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Journal of Neuromuscular Diseases 2021Duchenne muscular dystrophy (DMD) is a devastating, rare disease. While clinically described in the 19th century, the genetic foundation of DMD was not discovered until... (Review)
Review
Duchenne muscular dystrophy (DMD) is a devastating, rare disease. While clinically described in the 19th century, the genetic foundation of DMD was not discovered until more than 100 years later. This genetic understanding opened the door to the development of genetic treatments for DMD. Over the course of the last 30 years, the research that supports this development has moved into the realm of clinical trials and regulatory drug approvals. Exon skipping to therapeutically restore the frame of an out-of-frame dystrophin mutation has taken center stage in drug development for DMD. The research reviewed here focuses on the clinical development of exon skipping for the treatment of DMD. In addition to the generation of clinical treatments that are being used for patient care, this research sets the stage for future therapeutic development with a focus on increasing efficacy while providing safety and addressing the multi-systemic aspects of DMD.
Topics: Dystrophin; Exons; Genetic Therapy; Humans; Muscular Dystrophy, Duchenne; Mutation; Oligonucleotides, Antisense
PubMed: 34180420
DOI: 10.3233/JND-210682 -
Molecular Cell Aug 2022Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly...
Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.
Topics: Alternative Splicing; Animals; Exons; Mammals; Mice; Neurogenesis; Neurons; RNA Splicing; RNA-Binding Proteins
PubMed: 35914530
DOI: 10.1016/j.molcel.2022.06.036 -
Molecular Cell Jan 2023N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is...
N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we propose that m6A deposition is not selective. Instead, it is exclusion based: m6A consensus motifs are methylated by default, unless they are within a window of ∼100 nt from a splice junction. A simple model which we extensively validate, relying exclusively on presence of m6A motifs and exon-intron architecture, allows in silico recapitulation of experimentally measured m6A profiles. We provide evidence that exclusion from splice junctions is mediated by the exon junction complex (EJC), potentially via physical occlusion, and that previously observed associations between exon-intron architecture and mRNA decay are mechanistically mediated via m6A. Our findings establish a mechanism coupling nuclear mRNA splicing and packaging with the covalent installation of m6A, in turn controlling cytoplasmic decay.
Topics: RNA, Messenger; RNA Splicing; Transcriptome; RNA Stability; Exons
PubMed: 36599352
DOI: 10.1016/j.molcel.2022.12.026 -
Nature Biotechnology Jul 2022Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack...
Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample.
Topics: Alternative Splicing; Brain; Exons; Humans; Protein Isoforms; RNA; Sequence Analysis, RNA
PubMed: 35256815
DOI: 10.1038/s41587-022-01231-3 -
Nature Communications Dec 2022N6-methyladenosine (mA), the most abundant modification of mRNA, is essential for normal development and dysregulation promotes cancer. mA is highly enriched in the 3'...
N6-methyladenosine (mA), the most abundant modification of mRNA, is essential for normal development and dysregulation promotes cancer. mA is highly enriched in the 3' untranslated region (UTR) of a large subset of mRNAs to influence mRNA stability and/or translation. However, the mechanism responsible for the observed mA distribution remains enigmatic. Here we find the exon junction complex shapes the mA landscape by blocking METTL3-mediated mA modification close to exon junctions within coding sequence (CDS). Depletion of EIF4A3, a core component of the EJC, causes increased METTL3 binding and mA modification of short internal exons, and sites close to exon-exon junctions within mRNA. Reporter gene experiments further support the role of splicing and EIF4A3 deposition in controlling mA modification via the local steric blockade of METTL3. Our results explain how characteristic patterns of mA mRNA modification are established and uncover a role of the EJC in shaping the mA epitranscriptome.
Topics: RNA Splicing; Cell Nucleus; RNA, Messenger; Exons; RNA Stability
PubMed: 36550132
DOI: 10.1038/s41467-022-35643-1 -
Biomedica : Revista Del Instituto... Jun 2016The Li-Fraumeni syndrome is characterized clinically by the appearance of tumors in multiple organs generally at an early age. This hereditary condition is caused by...
The Li-Fraumeni syndrome is characterized clinically by the appearance of tumors in multiple organs generally at an early age. This hereditary condition is caused by germinal mutations in the TP53 gene, which codifies for the tumoural suppressor gene p53. We present the case of a patient aged 31 with clinical and molecular diagnosis of Li-Fraumeni syndrome who presented two synchronous tumors: a leiomyosarcoma on the forearm and a phyllodes breast tumour. She had a family history of cancer, including a son diagnosed with a cortical adrenal carcinoma when he was three years old, who died at five from the disease. Furthermore, her maternal grandmother and great-grandmother died of stomach cancer at 56 and 60 years old, respectively, while her other great-grandmother and a great aunt presented with breast cancer at the ages of 60 and 40, respectively. After genetic counseling, complete sequencing and analysis of duplications and deletions in the TP53 gene were ordered prior to diagnosis. The molecular analysis of a DNA sample taken from peripheral blood lymphocytes revealed the germinal mutation c.527G>T (p.Cys176Phe) on exon 5 of the TP53 gene, a deleterious mutation described previously in tumoural tissues. To our knowledge, this is the first published case in Colombia of Li-Fraumeni syndrome with confirmed molecular diagnosis. The diagnosis and management of Li-Fraumeni syndrome should be performed by a multidisciplinary team, and genetic counselling should be offered to patients and their relatives.
Topics: Breast Neoplasms; Exons; Genes, p53; Humans; Li-Fraumeni Syndrome; Mutation; Stomach Neoplasms; Tumor Suppressor Protein p53
PubMed: 27622479
DOI: 10.7705/biomedica.v36i3.2793 -
Nature Communications Jun 2023Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences....
Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo.
Topics: Mice; Animals; Gene Editing; DNA; DNA Breaks, Double-Stranded; Exons; Genome; CRISPR-Cas Systems
PubMed: 37291100
DOI: 10.1038/s41467-023-39137-6 -
Nucleic Acids Research Apr 2022Alternative splicing is frequently involved in the diversification of protein function and can also be modulated for therapeutic purposes. Here we develop a predictive...
Alternative splicing is frequently involved in the diversification of protein function and can also be modulated for therapeutic purposes. Here we develop a predictive model, called Exon ByPASS (predicting Exon skipping Based on Protein amino acid SequenceS), to assess the criticality of exon inclusion based solely on information contained in the amino acid sequence upstream and downstream of the exon junctions. By focusing on protein sequence, Exon ByPASS predicts exon skipping independent of tissue and species in the absence of any intronic information. We validate model predictions using transcriptomic and proteomic data and show that the model can capture exon skipping in different tissues and species. Additionally, we reveal potential therapeutic opportunities by predicting synthetically skippable exons and neo-junctions arising in cancer cells.
Topics: Alternative Splicing; Amino Acid Sequence; Exons; Introns; Proteomics
PubMed: 35286381
DOI: 10.1093/nar/gkac155 -
Human Molecular Genetics Apr 2011Muscular dystrophies are a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. Duchenne muscular dystrophy (DMD) is the most common... (Review)
Review
Muscular dystrophies are a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, and although the molecular mechanisms of the disease have been extensively investigated since the discovery of the gene in 1986, there is currently no effective treatment. However, new gene-based therapies have recently emerged with particular noted advances in using conventional gene replacement strategies, RNA-based technology and pharmacological approaches. While the proof of principle has been demonstrated in animal models, several clinical trials have recently been undertaken to investigate the feasibility of these strategies in patients. In particular, antisense-mediated exon skipping has shown encouraging results and holds promise for the treatment of dystrophic muscle. Here, we summarize the recent progress in therapeutic approaches to muscular dystrophies, with an emphasis on gene therapy and exon skipping for DMD.
Topics: Dystrophin; Exons; Genetic Therapy; Humans; Muscular Dystrophies
PubMed: 21436158
DOI: 10.1093/hmg/ddr105 -
PloS One 2013Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered...
Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.
Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.
Topics: Adolescent; Adult; Child; Child, Preschool; DNA Copy Number Variations; Exons; Female; Heterozygote; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Mutation; Pedigree; Retinal Dystrophies; Sequence Analysis, DNA; Young Adult
PubMed: 24265693
DOI: 10.1371/journal.pone.0078496