The LGMDs are genetic diseases characterized by progressive wasting of the pelvic and girdle muscles. There are over 17 autosomal recessive (AR) subtypes of LGMD. It is estimated that carrier frequency for AR LGMD is 1:150 to 1:211. Carriers of LGMD are haplosufficient, producing at least the minimum amount of functional protein to maintain a healthy state. We hypothesize that quantifying gene and functional protein in carriers can provide a benchmark for functional protein restoration in AR LGMDs. This will be vital information to inform dose selection for gene replacement therapeutic strategies including AAV-based approaches.
We will investigate three AR LGMD subtypes: LGMDR3 (SGCA), LGMDR1 (CAPN3), and LGMDR9 (FKRP). LGMDR3 is associated with insufficient production of functional structural protein, alpha-sarcoglycan. LGMDR1 and R9 are due to compromised enzymes calpain- 3 and fukutin-related protein, involved in glycosylation of alpha-dystroglycan (-DG) and muscle remodeling, respectively. We will assay three carriers and three affected individuals for each subtype, optimally including carriers harboring the same pathogenic variants as the affected individuals.
RNA and protein from skeletal muscle biopsies will be utilized to quantify gene expression and protein levels via Taqman qPCR and immunoblot, respectively. Relative levels in LGMD gene carriers will be compared to associated affected and a set of unaffected indivdiuals. To determine level of enzyme functionality LGMDR9 carriers, we will perform semi-quantitative western blotting to detect the ratio of glycosylated to un-glycosylated -dystroglycan (-DG) in participant muscle tissue. To assay enzyme functionality in LGMDR1 carriers, we will determine extent of calpain-3 autolysis by quantifying ratio of lysed to intact calpain-3 again with a semi-quantitative immunoblot.
Determining gene/protein expression and qualitative measures of protein functionality in carriers of LGMD pathogenic variants will provide a proxy measure for required protein restoration, further informing burgeoning LGMD gene therapies.