Defining Haplosufficiency in Autosomal Recessive Limb-Girdle Muscular Dystrophy Using Molecular Markers in Disease...

Autosomal recessive forms of limb-girdle muscular dystrophy are caused by biallelic loss of function variants. Haplosufficient carriers are hypothesized to have sufficient protein expression to maintain normal physical function. This study aims to determine relative protein expression in haplosufficient carriers of 3 subtypes of autosomal recessive LGMD: LGMDR1 (CAPN3, calpain-3), LGMDR9 (FKRP, FKRP), and LGMDR3 (SGCA, α-sarcoglycan). In addition, this study clarifies fukutin-related protein (FKRP) enzymatic functionality in LGMDR9 carriers. Individuals affected with LGMDR1, R9, and R3 and corresponding carriers provided a muscle biopsy following a physical examination. RNA-seq analysis was performed to determine global gene-expression patterns. Gene expression of CAPN3, FKRP, and SGCA were determined using RNA-seq normalized counts, and relative quantification of disease-associated proteins was determined by western blotting. Carriers of LGMDR1 were found to have approximately 25% total calpain-3 expression compared with noncarrier controls, while LGMDR9 carriers had nearly 50% functionality of FKRP. LGMDR3 carriers exhibited α-sarcoglycan and β-sarcoglycan protein levels comparable with controls. Carriers of several autosomal-recessive LGMDs have reduced expression of disease-related proteins compared with noncarrier controls with associated reduced protein functionality. Since these haplosufficient carriers have normal physical strength, this degree of protein expression may represent the threshold to restore haplosufficient status after gene replacement therapy.