RESUMO
Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy2J/dy2J mouse model of MDC1A. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy2J/dy2J mice display substantial improvement in muscle histopathology and function without signs of paralysis.
Assuntos
Reparo do DNA por Junção de Extremidades , Terapia Genética/métodos , Laminina/genética , Distrofias Musculares/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Animais , Western Blotting , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Imunofluorescência , Laminina/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Mutação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine.
Assuntos
Inteligência Artificial , Transtornos Globais do Desenvolvimento Infantil/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Estudo de Associação Genômica Ampla/métodos , Anotação de Sequência Molecular/métodos , Atrofia Muscular Espinal/genética , Splicing de RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Simulação por Computador , DNA/genética , Éxons/genética , Código Genético , Marcadores Genéticos , Variação Genética , Humanos , Íntrons/genética , Modelos Genéticos , Proteína 1 Homóloga a MutL , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Sítios de Splice de RNA/genética , Proteínas de Ligação a RNA/genéticaRESUMO
How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species.
