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Original Investigation |

Biochemical Characterization of Patients With In-Frame or Out-of-Frame DMD Deletions Pertinent to Exon 44 or 45 Skipping

Karen Anthony, PhD1; Virginia Arechavala-Gomeza, PhD1; Valeria Ricotti, MB BCh1; Silvia Torelli, PhD1; Lucy Feng, PhD1; Narinder Janghra, BSc1; Giorgio Tasca, MD2; Michela Guglieri, MD3; Rita Barresi, PhD3; Annarita Armaroli, MD4; Alessandra Ferlini, MD, PhD4; Katherine Bushby, MD3; Volker Straub, MD, PhD3; Enzo Ricci, MD5; Caroline Sewry, PhD1; Jennifer Morgan, PhD1; Francesco Muntoni, MD1
[+] Author Affiliations
1Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, England
2Don Carlo Gnocchi Onlus Foundation, Milan, Italy
3Institute of Genetic Medicine, Newcastle University, Newcastle, England
4Section of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy
5Institute of Neurology, Catholic University, Rome, Italy
JAMA Neurol. 2014;71(1):32-40. doi:10.1001/jamaneurol.2013.4908.
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Importance  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.

Design, Setting, and Participants  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.

Main Outcomes and Measures  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 and Relevance  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.

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Figure 1.
Comparative Analysis of Dystrophin Protein and Transcript Expression in Patients With In-Frame or Out-of-Frame DMD Deletions Around Exons 44 and 45

A, Transverse muscle sections were immunolabeled for β-spectrin, MANDYS106, MANEX50, and Dys2. Protein expression was quantified relative to control muscle in 40 muscle fibers and normalized to β-spectrin expression. Patients were grouped according to corresponding exon skipping models for Duchenne muscular dystrophy: control, exon 44 or 45 skippable out-of-frame (OOF) deletions (model 44 OOF and model 45 OOF), and in-frame (IF) deletions mimicking exon 45 skipping (model 45 IF). Data are presented as mean (SD) of the difference between sample means. Dotted lines indicate the mean dystrophin protein expression level from patients with Becker muscular dystrophy who have IF (blue lines) and OOF (gray lines) deletions modeling exon 51 skipping quantified in our previous study.21 B, Western blotting analysis of patients 7, 12, 14, 15, 16, and 20. Data are normalized to α-actinin and presented as the mean (SD) percentage of control. Dotted line indicates the mean dystrophin protein expression level from patients with Becker muscular dystrophy who have IF deletions modelling exon 51 skipping quantified in our previous study.21 C, The DMD messenger RNA transcript levels were quantified using 3 separate TaqMan assays (Applied Biosystems) targeting exon boundaries 19 and 20, 53 and 54, and 73 and 74. The mean polymerase chain reaction efficiency per amplicon (Table 2) and the mean threshold cycle (Ct) value per sample were used to calculate the starting concentration (N0) using the equation N0 = Nt/ECt where Nt is the fluorescence threshold and E is the efficiency. Data were normalized to myotilin, and dystrophin transcript expression is presented as mean (SD) relative to control.aP = .002.bP = .001.cP = .03.dP < .001.eP = .02.

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Figure 2.
Comparative Immunohistochemical Analysis of Dystrophin-Associated Protein Expression in Patients With In-Frame or Out-of-Frame DMD Deletions Around Exons 44 and 45

Transverse muscle sections were immunolabeled for β-spectrin, β-dystroglycan, neuronal nitric oxide synthase (nNOS), and utrophin. Expression was quantified relative to control muscle in 40 muscle fibers and normalized to β-spectrin expression. Patients were grouped according to corresponding exon skipping models for Duchenne muscular dystrophy: control, exon 44 or 45 skippable out-of-frame (OOF) deletions (model 44 OOF and model 45 OOF), and in-frame (IF) deletions mimicking exon 45 skipping (model 45 IF). Data are presented as mean (SD) of the difference between sample means. Dotted lines indicate the mean dystrophin-associated protein expression level from patients with Becker muscular dystrophy who have IF (blue lines) and OOF (gray lines) deletions modeling exon 51 skipping.21aP = .01.bP < .001.

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Figure 3.
Correlation of Dystrophin and Dystrophin-Associated Protein Expression With Clinical Severity in Patients With In-Frame DMD Deletions Around Exons 44 and 45

Patients with Becker muscular dystrophy were grossly grouped as having asymptomatic, mild, or severe Becker muscular dystrophy according to the severity of their skeletal muscle phenotype. Lines represent the mean expression in each group. BDG indicates β-dystroglycan; nNOS, neuronal nitric oxide synthase; and UTR, utrophin.

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