Antisense Morpholino-Based In Vitro Correction of a Pseudoexon-Generating Variant in the SGCB Gene

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.

Automated summary

Limb-girdle muscular dystrophies (LGMD) are characterized by clinically and genetically diverse presentations of predominantly proximal muscle weakness. Beta-sarcoglycanopathy (LGMDR4) is caused by biallelic defects in the SGCB gene and features pediatric onset with limb-girdle involvement, often accompanied by respiratory and heart dysfunction. In this study, a patient with high creatine kinase levels and cramps after strenuous exercise was diagnosed with beta-sarcoglycanopathy based on instrumental investigations. NGS panel sequencing revealed two variants in the SGCB gene, one of which (c.243+1548T>C) promoted the inclusion of a pseudoexon in the SGCB transcript. Interestingly, the same genotype was identified in a previously reported LGMDR4 patient. By delivering morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, the researchers observed correction of splicing and partial restoration of protein levels. These findings highlight the importance of analyzing the c.243+1548T>C variant in suspected LGMDR4 patients, especially those with monoallelic SGCB variants, and demonstrate the efficacy of antisense technology in correcting splicing defects resulting from molecular defects.