Genetic diagnosis involves identifying pathogenic variants in disease-causing genes, and the current success rate for rare neuromuscular diseases is approximately 40-60%. Rare variants discovered in disease genes that are difficult to interpret are classified as variants of unknown significance and developing methods to accurately classify these variants as either pathogenic or benign has the potential to increase the diagnosis rate. We have developed a high throughput functional assay to more accurately assess the pathogenicity of variants in two broad groups of neuromuscular diseases: collagenopathies and dystroglycanopathies.
The collagen VI related myopathies and the dystroglycanopathies are phenotypically diverse and range from severe forms apparent in infancy and milder forms which present through early to late adulthood. The collagenopathies are caused by mutations in genes encoding the three alpha chains of collagen type VI (COL6A1, COL6A2 and COL6A3) resulting in incomplete collagen VI biosynthesis and thus collagen VI remains internalized in the cell. We have optimized a flow cytometry assay to detect reduced levels at the cell surface and intracellular retention of collagen VI. For the dystroglycanopathies, mutations in genes involved in the glycosylation of a-dystroglycan results in hypoglycosylation and reduced binding to extracellular matrix. The hypoglycosylation of a-dystroglycan can be detected using flow cytometry with antibodies sensitive to glycosylation levels.
The flow cytometry assay was developed using patient cells harboring various pathogenic mutations to demonstrate its clinical relevance. We have also used CRISPR gene editing to create controls in relevant cell lines to better calibrate the assay. This involved creating a COL6A1 knock out for the collagenopathies and DAG1, LARGE, FKRP and ISPD knock outs for the dystroglycanopathies. The high throughput assay we have developed provides a framework for further functional genomics studies that will enable assessment of variants of unknown significance observed in neuromuscular disease patients.