Background:
Limb girdle muscular dystrophy type 2I/R9 (LGMD 2I/R9) is a rare autosomal recessive muscle wasting disorder with substantial unmet medical needs. It is caused by pathogenic variants in FKRP, which impair the glycosylation of α-dystroglycan (α-DG), leading to reduced muscle stability and progressive weakness.
Objectives:
Ribitol has shown therapeutic potential by enhancing α-DG glycosylation through substrate supplementation in cells, animal models, and patients. However, this effect has only been quantitated on a few FKRP variants. In our study, a high-throughput cell-based assay was developed to assess additional FKRP variants and their specific response to ribitol.
Results:
This assay involved creating a homozygous FKRP knockout MCF7 cell line (c.181Adup), which can be rescued by individual FKRP variants. In addition, a lentiviral entry assay was developed using the Lassa virus glycoprotein complex (LASV-GPC) pseudotyped lentivirus carrying a GFP and puromycin-resistant marker. Viral entry in this assay depends on FKRP-mediated α-DG glycosylation. This dependency enables a quantitative readout (i.e. GFP/antibiotic resistance) to assess how FKRP variants influence cell-surface α-DG glycosylation and evaluate ribitol enhancement of this process.
Our assay showed good reproducibility and a 2-fold ribitol response for the European founder mutation c.826C>A. Screening five additional FKRP variants demonstrated that ribitol substantially enhances the glycosylation across multiple founder mutations. The clinically relevant founder alleles, including the Chinese founder mutation c.545A>G showed 2-fold improvement following ribitol treatment. Most notably, the Mexican founder mutation c.1387A>G, which lacks glycosylation at baseline entirely, exhibited 53-fold restoration upon ribitol treatment, suggesting that certain severe mutations may retain residual activity that is strongly activated by ribitol.
Conclusion:
In summary, our study demonstrates how high-throughput assays used to determine damaging variants in disease genes (e.g. FKRP) can be adapted and calibrated to determine variant specific drug response that can be scaled to all possible variants.