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Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of â¼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as â¼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.
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Processos de Crescimento Celular/genética , Mapeamento de Híbridos Radioativos/métodos , Animais , Centrômero , Cricetinae , DNA , Doença/genética , Loci Gênicos , Células HEK293 , Humanos , Mitocôndrias , Mycoplasma/isolamento & purificação , Paclitaxel/farmacologiaRESUMO
PURPOSE: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications. METHODS: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide. RESULTS: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality. CONCLUSION: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.
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Doenças Genéticas Inatas/diagnóstico , Patologia Molecular , Doenças Raras/diagnóstico , Transcriptoma/genética , Exoma/genética , Doenças Genéticas Inatas/genética , Testes Genéticos/normas , Humanos , Mutação/genética , RNA-Seq/normas , Doenças Raras/genética , Análise de Sequência de DNA/normas , Sequenciamento do Exoma/normas , Sequenciamento Completo do Genoma/normasRESUMO
INTRODUCTION: In this study we investigate associations between genotypic and sociodemographic factors and the age of diagnosis of Duchenne muscular dystrophy (DMD). METHODS: Data were collected from the Duchenne Registry from 2007 to 2019, and then used to assess the impact genotype, race/ethnicity, neighborhood poverty levels, and other sociodemographics factors have on the age of diagnosis of DMD patients without a known family history, using univariate and multivariable linear regression. RESULTS: The mean age of diagnosis was 4.43 years. Non-Caucasian patients and patients from high-poverty neighborhoods were older at diagnosis (P < .01). Increased year of birth was associated with decreasing age of diagnosis (P < .001). Specific genetic mutation subtypes were associated with later ages of symptom onset and diagnosis (P = .005). DISCUSSION: After adjusting for genotype and year of birth, the average age of diagnosis was significantly later for traditionally at-risk patients.
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Diagnóstico Tardio , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Adolescente , Fatores Etários , Idade de Início , Criança , Pré-Escolar , Etnicidade , Genótipo , Humanos , Masculino , Distrofia Muscular de Duchenne/epidemiologia , Mutação/genética , Pobreza , Sistema de Registros , Fatores Socioeconômicos , Estados Unidos/epidemiologiaRESUMO
Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3-7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.
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Distrofina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Adolescente , Adulto , Fatores Etários , Biópsia , Códon sem Sentido/genética , Distrofina/antagonistas & inibidores , Éxons/genética , Feminino , Fibroblastos/patologia , Genótipo , Humanos , Estimativa de Kaplan-Meier , Tempo de Internação , Masculino , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mioblastos/patologia , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Cultura Primária de Células , Sistema de Registros , Deleção de Sequência/genética , Caracteres Sexuais , Adulto JovemRESUMO
PURPOSE OF REVIEW: This review aims to describe the benefits and limitations of using the Duchenne Connect patient registry to provide information particularly in regard to active treatment choices in Duchenne muscular dystrophy and their impact on disease progression. RECENT FINDINGS: Clinical trials and natural history studies are difficult for rare diseases like Duchenne muscular dystrophy. Using an online patient self-report survey model, Duchenne Connect provides relevant data that are difficult to gather in other ways. Validation of the overall dataset is supported by comparable mutational spectrum relative to other cohorts and demonstrated beneficial effect of corticosteroid use in prolonging ambulation. These types of analyses are provocative and allow multivariate analyses across the breadth of patient and physician medication and supplement practices. Because the data are self-reported and online, the barrier to participation is low and great potential exists for novel directions of further research in a highly participatory forum. SUMMARY: Patient registries for Duchenne and Becker muscular dystrophy (DBMD) are powerful tools for monitoring patient outcomes, comparing treatment options, and relating information between patients, researchers, and clinicians. Duchenne Connect is an online patient self-report registry for individuals with DBMD that facilitates aggregation of treatment modalities, outcomes, and genotype data and has played a vital role in furthering DBMD research, particularly in the USA, in a highly participatory and low-cost manner.
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Distrofia Muscular de Duchenne , Participação do Paciente , Sistema de Registros , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genéticaRESUMO
Using radiation hybrid genotyping data, 99% of all possible gene pairs across the mammalian genome were tested for interactions based on co-retention frequencies higher (attraction) or lower (repulsion) than chance. Gene interaction networks constructed from six independent data sets overlapped strongly. Combining the data sets resulted in a network of more than seven million interactions, almost all attractive. This network overlapped with protein-protein interaction networks on multiple measures and also confirmed the relationship between essentiality and centrality. In contrast to other biological networks, the radiation hybrid network did not show a scale-free distribution of connectivity but was Gaussian-like, suggesting a closer approach to saturation. The radiation hybrid (RH) network constitutes a platform for understanding the systems biology of the mammalian cell.
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Redes Reguladoras de Genes , Genoma Humano/genética , Mapeamento de Híbridos Radioativos , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Mapeamento de Interação de ProteínasRESUMO
Muscle damage and fibro-fatty replacement of skeletal muscles is a main pathologic feature of Duchenne muscular dystrophy (DMD) with more proximal muscles affected earlier and more distal affected later in the disease course, suggesting that different skeletal muscle groups possess distinctive characteristics that influence their susceptibility to disease. To explore transcriptomic factors driving differential gene expression and modulating DMD skeletal muscle severity, we characterized the transcriptome of vastus lateralis (VL), a more proximal and susceptible muscle, relative to tibialis anterior (TA), a more distal and protected muscle, in 15 healthy individuals using bulk RNA sequencing to identify gene expression differences that may mediate their relative susceptibility to damage with loss of dystrophin. Matching single nuclei RNA sequencing data was generated for 3 of the healthy individuals, to infer cell composition in the bulk RNA sequencing dataset and to improve mapping of differentially expressed genes to their cell source of expression. A total of 3,410 differentially expressed genes were identified and mapped to cell type using single nuclei RNA sequencing of muscle, including long non-coding RNAs and protein coding genes. There was an enrichment of genes involved in calcium release from the sarcoplasmic reticulum, particularly in the myofibers and these myofiber genes were higher in the VL. There was an enrichment of genes in "Collagen-Containing Extracellular Matrix" expressed by fibroblasts, endothelial, smooth muscle and pericytes, with most genes higher in the TA, as well as genes in "Regulation Of Apoptotic Process" expressed across all cell types. Previously reported genetic modifiers were also enriched within the differentially expressed genes. We also identify 6 genes with differential isoform usage between the VL and TA. Lastly, we integrate our findings with DMD RNA sequencing data from the TA, and identify "Collagen-Containing Extracellular Matrix" and "Negative Regulation Of Apoptotic Process" as differentially expressed between DMD compared to healthy. Collectively, these findings propose novel candidate mechanisms that may mediate differential muscle susceptibility in muscular dystrophies and provide new insight into potential therapeutic targets.
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BACKGROUND AND OBJECTIVES: Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder with a well-characterized disease phenotype but considerable interindividual heterogeneity that is not well understood. The aim of this study was to evaluate the effects of dystrophin variations and genetic modifiers of DMD on rate and age of muscle replacement by fat. METHODS: One hundred seventy-five corticosteroid treated participants from the ImagingDMD natural history study underwent repeated magnetic resonance spectroscopy (MRS) of the vastus lateralis (VL) and soleus (SOL) to determine muscle fat fraction (FF). MRS was performed annually in most instances; however, some individuals had additional visits at 3 or 6 monthss intervals. FF changes over time were modeled using nonlinear mixed effects to estimate disease trajectories based on the age that the VL or SOL reached half-maximum change in FF (mu) and the time required for FF change (sigma). Computed mu and sigma values were evaluated for dystrophin variations that have demonstrated the ability to lead to a mild phenotype as well as compared between different genetic polymorphism groups. RESULTS: Participants with dystrophin gene deletions amenable to exon 8 skipping (n = 4) had minimal increases in SOL FF and had an increase in VL mu value by 4.4 years compared with a reference cohort (p = 0.039). Participants with nonsense variations within exons that may produce milder phenotypes (n = 11) also had minimal increases in SOL and VL FFs. No differences in estimated FF trajectories were seen for individuals amenable to exon 44 skipping (n = 10). Modeling of the SPP1, LTBP4, and thrombospondin-1 (THBS1) genetic modifiers did not result in significant differences in muscle FF trajectories between genotype groups (p > 0.05); however, trends were noted for the polymorphisms associated with long-range regulation of LTBP4 and THBS1 that deserve further follow-up. DISCUSSION: The results of this study link the historically mild phenotypes seen in individuals amenable to exon 8 skipping and with certain nonsense variations with alterations in trajectories of lower extremity muscle replacement by fat.
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Distrofina , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico por imagem , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Éxons , Imageamento por Ressonância Magnética/métodos , Progressão da DoençaRESUMO
In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.
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Distrofina , Distrofia Muscular de Duchenne , Animais , Distrofina/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , TranscriptomaRESUMO
BACKGROUND: There is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped cis and trans regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs). RESULTS: Unexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an Xist independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained trans ceQTLs with -log10 P > 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements. CONCLUSION: Radiation hybrids are a useful tool for high resolution mapping of cis and trans loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.
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Variações do Número de Cópias de DNA , Genoma , Animais , Cromossomos Humanos X , Cricetinae , Mecanismo Genético de Compensação de Dose , Perfilação da Expressão Gênica , Ligação Genética , Glioblastoma/genética , Humanos , Células Híbridas , Camundongos , Locos de Características QuantitativasRESUMO
Meiotic mapping of quantitative trait loci regulating expression (eQTLs) has allowed the construction of gene networks. However, the limited mapping resolution of these studies has meant that genotype data are largely ignored, leading to undirected networks that fail to capture regulatory hierarchies. Here we use high resolution mapping of copy number eQTLs (ceQTLs) in a mouse-hamster radiation hybrid (RH) panel to construct directed genetic networks in the mammalian cell. The RH network covering 20,145 mouse genes had significant overlap with, and similar topological structures to, existing biological networks. Upregulated edges in the RH network had significantly more overlap than downregulated. This suggests repressive relationships between genes are missed by existing approaches, perhaps because the corresponding proteins are not present in the cell at the same time and therefore unlikely to interact. Gene essentiality was positively correlated with connectivity and betweenness centrality in the RH network, strengthening the centrality-lethality principle in mammals. Consistent with their regulatory role, transcription factors had significantly more outgoing edges (regulating) than incoming (regulated) in the RH network, a feature hidden by conventional undirected networks. Directed RH genetic networks thus showed concordance with pre-existing networks while also yielding information inaccessible to current undirected approaches.
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Hibridização Genômica Comparativa/métodos , Redes Reguladoras de Genes , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Mapeamento de Híbridos Radioativos , Animais , Cricetinae , Bases de Dados Genéticas , Camundongos , Modelos Genéticos , Modelos Estatísticos , Locos de Características Quantitativas , Análise de Regressão , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.
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BACKGROUND: Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient's phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100-300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations. METHODS: To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene. RESULTS: We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45-250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing. CONCLUSIONS: We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.
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Mapeamento Cromossômico , Genoma Humano , Variação Estrutural do Genoma , Distrofia Muscular de Duchenne/genética , Estudos de Coortes , Análise Mutacional de DNA , Distrofina/genética , Feminino , Deleção de Genes , Triagem de Portadores Genéticos , Humanos , Masculino , Mães , Distrofia Muscular de Duchenne/diagnóstico , Mutagênese Insercional , Inversão de SequênciaRESUMO
To assess the utility of online patient self-report outcomes in a rare disease, we attempted to observe the effects of corticosteroids in delaying age at fulltime wheelchair use in Duchenne muscular dystrophy (DMD) using data from 1,057 males from DuchenneConnect, an online registry. Data collected were compared to prior natural history data in regard to age at diagnosis, mutation spectrum, and age at loss of ambulation. Because registrants reported differences in steroid and other medication usage, as well as age and ambulation status, we could explore these data for correlations with age at loss of ambulation. Using multivariate analysis, current steroid usage was the most significant and largest independent predictor of improved wheelchair-free survival. Thus, these online self-report data were sufficient to retrospectively observe that current steroid use by patients with DMD is associated with a delay in loss of ambulation. Comparing commonly used steroid drugs, deflazacort prolonged ambulation longer than prednisone (median 14 years and 13 years, respectively). Further, use of Vitamin D and Coenzyme Q10, insurance status, and age at diagnosis after 4 years were also significant, but smaller, independent predictors of longer wheelchair-free survival. Nine other common supplements were also individually tested but had lower study power. This study demonstrates the utility of DuchenneConnect data to observe therapeutic differences, and highlights needs for improvement in quality and quantity of patient-report data, which may allow exploration of drug/therapeutic practice combinations impractical to study in clinical trial settings. Further, with the low barrier to participation, we anticipate substantial growth in the dataset in the coming years.
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Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.
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Dantroleno/farmacologia , Éxons/genética , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/farmacologia , Animais , Linhagem Celular , Dantroleno/administração & dosagem , Modelos Animais de Doenças , Sinergismo Farmacológico , Distrofina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Oligonucleotídeos Antissenso/administração & dosagem , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Sarcolema/patologiaRESUMO
We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse-hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The -2log10P support interval for the ceQTLs was <150 kb, containing an average of <2-3 genes. We identified 29,769 trans ceQTLs with -log10P > 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome.