P.171 Detection of alpha-dystroglycan glycation in muscle biopsies using a multiplexed western blot method

Abstract

Limb-girdle muscular dystrophy (LGMD) type 2I is an autosomal recessive muscle disease caused by partial loss of function mutations of the fukutin-related protein (FKRP) leading to hypo glycosylation of alpha dystroglycan (αDG). Diminished glycosylation of αDG leads to contraction induced injury in myocytes leading to impairment of muscle performance. Identify and develop a method for evaluating the extent of glycosylation of αDG in striated muscle biopsies from patients with LGMD2I. A multiplex western blot (WB) method was employed to assess the extent of glycosylation in αDG by detecting both total αDG and a specific glyco-epitope. In LGMD2I, the biochemical impairment of FKRP leads to reduced glycosylation, which can be measured by diminished levels of the glyco-epitope. The ability to detect both forms of αDG generates a ratio of αDG-glycan to total αDG as an estimate of the extent of glycosylation. Several commercially available antibodies were evaluated using several healthy control muscles to assess their utility in a multiplexed WB to detect total αDG and glycosylated αDG. Two suitable antibodies were identified that were compatible with the LiCor platform. The specificity of these antibodies was assessed using parental HEK293T and DAG1 HEK293T knockout cell lysates. The signal linearity in both detection channels was evaluated using a dilution series of the control tibialis anterior (TA) muscle and was found to have good linearity (r2 > 0.85) and dilutional linearity. A commercially available recombinantly produced human αDG (rh-αDG) with little to no detectable glyco-epitope was identified and evaluated. Healthy TA control muscle is present on each blot and used to normalize the calculated ratios from test articles. This WB method will be used to assess the extent of glycosylated αDG in a subset of participants in our ongoing LGMD2I natural history study and results will be presented. A multiplexed method was developed for determining the extent of glycosylation of αDG. This approach has the potential to inform on the extent of αDG glycosylation in LGMD2I patients and to assess cellular response to a therapeutic intervention.