ABSTRACT
Introduction: The unique red blood cell (RBC) properties that characterize the rare neuroacanthocytosis syndromes (NAS) have prompted the exploration of osmotic gradient ektacytometry (Osmoscan) as a diagnostic tool for these disorders. In this exploratory study, we assessed if Osmoscans can discriminate NAS from other neurodegenerative diseases. Methods: A comprehensive assessment was conducted using Osmoscan on a diverse group of patients, including healthy controls (n = 9), neuroacanthocytosis syndrome patients (n = 6, 2 VPS13A and 4 XK disease), Parkinson's disease patients (n = 6), Huntington's disease patients (n = 5), and amyotrophic lateral sclerosis patients (n = 4). Concurrently, we collected and analyzed RBC indices and patients' characteristics. Results: Statistically significant changes were observed in NAS patients compared to healthy controls and other conditions, specifically in osmolality at minimal elongation index (Omin), maximal elongation index (EImax), the osmolality at half maximal elongation index in the hyperosmotic part of the curve (Ohyper), and the width of the curve close to the osmolality at maximal elongation index (Omax-width). Discussion: This study represents an initial exploration of RBC properties from NAS patients using osmotic gradient ektacytometry. While specific parameters exhibited differences, only Ohyper and Omax-width yielded 100% specificity for other neurodegenerative diseases. Moreover, unique correlations between Osmoscan parameters and RBC indices in NAS versus controls were identified, such as osmolality at maximal elongation index (Omax) vs. mean cellular hemoglobin content (MCH) and minimal elongation index (EImin) vs. red blood cell distribution width (RDW). Given the limited sample size, further studies are essential to establish diagnostic guidelines based on these findings.
ABSTRACT
Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.
Subject(s)
Astrocytes , Gliosis , Animals , Gliosis/pathology , Gliosis/metabolism , Astrocytes/metabolism , Astrocytes/pathology , Mice , Chromatin/metabolism , Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/pathology , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/pathology , Chromatin Assembly and Disassembly , Microglia/metabolism , Microglia/pathology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mice, Inbred C57BL , Lipopolysaccharides/pharmacology , Humans , Mice, Knockout , Male , Cell ProliferationABSTRACT
The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD (n = 9 SCA, n = 5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p < 0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p = 0.0012), 9.6% (2.9%-15.1%; p = 0.0013), and 13.3% (5.7%-25.5%; p = 0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p = 0.4324), 58% (48%-72%; p = 0.0185), and 71% (49%-82%; p = 0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD.
ABSTRACT
Optimizing behavioral strategy requires belief updating based on new evidence, a process that engages higher cognition. In schizophrenia, aberrant belief dynamics may lead to psychosis, but the mechanisms underlying this process are unknown, in part, due to lack of appropriate animal models and behavior readouts. Here, we address this challenge by taking two synergistic approaches. First, we generate a mouse model bearing patient-derived point mutation in Grin2a (Grin2aY700X+/-), a gene that confers high-risk for schizophrenia and recently identified by large-scale exome sequencing. Second, we develop a computationally trackable foraging task, in which mice form and update belief-driven strategies in a dynamic environment. We found that Grin2aY700X+/- mice perform less optimally than their wild-type (WT) littermates, showing unstable behavioral states and a slower belief update rate. Using functional ultrasound imaging, we identified the mediodorsal (MD) thalamus as hypofunctional in Grin2aY700X+/- mice, and in vivo task recordings showed that MD neurons encoded dynamic values and behavioral states in WT mice. Optogenetic inhibition of MD neurons in WT mice phenocopied Grin2aY700X+/- mice, and enhancing MD activity rescued task deficits in Grin2aY700X+/- mice. Together, our study identifies the MD thalamus as a key node for schizophrenia-relevant cognitive dysfunction, and a potential target for future therapeutics.
ABSTRACT
Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.
Subject(s)
Genetic Therapy , Parkinson Disease , Animals , Humans , Mice , Corpus Striatum/metabolism , Levodopa/therapeutic use , Levodopa/genetics , Neurons/metabolism , Parkinson Disease/genetics , Parkinson Disease/therapy , Primates , Receptors, Dopamine D1/metabolism , Disease Models, AnimalABSTRACT
Novel developments in therapies for various hereditary hemolytic anemias reflect the pivotal role of pyruvate kinase (PK), a key enzyme of glycolysis, in red blood cell (RBC) health. Without PK catalyzing one of the final steps of the Embden-Meyerhof pathway, there is no net yield of adenosine triphosphate (ATP) during glycolysis, the sole source of energy production required for proper RBC function and survival. In hereditary hemolytic anemias, RBC health is compromised and therefore lifespan is shortened. Although our knowledge on glycolysis in general and PK function in particular is solid, recent advances in genetic, molecular, biochemical, and metabolic aspects of hereditary anemias have improved our understanding of these diseases. These advances provide a rationale for targeting PK as therapeutic option in hereditary hemolytic anemias other than PK deficiency. This review summarizes the knowledge, rationale, (pre)clinical trials, and future advances of PK activators for this important group of rare diseases.
Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic , Anemia, Hemolytic, Congenital , Anemia, Hemolytic , Humans , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , Anemia, Hemolytic/metabolism , Anemia, Hemolytic, Congenital Nonspherocytic/etiology , Anemia, Hemolytic, Congenital Nonspherocytic/therapy , Erythrocytes/metabolism , Anemia, Hemolytic, Congenital/therapy , Anemia, Hemolytic, Congenital/metabolismABSTRACT
Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population.
Subject(s)
De Lange Syndrome , Nuclear Proteins , Humans , Nuclear Proteins/genetics , De Lange Syndrome/diagnosis , De Lange Syndrome/genetics , De Lange Syndrome/pathology , Transcription Factors/genetics , Cell Cycle Proteins/genetics , Phenotype , Mutation , Genomics , Genetic Association Studies , Transcriptional Elongation Factors/genetics , Histone Deacetylases/genetics , Repressor Proteins/geneticsABSTRACT
In Diamond-Blackfan anaemia (DBA), iron overload (IO) is common in transfusion-dependent patients, yet has also been reported in non-transfusion-dependent patients. We explored the incidence of IO in transfusion-dependent and non-transfusion-dependent DBA patients. We observed hepatic IO in 65% of patients analysed with MRI, including three patients that were only treated with transfusions in the past. Whereas overall ferritin levels and liver iron content correlated, ferritin levels did not reflect total body iron adequately. Our data suggest that transfusion burden in the past plays an important role in IO in DBA, and should be taken into account during follow up.
ABSTRACT
Despite its wide-ranging benefits, whole-transcriptome or RNA exome profiling is challenging to implement in a clinical diagnostic setting. The Unified Assay is a comprehensive workflow wherein exome-enriched RNA-sequencing (RNA-Seq) assays are performed on clinical samples and analyzed by a series of advanced machine learning-based classifiers. Gene expression signatures and rare and/or novel genomic events, including fusions, mitochondrial variants, and loss of heterozygosity were assessed using RNA-Seq data generated from 120,313 clinical samples across three clinical indications (thyroid cancer, lung cancer, and interstitial lung disease). Since its implementation, the data derived from the Unified Assay have allowed significantly more patients to avoid unnecessary diagnostic surgery and have played an important role in guiding follow-up decisions regarding treatment. Collectively, data from the Unified Assay show the utility of RNA-Seq and RNA expression signatures in the clinical laboratory, and their importance to the future of precision medicine.
ABSTRACT
Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.
Subject(s)
DNA Repair/genetics , Gene Conversion , Rad51 Recombinase/metabolism , Alleles , Animals , Base Sequence , CRISPR-Associated Protein 9/metabolism , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromosomes, Mammalian/genetics , DNA Breaks, Double-Stranded , Embryo, Mammalian , Female , Genetic Loci , Homologous Recombination/genetics , Homozygote , Humans , INDEL Mutation/genetics , Mice, Inbred C57BL , Mosaicism , Nuclear Proteins/metabolism , Polymorphism, Single Nucleotide/genetics , Ribonucleoproteins/metabolism , Zygote/metabolismABSTRACT
Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival (i.e., hemolysis). In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA.
Subject(s)
Anemia/blood , Erythropoiesis/physiology , Iron/metabolism , Anemia/genetics , Homeostasis/physiology , Humans , Iron Overload/diagnosis , Iron Overload/metabolismABSTRACT
Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.
Subject(s)
Gene Editing/methods , Animals , Base Sequence , Embryo, Mammalian/metabolism , Exons/genetics , Fetus/metabolism , Liver/metabolism , Macaca fascicularis/embryology , Mutation/geneticsABSTRACT
BACKGROUND: Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet counts and often disproportionate bleeding with over 30 genes currently implicated. Previously the UK-GAPP study using whole exome sequencing (WES) identified a pathogenic variant in 19 of 47 (40%) patients of which 71% had variants in genes known to cause IT. AIMS: To employ a targeted next-generation sequencing platform to improve efficiency of diagnostic testing and reduce overall costs. METHODS: We have developed an IT-specific gene panel as a pre-screen for patients prior to WES using the Agilent SureSelectQXT transposon-based enrichment system. RESULTS: Thirty-one patients were analyzed using the panel-based sequencing, of which; 10% (3/31) were identified with a classified pathogenic variant, 16% (5/31) were identified with a likely pathogenic variant, 51% (16/31) were identified with variants of unknown significance, and 23% (7/31) were identified with either no variant or a benign variant. DISCUSSION AND CONCLUSION: Although requiring further clarification of the impact of the genetic variations, the application of an IT-specific next generation sequencing panel is an viable method of pre-screening patients for variants in known IT-causing genes prior to WES. With an added benefit of distinguishing IT from idiopathic thrombocytopenic purpura (ITP) and the potential to identify variants in genes known to have a predisposition to hematological malignancies, it could become a critical step in improving patient clinical management.
ABSTRACT
Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARß. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.
Subject(s)
Diencephalon/anatomy & histology , Diencephalon/metabolism , Signal Transduction/physiology , Tretinoin/metabolism , p300-CBP Transcription Factors/metabolism , Animals , Diencephalon/embryology , Enzyme Activation , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size/physiologyABSTRACT
Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.
Subject(s)
Exome/genetics , Genetic Variation , High-Throughput Nucleotide Sequencing , Thrombocytopenia/genetics , Blood Platelets/pathology , Genetic Predisposition to Disease , Humans , Mutation, Missense , Platelet CountABSTRACT
Myopathies decrease muscle functionality. Mutations in ryanodine receptor 1 (RyR1) are often associated with myopathies with microscopic core-like structures in the muscle fiber. In this study, we identify a mouse RyR1 model in which heterozygous animals display clinical and pathological hallmarks of myopathy with core-like structures. The RyR1 mutation decreases sensitivity to activated calcium release and myoplasmic calcium levels, subsequently affecting mitochondrial calcium and ATP production. Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis. Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology. We identify regulators of potassium homeostasis as biomarkers of disease that may reveal therapeutic targets in human patients with myopathy of central core disease (CCD). Altogether, our results suggest that amelioration of potassium leaks through potassium homeostasis mechanisms may minimize muscle damage of myopathies due to certain RyR1 mutations.
Subject(s)
Muscular Diseases/pathology , Potassium/metabolism , Animals , Biological Transport/drug effects , Biomarkers/metabolism , Biopsy , Calcium/metabolism , Diet , Ethylnitrosourea , Gene Expression Regulation/drug effects , Glyburide/pharmacology , Heterozygote , Homeostasis/drug effects , Humans , KATP Channels/metabolism , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/ultrastructure , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Muscular Diseases/genetics , Mutation/genetics , Myopathy, Central Core/genetics , Myopathy, Central Core/pathology , NAD/metabolism , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolismABSTRACT
The formation of the embryonic brain and spinal cord begins as the neural plate bends to form the neural folds, which meet and adhere to close the neural tube. The neural ectoderm and surrounding tissues also coordinate proliferation, differentiation, and patterning. This highly orchestrated process is susceptible to disruption, leading to neural tube defects (NTDs), a common birth defect. Here, we highlight genetic and epigenetic contributions to neural tube closure. We describe an online database we created as a resource for researchers, geneticists, and clinicians. Neural tube closure is sensitive to environmental influences, and we discuss disruptive causes, preventative measures, and possible mechanisms. New technologies will move beyond candidate genes in small cohort studies toward unbiased discoveries in sporadic NTD cases. This will uncover the genetic complexity of NTDs and critical gene-gene interactions. Animal models can reveal the causative nature of genetic variants, the genetic interrelationships, and the mechanisms underlying environmental influences.
Subject(s)
Brain/growth & development , Epigenesis, Genetic , Neural Tube/growth & development , Spinal Cord/growth & development , Animals , Brain/embryology , Female , Neural Crest/embryology , Neural Crest/growth & development , Neural Plate/embryology , Neural Plate/growth & development , Neural Tube/embryology , Spinal Cord/embryologyABSTRACT
Human genetic studies have established a link between a class of centrosome proteins and microcephaly. Current studies of microcephaly focus on defective centrosome/spindle orientation. Mutations in WDR62 are associated with microcephaly and other cortical abnormalities in humans. Here we create a mouse model of Wdr62 deficiency and find that the mice exhibit reduced brain size due to decreased neural progenitor cells (NPCs). Wdr62 depleted cells show spindle instability, spindle assembly checkpoint (SAC) activation, mitotic arrest and cell death. Mechanistically, Wdr62 associates and genetically interacts with Aurora A to regulate spindle formation, mitotic progression and brain size. Our results suggest that Wdr62 interacts with Aurora A to control mitotic progression, and loss of these interactions leads to mitotic delay and cell death of NPCs, which could be a potential cause of human microcephaly.
Subject(s)
Aurora Kinase A/metabolism , Brain/embryology , Cell Cycle Proteins/genetics , M Phase Cell Cycle Checkpoints/genetics , Microcephaly/embryology , Microtubule-Associated Proteins/genetics , Mitosis/genetics , Neural Stem Cells/metabolism , Spindle Apparatus/metabolism , Animals , Brain/metabolism , Brain/pathology , Embryonic Stem Cells/metabolism , Mice , Microcephaly/metabolism , Microcephaly/pathology , Mutation , Nerve Tissue Proteins , Organ SizeABSTRACT
A small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function.We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S-expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S-expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.