DOI
https://doi.org/10.25772/BT0J-Z594
Author ORCID Identifier
0009-0001-4838-087X
Defense Date
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Human and Molecular Genetics
First Advisor
Nicholas Johnson, MD
Abstract
Limb-girdle muscular dystrophy (LGMD) is a neuromuscular disorder clinically characterized by progressive proximal muscle wasting leading to significant loss of ambulation. There are 29 different subtypes (most inherited in an autosomal recessive pattern), each linked to a unique causative gene. Despite the immense symptom burden, there are currently no approved LGMD-specific gene therapies. Obtaining a genetic diagnosis is typically completed with next-generation sequencing (NGS) gene panels or whole exome sequencing (WES). However, genomic DNA testing often results in low diagnostic yield that is complicated by variants of unknown significance (VUS). To determine pathogenicity of these variants, we proposed performing RNA sequencing (RNA-seq) using patient muscle tissue to evaluate transcriptomic consequences of identified variants. To evaluate this assertion, we performed RNA-seq with muscle tissue derived from 12 genetically undiagnosed individuals with a limb-girdle phenotype. When paired with WES, RNA-seq enabled reclassification of one VUS to pathogenic and provided evidence to support reclassification based on aberrant splicing caused by another candidate disease VUS. Confirming pathogenicity of these VUS can confirm the disease gene, leading to appropriate patient diagnosis with a specific LGMD subtype. LGMD diagnosis is an essential qualifier that can permit a patient to be eligible for clinical trials evaluating impending gene replacement therapies, none of which have yet achieved FDA approval. These potentially curative therapies are incredibly powerful “one-and-done” treatments, meaning patients are infused/injected with the cloned vector only once. The therapies therefore require an optimal dose high enough for efficient treatment, but not enough to trigger adverse immune reactions. To elucidate the optimal dosing, we propose using asymptomatic carriers of autosomal recessive LGMD to define haplosufficiency in each disease state. We explored the transcriptomic and proteomic profiles for carriers of LGMD subtypes R1, R9, and R3 to determine relative protein expression and functionality for the respective causative gene. We determined carriers had about 1/3 CAPN3 and SGCA protein expression for LGMDR1 and R3, respectively, compared to healthy controls without any detectable pathogenic variations in any LGMD genes. LGMDR9 carriers had about 50% FKRP enzymatic functionality compared to healthy controls. These metrics could translate to the minimal effective dosing to restore a haplosufficient state in LGMD patients. Evaluation of gene therapy success also requires robust disease-specific gene markers that proxy protein restoration. To begin this exploration, I performed differential gene expression analysis for LGMDR1, R9 and R3 affected individuals. Each subtype was compared to a control group and normalized to a myotonic dystrophy background to provide disease specificity. I found several uniquely dysregulated genes, including sarcomeric and calcium-binding genes for LGMDR1, circadian clock genes for LGMDR9, and epidermal/connective tissue genes in LGMDR3. These results prompt further in vivo experimentation to evaluate gene candidacy as a disease marker.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
6-6-2025
Included in
Congenital, Hereditary, and Neonatal Diseases and Abnormalities Commons, Diagnosis Commons, Medical Genetics Commons, Musculoskeletal, Neural, and Ocular Physiology Commons, Neurology Commons, Therapeutics Commons, Translational Medical Research Commons