Abstract
Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophinΔR4-R23/ΔCT with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophinΔR4-R23/ΔCT led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophinΔR4-R23/ΔCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid α-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.
Highlights
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease caused by mutations in the 2.2 MB dystrophin gene [1,2,3]
We used a sub-optimal dose of rAAV6microdystrophins so that we could examine whether changing the hinge domain increased or decreased the functional capacity of microdystrophin
We found that sub-optimal doses of both rAAV vectors pseudotyped with serotype 6 capsid (rAAV6)-microdystrophinDR4-R23/DCT and rAAV6- microdystrophinDH2-R23+H3/DCT maintained the peak force producing capacity of mdx gastrocnemius and tibialis anterior muscles (Figure 4A)
Summary
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease caused by mutations in the 2.2 MB dystrophin gene [1,2,3]. Most frame-shift mutations in dystrophin lead to DMD whereas internal truncations (in-frame deletions) lead to a milder form of the disease called Becker muscular dystrophy (BMD) [7,8,9,10,11,12,13,14]. Dystrophin consists of a N-terminal actin-binding domain, a large central rod domain, a cysteine rich region and a C-terminal domain (Figure 1A) [15,16]. The locus encoding the N-terminal actin-binding domain and the region near hinge 3 of dystrophin are more susceptible to deletion mutations [7,8,9,10,11,12,13]. In-frame deletions of the central rod domain typically lead to a mild BMD [8,9,10,11,12,13]
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