Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy

Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies, as pathological control group, and four age-matched controls. Fluorescence data showed a significantly reduced expression of the mutated sarcoglycan in all patients when compared to their respective age-matched healthy controls, and a variable reduction of the other sarcoglycans. The reduction is due to the effect of gene mutation and not to the increasing age of controls. Fluorescence normalized data analyzed in relation to the age of onset of the disease, showed a negative correlation of a-sarcoglycan fluorescence signal vs fibrosis in patients with an early age of onset and a negative correlation between d-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful also in view of possible inferences on disease prognosis. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.