FP.14 Dystrophin and satellite cell quantification in Duchenne and Becker muscular dystrophies

Abstract

The dystrophinopathies are a group of muscular dystrophies stemming from mutations in the <i>DMD</i> gene. We have developed a series of sensitive and reproducible dystrophin and satellite cell quantification methods in order to better define associations between dystrophin expression and disease severity, as well as the molecular mechanisms of mutations in the N-terminal region of dystrophin. In a pilot study of 18 dystrophinopathy patient muscle biopsies, we employed western blot, capillary western immunoassay, and automated quantitative immunofluorescence analysis to quantify dystrophin expression and Pax7-positive satellite cells. Among 9 patients with a Duchenne muscular dystrophy phenotype, the mean dystrophin protein level by capillary western was 1.8% of normal. These biopsies displayed a mean of 6.8% immunofluorescence staining intensity of individual fibers and 26% dystrophin positive fibers (PDPF). This high PDPF reflects the use of a sensitive cutoff to improve the discriminating power of the assay at low expression levels. By contrast, patients with intermediate or Becker muscular dystrophy had 11% dystrophin expression by capillary western and 70% PDPF. Several truncating mutations in the first 4 exons of DMD were associated with milder phenotypes and significantly higher dystrophin expression (16% by capillary western and 79% PDPF), in keeping with activation of an internal ribosome entry site in exon 5. There was poor overall correlation between Pax7-positive satellite cell count and dystrophin expression. Analysis of individual muscle fibers however demonstrated that fibers with one or more adjacent Pax7-positive satellite cells had a lower mean dystrophin expression, indicating concentration of satellite cells around dystrophin-deficient fibers. This study lays the groundwork for a comprehensive characterization of genotype, phenotype, and molecular features in a broader cohort of dystrophinopathy patients.