Muscular dystrophy (MD) encompasses genetic disorders marked by progressive muscle weakness, impacting mobility and organ function. Despite identifying over 100 genes associated with MD, many cases remain undiagnosed. This study focuses on a 48-year-old patient with undiagnosed MD, carrying a homozygous variant of unknown significance in the PLECTIN gene (PLEC1). Using hiPSC-derived skeletal muscle cells (SkMCs) and innervated muscle fibers (iMFs), we aim to characterize phenotypic and functional abnormalities, and explore the pathogenicity of the PLEC1 mutation with CRISPR/Cas9-corrected lines.

HiPSCs from the MD patient (UND18), healthy controls (AG09, AG108, AG08) including an unaffected sibling (UND25) and control lines with known MDs (DM1, DMD, SMA), were differentiated into SkMCs or iMFs and characterized by immunofluorescence, Western blotting, and RT-qPCR. RNA-sequencing profiled transcriptomic changes in patient hiPSC-iMFs. Specifically, transcriptomic analysis identified dysregulated genes (DEGs) in the patient’s hiPSC-iMFS, including significant downregulation of the mitochondrial genes CHCHD2 and SLC15A4, and the metabolic gene NAPRT in patient cells, indicating potential mitochondrial and metabolic dysfunctions. Consistently, Seahorse Mito Stress Test revealed reduced mitochondrial respiration in patient hiPSC-derived SkMCs, confirming impaired mitochondrial function.

Using a custom MATLAB algorithm, functional analysis of hiPSC-iMFs revealed calium signaling defects in the patient’s cells. Western blot quantification showed reduced Plectin protein levels in patient-derived myotubes compared to controls, suggesting the PLEC1 variant's impact. CRISPR/Cas9 technology is now being employed to correct the PLEC1 mutation in the patient’s hiPSC to validate the pathogenicity of the identified variant.

Our study provides insights into the pathogenesis of an undiagnosed MD, revealing distinct contractile differences in patient-derived myotubes and identifying potential targets for further research.The discovery of the PLECTIN gene variant expands knowledge of MD-related genetic factors. Our MATLAB algorithm for calcium flux analysis offers a useful tool for studying contractile function. Additionally, downregulated mitochondrial genes suggest new molecular mechanisms to explore. This integrative approach enhances our understanding of MD and may guide future therapeutic strategies and diagnostic improvements.