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Background Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full‐length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not. Methods Functional outcome data from 387 DMD boys aged 4–15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild‐type and DMD models). Grip strength and rotarod running test were studied in wild‐type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD‐null (lacking all isoforms). Results In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four‐limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD‐null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild‐type, mdx52 or DMD‐null mice. Conclusions Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.

Introduction/Aims Mutations amenable to skipping of specific exons have been associated with different motor progression in Duchenne muscular dystrophy (DMD). Less is known about their association with long‐term respiratory function. In this study we investigated the features of respiratory progression in four DMD genotypes relevant in ongoing exon‐skipping therapeutic strategies. Methods This was a retrospective longitudinal study including DMD children followed by the UK NorthStar Network and international AFM Network centers (May 2003 to October 2020). We included boys amenable to skip exons 44, 45, 51, or 53, who were older than 5 years of age and ambulant at first recorded visit. Subjects who were corticosteroid‐naive or enrolled in interventional clinical trials were excluded. The progression of respiratory function (absolute forced vital capacity [FVC] and calculated as percent predicted [FVC%]) was compared across the four subgroups (skip44, skip45, skip51, skip53). Results We included 142 boys in the study. Mean (standard deviation) age at first visit was 8.6 (2.5) years. Median follow‐up was 3 (range, 0.3‐8.3) years. In skip45 and skip51, FVC% declined linearly from the first recorded visit. From the age of 9 years, FVC% declined linearly in all genotypes. Skip44 had the slowest (2.7%/year) and skip51 the fastest (5.9%/year) annual FVC% decline. The absolute FVC increased progressively in skip44, skip45, and skip51. In skip53, FVC started declining from 14 years of age. Discussion The progression of respiratory dysfunction follows different patterns for specific genotype categories. This information is valuable for prognosis and for the evaluation of exon‐skipping therapies.

Background Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full length Dp427 is the primary dystrophin isoform expressed in skeletal muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS co-morbidities is well known, relationships between lack of Dp140 and Dp71 and DMD motor outcomes are not. We have conducted a series of investigations addressing this. Methods Functional outcome data from 387 DMD boys aged 4.0-15.4 years was subdivided by DMD mutation expected effect on isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n=201); group 2 (Dp427/Dp140 absent, Dp71 present, n=152); and group 3 (Dp427/Dp140/Dp71 absent, n=34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10m walk/run and rise times were explored using regression analysis. We used Capillary Western immunoassay (Wes) analysis to study Dp427, Dp140 and Dp71 production in wild-type and DMD skeletal muscle and myogenic cultures. Grip strength was studied in wild-type, mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms) mice. Results In DMD boys, we found a strong association between isoform group and motor function. In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in group 3 than group 1 (p<0.01) and 4.9 points lower in group 3 than group 2 (p=0.05). Mean peak NSAA scores were 4.0 points lower in group 3 than group 1 (p<0.01), 2.4 points lower in group 3 than group 2 (p=0.09) and 1.6 points lower in group 2 than group 1 (p=0.04). Average grip strength in peak force at 3 months of age was higher in mdx than mdx52 mice (p=0.01). Dp427, but not Dp71, was produced in normal skeletal muscle; low levels of Dp71 were detected in DMD skeletal muscle. High Dp71 levels were present in wild-type and DMD myogenic cultures. Conclusions DMD boys lacking Dp140 and Dp140/Dp71 displayed worse motor function with a cumulative effect of isoform loss. DMD mouse models lacking Dp427 and Dp140 had lower grip strength than those lacking Dp427 but not Dp140. Our results highlight the importance of considering the effects of dystrophin isoform loss on DMD motor impairment, with important implications for understanding the complex relationship between brain and muscle function in DMD and patient stratification for clinical trials.

Aim: To perform cross-sectional and longitudinal miRNA profiling in plasma from Duchenne muscular dystrophy (DMD) subjects and find non-invasive biomarkers in DMD. Subjects/materials & methods: Plasma was collected from 14 age and sex matched controls and 46 DMD subjects. Free-circulating and extracellular vesicle (EV)-derived miRNA expression was measured by RT-qPCR. Results: Free-circulating and EVs derived miR-29c-3p and miR-133a-3p are dysregulated in DMD subjects. Free-circulating and EV-derived miR-29c-3p are reduced in DMD subjects undergoing daily corticosteroid treatment. Free-circulating miR-1-3p and miR-122-5p are longitudinally upregulated in ambulant DMD subjects. Conclusion: We detected novel free-circulating and EV-derived dysregulated miRNAs in plasma from DMD subjects and characterized the longitudinal profile of free-circulating miRNA on plasma from DMD subjects.

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Duchenne muscular dystrophy is characterised by loss of dystrophin in muscle, however patients also have variable degree of intellectual disability and neurobehavioural co-morbidities. In contrast to muscle, in which a single full-length dystrophin isoform (Dp427) is produced, multiple isoforms are produced in the brain, and their deficiency accounts for the variability of CNS manifestations, with increased risk of comorbidities in patients carrying mutations affecting the 3’ end of gene, which disrupt expression of shorter Dp140 and Dp71 isoforms. A mouse model (mdx mouse) lacks Dp427 in muscle and CNS and exhibits exaggerated startle responses to threat, linked to the deficiency of dystrophin in limbic structures such as the amygdala, which normalise with postnatal brain dystrophin-restoration therapies. A pathological startle response is not a recognised feature of DMD, and its characterisation has implications for improved clinical management and translational research. To investigate startle responses in Duchenne muscular dystrophy, we used a novel fear-conditioning task in an observational study of 56 males aged 7-12 years (31 affected boys, mean age 9.7 ± 1.8 years; 25 controls, mean age 9.6 ± 1.4 years). Trials of two neutral visual stimuli were presented to participants: one ‘safe’ cue presented alone; one ‘threat’ cue paired with an aversive noise to enable conditioning of physiological startle responses (skin conductance response and heart rate). Retention of conditioned physiological responses was subsequently tested by presenting both cues without the aversive noise in an ‘Extinction’ phase. Primary outcomes were the initial unconditioned skin conductance and change in heart rate responses to the aversive ‘threat’ and acquisition and retention of conditioned responses after conditioning. Secondary and exploratory outcomes were neuropsychological measures and genotype associations. The mean unconditioned skin conductance response was greater in the Duchenne group than Controls (mean difference 3.0µS (1.0, 5.1); P = .004), associated with a significant threat-induced bradycardia only in the patient group (mean difference -8.7bpm (-16.9, -0.51); P = .04). Duchenne participants found the task more aversive than Controls, with increased early termination rates during the Extinction phase (26% in Duchenne group vs. 0% Controls; P = .007). This study provides the first evidence that boys with Duchenne muscular dystrophy show similar increased unconditioned startle responses to threat to the mdx mouse, which in the mouse respond to brain dystrophin restoration. Our study provides new insights into the neurobiology underlying the complex neuropsychiatric co-morbidities in Duchenne muscular dystrophy and defines an objective measure of this CNS phenotype, which will be valuable for future CNS-targeted dystrophin-restoration studies.

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