A Recurrent 3′ UTR Variant in CAPN3 Highlights the Diagnostic Impact of Non‐Coding Variants in Limb‐Girdle Muscular Dystrophies

This is the authors' abstract. We don't add key points for this paper.

Objective A substantial proportion of individuals with suspected genetic conditions, including neuromuscular disorders, lack a definitive molecular diagnosis. Non‐coding variants, particularly those in the 3′ untranslated region (3´UTR), which regulate mRNA stability, translation, and localization, may underlie part of this gap. We investigated the clinical, genetic, and functional relevance of a novel 3′UTR variant in CAPN3 in individuals with persistent hyperCKemia, most of whom also presented with proximal muscle weakness. Methods Clinical assessment, including muscle biopsy and muscle magnetic resonance imaging (MRI), was performed in individuals harboring the CAPN3 c.*534T>C variant. In silico analyses included the assessment of miRNA binding, mRNA secondary structure, creation or disruption of a poly‐A tail, and RNA‐binding protein (RBP) motifs. A luciferase reporter assay with the c.*534T>C and reference sequence, in the presence or absence of RBFOX1 co‐expression, was performed. Results We identified 13 individuals from 11 unrelated families presenting with persistent hyperCKemia, commonly accompanied by proximal muscle weakness and myalgia, all harboring the c.*534T>C variant in combination with a previously identified heterozygous CAPN3 variant. Western blot analyses consistently demonstrated absent or markedly reduced calpain‐3 expression, and muscle RNA sequencing (RNAseq) showed reduced CAPN3 levels. In silico analyses indicated that the c.*534T>C variant creates a novel binding site for the RNA‐binding protein RBFOX1. Luciferase reporter assays supported a regulatory effect of the identified variant on calpain‐3 expression. Interpretation Our results show that a recurrent non‐coding CAPN3 variant causes disease by disrupting post‐transcriptional regulation and demonstrate a generalizable principle, as 3′UTR variants can affect mRNA stability and protein expression across neuromuscular and other Mendelian diseases. Although often difficult to interpret as pathogenic, these variants can be therapeutically actionable, highlighting the importance of systematically investigating non‐coding regions in unsolved genetic cases.