Biology subjects

Nelson, S. F.

Publications and source records attributed to Nelson, S. F..

7 recordsLinked to original sources

bioRxiv2026

Structural engineering of dual-targeting antisense oligonucleotides enhances exon 44 skipping for treating Duchenne muscular dystrophy

Antisense oligonucleotides (ASOs) inducing exon-skipping during pre-mRNA splicing offer promising treatment for diseases such as Duchenne muscular dystrophy. Recently, dual-targeting ASOs that simultaneously bind two splicing regulatory sequences have been shown to improve exon-skipping efficiency. Current ASOs are designed to interact with target RNAs solely based on Watson-Crick base pairing. Here we report the development of dual-targeting three-dimensional bridge (3D-bridge) ASOs in which tertiary interactions are engineered to increase target-binding affinity and the number of target residues occupied. We designed a 3D-bridge ASO for skipping exon 44 of dystrophin mRNA and biochemically validated its enhanced binding affinity for the target RNA over a similar ASO with the tertiary interactions disrupted. We subsequently optimized the 3D-bridge ASO by varying its ends and ribose chemical modifications using patient-derived induced directly reprogrammed myotubes and identified an LNA/DNA mixmer as the most potent. We further demonstrate enhanced exon-skipping efficacy and potency of the 3D-bridge ASO compared to previously reported ASOs in myotubes differentiated from patient-derived primary myoblasts and MyoD-reprogrammed fibroblasts harboring mutations amenable to reframing through exon 44 skipping. In conclusion, the 3D-bridge ASO technology improves the current dual-targeting design, demonstrating the utility and power of structure-based ASO design methods.

pharmacology and toxicology↗

bioRxiv2023

Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A

ObjectivesTranscript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. MethodsWe applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. ResultsWe identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). DiscussionThis case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

genetics↗

bioRxiv2022

De Novo Variants in MRTFB have gain of function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

Myocardin-Related Transcription Factor B (MRTFB) is an important transcriptional regulator which promotes the activity of an estimated 300 genes during different stages of development. Here we report two pediatric probands with de novo variants in MRTFB (R104G and A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. As the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Expression of the human MRTFBR104G variant using a mrtf-T2A-GAL4 line proved to be embryonic lethal. Additional phenotypes were also identified by expressing the MRTFBR104G and MRTFBA91P variant in a subset of Drosophila tissues. Notably, expression within wing tissues resulted in an expansion of intervein tissue, wing vein thickening, shortening or loss of wing veins, and blistering. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the Actin cytoskeleton. These changes were not observed in flies expressing two additional candidate variants, MRTFBN95Sand MRTFBR109Q, highlighting that the location of the mutation within the 2nd RPEL domain is critical to the pathogenicity of the variant. These changes suggest that the MRTFBR104G and MRTFBA91P alleles we have identified affect the regulation of the protein and that these variants in MRTFB underly a novel neurodevelopmental disorder.

genetics↗

bioRxiv2022

Single nuclei transcriptomics of muscle reveals intra-muscular cell dynamics linked to dystrophin loss and rescue

In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples from small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing treatment effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of a novel MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1+ fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair.

genetics↗

bioRxiv2020

INTS13 Mutations Causing a Developmental Ciliopathy Disrupt Integrator Complex Assembly

Oral-facial-digital syndromes (OFD) are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. To date, mutations in 12 ciliary-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline mutations in INTS13, a subunit of the Integrator complex. This 14-component complex associates with RNAPII and can cleave nascent RNA to modulate gene expression. We determined that INTS13 utilizes a discrete domain within its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline INTS13 mutations. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos lead to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions within Integrator components.

biochemistry↗

bioRxiv2019

Identification of Pathogenic Structural Variants in Rare Disease Patients through Genome Sequencing

PurposeClinical whole genome sequencing is becoming more common for determining the molecular diagnosis of rare disease. However, standard clinical practice often focuses on small variants such as single nucleotide variants and small insertions/deletions. This leaves a wide range of larger \"structural variants\" that are not commonly analyzed in patients.\n\nMethodsWe developed a pipeline for processing structural variants for patients who received whole genome sequencing through the Undiagnosed Diseases Network (UDN). This pipeline called structural variants, stored them in an internal database, and filtered the variants based on internal frequencies and external annotations. The remaining variants were manually inspected and then interesting findings were reported as research variants to clinical sites in the UDN.\n\nResultsOf 477 analyzed UDN cases, 286 cases ({approx} 60%) received at least one structural variant as a research finding. The variants in 16 cases ({approx} 4%) are considered \"Certain\" or \"Highly likely\" molecularly diagnosed and another 4 cases are currently in review. Of those 20 cases, at least 13 were identified originally through our pipeline with one finding leading to identification of a new disease. As part of this paper, we have also released the collection of variant calls identified in our cohort along with heterozygous and homozygous call counts. This data is available at https://github.com/HudsonAlpha/UDN_SV_export.\n\nConclusionStructural variants are key genetic features that should be analyzed during routine clinical genomic analysis. For our UDN patients, structural variants helped solve {approx} 4% of the total number of cases ({approx} 13% of all genome sequencing solves), a success rate we expect to improve with better tools and greater understanding of the human genome.

genomics↗

bioRxiv2017

Calculating the statistical significance of rare variants causal for Mendelian and complex disorders

With the expanding use of next-gen sequencing (NGS) to diagnose the thousands of rare Mendelian genetic diseases, it is critical to be able to interpret individual DNA variation. We developed a general method to better interpret the likelihood that a rare variant is disease causing if observed in a given gene or genic region mapping to a described protein domain, using genome-wide information from a large control sample. We implemented these methods as a web tool and demonstrate application to 19 relevant but diverse next-gen sequencing studies. Additionally, we calculate the statistical significance of findings involving multi-family studies with rare Mendelian disease and studies of large-scale complex disorders such as autism spectrum disorder.

genetics↗