Using high-thoughput assays to improve the interpretation of rare variants in the context of the dystroglycanopathies



Poster Number: Virtual


Kaiyue Ma, PhD candidate, Yale University, Shushu Huang, MD, Yale University, Angela Lek, PhD, Yale University; Department of Genetics, Keryn Woodman, PhD, Yale University; Department of Genetics, Monkol Lek, PhD, Yale University

Dystroglycanopathies are caused by defective glycosylation of ?-dystroglycan (?-DG). Pathogenic variants in related enzymes are typically ultra-rare with many unique to affected families. This results in challenges in interpreting pathogenicity, where typically over 50% of variants are variants of uncertain significance (VUS). We aim at developing a high-throughput functional assay to characterize all possible variants of these enzymes with FKRP as our initial focus.

FKRP participates in ?-DG glycosylation by catalyzing ribitol 5-phosphate addition. FKRP associated muscular dystrophies range from late-onset limb girdle muscular dystrophy 2I (LGMD2I) to severe congenital muscular dystrophies (CMDs). We developed a platform cell line by knocking out FKRP and overexpressing ?-DG in the HAP1 cell line. Employing the IIH6C4 antibody that detects glycosylated ?-DG, we developed a flow-cytometry based assay to characterize the functional impact on ?-DG glycosylation. Using 8 FKRP variants with ClinVar interpretations, we demonstrated that our assay could distinguish between pathogenic and benign variants. We extended this assay to be high throughput by building a lentivirus pool carrying all possible FKRP single nucleotide variants (SNVs) to transduce our platform cell line followed by sorting cells into high and low ?-DG glycosylation groups, which were then examined by Next Generation Sequencing (NGS). We developed a computational pipeline to determine the function score of each variant based on its enrichments in both groups.

Our result showed the expected trend for nonsense variants to be the most damaging. Among the missense variants, known pathogenic and benign variants clustered closer to nonsense variants and synonymous variants, respectively. Thus, we anticipate our function scores can be used as another line of evidence to interpret VUS. Additionally, we developed an immunofluorescence assay to validate the variants individually in a myogenic cell line. Future work will involve validation by orthogonal functional assays independent of the IIH6C4 antibody.