The discovery of dystrophin, the protein product of the Duchenne muscular dystrophy gene

Abstract

Duchenne muscular dystrophy was a well-established medical and genetic enigma by the 1970s. Why was the new mutation rate so high in all world populations? Why were affected boys doing well in early childhood, but then showed relentless progression of muscle wasting? What was wrong with the muscle? The identification of the first fragments of DMD gene cDNA in 1986, prediction of the entire 3685 amino acid protein sequence, and production of antibodies to dystrophin, both in 1987, provided key tools to understand DMD genetics and molecular pathology. The identification of dystrophin nucleated extensive research on myofiber membrane cytoskeleton, membrane repair, muscle regeneration, and failure of regeneration. This in turn led to molecular therapeutics based on understanding of dystrophin structure and function. This historical perspective describes the events surrounding the initial identification of the dystrophin protein.

Keywords: Duchenne muscular dystrophy; dystrophin; membrane cytoskeleton; skeletal muscle.

Fig. 1

Timelines. (A) Timeline of DMD gene and dystrophin protein discovery. Shown is a timeline of key milestones in the identification of the DMD gene (top) and dystrophin protein (bottom). Citations relevant to each milestone are provided in the text. Figure inserts. DMD genomic locus (upper right) shows a schematic of the genomic locus and chromosomal walks (top), cDNA/mRNA map (middle), and patient gene deletions (bottom). Taken from Koenig et al. (1987) (fig. 3) [7]. TrpE fusions to dystrophin for antibodies. Affinity‐purified, region‐specific dystrophin antibodies produced against dystrophin. A schematic of the 427 kDa dystrophin protein, with its four constituent domains, is shown. Taken from Hoffman et al. (1990) (fig. 1) [41]. Dystrophin immunostaining. Dystrophin immunofluorescence showing membrane localization in normal skeletal muscle and loss of dystrophin in DMD muscle. Taken from Bonilla et al. (1989) figs 1a, 2a). Bar = 50 µm [11]. (B) Timeline of dystrophin‐enabled pathophysiology and therapeutics. Shown is a timeline of increased knowledge of the pathophysiological consequences of dystrophin deficiency and the emergence of therapeutic approaches. Citations relevant to each milestone are provided in the text. Figure inserts. Dystrophin‐deficient cat. Spontaneously occurring dystrophin deficiency in domestic cats; cats show lethal muscle hypertrophy. Taken from Gaschen et al. (1992) (fig. 1) [33]. Early‐onset NF‐κB inflammation. DMD muscle shows strong activation of NF‐κB ‘cell danger signal’ pathways from 8 to 10 months of age, long before obvious clinical symptoms. Taken from Chen et al. (2005) (fig. 1) [30]. Systemic morpholino exon skipping in dog. Rescue of dystrophin in the CXMD dog model using morpholino oligonucleotides. Taken from Yokota et al. (2009) (fig. 3). Bar = 100 µm [25]. Vamorolone suppression of inflammation. DMD patient sera show dose–response suppression of inflammation‐associated biomarkers. Taken from Conklin et al. (2019) (fig. 3) [39].

Fig. 2

Dystrophin rescue by exon skipping in a viltolarsen clinical trial in DMD. (A) reverse transcription–polymerase chain reaction (RT‐PCR) of participant muscle biopsies taken before treatment and after treatment with viltolarsen. RT‐PCR products showing unskipped ‘out‐of‐frame’ mRNA transcript were seen pretreatment, while viltolarsen‐induced exon skipping led to a smaller skipped ‘in‐frame’ mRNA. (B) immunoblots for dystrophin [D], with protein loading controls for myosin heavy chain, M and alpha‐actinin, A. Standard curves for dystrophin are shown from mixed normal and DMD skeletal muscle samples. Clinical trial participant muscle biopsies, pretreatment and post‐viltolarsen treatment, were tested in a blinded manner. Pretreatment biopsies showed no dystrophin, whereas post‐treatment biopsies showed viltolarsen‐induced ‘Becker‐like’ dystrophin rescue. Taken from Clemens et al. (2020) (fig. 2) [27].