Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping.
OBJECTIVE
In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping.


OBJECTIVE
To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping.


METHODS
Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype.


METHODS
Dystrophin mRNA and protein expression.


RESULTS
Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes).


CONCLUSIONS
Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Karen
K
Anthony
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Virginia
V
Arechavala-Gomeza
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Valeria
V
Ricotti
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Silvia
S
Torelli
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Lucy
L
Feng
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Narinder
N
Janghra
Don Carlo Gnocchi Onlus Foundation, Milan, Italy.
Giorgio
G
Tasca
Institute of Genetic Medicine, Newcastle University, Newcastle, England.
Michela
M
Guglieri
Institute of Genetic Medicine, Newcastle University, Newcastle, England.
Rita
R
Barresi
Section of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy.
Annarita
A
Armaroli
Section of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy.
Alessandra
A
Ferlini
Institute of Genetic Medicine, Newcastle University, Newcastle, England.
Katherine
K
Bushby
Institute of Genetic Medicine, Newcastle University, Newcastle, England.
Volker
V
Straub
Institute of Neurology, Catholic University, Rome, Italy.
Enzo
E
Ricci
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Caroline
C
Sewry
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Jennifer
J
Morgan
Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England.
Francesco
F
Muntoni
D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D006801:Humans; D008297:Male; D018482:Muscle, Skeletal / Q000737:chemistry / Q000473:pathology; D009136:Muscular Dystrophies / Q000145:classification / Q000235:genetics* / Q000473:pathology; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics*; D012189:Retrospective Studies; D017384:Sequence Deletion / Q000235:genetics*; D055815:Young Adult
D015415:Biomarkers; D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger