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Duchenne muscular dystrophy (DMD), an X-linked childhood-onset muscular dystrophy caused by loss of the protein dystrophin, can be associated with neurodevelopmental, emotional and behavioural problems. A DMD mouse model also displays a neuropsychiatric phenotype, including increased startle responses to threat which normalise when dystrophin is restored in the brain. We hypothesised that startle responses may also be increased in humans with DMD, which would have potential translational therapeutic implications. To investigate this, we first designed a novel discrimination fear-conditioning task and tested it in six healthy volunteers, followed by male DMD (n = 11) and Control (n = 9) participants aged 7–12 years. The aims of this methodological task development study were to: i) confirm the task efficacy; ii) optimise data processing procedures; iii) determine the most appropriate outcome measures. In the task, two neutral visual stimuli were presented: one ‘safe’ cue presented alone; one ‘threat’ cue paired with a threat stimulus (aversive noise) to enable conditioning of physiological startle responses (skin conductance response, SCR, and heart rate). Outcomes were the unconditioned physiological startle responses to the initial threat, and retention of conditioned responses in the absence of the threat stimulus. We present the protocol development and optimisation of data processing methods based on empirical data. We found that the task was effective in producing significantly higher physiological startle SCR in reinforced ‘threat’ trials compared to ‘safe’ trials ( P < .001). Different data extraction methods were compared and optimised, and the optimal sampling window was derived empirically. SCR amplitude was the most effective physiological outcome measure when compared to SCR area and change in heart rate, with the best profile on data processing, the least variance, successful conditioned response retention ( P = .01) and reliability assessment in test-retest analysis ( rho = .86). The definition of this novel outcome will allow us to study this response in a DMD population.

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.

Exon skipping is a novel, mutation-specific approach to treating patients with Duchenne muscular dystrophy (DMD). Phosphorodiamidate morpholino oligomers are nucleic acid analogues that selectively redirect pre-mRNA splicing to enable production of internally truncated dystrophin.In exon 51 skipping (eteplirsen; n=36) and exon 53 skipping (golodirsen; n=25) clinical studies, internally shortened dystrophin mRNA was observed in all treated patients (per reverse transcription polymerase chain reaction). Eteplirsen increased dystrophin expression 15.5-fold, 11.6-fold, and 2.4-fold vs untreated controls (percent dystrophin-positive fibres, Western blot, and immunohistochemistry intensity, respectively; all, p≤0.007) in a 180 week study, and 2.8-fold (Western blot; p=0.008) in a 48 week study. Golodirsen increased dystrophin expression 10.7-fold (Western blot) over baseline following 48 weeks of treatment. Over 4 years, versus comparable external controls, eteplirsen slowed ambulatory decline (6 min walk test difference, 165 m; p=0.001) and cumulative risk of losing ambulation (83% vs 17%). In 2 clinical studies that included non-ambulatory patients, eteplirsen slowed pulmonary decline versus natural history data (assessed by spirometry).Eteplirsen and golodirsen demonstrated clinical and biochemical effects in patients with DMD; ongoing studies of these compounds are further characterising their effects in various patient populations.

Dystrophin is expressed in brain as well as muscle, and its loss in Duchenne muscular dystrophy (DMD) causes CNS disturbance in addition to muscle pathology. A DMD mouse model (mdx) exhibits exagger- ated fear responses to threat, which normalise with postnatal dystrophin-restoration therapies.To investigate whether fear responses are abnormal in DMD patients, we tested 31 DMD and 25 male control participants aged 7–12 years using a fear conditioning task. Participants viewed trials of two neutral visual stimuli: one ‘safe’ stimulus and one paired with a ‘threat’ (aversive noise) to enable fear condi- tioning. Both stimuli were subsequently presented without the noise to assess extinction of conditioned autonomic fear responses (skin conductance).The initial (unconditioned) response to threat was significantly greater in DMD than controls (p=0.016). Both groups similarly acquired conditioned fear responses (p=0.55). These were retained in controls during early extinction, and later extinguished, however DMD participants showed no significant discrimination between threat/safe stimuli in extinction.This study is the first to investigate fear responses in DMD boys, showing exaggerated unconditioned responses to threat, similar to mdx mice, and abnormalities of fear extinction. Establishing this objective measure of a potentially reversible CNS phenotype will benefit clinical trials of CNS-targeted dystrophin- restoration therapies.drkatemaresh@gmail.com

Duchenne muscular dystrophy (DMD) is an X-linked, life-limiting muscle-wasting disorder caused by a loss of the protein dystrophin. Approximately half of DMD patients have cognitive and neurobehavioural symptoms, which can be related to mutation site. A mouse model of DMD (mdx) displays a central nervous system (CNS) phenotype, including increased fear responses. Abnormal neurodevelopment has been implicated in DMD pathogenesis, with white matter microstructural abnormalities and reduced grey matter volume on magnetic resonance imaging (MRI). Absence of dystrophin also causes functional abnormalities in the hippocampus, amygdala, and prefrontal cortex due to dysfunction in inhibitory GABA-ergic synapses; areas implicated in neurodevelopmental disorders, memory, emotional reactivity, and fear. Abnormal synaptic dysfunction and fear responses are reversed with dystrophin-restoration in mdx mice, thus raising the prospect of future trials for therapeutic interventions for which sensitive CNS outcome measures would be needed. In this doctoral thesis I have addressed two broad themes: 1) a deeper exploration of the DMD CNS phenotype and genotype-phenotype associations; 2) an evaluation of existing and novel CNS outcome measures. 1) I determined estimates of neurobehavioural co-morbidities in dystrophinopathies (DMD and Becker muscular dystrophy) in a systematic review and meta-analysis. I analysed neurobehavioural data from 140 DMD patients to further evaluate phenotype-genotype associations. I explored anxiety symptoms directly with DMD boys and their parents in a qualitative study and evaluated anxiety assessment instruments. 2) As fear, or startle, responses have not previously been studied in DMD, I developed a novel task to investigate physiological startle responses in a cohort of DMD and control boys, finding that they were indeed increased in DMD, and that the startle response could be a useful biomarker of dystrophin deficiency. Finally, I conducted an imaging study using advanced MRI techniques, and identified a diffusion imaging metric that could also be a useful CNS biomarker.

To perform cross-sectional and longitudinal miRNA profiling in plasma from Duchenne muscular dystrophy (DMD) subjects and find non-invasive biomarkers in DMD. 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. 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. 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.

Elevated CUB-domain containing protein 1 (CDCP1) is predictive of colorectal cancer (CRC) recurrence and poor patient survival. While CDCP1 expression identifies stem cell populations that mediate lung metastasis, mechanisms underlying the role of this cell surface receptor in CRC have not been defined. We sought to identify CDCP1 regulated processes in CRC using stem cell populations, enriched from primary cells and cell lines, in extensive in vitro and in vivo assays. These experiments, demonstrating that CDCP1 is functionally important in CRC tumor initiation, growth and metastasis, identified CDCP1 as a positive regulator of Wnt signaling. Detailed cell fractionation, immunoprecipitation, microscopy, and immunohistochemical analyses demonstrated that CDCP1 promotes translocation of the key regulators of Wnt signaling, β-catenin, and E-cadherin, to the nucleus. Of functional importance, disruption of CDCP1 reduces nuclear localized, chromatin-associated β-catenin and nuclear localized E-cadherin, increases sequestration of these proteins in cell membranes, disrupts regulation of CRC promoting genes, and reduces CRC tumor burden. Thus, disruption of CDCP1 perturbs pro-cancerous Wnt signaling including nuclear localization of β-catenin and E-cadherin.