Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies

Abstract

Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5-15 ng protein (per 5 µg total protein per lane), precision of 3.3 to 25.5%, and accuracy of - 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at - 70°C. Furthermore, our WB method was compared to the results from our recently published LC-MS method. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissues (created in Biorender.com). Step 1 involves protein extraction from skeletal muscle tissue lysates from control, DMD, or BMD biospecimen. Step 2 measures total protein concentrations. Step 3 involves running gel electrophoresis with wild-type dystrophin (wt-DYS) from muscle tissue extracts alongside mini-dystrophin STD curve and mini-DYS and protein normalization with housekeeping GAPDH.

Keywords: AAV9; Duchenne muscular dystrophy; Gene therapy; Quantitative; Western blot.

Fig. 1

Quantitation of dystrophin using mini-dystrophin STD curve. A representative scheme of a validation run. A Image acquisition and quantitation of total protein from tissue extracts using dot-blot followed by B construction of standard curve. C An image of a WB gel showing the wt-DYS, mini-DYS standard curve, quality controls (QCs) detected through the green fluorescent channel (800 nm), and protein normalization with GAPDH using the 700 nm channel (in red) is shown

Fig. 2

Quantitation of dystrophin in healthy and diseased human muscle tissues. A Gel electrophoresis images of wt-DYS from 20 normal, 18 BMD, and 20 DMD donors. B Dystrophin concentration quantified by Western blot in 3 cohorts with 20 subjects each in DMD and non-dystrophic control, and 18 subjects in the BMD group. The lower and upper hinges of the boxplot correspond to the first and third quartiles (the 25th and 75th percentiles); the upper whisker extends from the hinge to the largest value no further than 1.5*IQR (inter-quartile range) from the hinge, and the converse is the case for the lower whiskers. Data beyond the end of the whiskers are outliers and are plotted individually. Blue bars are the 95% confidence intervals for the mean in each group (blue diamonds), based on 10,000 bootstrap samples of the sample size in each cohort (40, 54) (Table V)

Fig. 3

Relationship between the dystrophin concentration by Western blot and LC–MS. Linear regression for samples (n = 32) with quantifiable dystrophin concentrations by Western blot (WB_DYS) and LC–MS %normal dystrophin (LC_DYS): %normal LC_DYS = 28.8 + 145.1 WB_DYS. Adjusted R² = 0.43, p-value for the regression coefficient of WB_DYS < 0.0001. The vertical gray line is the LLOQ of 0.1 ng/μg obtained by WB. Imputed values of WB_DYS (n = 21) below the LLOQ were overlaid with +. The shaded region is the 95% confidence band