Expanded precision splice correction: multiple pathogenic variants in a deep intronic hotspot in IGHMBP2 can be addressed with a single ASO

Topic:

Translational Research

Poster Number: 103 S

Author(s):

Sarah Silverstein, NIH, Rotem Orbach, MD, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Sandra Donkervoort, MS, Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Healt, Thomas Cassini, Vanderbilt University Medical Center, Mary Koziura, Vanderbilt University Medical Center, Véronique Bolduc, PhD, National Institute of Neurological Disorders and Stroke, National Institutes of Health, JJ Busgang, Neurogenetics Branch, NINDS, NIH, Jahan Misra, Neurogenetics Branch, NINDS, NIH, Jordan Bontrager, MS, CGC, University Rochester Medical Center, David Herrmann, MBBCh, University of Rochester. Medical Centre Neurology, Francesco Vetrini, Undiagnosed Rare Disease Clinic (URDC), Department of Medical and Molecular Genetics, Indiana Unive, Erin Conboy, Undiagnosed Rare Disease Clinic (URDC), Department of Medical and Molecular Genetics, Indiana Unive, Adam Comer, MD, Department of Neurology, Indiana University School of Medicine,, Kayla Treat, Undiagnosed Rare Disease Clinic (URDC), Department of Medical and Molecular Genetics, Indiana Unive, Katelyn Payne, Department of Neurology, Indiana University School of Medicine,, Khurram Liaqat, Undiagnosed Rare Disease Clinic (URDC), Department of Medical and Molecular Genetics, Indiana Unive, Aneesh Patankar, Neurogenetics Branch, NINDS, NIH, Alayne Meyer, MS, CGC, Nationwide Children's Hospital, Daniel Koboldt, MS, Nationwide Children's Hospital, Pimchanok Kulsirichawaroj, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Oranee Sanmaneechai, MD, Siriraj Hospital, Mahidol University, Kullasate Sakpichaisakul, Siriraj Center of Research Excellence in Neuromuscular Disease, Faculty of Medicine Siriraj Hospital, Kye-yoon Park, Stem Cell Unit, NINDS,NIH, Yan Li, Proteomics Core, NINDS, NIH, Diana Bharhucha-Goebel, MD, Nationwide Children's Hospital, The Ohio State University College of Medicine, William Macken, Department of Neuromuscular diseases, UCL Queen Square Institute of Neurology, Queen Square House, Anna Sarkozy, MD, UCL Great Ormond Street Institute of Child Health, James Polke, National Hospital for Neurology and Neurosurgery and North Thames Genomics Laboratory Hub Rare Disea, Adnan Y Manzur, FRCPCH, Department of Neuromuscular diseases, UCL Queen Square Institute of Neurology, Queen Square House, A. Reghan Foley, MD, MD(Res), Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health, Katherine R Chao, BS, Broad Insitute, Sarah Neuhaus, National Institute of Neurological Disorders and Stroke (NINDS)/NIH, David R Adams, MD, PhD, Office of the Clinical Director, NHGRI, NIH, Christopher Grunseich, MD, Neurogenetics Branch, NINDS, NIH, Carsten G. Bönnemann, MD, habil., Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health

Biallelic disease causing variants in IGHMBP2 cause spinal muscular atrophy with respiratory distress type I (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S). We present 12 unrelated patients with suspected IGHMBP2-related disease, each carrying a variant of uncertain significance deep in intron 8 of IGHMBP2 (c.1235+1076G>A (n=6), c.1235+450G>A (n=5), and c.1235+894C>A(n=1), along with a deleterious variant in trans.

To assess the pathogenicity of these deep intronic variants, patient-derived induced pluripotent stem cells (iPSCs) were differentiated into motor neurons (iMNs). Long-read RNA sequencing for c.1235+450G>A revealed a 626bp pseudoexon inclusion (allele-specific percent spliced in (asPSI) 67.9%). For c.1235+1076G>A, two pseudoexons were detected, 112bp (asPSI 26.9%) and 77bp (asPSI 27.5%). A 182bp pseudoexon was identified for c.1235+894C>A (asPSI 50%). Although each variant utilizes a unique splice acceptor site, all activate the same cryptic donor site, enabling a shared antisense oligonucleotide (ASO) therapeutic approach.

A single ASO was thus developed to correct aberrant splicing. Treatment of iMNs restored full-length IGHMBP2 protein in c.1235+894G>A and c.1235+1076G>A (p<0.001) compared to a non-targeted control, with no effect in c.1235+450G>A or control iMNs. RNAseq of ASO-treated iMNs showed decreased use of novel acceptor sites in c.1235+894C>A and c.1235+1076G>A samples. In c.1235+450G>A, targeting the shared donor led to intron retention without reducing novel acceptor use, reflecting a limitation of this approach.

Pre-treatment differential expression analysis revealed dysregulation of pathways related to local translation at the nerve terminus, with successful treatment resulting in pathway restoration. Immunofluorescence staining of beta actin, a locally translated neuronal protein, was reduced at growth cones in IGHMBP2 KO and c.1235+1076G>A iMNs versus controls (p<0.0001), and experiments testing ASO treatment effect are underway. Additional characterization with proteomics data is ongoing. This study highlights the importance of characterizing deep intronic variants in disease-relevant cells to assist the diagnostic process and inform therapeutics development.