RESUMO
Heterozygous carriers of the survival of motor neuron 1 (SMN1) gene deletion in parents account for approximately 95% of neonatal spinal muscular atrophy cases. Given the severity of the disease, professional organizations have recommended periconceptional spinal muscular atrophy carrier screening to all couples, regardless of race or ethnicity. However, the prevalence of screening activities in mainland China remains suboptimal, mainly attributed to the limitations of the existing carrier screening methods. Herein, we aimed to develop a low-cost, accessible, and accurate carrier screening method based on duplex droplet digital PCR (ddPCR), to cover a wider population in developing countries, including China. The receiver operating characteristic curve was used to determine the cut-off value of SMN1 copy numbers. Performance validation was conducted for linearity, precision, and accuracy. In total, 482 cases were considered to validate the concordance between the developed ddPCR assay and multiplex ligation-dependent probe amplification. Linear correlations were excellent between the expected concentration of the reference gene and the observed values (R2 > 0.99). Both the intra- and inter-assay precision of our ddPCR assays were less than 6.0%. The multiplex ligation-dependent probe amplification and ddPCR results were consistent in 480 of the 482 cases (99.6%). Two cases with multiplex ligation-dependent probe amplification, suggestive of two copies of SMN1 exon 7, were classified into three copies by ddPCR analysis. The overall correct classification of the samples included in our ddPCR assay was 100%. This study demonstrates that an appropriate cut-off value is an important prerequisite for establishing a semi-quantitative method to determine the SMN1 copy numbers. Compared to conventional methods, our ddPCR assay is low-cost, highly accurate, and has full potential for application in population spinal muscular atrophy carriers screening.
Assuntos
Países em Desenvolvimento , Atrofia Muscular Espinal , Recém-Nascido , Humanos , Deleção de Genes , Heterozigoto , Reação em Cadeia da Polimerase Multiplex/métodos , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by degeneration of lower motor neurons (LMNs), causing muscle weakness, atrophy, and paralysis. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and can be classified into four subgroups, depending on its severity. Even though the genetic component of SMA is well known, the precise mechanisms underlying its pathophysiology remain elusive. Thus far, there are three FDA-approved drugs for treating SMA. While these treatments have shown promising results, their costs are extremely high and unaffordable for most patients. Thus, more efforts are needed in order to identify novel therapeutic targets. In this context, zebrafish (Danio rerio) stands out as an ideal animal model for investigating neurodegenerative diseases like SMA. Its well-defined motor neuron circuits and straightforward neuromuscular structure offer distinct advantages. The zebrafish's suitability arises from its low-cost genetic manipulation and optical transparency exhibited during larval stages, which facilitates in vivo microscopy. This review explores advancements in SMA research over the past two decades, beginning with the creation of the first zebrafish model. Our review focuses on the findings using different SMA zebrafish models generated to date, including potential therapeutic targets such as U snRNPs, Etv5b, PLS3, CORO1C, Pgrn, Cpg15, Uba1, Necdin, and Pgk1, among others. Lastly, we conclude our review by emphasizing the future perspectives in the field, namely exploiting zebrafish capacity for high-throughput screening. Zebrafish, with its unique attributes, proves to be an ideal model for studying motor neuron diseases and unraveling the complexity of neuromuscular defects.
Assuntos
Doença dos Neurônios Motores , Atrofia Muscular Espinal , Doenças Neurodegenerativas , Animais , Humanos , Peixe-Zebra/genética , Atrofia Muscular Espinal/terapia , Neurônios Motores , Proteína 1 de Sobrevivência do Neurônio Motor , Modelos Animais de DoençasRESUMO
Alternative splicing of the messenger RNA plays a fundamental role in the flow of genetic information from DNA to proteins by expanding the coding capacity of the genome. The regulation of alternative splicing is as important as the regulation of transcription to determine the specific characteristics of cells and tissues, the normal functioning of cells and the responses of eukaryotic cells to external signals. Basic knowledge of the pre-mRNA sequences and splicing factors that recognize them has allowed scientists to design a therapeutic synthetic oligonucleotide for spinal muscular atrophy. This is an autosomal recessive inherited disease in which the SMN1 gene is mutated and affects one in 10,000 births. By blocking the binding of a negative splicing factor to the mRNA of a paralogue of the SMN1 gene, called SMN2, the Spinraza oligonucleotide corrects an abnormal alternative splicing event of the SMN2 gene and allows the synthesis of high levels of the SMN protein, constituting the first successful case of cure of a neurodegenerative disease.
El splicing alternativo del ácido ribonucleico mensajero (mRNA) juega un papel fundamental en el flujo de información genética desde el ADN a las proteínas al expandir la capacidad de codificación de los genomas. La regulación del splicing alternativo es tan importante como la regulación de la transcripción para determinar las características específicas de las células y los tejidos, el funcionamiento normal de las células y las respuestas de las células eucarióticas a las señales externas. El conocimiento básico de las secuencias del pre-mRNA y de los factores de splicing que las reconocen ha permitido a científicos diseñar un oligonucleótido sintético terapéutico para la atrofia muscular espinal. ésta es una enfermedad hereditaria autosómica recesiva en que el gen SMN1 se encuentra mutado y que afecta a uno de cada 10 000 nacimientos. Al bloquear la unión de un factor de splicing negativo al mRNA del gen parálogo del gen SMN1, denominado SMN2, el oligonucleótido Spinraza corrige un evento de splicing alternativo anormal del gen SMN2 y permite que se sinteticen altos niveles de la proteína SMN, constituyéndose en el primer caso exitoso de cura de una enfermedad neurodegenerativa.
Assuntos
Processamento Alternativo/genética , Atrofia Muscular Espinal/terapia , Splicing de RNA/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Transcrição Gênica/genética , Humanos , Atrofia Muscular Espinal/genética , RNA Mensageiro/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Spinal muscular atrophy (SMA) type 0 is the most severe form of SMA, associated with the SMN1 gene and manifesting at birth. Most patients die in the first weeks of life. In this work, we present 3 patients with SMA type 0 who survived >1 year and presented diffuse and progressive brain abnormalities on magnetic resonance imaging, which are not usually seen in patients with SMA. Thus, severe brain involvement may likely be the full end manifestation of an already extreme SMA phenotype caused by substantial reduction of the SMN protein in the brain. ANN NEUROL 2019;86:458-462.
Assuntos
Encéfalo/patologia , Atrofia Muscular Espinal/patologia , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Atrofia Muscular Espinal/genética , Neuroimagem , Fenótipo , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
ANTECEDENTES: En el marco de la metodología ad hoc para evaluar solicitudes de tecnologías sanitarias, aprobada mediante Resolución de Instituto de Evaluación de Tecnologías en Salud e Investigación N° 111-IETSI-ESSALUD-2021 y ampliada mediante Resolución de Instituto de Evaluación de Tecnologías en Salud e Investigación N° 97-IETSI-ESSALUD2022, se ha elaborado el presente dictamen, el cual expone la evaluación de la eficacia y seguridad de risdiplam en pacientes pediátricos con atrofia muscular espinal 5q tipo 2 y tipo 3. Así, Elizabeth Ruth Espíritu Rojas y Katherine Joyce Ramos, médicos especialistas en neurología pediátrica del Hospital Nacional Edgardo Rebagliati Martins (HNERM) siguiendo la Directiva N° 003-IETSI-ESSALUD-2016, enviaron a través del comité farmacoterapéutico del HNERM al Instituto de Evaluación de Tecnologías en Salud e Investigación - IETSI la solicitud de autorización de uso del producto farmacéutico risdiplam no incluido en el Petitorio Farmacológico del EsSalud. ASPECTOS GENERALES: La atrofia muscular espinal (AME) es un conjunto de enfermedades neuromusculares raras con una base genética caracterizada por la degeneración de las células de la asta anterior de la médula espinal y de los núcleos motores en el tronco encefálico inferior, que lleva a una debilidad muscular proximal simétrica difusa y atrofia progresiva. La condición genética del AME se asocia al cromosoma 5 brazo q, y se hereda de forma autosómica recesiva. Los diferentes tipos del AME son causados por deleciones o mutaciones puntuales en el gen SMN1, lo que ocasiona un déficit de la proteína SMN, generando alteración en la síntesis de ARNm en tejido cerebral, motoneuronas y en la médula espinal, estos cambios se relacionan directamente con la fisiopatología del AME. Sin embargo, debido a que los genes SMN1 y SMN2 presentan homología en un 99 % en la secuencia de sus nucleátidos, la proteína SMN se compensa parcialmente mediante la síntesis de la SMN2 (mayo, ©2023 UpToDate) (Ferrari et al. 2010). Así, el inicio y la gravedad de la enfermedad se correlacionan principalmente con el número de copias del gen SMN2, lo cual también permite la clasificación clínica de la AME en fenotipos del O al 4 (Russman B, 2007; Munsat T, 1992). METODOLOGÍA: Se llevó a cabo una búsqueda bibliográfica exhaustiva, sistemática y jerárquica de la literatura con el objetivo de identificar la mejor evidencia sobre la eficacia y seguridad de risdiplam en pacientes pediátricos con AME 5q tipo 2 o tipo 3. La búsqueda bibliográfica se realizó en las bases de datos PubMed, The Cochrane Library y LILACS. Asimismo, se realizó una búsqueda manual dentro de las páginas web pertenecientes a grupos que realizan evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC) incluyendo National Institute for Health and Care Excellence (NICE), International HTA database, Institut national d'excellence en santé et en services sociaux (INESSS), Canadian Agency for Drugs and Technologies in Health (CADTH), Scottish Medicines Consortium (SMC), Scottish Intercollegiate Guidelines Network (SIGN), Institute for Quality and Efficiency in Healthcare (IQWiG por sus siglas en alemán), International Database of GRADE Guideline, Centro Nacional de Excelencia Tecnológica en Salud (CENETEC), Guidelines International Network (GIN), National Health and Medical Research Council (NHMRC), Comissáo Nacional de IncorporaQáo de Tecnologias no Sistema Único de Saúde (CONITEC), Instituto de Evaluación Tecnológica en Salud (IETS), Red Española de Agencias de Evaluación de Tecnologías Sanitarias y Prestaciones del Sistema Nacional de Salud, Instituto de Efectividad Clínica y Sanitaria (IECS), Base Regional de Informes de Evaluación de Tecnologías en Salud de las Américas (BRISA), Organización Mundial de la Salud (OMS), Ministerio de Salud del Perú (MINSA) e Instituto de Evaluación de Tecnologías en Salud e Investigación (IETSI). Finalmente, se realizó una búsqueda en la página web de registro de ensayos clínicos (EC) www.clinicaltrials.gov, para identificar EC en curso o que no hayan sido publicados aún. Se seleccionaron GPC, ETS, revisiones sistemáticas (RS), y ECA de fase III que abordaran la pregunta PICO del presente dictamen. La selección de documentos se realizó en dos fases. En la primera, se realizó la revisión de títulos y resúmenes de las publicaciones, a través del aplicativo web Rayyan (https://rawan.ai/), por parte de dos evaluadores independientes. En la segunda, uno de los evaluadores revisó los documentos a texto completo incluidos en la primera fase y realizó la selección final de los estúdios. RESULTADOS: Luego de la búsqueda bibliográfica, se logró identificar: una GPC de la Sociedad Peruana de Neurología (GPC- AME 2021); seis ETS elaboradas por HAS 2021, NICE 2021, IQWiG 2021, CADTH 2021, SMC 2022 y CONITEC 2022, y finalmente un ECA III, el estudio SUNFISH (NCT02908685). No se contó con alguna RS que se enfocara en la PICO de interés. CONCLUSIÓN: Por lo expuesto, el Instituto de Evaluación de Tecnologías en Salud e Investigación no aprueba el uso de risdiplam para pacientes pediátricos con AME tipo 2 y tipo 3, como producto farmacéutico no incluido en el Petitorio Farmacológico de EsSalud.
Assuntos
RNA Mensageiro/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Proteína 1 de Sobrevivência do Neurônio Motor/uso terapêutico , Eficácia , Análise Custo-BenefícioRESUMO
Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.
Assuntos
DNA Antissenso/administração & dosagem , Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Oligonucleotídeos/administração & dosagem , Proteína 1 de Sobrevivência do Neurônio Motor/administração & dosagem , Humanos , Injeções Espinhais , Mutação , FenótipoRESUMO
ABSTRACT Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.
RESUMO A atrofia muscular espinhal (AME) é uma grave doença dos neurônios motores, de grande variabilidade clínica e causada na maioria dos casos por mutação em homozigose no gene SMN1. Pelo menos quatro fenótipos clínicos distintos são reconhecidos com base na idade de início e no grau de envolvimento motor. Tal variabilidade clínica é em parte relacionada com o número de cópias do gene SMN2. Até recentemente, apenas terapias de suporte estavam disponíveis. Atualmente, terapias especificas estão sendo desenvolvidas com base em diferentes mecanismos para aumentar o nível de proteína SMN; em particular oligonucleotídeos antissenso por via intratecal e inserção de cópia do gene SMN1, via endovenosa, usando vetor viral. Nesta revisão, objetivamos discutir as mais recentes e promissoras estratégias terapêuticas, com consideração especial aos aspectos patogênicos da doença e aos mecanismos de ação de tais terapias.
Assuntos
Humanos , Oligonucleotídeos/administração & dosagem , Atrofia Muscular Espinal/terapia , Terapia Genética/métodos , DNA Antissenso/administração & dosagem , Proteína 1 de Sobrevivência do Neurônio Motor/administração & dosagem , Fenótipo , Injeções Espinhais , MutaçãoRESUMO
OBJECTIVE: To demonstrate the feasibility of presymptomatic diagnosis of spinal muscular atrophy (SMA) through newborn screening (NBS). STUDY DESIGN: We performed a screening trial to assess all newborns who underwent routine newborn metabolic screening at the National Taiwan University Hospital newborn screening center between November 2014 and September 2016. A real-time polymerase chain reaction (RT-PCR) genotyping assay for the SMN1/SMN2 intron 7 c.888+100A/G polymorphism was performed to detect homozygous SMN1 deletion using dried blood spot (DBS) samples. Then the exon 7 c.840C>T mutation and SMN2 copy number were determined by both droplet digital PCR (ddPCR) using the original screening DBS and multiplex ligation-dependent probe amplification (MLPA) using a whole blood sample. RESULTS: Of the 120 267 newborns, 15 tested positive according to the RT-PCR assay. The DBS ddPCR assay excluded 8 false-positives, and the other 7 patients were confirmed by the MLPA assay. Inclusion of the second-tier DBS ddPCR screening assay resulted in a positive prediction value of 100%. The incidence of SMA was 1 in 17 181 (95% CI, 1 in 8323 to 1 in 35 468). Two of the 3 patients with 2 copies of SMN2 and all 4 patients with 3 or 4 copies of SMN2 were asymptomatic at the time of diagnosis. Five of the 8 false-positives were caused by intragenic recombination between SMN1 and SMN2. CONCLUSION: Newborn screening can detect patients affected by SMA before symptom onset and enable early therapeutic intervention. A combination of a RT-PCR and a second-tier ddPCR can accurately diagnose SMA from DBS samples with no false-positives. TRIAL REGISTRATION: ClinicalTrials.gov NCT02123186.
Assuntos
Atrofia Muscular Espinal/diagnóstico , Triagem Neonatal/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteína 2 de Sobrevivência do Neurônio Motor/genética , TaiwanRESUMO
BACKGROUND/PURPOSE: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, considered one of the leading causes of infant mortality. It is caused by mutations in the SMN1 gene. A highly homologous copy of this gene named SMN2 and other neighbouring genes, SERF1A and NAIP, are considered phenotypic modifiers of the disease. In recent years, notable advances have been made in SMA research regarding evaluation, prognosis, and therapeutic options. Thus, genotype-phenotype studies in SMA are important to stratify patients for motor function tests and for envisaged clinical trials. The aim of this study was to provide clinical and molecular data of a series of Argentinean children with SMA to establish a comprehensive genotype-phenotype correlation. METHODS: 144 Argentinean children with SMA (56 children with type I, 58 with type II, and 30 with type III) were evaluated. The copy number of SMN2, SERF1A, and NAIP genes was established using MLPA (Multiplex Ligation-dependent Probe Amplification) and then correlated with the patients clinical subtypes. To improve clinical characterization we considered the initial symptoms that prompted the consultation, age of acquisition of motor abilities to independent walking and age at loss of gait. We also evaluated clinical and molecular features of sibling pairs in seven families. RESULTS: A strong correlation was observed between the SMN2 copy number and SMA phenotype while SERF1A and NAIP copy number showed a moderate correlation. We observed intra- and inter-family differences among the SMA types. CONCLUSION: This first genotype-phenotype correlation study in Argentinean SMA children provides data to improve patient stratification and define more adequate follow-up parameters.