Leukocyte Adhesion and Chemotaxis Contributes to Combined Immunodeficiency in Humans with Autosomal Recessive MST1 Deficiency.

PURPOSE: To investigate the clinical and functional aspects of MST1 (STK4) deficiency in a profoundly CD4-lymphopenic kindred with a novel homozygous nonsense mutation in STK4. Although recent studies have described the cellular effects of murine Mst1 deficiency, the phenotype of MST1-deficient human lymphocytes has yet to be fully explored. Patient lymphocytes were therefore investigated in the context of current knowledge of murine Mst1 deficiency. METHODS: Genetic etiology was identified by whole exome sequencing of genomic DNA from two siblings, combined with linkage analysis in the wider family. MST1 protein expression was assessed by immunoblotting. The ability of patient lymphocytes to adhere to ICAM-1 under flow conditions was measured, and transwell assays were used to assess chemotaxis. Chemokine receptor expression was examined by flow cytometry and receptor signalling by immunoblotting. RESULTS: A homozygous nonsense mutation in STK4 (c.442C > T, p.Arg148Stop) was found in the patients, leading to a lack of MST1 protein expression. Patient leukocytes exhibited deficient chemotaxis after stimulation with CXCL11, despite preserved expression of CXCR3. Patient lymphocytes were also unable to bind effectively to immobilised ICAM-1 under flow conditions, in keeping with a failure to develop high affinity binding. CONCLUSION: The observed abnormalities of adhesion and migration imply a profound trafficking defect among human MST1-deficient lymphocytes. By analogy with murine Mst1 deficiency and other defects of leucocyte trafficking, this is likely to contribute to immunodeficiency by impairing key aspects of T-cell development and function such as positive selection in the thymus, thymic egress and immune synapse formation in the periphery.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.