RESUMO
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that ligate tRNA molecules to cognate amino acids. Heterozygosity for missense variants or small in-frame deletions in six ARS genes causes dominant axonal peripheral neuropathy. These pathogenic variants reduce enzyme activity without significantly decreasing protein levels and reside in genes encoding homo-dimeric enzymes. These observations raise the possibility that neuropathy-associated ARS variants exert a dominant-negative effect, reducing overall ARS activity below a threshold required for peripheral nerve function. To test such variants for dominant-negative properties, we developed a humanized yeast assay to co-express pathogenic human alanyl-tRNA synthetase (AARS1) mutations with wild-type human AARS1. We show that multiple loss-of-function AARS1 mutations impair yeast growth through an interaction with wild-type AARS1, but that reducing this interaction rescues yeast growth. This suggests that neuropathy-associated AARS1 variants exert a dominant-negative effect, which supports a common, loss-of-function mechanism for ARS-mediated dominant peripheral neuropathy.
Assuntos
Alanina-tRNA Ligase , Aminoacil-tRNA Sintetases , Doenças do Sistema Nervoso Periférico , Humanos , Alanina-tRNA Ligase/genética , Doenças do Sistema Nervoso Periférico/patologia , Mutação , Aminoacil-tRNA Sintetases/genética , Nervos Periféricos/metabolismoRESUMO
BACKGROUND AND AIMS: Pathogenic variants in the NARS1 gene, which encodes for the asparaginyl-tRNA synthetase1 (NARS1) enzyme, were associated with complex central and peripheral nervous system phenotypes. Recently, Charcot-Marie-Tooth (CMT) disease has been linked to heterozygous pathogenic variants in NARS1 in nine patients. Here, we report two brothers and their mother from a French family with distal hereditary motor neuropathy (dHMN) carrying a previously unreported NARS1 variant. METHODS: The NARS1 variant (c.1555G>C; p.(Gly519Arg)) was identified through whole-genome sequencing (WGS) performed on the family members. Clinical findings, nerve conduction studies (NCS), needle electromyography (EMG), and functional assays in yeast complementation assays are reported here. RESULTS: The family members showed symptoms of dHMN, including distal weakness and osteoarticular deformities. They also exhibited brisk reflexes suggestive of upper motor neuron involvement. All patients were able to walk independently at the last follow-up. NCS and EMG confirmed pure motor neuropathy. Functional assays in yeast confirmed a loss-of-function effect of the variant on NARS1 activity. INTERPRETATION: Our findings expand the clinical spectrum of NARS1-associated neuropathies, highlighting the association of NARS1 mutations with dHMN. The benign disease course observed in our patients suggests a slowly progressive phenotype. Further reports could contribute to a more comprehensive understanding of the spectrum of NARS1-associated neuropathies.
Assuntos
Aminoacil-tRNA Sintetases , Neuropatia Hereditária Motora e Sensorial , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , França , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , LinhagemRESUMO
Aminoacyl-tRNA synthetases (ARS) are ubiquitously expressed, essential enzymes that charge tRNA with cognate amino acids. Variants in genes encoding ARS enzymes lead to myriad human inherited diseases. First, missense alleles cause dominant peripheral neuropathy. Second, missense, nonsense, and frameshift alleles cause recessive multisystem disorders that differentially affect tissues depending on which ARS is mutated. A preponderance of evidence has shown that both phenotypic classes are associated with loss-of-function alleles, suggesting that tRNA charging plays a central role in disease pathogenesis. However, it is currently unclear how perturbation in the function of these ubiquitously expressed enzymes leads to tissue-specific or tissue-predominant phenotypes. Here, we review our current understanding of ARS-associated disease phenotypes and discuss potential explanations for the observed tissue specificity.
Assuntos
Aminoacil-tRNA Sintetases/genética , Predisposição Genética para Doença , Doenças do Sistema Nervoso Periférico/genética , RNA de Transferência/genética , Alelos , Aminoácidos/genética , Humanos , Mutação/genética , Doenças do Sistema Nervoso Periférico/patologia , Fenótipo , Aminoacilação de RNA de Transferência/genéticaRESUMO
Heterozygosity for missense variants and small in-frame deletions in GARS1 has been reported in patients with a range of genetic neuropathies including Charcot-Marie-Tooth disease type 2D (CMT2D), distal hereditary motor neuropathy type V (dHMN-V), and infantile spinal muscular atrophy (iSMA). We identified two unrelated patients who are each heterozygous for a previously unreported missense variant modifying amino-acid position 336 in the catalytic domain of GARS1. One patient was a 20-year-old woman with iSMA, and the second was a 41-year-old man with CMT2D. Functional studies using yeast complementation assays support a loss-of-function effect for both variants; however, this did not reveal variable effects that might explain the phenotypic differences. These cases expand the mutational spectrum of GARS1-related disorders and demonstrate phenotypic variability based on the specific substitution at a single residue.
Assuntos
Doença de Charcot-Marie-Tooth , Glicina-tRNA Ligase , Humanos , Doença de Charcot-Marie-Tooth/genética , Códon , Glicina-tRNA Ligase/genética , Mutação , FenótipoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.
Assuntos
Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/etiologia , Deficiências do Desenvolvimento/etiologia , Doenças do Cabelo/etiologia , Microcefalia/etiologia , Mutação , Doenças da Unha/etiologia , Adulto , Sequência de Aminoácidos , Doença de Charcot-Marie-Tooth/enzimologia , Doença de Charcot-Marie-Tooth/patologia , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/patologia , Feminino , Genes Recessivos , Predisposição Genética para Doença , Doenças do Cabelo/enzimologia , Doenças do Cabelo/patologia , Humanos , Masculino , Microcefalia/enzimologia , Microcefalia/patologia , Doenças da Unha/enzimologia , Doenças da Unha/patologia , Linhagem , Fenótipo , Prognóstico , Homologia de Sequência , Adulto JovemRESUMO
Inherited neuromuscular diseases are a heterogeneous group of developmental and degenerative disorders that affect motor unit function. Major challenges toward developing therapies for these diseases include heterogeneity with respect to clinical severity, age of onset and the primary cell type that is affected (e.g. motor neurons, skeletal muscle and Schwann cells). Here, we review recent progress toward the establishment of genetic therapies to treat inherited neuromuscular disorders that affect both children and adults with a focus on spinal muscular atrophy, Charcot-Marie-Tooth disease and spinal and bulbar muscular atrophy. We discuss clinical features, causative mutations and emerging approaches that are undergoing testing in preclinical models and in patients or that have received recent approval for clinical use. Many of these efforts employ antisense oligonucleotides to alter pre-mRNA splicing or diminish target gene expression and use viral vectors to replace expression of mutant genes. Finally, we discuss remaining challenges for optimizing the delivery and effectiveness of these approaches. In sum, therapeutic strategies for neuromuscular diseases have shown encouraging results, raising hope that recent strides will translate into significant clinical benefits for patients with these disorders.
Assuntos
Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Predisposição Genética para Doença , Terapia Genética , Doenças Neuromusculares/genética , Doenças Neuromusculares/terapia , Animais , Gerenciamento Clínico , Doenças Genéticas Inatas/diagnóstico , Terapia Genética/métodos , Humanos , Doenças Neuromusculares/diagnóstico , Resultado do TratamentoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are critical for protein translation. Pathogenic variants of ARSs have been previously associated with peripheral neuropathy and multisystem disease in heterozygotes and homozygotes, respectively. We report seven related children homozygous for a novel mutation in tyrosyl-tRNA synthetase (YARS, c.499C > A, p.Pro167Thr) identified by whole exome sequencing. This variant lies within a highly conserved interface required for protein homodimerization, an essential step in YARS catalytic function. Affected children expressed a more severe phenotype than previously reported, including poor growth, developmental delay, brain dysmyelination, sensorineural hearing loss, nystagmus, progressive cholestatic liver disease, pancreatic insufficiency, hypoglycemia, anemia, intermittent proteinuria, recurrent bloodstream infections and chronic pulmonary disease. Related adults heterozygous for YARS p.Pro167Thr showed no evidence of peripheral neuropathy on electromyography, in contrast to previous reports for other YARS variants. Analysis of YARS p.Pro167Thr in yeast complementation assays revealed a loss-of-function, hypomorphic allele that significantly impaired growth. Recombinant YARS p.Pro167Thr demonstrated normal subcellular localization, but greatly diminished ability to homodimerize in human embryonic kidney cells. This work adds to a rapidly growing body of research emphasizing the importance of ARSs in multisystem disease and significantly expands the allelic and clinical heterogeneity of YARS-associated human disease. A deeper understanding of the role of YARS in human disease may inspire innovative therapies and improve care of affected patients.
Assuntos
Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Mutação com Perda de Função/genética , Tirosina-tRNA Ligase/genética , Adulto , Domínio Catalítico/genética , Pré-Escolar , Feminino , Doenças Genéticas Inatas/fisiopatologia , Perda Auditiva Neurossensorial/diagnóstico por imagem , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/fisiopatologia , Heterozigoto , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Linhagem , Fenótipo , Índice de Gravidade de Doença , Sequenciamento do Exoma , Leveduras/genéticaRESUMO
Aminoacyl-tRNA synthetases (aaRSs) are a conserved family of enzymes with an essential role in protein synthesis: ligating amino acids to cognate tRNA molecules for translation. In addition to their role in tRNA charging, aaRSs have acquired non-canonical functions, including post-transcriptional regulation of mRNA expression. Yet, the extent and mechanisms of these post-transcriptional functions are largely unknown. Herein, we performed a comprehensive transcriptome analysis to define the mRNAs that are associated with almost all aaRSs present in S. cerevisiae cytosol. Nineteen (out of twenty) isogenic strains of GFP-tagged cytosolic aaRSs were subjected to immunoprecipitation with anti-GFP beads along with an untagged control. mRNAs associated with each aaRS were then identified by RNA-seq. The extent of mRNA association varied significantly between aaRSs, from MetRS in which none appeared to be statistically significant, to PheRS that binds hundreds of different mRNAs. Interestingly, many target mRNAs are bound by multiple aaRSs, suggesting co-regulation by this family of enzymes. Gene Ontology analyses for aaRSs with a considerable number of target mRNAs discovered an enrichment for pathways of amino acid metabolism and of ribosome biosynthesis. Furthermore, sequence and structure motif analysis revealed for some aaRSs an enrichment for motifs that resemble the anticodon stem loop of cognate tRNAs. These data suggest that aaRSs coordinate mRNA expression in response to amino acid availability and may utilize RNA elements that mimic their canonical tRNA binding partners.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Citosol/enzimologia , Regulação Fúngica da Expressão Gênica , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoacil-tRNA Sintetases/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mutations in histidyl-tRNA synthetase (HARS1), an enzyme that charges transfer RNA with the amino acid histidine in the cytoplasm, have only been associated to date with autosomal recessive Usher syndrome type III and autosomal dominant Charcot-Marie-Tooth disease type 2W. Using massive parallel sequencing, we identified bi-allelic HARS1 variants in a child (c.616G>T, p.Asp206Tyr and c.730delG, p.Val244Cysfs*6) and in two sisters (c.1393A>C, p.Ile465Leu and c.910_912dupTTG, p.Leu305dup), all characterized by a multisystem ataxic syndrome. All mutations are rare, segregate with the disease, and are predicted to have a significant effect on protein function. Functional studies helped to substantiate their disease-related roles. Indeed, yeast complementation assays showing that one out of two mutations in each patient is loss-of-function, and the reduction of messenger RNA and protein levels and enzymatic activity in patient's skin-derived fibroblasts, together support the pathogenicity of the identified HARS1 variants in the patient phenotypes. Thus, our efforts expand the allelic and clinical spectrum of HARS1-related disease.
Assuntos
Ataxia/genética , Histidina-tRNA Ligase/genética , Adulto , Alelos , Criança , Feminino , Humanos , Masculino , Mutação de Sentido IncorretoRESUMO
BACKGROUND: Multicellular organisms adopt various strategies to tailor gene expression to cellular contexts including the employment of multiple promoters (and the associated transcription start sites (TSSs)) at a single locus that encodes distinct gene isoforms. Schwann cells-the myelinating cells of the peripheral nervous system (PNS)-exhibit a specialized gene expression profile directed by the transcription factor SOX10, which is essential for PNS myelination. SOX10 regulates promoter elements associated with unique TSSs and gene isoforms at several target loci, implicating SOX10-mediated, isoform-specific gene expression in Schwann cell function. Here, we report on genome-wide efforts to identify SOX10-regulated promoters and TSSs in Schwann cells to prioritize genes and isoforms for further study. RESULTS: We performed global TSS analyses and mined previously reported ChIP-seq datasets to assess the activity of SOX10-bound promoters in three models: (i) an adult mammalian nerve; (ii) differentiating primary Schwann cells, and (iii) cultured Schwann cells with ablated SOX10 function. We explored specific characteristics of SOX10-dependent TSSs, which provides confidence in defining them as SOX10 targets. Finally, we performed functional studies to validate our findings at four previously unreported SOX10 target loci: ARPC1A, CHN2, DDR1, and GAS7. These findings suggest roles for the associated SOX10-regulated gene products in PNS myelination. CONCLUSIONS: In sum, we provide comprehensive computational and functional assessments of SOX10-regulated TSS use in Schwann cells. The data presented in this study will stimulate functional studies on the specific mRNA and protein isoforms that SOX10 regulates, which will improve our understanding of myelination in the peripheral nerve.
Assuntos
Fatores de Transcrição SOXE , Células de Schwann , Animais , Expressão Gênica , Bainha de Mielina/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/metabolismoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.
Assuntos
Alanina-tRNA Ligase/genética , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , RNA de Transferência/genética , Adulto , Alelos , Aminoácidos/genética , Animais , Doença de Charcot-Marie-Tooth/patologia , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Heterogeneidade Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Leveduras/genética , Peixe-Zebra/genéticaRESUMO
Mutations in the mitochondrial alanyl-tRNA synthetase gene, AARS2, have been reported to cause leukoencephalopathy associated with early ovarian failure, a clinical presentation described as "ovarioleukodystrophy." We present a sibling pair: one with cerebellar ataxia and one with vision loss and cognitive impairment in addition to ataxia. Neither shows evidence of leukoencephalopathy on MRI imaging. Exome sequencing revealed that both siblings are compound heterozygous for AARS2 variants (p.Phe131del and p.Ile328Met). Yeast complementation assays indicate that p.Phe131del AARS2 dramatically impairs gene function and that p.Ile328Met AARS2 is a hypomorphic allele. This work expands the phenotypic spectrum of AARS2-associated disease to include ataxia without leukoencephalopathy.
Assuntos
Alanina-tRNA Ligase/genética , Ataxia/diagnóstico por imagem , Ataxia/genética , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , Adulto , Sequência de Aminoácidos , Feminino , Humanos , Masculino , Linhagem , IrmãosRESUMO
The majority of patients with spinal muscular atrophy (SMA) identified to date harbor a biallelic exonic deletion of SMN1. However, there have been reports of SMA-like disorders that are independent of SMN1, including those due to pathogenic variants in the glycyl-tRNA synthetase gene (GARS1). We report three unrelated patients with de novo variants in GARS1 that are associated with infantile-onset SMA (iSMA). Patients were ascertained during inpatient hospital evaluations for complications of neuropathy. Evaluations were completed as indicated for clinical care and management and informed consent for publication was obtained. One newly identified, disease-associated GARS1 variant, identified in two out of three patients, was analyzed by functional studies in yeast complementation assays. Genomic analyses by exome and/or gene panel and SMN1 copy number analysis of three patients identified two previously undescribed de novo missense variants in GARS1 and excluded SMN1 as the causative gene. Functional studies in yeast revealed that one of the de novo GARS1 variants results in a loss-of-function effect, consistent with other pathogenic GARS1 alleles. In sum, the patients' clinical presentation, assessments of previously identified GARS1 variants and functional assays in yeast suggest that the GARS1 variants described here cause iSMA. GARS1 variants have been previously associated with Charcot-Marie-Tooth disease (CMT2D) and distal SMA type V (dSMAV). Our findings expand the allelic heterogeneity of GARS-associated disease and support that severe early-onset SMA can be caused by variants in this gene. Distinguishing the SMA phenotype caused by SMN1 variants from that due to pathogenic variants in other genes such as GARS1 significantly alters approaches to treatment.
Assuntos
Predisposição Genética para Doença , Glicina-tRNA Ligase/genética , Atrofias Musculares Espinais da Infância/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Mutação de Sentido Incorreto/genética , Fenótipo , Atrofias Musculares Espinais da Infância/diagnóstico por imagem , Atrofias Musculares Espinais da Infância/fisiopatologiaRESUMO
Aminoacyl-tRNA synthetases (ARSs) are responsible for charging amino acids to cognate tRNA molecules, which is the essential first step of protein translation. Interestingly, mutations in genes encoding ARS enzymes have been implicated in a broad spectrum of human inherited diseases. Bi-allelic mutations in ARSs typically cause severe, early-onset, recessive diseases that affect a wide range of tissues. The vast majority of these mutations show loss-of-function effects and impair protein translation. However, it is not clear how a subset cause tissue-specific phenotypes. In contrast, dominant ARS-mediated diseases specifically affect the peripheral nervous system-most commonly causing axonal peripheral neuropathy-and usually manifest later in life. These neuropathies are linked to heterozygosity for missense mutations in five ARS genes, which points to a shared mechanism of disease. However, it is not clear if a loss-of-function mechanism or a toxic gain-of-function mechanism is responsible for ARS-mediated neuropathy, or if a combination of these mechanisms operate on a mutation-specific basis. Here, we review our current understanding of recessive and dominant ARS-mediated disease. We also propose future directions for defining the molecular mechanisms of ARS mutations toward designing therapies for affected patient populations.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/fisiologia , Alelos , Genes Dominantes/genética , Genes Recessivos/genética , Heterozigoto , Humanos , Mutação , Fenótipo , Biossíntese de Proteínas/genéticaRESUMO
We found a p.Gly327Arg mutation in GARS in two unrelated women, both of whom had a similar phenotype - motor weakness that began in late childhood, distal weakness in the arms and legs, a motor greater than sensory neuropathy with slowing of motor and not sensory conduction velocities. A de novo mutation was proven in one patient and suspected in the other. The p.Gly327Arg GARS variant did not support yeast growth in a complementation assay, showing that this variant severely impairs protein function. Thus, the p.Gly327Arg GARS mutation causes a distal motor neuropathy.
Assuntos
Glicina-tRNA Ligase/genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Adolescente , Adulto , Feminino , Humanos , Mutação , Adulto JovemRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in phenotypically diverse dominant and recessive human diseases. The charging of tRNAPHE with phenylalanine is performed by a tetrameric enzyme that contains two alpha (FARSA) and two beta (FARSB) subunits. To date, mutations in the genes encoding these subunits (FARSA and FARSB) have not been implicated in any human disease. Here, we describe a patient with a severe, lethal, multisystem, developmental phenotype who was compound heterozygous for FARSB variants: p.Thr256Met and p.His496Lysfs*14. Expression studies using fibroblasts isolated from the proband revealed a severe depletion of both FARSB and FARSA protein levels. These data indicate that the FARSB variants destabilize total phenylalanyl-tRNA synthetase levels, thus causing a loss-of-function effect. Importantly, our patient shows strong phenotypic overlap with patients that have recessive diseases associated with other ARS loci; these observations strongly support the pathogenicity of the identified FARSB variants and are consistent with the essential function of phenylalanyl-tRNA synthetase in human cells. In sum, our clinical, genetic, and functional analyses revealed the first FARSB variants associated with a human disease phenotype and expand the locus heterogeneity of ARS-related human disease.
Assuntos
Aminoacil-tRNA Sintetases/genética , Predisposição Genética para Doença , Mutação com Perda de Função/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/deficiência , Regulação da Expressão Gênica , Humanos , Masculino , Fenótipo , Fenilalanina-tRNA Ligase/genéticaRESUMO
Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.
Assuntos
Axônios/patologia , Histidina-tRNA Ligase/metabolismo , Doenças do Sistema Nervoso Periférico/enzimologia , Doenças do Sistema Nervoso Periférico/patologia , Sequência de Aminoácidos , Aminoacilação , Biocatálise , Domínio Catalítico , Sequência Conservada , Feminino , Teste de Complementação Genética , Histidina-tRNA Ligase/química , Histidina-tRNA Ligase/genética , Histidina-tRNA Ligase/isolamento & purificação , Humanos , Cinética , Masculino , Mutação/genética , Linhagem , Doenças do Sistema Nervoso Periférico/genética , Multimerização Proteica , Especificidade por SubstratoRESUMO
BACKGROUND: Identifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells. RESULTS: We identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells-followed by differential gene expression studies-revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts. CONCLUSIONS: Our efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations.
Assuntos
Alelos , Genômica , Nervos Periféricos/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Neurônios Motores/metabolismo , Células Musculares/metabolismo , Nervos Periféricos/citologia , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Charcot-Marie-Tooth (CMT) disease is a genetically heterogeneous group of peripheral neuropathies. Mutations in several aminoacyl-tRNA synthetase (ARS) genes have been implicated in inherited CMT disease. There are 12 reported CMT-causing mutations dispersed throughout the primary sequence of the human glycyl-tRNA synthetase (GARS). While there is strong genetic evidence linking GARS mutations to CMT disease, the molecular pathology underlying the neuromuscular and sensory phenotypes is still not fully understood. In particular, it is unclear whether the mutations result in a toxic gain of function, a partial loss of activity related to translation, or a combination of these mechanisms. We identified a zebrafish allele of gars (gars(s266)). Homozygous mutant embryos carry a C->A transversion, that changes a threonine to a lysine, in a residue next to a CMT-associated human mutation. We show that the neuromuscular phenotype observed in animals homozygous for T209K Gars (T130K in GARS) is due to a loss of dimerization of the mutated protein. Furthermore, we show that the loss of function, dimer-deficient and human disease-associated G319R Gars (G240R in GARS) mutant protein is unable to rescue the above phenotype. Finally, we demonstrate that another human disease-associated mutant G605R Gars (G526 in GARS) dimerizes with the remaining wild-type protein in animals heterozygous for the T209K Gars and reduces the function enough to elicit a neuromuscular phenotype. Our data indicate that dimerization is required for the dominant neurotoxicity of disease-associated GARS mutations and provide a rapid, tractable model for studying newly identified GARS variants for a role in human disease.
Assuntos
Doença de Charcot-Marie-Tooth/patologia , Glicina-tRNA Ligase/química , Glicina-tRNA Ligase/genética , Mutação , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glicina-tRNA Ligase/metabolismo , Humanos , Modelos Biológicos , Fenótipo , Multimerização Proteica , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Schwann cells are myelinating glia in the peripheral nervous system that form the myelin sheath. A major cause of peripheral neuropathy is a copy number variant involving the Peripheral Myelin Protein 22 (PMP22) gene, which is located within a 1.4-Mb duplication on chromosome 17 associated with the most common form of Charcot-Marie-Tooth Disease (CMT1A). Rodent models of CMT1A have been used to show that reducing Pmp22 overexpression mitigates several aspects of a CMT1A-related phenotype. Mechanistic studies of Pmp22 regulation identified enhancers regulated by the Sox10 (SRY sex determining region Y-box 10) and Egr2/Krox20 (Early growth response protein 2) transcription factors in myelinated nerves. However, relatively little is known regarding how other transcription factors induce Pmp22 expression during Schwann cell development and myelination. Here, we examined Pmp22 enhancers as a function of cell type-specificity, nerve injury and development. While Pmp22 enhancers marked by active histone modifications were lost or remodeled after injury, we found that these enhancers were permissive in early development prior to Pmp22 upregulation. Pmp22 enhancers contain binding motifs for TEA domain (Tead) transcription factors of the Hippo signaling pathway. We discovered that Tead1 and co-activators Yap and Taz are required for Pmp22 expression, as well as for the expression of Egr2 Tead1 directly binds Pmp22 and Egr2 enhancers early in development and Tead1 binding is induced during myelination, correlating with Pmp22 expression. The data identify Tead1 as a novel regulator of Pmp22 expression during development in concert with Sox10 and Egr2.