Low dystrophin variability between muscles and stable expression over time in Becker muscular dystrophy using capillary Western immunoassay

Figures

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  Figure 1. Illustration to explain the various steps and nomenclature from the biopsy procedure using a conchotome (performed in either the TA and/or the VL muscle), via 3–4 samples taken per biopsy and frozen on site, to sections cut in the laboratory, and finally the preparation of lysates that were all analyzed in duplo.

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  Figure 2. Overview of muscle biopsies and cohorts. Each horizontal line represents a study participant and participants are ordered by cohorts. Blue symbols represent biopsies obtained from the right tibialis anterior (TA) muscle of participants from either the 2011 or 2014 study, green symbols represent biopsies obtained from the right TA muscle from pediatric BMD patients, and red symbols represent biopsies obtained from the right vastus lateralis (VL) muscle of participants from the 2014 study. Two similar symbols indicate two samples analyzed from the same biopsy. Cohort 1 (cross-sectional analysis): participants of whom a biopsy from at least one point in time was available. Cohort 2 (longitudinal analysis): participants of whom a right TA biopsy was available from the 2011 and 2014 study. Cohort 3 (intermuscular analysis): participants from the 2014 study of whom biopsies formthe right TA and VL muscle obtained on the same day were available.

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  Figure 3. Dystrophin levels quantified with Mandys106 and ab154168. (A) Scatter plot showing the correlation between dystrophin quantification in two technical replicates with Mandys106 (Pearson’s correlation = 0.897; p < 0.0001). (B) Scatter plot showing the correlation between dystrophin quantification in two technical replicates with ab154168 (Pearson’s correlation = 0.940; p < 0.001). (C) Scatter plot showing the correlation between dystrophin quantification with Mandys106 and ab154168 (Pearson’s correlation = 0.972; p < 0.0001).

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  Figure 4. Variation in dystrophin levels. Bar graphs are presented as mean and standard deviation. (A) Bar graphs showing the mean and standard deviation of dystrophin levels quantified in two adjacent muscle samples obtained during the same biopsy procedure from the right TA muscle of participants from the 2011 (light blue bars) and 2014 (dark blue bars) study. (B) Bar graphs showing the mean and standard deviation of dystrophin levels over time [2011 (light blue bars) versus 2014 (dark blue bars) study] in right TA muscle samples. For each study participant the time lag between the two biopsy procedures are presented along the x-axis. (C) Bar graphs showing the mean and standard deviation of dystrophin levels of the right TA (blue) and right VL muscle (red).

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  Figure 5. Association between baseline dystrophin levels and muscle strength and function. Scatter plots show individual assessments of change in knee strength and North Star Ambulatory Assessment (NSAA) over time. Longitudinal assessments within the same patients are represented by connecting the individual dots. The black dots represent patients with variable types of mutations and the red dots represent a subset of participants with a deletion of exon 45–47. Even in the deletion 45–47 group dystrophin levels and disease severity varied. (A) Change in knee strength over time grouped by dystrophin range. (B) Change in NSAA over time grouped by dystrophin range. NSAA results are limited as this test was only performed during the 2014 study.