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RATIONALE: Cannabis-based medicinal products (CBMPs) have been identified as novel therapeutics for generalised anxiety disorder (GAD) based on pre-clinical models; however, there is a paucity of high-quality evidence on their effectiveness and safety. OBJECTIVES: This study aimed to evaluate the clinical outcomes of patients with GAD treated with dried flower, oil-based preparations, or a combination of both CBMPs. METHODS: A prospective cohort study of patients with GAD (n = 302) enrolled in the UK Medical Cannabis Registry prescribed oil or flower-based CBMPs was performed. Primary outcomes were changes in generalised anxiety disorder-7 (GAD-7) questionnaires at 1, 3, and 6 months compared to baseline. Secondary outcomes were single-item sleep quality scale (SQS) and health-related quality of life index (EQ-5D-5L) questionnaires at the same time points. These changes were assessed by paired t-tests. Adverse events were assessed in line with CTCAE (Common Terminology Criteria for Adverse Events) v4.0. RESULTS: Improvements in anxiety, sleep quality and quality of life were observed at each time point (p < 0.001). Patients receiving CBMPs had improvements in GAD-7 at all time points (1 month: difference -5.3 (95% CI -4.6 to -6.1), 3 months: difference -5.5 (95% CI -4.7 to -6.4), 6 months: difference -4.5 (95% CI -3.2 to -5.7)). Thirty-nine participants (12.9%) reported 269 adverse events in the follow-up period. CONCLUSIONS: Prescription of CBMPs in those with GAD is associated with clinically significant improvements in anxiety with an acceptable safety profile in a real-world setting. Randomised trials are required as a next step to investigate the efficacy of CBMPs.
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Cannabis , Maconha Medicinal , Humanos , Maconha Medicinal/efeitos adversos , Qualidade de Vida , Estudos de Coortes , Estudos Prospectivos , Transtornos de Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/induzido quimicamente , Ansiedade/tratamento farmacológico , Reino UnidoRESUMO
Human genetic studies of smoking behavior have been thus far largely limited to common variants. Studying rare coding variants has the potential to identify drug targets. We performed an exome-wide association study of smoking phenotypes in up to 749,459 individuals and discovered a protective association in CHRNB2, encoding the ß2 subunit of the α4ß2 nicotine acetylcholine receptor. Rare predicted loss-of-function and likely deleterious missense variants in CHRNB2 in aggregate were associated with a 35% decreased odds for smoking heavily (odds ratio (OR) = 0.65, confidence interval (CI) = 0.56-0.76, P = 1.9 × 10-8). An independent common variant association in the protective direction ( rs2072659 ; OR = 0.96; CI = 0.94-0.98; P = 5.3 × 10-6) was also evident, suggesting an allelic series. Our findings in humans align with decades-old experimental observations in mice that ß2 loss abolishes nicotine-mediated neuronal responses and attenuates nicotine self-administration. Our genetic discovery will inspire future drug designs targeting CHRNB2 in the brain for the treatment of nicotine addiction.
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Nicotina , Tabagismo , Humanos , Animais , Camundongos , Fumar/genética , Tabagismo/genética , Fenótipo , Razão de ChancesRESUMO
BACKGROUND: The current paucity of clinical evidence limits the use of cannabis-based medicinal products (CBMPs) in post-traumatic stress disorder (PTSD). This study investigates health-related quality of life (HRQoL) changes and adverse events in patients prescribed CBMPs for PTSD. METHODS: A case-series of patients from the UK Medical Cannabis Registry was analyzed. HRQoL was assessed at 1-, 3-, and 6-months using validated patient reported outcome measures (PROMs). Adverse events were analyzed according to the Common Terminology Criteria for Adverse Events version 4.0. Statistical significance was defined as p < 0.050. RESULTS: Of 162 included patients, 88.89% (n = 144) were current/previous cannabis users. Median daily CBMP dosages were 5.00 (IQR: 0.00-70.00) mg of cannabidiol and 145.00 (IQR: 100.00-200.00) mg of Δ9-tetrahydrocannabinol. Significant improvements were observed in PTSD symptoms, sleep, and anxiety across all follow-up periods (p < 0.050). There were 220 (135.8%) adverse events reported by 33 patients (20.37%), with the majority graded mild or moderate in severity (n = 190, 117.28%). Insomnia and fatigue had the greatest incidence (n = 20, 12.35%). CONCLUSIONS: Associated improvements in HRQoL were observed in patients who initiated CBMP therapy. Adverse events analysis suggests acceptability and safety up to 6 months. This study may inform randomized placebo-controlled trials, required to confirm causality and determine optimal dosing.
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Alucinógenos , Maconha Medicinal , Transtornos de Estresse Pós-Traumáticos , Humanos , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológico , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Maconha Medicinal/efeitos adversos , Qualidade de Vida , Ansiedade , Alucinógenos/uso terapêutico , Reino Unido/epidemiologiaRESUMO
BACKGROUND: Although pre-clinical experiments associate cannabinoids with reduced depressive symptoms, there is a paucity of clinical evidence. This study aims to analyze the health-related quality of life changes and safety outcomes in patients prescribed cannabis-based medicinal products (CBMPs) for depression. METHODS: A series of uncontrolled cases from the UK Medical Cannabis Registry were analyzed. The primary outcomes were changes from baseline in the Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7), Sleep Quality Scale (SQS), and EQ-5D-5 L at 1, 3, and 6 months. Secondary outcomes included adverse events incidence. RESULTS: 129 patients were identified for inclusion. Median PHQ-9 at baseline was 16.0 (IQR: 9.0-21.0). There were reductions in PHQ-9 at 1-month (median: 8.0; IQR: 4.0-14.0; p < 0.001), 3-months (7.0; 2.3-12.8; p < 0.001), and 6-months (7.0; 2.0-9.5; p < 0.001). Improvements were also observed in GAD-7, SQS, and EQ-5D-5L Index Value at 1, 3, and 6 months (p < 0.050). 153 (118.6%) adverse events were recorded by 14.0% (n = 18) of participants, 87% (n = 133) of which were mild or moderate. CONCLUSION: CBMP treatment was associated with reductions in depression severity at 1, 3, and 6 months. Limitations of the study design mean that a causal relationship cannot be proven. This analysis provides insights for further study within clinical trial settings.
Depression is a highly prevalent mental health condition with approximately one in five people affected by at least one episode of depression in their lifetime. Two cardinal symptoms of depression are low mood and loss of interest. Since depression is such a debilitating condition, improving quality of life is an important part of treatment.Antidepressant medications are currently an important part of treating depression, but the variability in their effectiveness means that there is a need for alternative treatments. Medicinal cannabis, which contains certain chemicals from the cannabis plant, has received growing interest as a potential novel treatment for depression. Due to the lack of clinical studies on the use of medicinal cannabis to treat depression, this study aims to assess the effects of medicinal cannabis on quality of life in patients suffering from depression.The study included 129 patients treated with medicinal cannabis for depression at Sapphire Medical Clinics. The results showed that medicinal cannabis was associated with improvements in depression and anxiety symptoms, as well as health-related quality of life, and sleep quality after 1, 3, and 6 months of treatment. Although there were numerous adverse events in a small number of patients, most of these were mild or moderate. A major limitation is that this study cannot determine the extent to which medicinal cannabis is directly responsible for the improvements in depression symptoms that were observed. Future studies should focus on conducting clinical trials which can better evaluate the true treatment effects of medicinal cannabis for depression.
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Maconha Medicinal , Humanos , Maconha Medicinal/uso terapêutico , Depressão/tratamento farmacológico , Qualidade de Vida , Sistema de Registros , Reino UnidoRESUMO
Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases the IOP. We also show that acute Angptl7 silencing in adult mice lowers the IOP (~2-4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.
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Glaucoma , Pressão Intraocular , Adulto , Proteína 7 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/genética , Animais , Cegueira , Glaucoma/tratamento farmacológico , Glaucoma/genética , Humanos , Camundongos , Camundongos KnockoutRESUMO
Introduction: Cannabis-based medicinal products (CBMPs) have been identified as a promising novel therapeutic for symptoms and comorbidities related to autism spectrum disorder (ASD). However, there is a paucity of clinical evidence of their efficacy and safety. Objective: This case series aims to assess changes to health-related quality of life and the incidence of adverse events in patients treated with CBMPs for associated symptoms of ASD enrolled on the UK Medical Cannabis Registry (UKMCR). Methods: Patients treated with CBMPs for ASD-related symptoms for a minimum of 1 month were identified from the UKMCR. Primary outcomes were changes in validated patient-reported outcome measures [Generalised Anxiety Disorder-7 (GAD-7), Single-Item Sleep Quality Scale (SQS), 5-level version of the EQ-5D (EQ-5D-5L) index values] at 1, 3 and 6 months compared with baseline. Adverse events were recorded and analysed. Statistical significance was determined by p < 0.050. Results: Seventy-four patients with ASD were included in the analysis. The mean age of participants was 32.7 (±11.6) years. There were significant improvements in general health-related quality of life and sleep as assessed by the EQ-5D-5L, SQS and GAD-7 at 1 and 3 months, with sustained changes in EQ-5D-5L and SQS at 6 months (p < 0.010). There were 180 (243.2%) adverse events reported by 14 (18.9%) participants. If present, adverse events were commonly mild (n = 58; 78.4%) or moderate (n = 81; 109.5%), rather than severe (n = 41; 55.4%). Conclusion: This study demonstrated an associated improvement in general health-related quality of life, and anxiety- and sleep-specific symptoms following initiation of treatment with CBMPs in patients with ASD. These findings, while promising, are limited by the confines of the study which lacks a control arm and is subject to attrition bias. Therefore, further evaluation is required with randomised controlled trials.
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To better understand the genetics of hearing loss, we performed a genome-wide association meta-analysis with 125,749 cases and 469,497 controls across five cohorts. We identified 53/c loci affecting hearing loss risk, including common coding variants in COL9A3 and TMPRSS3. Through exome sequencing of 108,415 cases and 329,581 controls, we observed rare coding associations with 11 Mendelian hearing loss genes, including additive effects in known hearing loss genes GJB2 (Gly12fs; odds ratio [OR] = 1.21, P = 4.2 × 10-11) and SLC26A5 (gene burden; OR = 1.96, P = 2.8 × 10-17). We also identified hearing loss associations with rare coding variants in FSCN2 (OR = 1.14, P = 1.9 × 10-15) and KLHDC7B (OR = 2.14, P = 5.2 × 10-30). Our results suggest a shared etiology between Mendelian and common hearing loss in adults. This work illustrates the potential of large-scale exome sequencing to elucidate the genetic architecture of common disorders where both common and rare variation contribute to risk.
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Estudo de Associação Genômica Ampla , Perda Auditiva , Exoma/genética , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Perda Auditiva/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Serina Endopeptidases/genética , Sequenciamento do ExomaRESUMO
Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
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Nucleotídeos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas do Citoesqueleto/deficiência , Situs Inversus/genética , Adolescente , Adulto , Animais , Astenozoospermia/patologia , Axonema/ultraestrutura , Sistemas CRISPR-Cas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Epididimo/patologia , Feminino , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Planárias/citologia , Planárias/genética , Planárias/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia , Motilidade dos Espermatozoides/genética , Tomografia Computadorizada por Raios X , Sequenciamento do ExomaRESUMO
The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world1. Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, including PIEZO1 on varicose veins, COL6A1 on corneal resistance, MEPE on bone density, and IQGAP2 and GMPR on blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenic BRCA1 and BRCA2 variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.
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Bases de Dados Genéticas , Sequenciamento do Exoma , Exoma/genética , Mutação com Perda de Função/genética , Fenótipo , Idoso , Densidade Óssea/genética , Colágeno Tipo VI/genética , Demografia , Feminino , Genes BRCA1 , Genes BRCA2 , Genótipo , Humanos , Canais Iônicos/genética , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Penetrância , Fragmentos de Peptídeos/genética , Reino Unido , Varizes/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
PurposeWe integrated whole-exome sequencing (WES) and chromosomal microarray analysis (CMA) into a clinical workflow to serve an endogamous, uninsured, agrarian community.MethodsSeventy-nine probands (newborn to 49.8 years) who presented between 1998 and 2015 remained undiagnosed after biochemical and molecular investigations. We generated WES data for probands and family members and vetted variants through rephenotyping, segregation analyses, and population studies.ResultsThe most common presentation was neurological disease (64%). Seven (9%) probands were diagnosed by CMA. Family WES data were informative for 37 (51%) of the 72 remaining individuals, yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, negative WES decreased the likelihood of genetic disease. Compared to trio analysis, "family" WES (average seven exomes per proband) reduced filtered candidate variants from 22 ± 6 to 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 (46%) inherited; the latter added to a population-based diagnostic panel. We found actionable secondary variants in 21 (4.2%) of 502 subjects, all of whom opted to be informed.ConclusionCMA and family-based WES streamline and economize diagnosis of rare genetic disorders, accelerate novel gene discovery, and create new opportunities for community-based screening and prevention in underserved populations.
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Testes Genéticos/estatística & dados numéricos , Genética Médica/métodos , Genética Médica/estatística & dados numéricos , Genômica/estatística & dados numéricos , Disparidades em Assistência à Saúde/estatística & dados numéricos , Populações Vulneráveis , Adolescente , Adulto , Algoritmos , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Humanos , Achados Incidentais , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Linhagem , Vigilância da População , Fluxo de Trabalho , Adulto JovemRESUMO
The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery.
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Prestação Integrada de Cuidados de Saúde , Doença/genética , Registros Eletrônicos de Saúde , Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Adulto , Desenho de Fármacos , Frequência do Gene , Genômica , Humanos , Hipolipemiantes/farmacologia , Mutação INDEL , Lipídeos/sangue , Terapia de Alvo Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies.
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Drosophila/metabolismo , Atrofia Muscular Espinal/genética , Mutação de Sentido Incorreto , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Transcriptoma , Processamento Alternativo , AnimaisRESUMO
Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.
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Dineínas do Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Pulmão/metabolismo , Sequência de Bases , Cílios/ultraestrutura , Dineínas/ultraestrutura , Homozigoto , Humanos , Síndrome de Kartagener/genética , Mutação/genética , Transporte ProteicoRESUMO
Primary ciliary dyskinesia (PCD) is a ciliopathy, but represents the sole entity from this class of disorders that results from the dysfunction of motile cilia. Characterized by respiratory problems appearing in childhood, infertility, and situs defects in ~50% of individuals, PCD has an estimated prevalence of approximately 1 in 10,000 live births. The diagnosis of PCD can be prolonged due to a lack of disease awareness, coupled with the fact that symptoms can be confused with other more common genetic disorders, such as cystic fibrosis, or environmental insults that result in frequent respiratory infections. A primarily autosomal recessive disorder, PCD is genetically heterogeneous with >30 causal genes identified, posing significant challenges to genetic diagnosis. Here, we provide an overview of PCD as a disorder underscored by impaired ciliary motility; we discuss the recent advances towards uncovering the genetic basis of PCD; we discuss the molecular knowledge gained from PCD gene discovery, which has improved our understanding of motile ciliary assembly; and we speculate on how accelerated diagnosis, together with detailed phenotypic data, will shape the genetic and functional architecture of this disorder.
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Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.
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Proteínas de Drosophila/genética , Drosophila/genética , Atrofia Muscular Espinal/genética , Proteínas de Ligação a RNA/genética , Proteínas do Complexo SMN/genética , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/química , Humanos , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Mutação de Sentido Incorreto/genética , Fenótipo , Multimerização Proteica/genética , Proteínas de Ligação a RNA/química , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN/química , Relação Estrutura-AtividadeRESUMO
Reduced levels of survival motor neuron (SMN) protein lead to a neuromuscular disease called spinal muscular atrophy (SMA). Animal models of SMA recapitulate many aspects of the human disease, including locomotion and viability defects, but have thus far failed to uncover the causative link between a lack of SMN protein and neuromuscular dysfunction. While SMN is known to assemble small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing, it remains unclear whether disruptions in splicing are etiologic for SMA. To investigate this issue, we carried out RNA deep-sequencing (RNA-seq) on age-matched Drosophila Smn-null and wild-type larvae. Comparison of genome-wide mRNA expression profiles with publicly available data sets revealed the timing of a developmental arrest in the Smn mutants. Furthermore, genome-wide differences in splicing between wild-type and Smn animals did not correlate with changes in mRNA levels. Specifically, we found that mRNA levels of genes that contain minor introns vary more over developmental time than they do between wild-type and Smn mutants. An analysis of reads mapping to minor-class intron-exon junctions revealed only small changes in the splicing of minor introns in Smn larvae, within the normal fluctuations that occur throughout development. In contrast, Smn mutants displayed a prominent increase in levels of stress-responsive transcripts, indicating a systemic response to the developmental arrest induced by loss of SMN protein. These findings not only provide important mechanistic insight into the developmental arrest displayed by Smn mutants, but also argue against a minor-intron-dependent etiology for SMA.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Atrofia Muscular Espinal/genética , Proteínas de Ligação a RNA/genética , Animais , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Larva/genética , Atrofia Muscular Espinal/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Análise de Sequência de RNARESUMO
We aimed to refine the phenotypic spectrum and map the causative gene in two families with familial focal epilepsy with variable foci (FFEVF). A new five-generation Australian FFEVF family (A) underwent electroclinical phenotyping, and the original four-generation Australian FFEVF family (B) (Ann Neurol, 44, 1998, 890) was re-analyzed, including new affected individuals. Mapping studies examined segregation at the chromosome 22q12 FFEVF region. In family B, the original whole genome microsatellite data was reviewed. Five subjects in family A and 10 in family B had FFEVF with predominantly awake attacks and active EEG studies with a different phenotypic picture from other families. In family B, reanalysis excluded the tentative 2q locus reported. Both families mapped to chromosome 22q12. Our results confirm chromosome 22q12 as the solitary locus for FFEVF. Both families show a subtly different phenotype to other published families extending the clinical spectrum of FFEVF.
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Cromossomos Humanos 21-22 e Y/genética , Epilepsias Parciais/genética , Adolescente , Adulto , Criança , Pré-Escolar , Mapeamento Cromossômico , Eletroencefalografia , Epilepsias Parciais/fisiopatologia , Feminino , Ligação Genética/genética , Genótipo , Humanos , Lactente , Escore Lod , Masculino , Repetições de Microssatélites/genética , Linhagem , Fenótipo , Adulto JovemRESUMO
The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Locomoção , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sobrevivência Celular , Modelos Animais de Doenças , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Humanos , Íntrons/genética , Larva , Dados de Sequência Molecular , Mutação/genética , Estrutura Quaternária de Proteína , Splicing de RNA/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Sm and Sm-like proteins are RNA-binding factors found in all three domains of life. Eukaryotic Sm proteins play essential roles in pre-mRNA splicing, forming the cores of spliceosomal small nuclear ribonucleoproteins (snRNPs). Recently, Sm proteins have been implicated in the specification of germ cells. However, a mechanistic understanding of their involvement in germline specification is lacking and a germline-specific RNA target has not been identified. We demonstrate that Drosophila SmB and SmD3 are specific components of the oskar messenger ribonucleoprotein (mRNP), proper localization of which is required for establishing germline fate and embryonic patterning. Importantly, oskar mRNA is delocalized in females harboring a hypomorphic mutation in SmD3, and embryos from mutant mothers are defective in germline specification. We conclude that Sm proteins function to establish the germline in Drosophila, at least in part by mediating oskar mRNA localization.
Assuntos
RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Eucariotos , Feminino , Células Germinativas/metabolismo , Mutação , Proteínas/genética , Proteínas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Ribonucleoproteínas , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
The principles of self-assembly and self-organization are major tenets of molecular and cellular biology. Governed by these principles, the eukaryotic nucleus is composed of numerous subdomains and compartments, collectively described as nuclear bodies. Emerging evidence reveals that associations within and between various nuclear bodies and genomic loci are dynamic and can change in response to cellular signals. This review will discuss recent progress in our understanding of how nuclear body components come together, what happens when they form, and what benefit these subcellular structures may provide to the tissues or organisms in which they are found.