Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.052
Filtrar
1.
Biomolecules ; 11(7)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34356630

RESUMO

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC)-the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.


Assuntos
Códon sem Sentido , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Biossíntese de Proteínas , RNA Mensageiro , Células HeLa , Humanos , Transtornos Peroxissômicos/terapia , Receptor 1 de Sinal de Orientação para Peroxissomos/biossíntese , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
Pan Afr Med J ; 39: 21, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394812

RESUMO

Split-hand foot malformation (SHFM) is a clinically heterogeneous congenital limb defect affecting predominantly the central rays of hands and/or feet. The clinical expression varies in severity between patients as well between the limbs in the same individual. SHFM might be non-syndromic with limb-confined manifestations or syndromic with extra-limb manifestations. Isolated SHFM is a rare condition with an incidence of about 1 per 18,000 live born infants and accounts for 8-17 % of all limb malformations. To date, many chromosomal loci and genes have been described as associated with isolated SHFM, i.e., SHFM1 to 6. SHFM6 is one of the rarest forms of SHFM, and is caused by mutations in WNT10B gene. Less than ten pathogenic variants have been described. We have investigated a large consanguineous Moroccan family with three affected members showing feet malformations with or without split hand malformation phenotypes. Using an exome sequencing approach, we identified a homozygous nonsense variant p.Arg115* of WNT10B gene retaining thereby the diagnosis of SHFM6. This homozygous nonsense mutation identified by exome sequencing in a large family of split hand foot malformation highlights the importance of exome sequencing in genetically heterogeneous entities.


Assuntos
Deformidades Congênitas dos Membros/diagnóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Criança , Códon sem Sentido , Exoma/genética , Feminino , Homozigoto , Humanos , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/fisiopatologia , Marrocos
3.
J Genet ; 1002021.
Artigo em Inglês | MEDLINE | ID: mdl-34282730

RESUMO

Choroideraemia (CHM) is a rare X-linked progressive-inherited retinal disease. In this study, we diagnosed and explored the genetic cause in a Chinese pedigree exhibiting nyctalopia and decreased visual acuity in early life. Clinical data and peripheral blood samples were collected from available family members. Sanger sequencing of RPGR and RP2 genes, and subsequently whole-exome sequencing was carried out to investigate the molecular cause. The proband was initially diagnosed as retinitis pigmentosa and experienced night blindness at an early age and decreased visual acuity in teens. The other affected males in this family suffered from the same problem. Direct sequencing failed to reveal the genetic cause and hence a novel hemizygous mutation c.861_862insGCTT was detected by WES in CHM gene resulting in a premature stop codon and a truncated protein. Subsequently, it was confirmed by Sanger sequencing and cosegregation analysis. We describe a novel mutation c.861_862insGCTT in CHM gene in a Chinese pedigree with choroideraemia. Our study emphasizes the utilization of next-generation sequencing in the diagnosis and genetic analysis of retinal diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Coroideremia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Predisposição Genética para Doença , Adolescente , Adulto , Coroideremia/patologia , Códon sem Sentido/genética , Proteínas do Olho/genética , Genes Ligados ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Testes Genéticos , Humanos , Masculino , Mutação/genética , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
4.
Am J Hum Genet ; 108(8): 1401-1408, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216550

RESUMO

Precise interpretation of the effects of rare protein-truncating variants (PTVs) is important for accurate determination of variant impact. Current methods for assessing the ability of PTVs to induce nonsense-mediated decay (NMD) focus primarily on the position of the variant in the transcript. We used RNA sequencing of the Genotype Tissue Expression v.8 cohort to compute the efficiency of NMD using allelic imbalance for 2,320 rare (genome aggregation database minor allele frequency ≤ 1%) PTVs across 809 individuals in 49 tissues. We created an interpretable predictive model using penalized logistic regression in order to evaluate the comprehensive influence of variant annotation, tissue, and inter-individual variation on NMD. We found that variant position, allele frequency, the inclusion of ultra-rare and singleton variants, and conservation were predictive of allelic imbalance. Furthermore, we found that NMD effects were highly concordant across tissues and individuals. Due to this high consistency, we demonstrate in silico that utilizing peripheral tissues or cell lines provides accurate prediction of NMD for PTVs.


Assuntos
Códon sem Sentido/genética , Regulação da Expressão Gênica , Doenças Genéticas Inatas/patologia , Variação Genética , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/genética , Frequência do Gene , Doenças Genéticas Inatas/genética , Humanos
5.
Eur J Med Genet ; 64(9): 104282, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34284163

RESUMO

The Forkhead transcription factor FOXG1 is a prerequisite for telencephalon development in mammals and is an essential factor controlling expansion of the dorsal telencephalon by promoting neuron and interneuron production. Heterozygous FOXG1 gene mutations cause FOXG1 syndrome characterized by severe intellectual disability, motor delay, dyskinetic movements and epilepsy. Neuroimaging studies in patients disclose constant features including microcephaly, corpus callosum dysgenesis and delayed myelination. Currently, investigative research on the underlying pathophysiology relies on mouse models only and indicates that de-repression of FOXG1 target genes may cause premature neuronal differentiation at the expense of the progenitor pool, patterning and migration defects with impaired formation of cortico-cortical projections. It remains an open question to which extent this recapitulates the neurodevelopmental pathophysiology in FOXG1-haploinsufficient patients. To close this gap, we performed neuropathological analyses in two foetal cases with FOXG1 premature stop codon mutations interrupted during the third trimester of the pregnancy for microcephaly and corpus callosum dysgenesis. In these foetuses, we observed cortical lamination defects and decreased neuronal density mainly affecting layers II, III and V that normally give rise to cortico-cortical and inter-hemispheric axonal projections. GABAergic interneurons were also reduced in number in the cortical plate and persisting germinative zones. Additionally, we observed more numerous PDGFRα-positive oligodendrocyte precursor cells and fewer Olig2-positive pre-oligodendrocytes compared to age-matched control brains, arguing for delayed production and differentiation of oligodendrocyte lineage leading to delayed myelination. These findings provide key insights into the human pathophysiology of FOXG1 syndrome.


Assuntos
Agenesia do Corpo Caloso/genética , Axônios/patologia , Fatores de Transcrição Forkhead/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Neurogênese , Oligodendroglia/patologia , Feto Abortado/metabolismo , Feto Abortado/patologia , Adulto , Agenesia do Corpo Caloso/patologia , Axônios/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/patologia , Códon sem Sentido , Feminino , Fatores de Transcrição Forkhead/metabolismo , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Humanos , Interneurônios/metabolismo , Interneurônios/patologia , Microcefalia/patologia , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Oligodendroglia/metabolismo , Linhagem , Gravidez , Síndrome
6.
Vasc Health Risk Manag ; 17: 415-419, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34321884

RESUMO

The "Lebanese allele" {LDLR c.2043 C>A (p.cys681X)} is a nonsense mutation in the low-density lipoprotein receptor (LDLR) gene that results in a truncated non-functioning LDLR protein. We report two sisters of Lebanese descent who presented with familial hypercholesterolemia (FH) and were both heterozygous for the Lebanese allele, but had very distinct LDL-C levels and clinical phenotypes. Whereas one of the sisters had LDL-C in the expected range of Heterozygous FH (HeFH) with the Lebanese allele (LDL-C of 292 mg/dl), the other sister had a more severe LDL-C phenotype in the Homozygous FH (HoFH) range (LDL-C of 520 mg/dl) along with manifest atherosclerosis. Surprisingly, she did not demonstrate a compound heterozygote or double heterozygote status. We discuss different mechanisms that are purported to play a role in modifying the phenotype of FH, including different variants and polygenic modifiers. HeFH patients with the Lebanese allele can have a wide spectrum of LDL-C levels that range from the typical heterozygous to homozygous phenotypes.


Assuntos
LDL-Colesterol/sangue , Códon sem Sentido , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Irmãos , Feminino , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/diagnóstico , Pessoa de Meia-Idade , Linhagem , Fenótipo , Índice de Gravidade de Doença
7.
Nat Commun ; 12(1): 4358, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272367

RESUMO

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.


Assuntos
Códon sem Sentido/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Degradação do RNAm Mediada por Códon sem Sentido , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Terminação de Peptídeos/metabolismo , Aminoglicosídeos/metabolismo , Códon sem Sentido/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Genes Reporter , Gentamicinas/farmacologia , Células HEK293 , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Fatores de Terminação de Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ribossomos/metabolismo , Relação Estrutura-Atividade
8.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
9.
Nat Commun ; 12(1): 4544, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315873

RESUMO

Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.


Assuntos
Ribossomos Mitocondriais/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Linhagem Celular , Códon sem Sentido/genética , Microscopia Crioeletrônica , RNA Helicases DEAD-box , Humanos , Metiltransferases/metabolismo , Ribossomos Mitocondriais/ultraestrutura , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/ultraestrutura , RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores/ultraestrutura
10.
Cytogenet Genome Res ; 161(3-4): 153-159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34229322

RESUMO

Terminal deletions in the long arm of chromosome 4 are an uncommon event, with a worldwide incidence of approximately 0.001%. The majority of these deletions occur de novo. Terminal deletion cases are usually accompanied by clinical findings that include facial and cardiac anomalies, as well as intellectual disability. In this study, we describe the case of a 2-year-old girl, the fourth child born to consanguineous parents. While her karyotype was normal, a homozygous deletion was identified in the chromosome 4q35.2 region by subtelomeric FISH. A heterozygous deletion of the chromosome 4q35.2 region was observed in both parents. According to the literature, this is the first report of a case that has inherited a homozygous deletion of chromosome 4qter from carrier parents. Subsequent array-CGH analyses were performed on both the case and her parents. Whole-exome sequencing was also carried out to determine potential variants. We detected a NM_001111125.3:c.2329G>T (p.Glu777Ter) nonsense variant of the IQSEC2 gene in the girl, a variant that is related to X-linked intellectual disability.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 4/genética , Códon sem Sentido , Fatores de Troca do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Pré-Escolar , Consanguinidade , Feminino , Genes Ligados ao Cromossomo X/genética , Homozigoto , Humanos , Cariotipagem , Telômero/genética , Sequenciamento Completo do Exoma
11.
F1000Res ; 10: 148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34164111

RESUMO

Neurofibromatosis 1 (NF1) is a neurocutaneous syndrome characterized by multiple café-au-lait macules, cutaneous neurofibromas or plexiform neurofibromas, iris Lisch nodules, axillary and inguinal freckling. Mosaicism in NF1 can either present as a generalized disease, or in a localized (segmental) manner. Mosaic generalized NF1 may have presentations that are similar to generalized NF1 or have a milder phenotype and hence may be under-recognised in clinical practice. We report a nonsense mutation in the NF1 gene in a 55-year old Chinese male with the mosaic generalized phenotype. He reported noticing increasing numbers of skin-colored papules over his face, neck, back and abdomen when he was about 40 years old. From both next-generation and Sanger sequencing data, the variant appeared to be mosaic and present at about 24%. It is in exon 39 and has not been reported in any database or published literature.


Assuntos
Neurofibromatose 1 , Neurofibromina 1 , Adulto , Códon sem Sentido , Genes da Neurofibromatose 1 , Humanos , Masculino , Pessoa de Meia-Idade , Mosaicismo , Neurofibromatose 1/genética , Neurofibromina 1/genética , Fenótipo
12.
Eur J Med Genet ; 64(8): 104260, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34118472

RESUMO

Fanconi anemia (FA) due to biallelic mutations in the BRCA2 gene is very rare and associated with an extremely high risk of early-onset of aggressive childhood malignancies, predominantly brain tumors, leukemia, and nephroblastoma. Here, we present a consanguineous family with three affected children of the D1 subtype of FA and describe the clinical consequences of the earliest known biallelic nonsense/stop-gain germ-line mutation in BRCA2, exon 5 c.469A>T, that leads to a premature stop of translation, p.Lys157*. The three patients were born with severe intrauterine growth restrictions and different degrees of congenital malformations. Altogether, they developed eight distinct malignancies and died within their first three years of life. Treatment with a reduced chemotherapy regimen was only performed in patient 2 for his first tumor, a nephroblastoma, which the patient tolerated well for eight months, until he developed myelodysplastic syndrome (MDS) and then acute myeloid leukemia (AML). Finally, the third patient experienced a hepatoblastoma, an unclassified brain tumor and MDS in parallel and died in her second year of life. Our report re-emphasizes the aggressiveness and fatality of the FA-D1 children with biallelic BRCA2 nonsense mutations, that are both located before exon 11, which contains binding domains for the RAD51 recombinase.


Assuntos
Proteína BRCA2/genética , Anemia de Fanconi/genética , Fenótipo , Adulto , Criança , Pré-Escolar , Códon sem Sentido , Éxons , Anemia de Fanconi/patologia , Feminino , Homozigoto , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem
13.
Medicine (Baltimore) ; 100(23): e26093, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34114993

RESUMO

RATIONALE: Mutations of connector enhancer of kinase suppressor of Ras-2 (CNKSR2) gene were identified as the cause of Houge type of X-linked syndromic mental retardation. The mutations of CNKSR2 gene are rare, we reporta patient carrying a novel nonsense mutation of CNKSR2,c.625C > T(p.Gln209∗) and review the clinical features and mutations of CNKSR2 gene for this rare condition considering previous literature. PATIENT CONCERNS: We report a case of a 7-year and 5-month-old Chinese patient with clinical symptoms of intellectual disability, language defect, epilepsy and hyperactivity. Genetic study revealed a novel nonsense variant of CNKSR2, which has not been reported yet. DIAGNOSIS: According to clinical manifestations, genetic pattern and ACMG classification of mutation site as Class 1-cause disease, the patient was diagnosed as Houge type of X-linked syndromic mental retardation caused by CNKSR2 gene mutation. INTERVENTIONS: The patient was administrated with a gradual titration of valproic acid (VPA). OUTCOMES: On administration of valproic acid, he had no further seizures. LESSONS: This is the first time to report a nonsense variant in CNKSR2, c.625C > T(p.Gln209∗), this finding could expand the spectrum of CNKSR2 mutations and might also support the further study of Houge type of X-linked syndromic mental retardation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Epilepsia , Deficiência Intelectual , Transtornos da Linguagem , Retardo Mental Ligado ao Cromossomo X , Agitação Psicomotora , Ácido Valproico/administração & dosagem , Anticonvulsivantes/administração & dosagem , Criança , Códon sem Sentido , Epilepsia/diagnóstico , Epilepsia/genética , Epilepsia/prevenção & controle , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Transtornos da Linguagem/diagnóstico , Transtornos da Linguagem/genética , Masculino , Retardo Mental Ligado ao Cromossomo X/diagnóstico , Retardo Mental Ligado ao Cromossomo X/tratamento farmacológico , Retardo Mental Ligado ao Cromossomo X/genética , Mutação , Agitação Psicomotora/diagnóstico , Agitação Psicomotora/etiologia , Avaliação de Sintomas , Resultado do Tratamento
14.
Nat Commun ; 12(1): 3850, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158503

RESUMO

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.


Assuntos
Códon sem Sentido/genética , Códon de Terminação/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Ribossomos/genética , Sequência de Bases , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Supressão Genética
15.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34066907

RESUMO

Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.


Assuntos
Aminoglicosídeos/farmacologia , Transtornos da Motilidade Ciliar/genética , Códon sem Sentido/genética , Mutação/genética , Biossíntese de Proteínas/genética , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Nariz/patologia , Biossíntese de Proteínas/efeitos dos fármacos
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(6): 549-552, 2021 Jun 10.
Artigo em Chinês | MEDLINE | ID: mdl-34096023

RESUMO

OBJECTIVE: To explore the genetic basis for a pedigree affected with hereditary multiple osteochondroma (HMO). METHODS: Peripheral blood samples were collected from the proband and members of his pedigree with informed consent. Following extraction of genomic DNA, all coding exons and flanking intronic sequences (-10 bp) of the EXT1 and EXT2 genes were subjected to targeted capture and next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing. RESULTS: A heterozygous nonsense variant (c.1911C>A) was found in exon 10 of the EXT1 gene in the proband and his affected father but not in a healthy sister and normal controls. The variant was classified as a pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (PVS1+PM2+PP1). Bioinformatic analysis predicted that the c.1911C>A variant may be disease-causing via nonsense-mediated mRNA decay and anomalous splicing. CONCLUSION: The c.1911C>A variant probably underlay the disease in this pedigree. Discovery of this variant enriched the variant spectrum of HMO.


Assuntos
Exostose Múltipla Hereditária , Códon sem Sentido , Éxons/genética , Exostose Múltipla Hereditária/genética , Heterozigoto , Humanos , Linhagem
17.
Eur J Med Genet ; 64(7): 104248, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34000439

RESUMO

Smith-McCort dysplasia 2 (SMC2) is a rare spondylo-epiphyseal-metaphyseal dysplasia caused by biallelic RAB33B variants. Short trunk dwarfism and radiological findings including the lacy ilia appearance and double bumps of the vertebral bodies are typical features. To date, only eight patients with SMC2 had been reported. The aim of this study is to evaluate the follow-up findings of seven patients from five families with SMC2 and to present four novel variants in RAB33B. The age of diagnosis of the patients was between 4 and 18 years. All patients had variable degrees of short trunk dwarfism with barrel chest, waddling gait, hyperlordosis, genu valgum, elbow and finger joint stiffness, which became more evident with growth. Lacy iliac crest, short ilia with basilar hypoplasia, platyspondyly, dysplastic acetabulum with small and/or laterally displaced femoral heads, and small, irregular carpal bones were detected on skeletal radiographies of all patients. Typical double hump appearance of vertebral bodies was present in patients under 12 years of age, which disappeared after puberty and development of elongated vertebral bodies was also observed. At the time of diagnosis, six patients were able to walk independently; patients who were followed for five to nine years, developed severe hip pain, hip and knee joints stiffness and difficultly of walking after 10 years of age. Only two patients could walk independently during final examination. We detected four novel nonsense variants (p.Gln85Ter, p. Cys48Ter, p. Arg94Ter and p. Gln134Ter) in RAB33B. This study provides important data on long-term skeletal findings of the patients with SMC2.


Assuntos
Osteocondrodisplasias/genética , Fenótipo , Proteínas rab de Ligação ao GTP/genética , Adolescente , Criança , Pré-Escolar , Códon sem Sentido , Feminino , Humanos , Masculino , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/patologia
18.
PLoS Biol ; 19(5): e3001221, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33939688

RESUMO

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.


Assuntos
Aminoglicosídeos/farmacologia , Códon sem Sentido/genética , Ribossomos/metabolismo , Aminoglicosídeos/metabolismo , Aminoglicosídeos/fisiologia , Linhagem Celular , Quimiocinas CXC/efeitos dos fármacos , Quimiocinas CXC/metabolismo , Códon sem Sentido/metabolismo , Códon de Terminação , Gentamicinas/farmacologia , Humanos , Mutação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas , Ribossomos/efeitos dos fármacos
20.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801686

RESUMO

Forward genetic screens have shown the consequences of deleterious mutations; however, they are best suited for model organisms with fast reproductive rates and large broods. Furthermore, investigators must faithfully identify changes in phenotype, even if subtle, to realize the full benefit of the screen. Reverse genetic approaches also probe genotype to phenotype relationships, except that the genetic targets are predefined. Until recently, reverse genetic approaches relied on non-genomic gene silencing or the relatively inefficient, homology-dependent gene targeting for loss-of-function generation. Fortunately, the flexibility and simplicity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has revolutionized reverse genetics, allowing for the precise mutagenesis of virtually any gene in any organism at will. The successful integration of insertions/deletions (INDELs) and nonsense mutations that would, at face value, produce the expected loss-of-function phenotype, have been shown to have little to no effect, even if other methods of gene silencing demonstrate robust loss-of-function consequences. The disjunction between outcomes has raised important questions about our understanding of genotype to phenotype and highlights the capacity for compensation in the central dogma. This review describes recent studies in which genomic compensation appears to be at play, discusses the possible compensation mechanisms, and considers elements important for robust gene loss-of-function studies.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Estudos de Associação Genética , Mutação , Degradação do RNAm Mediada por Códon sem Sentido/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Códon sem Sentido , Técnicas Genéticas , Genômica , Genótipo , Mutação INDEL , Mutagênese , Fenótipo , Peixe-Zebra
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...