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BACKGROUND: Dilated cardiomyopathy (DCM) is a major complication of, and leading cause of mortality in Duchenne muscular dystrophy (DMD). Its severity, age at onset, and rate of progression display wide variability, whose molecular bases have been scarcely elucidated. Potential DCM-modifying factors include glucocorticoid (GC) and cardiological treatments, DMD mutation type and location, and variants in other genes. METHODS AND RESULTS: We retrospectively collected 3138 echocardiographic measurements of left ventricular ejection fraction (EF), shortening fraction (SF), and end-diastolic volume (EDV) from 819 DMD participants, 541 from an Italian multicentric cohort and 278 from the Cooperative International Neuromuscular Group Duchenne Natural History Study (CINRG-DNHS). Using generalized estimating equation (GEE) models, we estimated the yearly rate of decrease of EF (-0.80%) and SF (-0.41%), while EDV increase was not significantly associated with age. Utilizing a multivariate generalized estimating equation (GEE) model we observed that mutations preserving the expression of the C-terminal Dp71 isoform of dystrophin were correlated with decreased EDV (-11.01 mL/m2, p = 0.03) while for dp116 were correlated with decreased EF (-4.14%, p =

BACKGROUND: Becker muscular dystrophy is an X-linked, genetic disorder causing progressive degeneration of skeletal and cardiac muscle, with a widely variable phenotype. OBJECTIVE: A 3-year, longitudinal, prospective dataset contributed by patients with confirmed Becker muscular dystrophy was analyzed to characterize the natural history of this disorder. A better understanding of the natural history is crucial to rigorous therapeutic trials. METHODS: A cohort of 83 patients with Becker muscular dystrophy (5-75 years at baseline) were followed for up to 3 years with annual assessments. Muscle and pulmonary function outcomes were analyzed herein. Age-stratified statistical analysis and modeling were conducted to analyze cross-sectional data, time-to-event data, and longitudinal data to characterize these clinical outcomes. RESULTS: Deletion mutations of dystrophin exons 45-47 or 45-48 were most common. Subgroup analysis showed greater pairwise association between motor outcomes at baseline than association between these outcomes and age. Stronger correlations between outcomes for adults than for those under 18 years were also observed. Using cross-sectional binning analysis, a ceiling effect was seen for North Star Ambulatory Assessment but not for other functional outcomes. Longitudinal analysis showed a decline in percentage predicted forced vital capacity over the life span. There was relative stability or improved median function for motor functional outcomes through childhood and adolescence and decreasing function with age thereafter. CONCLUSIONS: There is variable progression of outcomes resulting in significant heterogeneity of the clinical phenotype of Becker muscular dystrophy. Disease progression is largely manifest in adulthood. There are implications for clinical trial design revealed by this longitudinal analysis of a Becker natural history dataset.

Recent genomic data points to a growing role for somatic mutations altering core histone and linker histone-encoding genes in cancer. However, the prevalence and the clinical and biological implications of histone gene mutations in malignant tumors remain incompletely defined. To address these knowledge gaps, we analyzed somatic mutations in 88 linker and core histone genes across 12,743 tumors from pediatric, adolescent and young adult (AYA), and adult cancer patients. We established a pan-cancer histone mutation atlas contextualized by patient age, survival outcome, and tumor location. Overall, 11% of tumors harbored somatic histone mutations, with the highest rates observed among chondrosarcoma (67%), pediatric high-grade glioma (pHGG, >60%), and lymphoma (>30%). Previously unreported histone mutations were discovered in pHGG and other pediatric brain tumors, extending the spectrum of histone gene alterations associated with these cancers. Histone mutation status predicted patient survival outcome in tumor entities including adrenocortical carcinoma. Recurrent pan-cancer histone mutation hotspots were defined and shown to converge on evolutionarily conserved and functional residues. Moreover, we studied histone gene mutations in 1700 pan-cancer cell lines to validate the prevalence and spectrum of histone mutations seen in primary tumors and derived histone-associated drug response profiles, revealing candidate drugs targeting histone mutant cancer cells. This study presents the first-of-its-kind atlas of both core and linker histone mutations across pediatric, AYA, and adult cancers, providing a framework by which specific cancers may be redefined in the context of histone and chromatin alterations.

OBJECTIVE: Strategic Targeting of Registries and International Database of Excellence (STRIDE) is an ongoing, international, multicenter registry of real-world ataluren use in individuals with nonsense mutation Duchenne muscular dystrophy (nmDMD) in clinical practice. This updated interim report (data cut-off: January 31, 2022), describes STRIDE patient characteristics and ataluren safety data, as well as the effectiveness of ataluren plus standard of care (SoC) in STRIDE versus SoC alone in the Cooperative International Neuromuscular Research Group (CINRG) Duchenne Natural History Study (DNHS). METHODS: Patients are followed up from enrollment for at least 5 years or until study withdrawal. Propensity score matching was performed to identify STRIDE and CINRG DNHS patients who were comparable in established predictors of disease progression. RESULTS: As of January 31, 2022, 307 patients were enrolled from 14 countries. Mean (standard deviation [SD]) ages at first symptoms and at genetic diagnosis were 2.9 (1.7) years and 4.5 (3.7) years, respectively. Mean (SD) duration of ataluren exposure was 1671 (56.8) days. Ataluren had a favorable safety profile; most treatment-emergent adverse events were mild or moderate and unrelated to ataluren. Kaplan-Meier analyses demonstrated that ataluren plus SoC significantly delayed age at loss of ambulation by 4 years (p

Genetic modifiers of Duchenne muscular dystrophy (DMD) are variants located in genes different from the disease-causing gene DMD, but associated with differences in disease onset, progression, or response to treatment. Modifiers described so far have been tested mainly for associations with ambulatory function, while their effect on upper limb function, which is especially relevant for quality of life and independence in non-ambulatory patients, is unknown. We tested genotypes at several known modifier loci (SPP1, LTBP4, CD40, ACTN3) for association with Performance Upper Limb version 1.2 score in an Italian multicenter cohort, and with Brooke scale score in the Cooperative International Neuromuscular Group Duchenne Natural History Study (CINRG-DNHS), using generalized estimating equation (GEE) models of longitudinally collected data, with age and glucocorticoid treatment as covariates. CD40 rs1883832, previously linked to earlier loss of ambulation, emerged as a modifier of upper limb function, negatively affecting shoulder and distal domains of PUL (p = 0.023 and 0.018, respectively) in the Italian cohort, as well as of Brooke score (p = 0.018) in the CINRG-DNHS. These findings will be useful for the design and interpretation of clinical trials in DMD, especially for non-ambulatory populations.

Introduction/aimsThere is debate about whether and to what extent either respiratory or cardiac dysfunction occurs in patients with dysferlinopathy. This study aimed to establish definitively whether dysfunction in either system is part of the dysferlinopathy phenotype.MethodsAs part of the Jain Foundation's International Clinical Outcome Study (COS) for dysferlinopathy, objective measures of respiratory and cardiac function were collected twice, with a 3-y interval between tests, in 188 genetically confirmed patients aged 11-86 y (53% female). Measures included forced vital capacity (FVC), electrocardiogram (ECG), and echocardiogram (echo).ResultsMean FVC was 90% predicted at baseline, decreasing to 88% at year 3. FVC was less than 80% predicted in 44 patients (24%) at baseline and 48 patients (30%) by year 3, including ambulant participants. ECGs showed P-wave abnormalities indicative of delayed trans-atrial conduction in 58% of patients at baseline, representing a risk for developing atrial flutter or fibrillation. The prevalence of impaired left ventricular function or hypertrophy was comparable to that in the general population.DiscussionThese results demonstrate clinically significant respiratory impairment and abnormal atrial conduction in some patients with dysferlinopathy. Therefore, we recommend that annual or biannual follow-up should include FVC measurement, enquiry about arrhythmia symptoms and peripheral pulse palpation to assess cardiac rhythm. However, periodic specialist cardiac review is probably not warranted unless prompted by symptoms or abnormal pulse findings.

Aim: We investigated the effect of ataluren plus standard of care (SoC) on age at loss of ambulation (LoA) and respiratory decline in patients with nonsense mutation Duchenne muscular dystrophy (nmDMD) versus patients with DMD on SoC alone. Patients & methods: Study 019 was a long-term Phase III study of ataluren safety in nmDMD patients with a history of ataluren exposure. Propensity score matching identified Study 019 and CINRG DNHS patients similar in disease progression predictors. Results & conclusion: Ataluren plus SoC was associated with a 2.2-year delay in age at LoA (p = 0.0006), and a 3.0-year delay in decline of predicted forced vital capacity to

BACKGROUND: Duchenne muscular dystrophy (DMD) is a rare x-linked recessive genetic disorder affecting 1 in every 5000-10000 [1, 2]. This disease leads to a variable but progressive sequential pattern of muscle weakness that eventually causes loss of important functional milestones such as the ability to walk. With promising drugs in development to ameliorate the effects of muscle weakness, these treatments must be associated with a clinically meaningful functional change. OBJECTIVE: The objective of this analysis is to determine both distribution, minimal detectable change (MDC), and anchor-based, minimal clinically important difference, (MCID) of 12 month change values in standardized time function tests (TFT) used to monitor disease progression in DMD. METHOD: This is a retrospective analysis of prospectively collected data from a multi-center prospective natural history study with the Cooperative International Neuromuscular Research Group (CINRG). This study calculated MDC and MCID values for 3 commonly used timed function tests typically used to monitor disease progression; supine to stand (STS), 10 meter walk/run (10MWT), and 4 stair climb (4SC). MDC used standard error of measurement (SEM) while MCID measurements used the Vignos scale as an anchor to determine clinical change in functional status. RESULTS: All 3 TFT were significantly important clinical endpoints to detect MDC and MCID changes. MDC and MCID 12 month changes were significant in 10MWT (-0.138, -0.212), Supine to Stand (-0.026, -0.023) and 4 stair climb (-0.034, -0.035) with an effect size greater or close to 0.2. CONCLUSION: The 3 TFT are clinically meaningful endpoints used to establish change in DMD. MCID values were higher than MDC values indicating that an anchor-based approach using Vignos as a clinically meaningful loss of lower extremity abilities is appropriate to assess change in boys with DMD.

The development of therapeutics for muscle diseases such as facioscapulohumeral dystrophy (FSHD) is impeded by a lack of objective, minimally invasive biomarkers. Here we identify circulating miRNAs and proteins that are dysregulated in early-onset FSHD patients to develop blood-based molecular biomarkers. Plasma samples from clinically characterized individuals with early-onset FSHD provide a discovery group and are compared to healthy control volunteers. Low-density quantitative polymerase chain reaction (PCR)-based arrays identify 19 candidate miRNAs, while mass spectrometry proteomic analysis identifies 13 candidate proteins. Bioinformatic analysis of chromatin immunoprecipitation (ChIP)-seq data shows that the FSHD-dysregulated DUX4 transcription factor binds to regulatory regions of several candidate miRNAs. This panel of miRNAs also shows ChIP signatures consistent with regulation by additional transcription factors which are up-regulated in FSHD (FOS, EGR1, MYC, and YY1). Validation studies in a separate group of patients with FSHD show consistent up-regulation of miR-100, miR-103, miR-146b, miR-29b, miR-34a, miR-454, miR-505, and miR-576. An increase in the expression of S100A8 protein, an inflammatory regulatory factor and subunit of calprotectin, is validated by Enzyme-Linked Immunosorbent Assay (ELISA). Bioinformatic analyses of proteomics and miRNA data further support a model of calprotectin and toll-like receptor 4 (TLR4) pathway dysregulation in FSHD. Moving forward, this panel of miRNAs, along with S100A8 and calprotectin, merit further investigation as monitoring and pharmacodynamic biomarkers for FSHD.

OBJECTIVE: To evaluate the suitability of real-world data (RWD) and natural history data (NHD) for use as external controls in drug evaluations for ambulatory Duchenne muscular dystrophy (DMD). METHODS: The consistency of changes in the 6-minute walk distance (Δ6MWD) was assessed across multiple clinical trial placebo arms and sources of NHD/RWD. Six placebo arms reporting 48-week Δ6MWD were identified via literature review and represented 4 sets of inclusion/exclusion criteria (n = 383 patients in total). Five sources of RWD/NHD were contributed by Universitaire Ziekenhuizen Leuven, DMD Italian Group, The Cooperative International Neuromuscular Research Group, ImagingDMD, and the PRO-DMD-01 study (n = 430 patients, in total). Mean Δ6MWD was compared between each placebo arm and RWD/NHD source after subjecting the latter to the inclusion/exclusion criteria of the trial for baseline age, ambulatory function, and steroid use. Baseline covariate adjustment was investigated in a subset of patients with available data. RESULTS: Analyses included ∼1,200 patient-years of follow-up. Differences in mean Δ6MWD between trial placebo arms and RWD/NHD cohorts ranged from -19.4 m (i.e., better outcomes in RWD/NHD) to 19.5 m (i.e., worse outcomes in RWD/NHD) and were not statistically significant before or after covariate adjustment. CONCLUSIONS: We found that Δ6MWD was consistent between placebo arms and RWD/NHD subjected to equivalent inclusion/exclusion criteria. No evidence for systematic bias was detected. These findings are encouraging for the use of RWD/NHD to augment, or possibly replace, placebo controls in DMD trials. Multi-institution collaboration through the Collaborative Trajectory Analysis Project rendered this study feasible.

We performed an observational, natural history study of males with in-frame dystrophin gene deletions causing Becker muscular dystrophy (BMD). A prospective natural history study collected longitudinal medical, strength, and timed function assessments. Eighty-three participants with genetically confirmed BMD were enrolled (age range 5.6-75.4 years). Lower extremity function and the percentage of participants who retained ambulation declined across the age span. The largest single group of participants had in-frame deletions that corresponded to an out-of-frame deletion treated with an exon 45 skip to restore the reading frame. This group of 54 participants showed similarities in baseline motor functional assessments when compared to the group of all others in the study. A prospective natural history cohort with in-frame dystrophin gene deletions offers the potential to contribute to clinical trial readiness for BMD and to analyze therapeutic benefit of exon skipping for Duchenne muscular dystrophy.

BACKGROUND: Treatment with corticosteroids is recommended for Duchenne muscular dystrophy (DMD) patients to slow the progression of weakness. However, chronic corticosteroid treatment causes significant morbidities. Vamorolone is a first-in-class anti-inflammatory investigational drug that has shown evidence of efficacy in DMD after 24 weeks of treatment at 2.0 or 6.0 mg/kg/day. Here, open-label efficacy and safety experience of vamorolone was evaluated over a period of 18 months in trial participants with DMD. METHODS AND FINDINGS: A multicenter, open-label, 24-week trial (VBP15-003) with a 24-month long-term extension (VBP15-LTE) was conducted by the Cooperative International Neuromuscular Research Group (CINRG) and evaluated drug-related effects of vamorolone on motor outcomes and corticosteroid-associated safety concerns. The study was carried out in Canada, US, UK, Australia, Sweden, and Israel, from 2016 to 2019. This report covers the initial 24-week trial and the first 12 months of the VBP15-LTE trial (total treatment period 18 months). DMD trial participants (males, 4 to

OBJECTIVE: Respiratory insufficiency is a major complication of Duchenne muscular dystrophy (DMD). Its progression shows considerable interindividual variability, which has been less thoroughly characterized and understood than in skeletal muscle. We collected pulmonary function testing (PFT) data from a large retrospective cohort followed at Centers collaborating in the Italian DMD Network. Furthermore, we analyzed PFT associations with different DMD mutation types, and with genetic variants in SPP1, LTBP4, CD40, and ACTN3, known to modify skeletal muscle weakness in DMD. Genetic association findings were independently validated in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). METHODS AND RESULTS: Generalized estimating equation analysis of 1852 PFTs from 327 Italian DMD patients, over an average follow-up time of 4.5 years, estimated that forced vital capacity (FVC) declined yearly by -4.2%, forced expiratory volume in 1 sec by -5.0%, and peak expiratory flow (PEF) by -2.9%. Glucocorticoid (GC) treatment was associated with higher values of all PFT measures (approximately + 15% across disease stages). Mutations situated 3 of DMD intron 44, thus predicted to alter the expression of short dystrophin isoforms, were associated with lower (approximately -6%) PFT values, a finding independently validated in the CINRG-DNHS. Deletions amenable to skipping of exon 51 and 53 were independently associated with worse PFT outcomes. A meta-analysis of the two cohorts identified detrimental effects of SPP1 rs28357094 and CD40 rs1883832 minor alleles on both FVC and PEF. INTERPRETATION: These findings support GC efficacy in delaying respiratory insufficiency, and will be useful for the design and interpretation of clinical trials focused on respiratory endpoints in DMD.

Aim: Strategic Targeting of Registries and International Database of Excellence (STRIDE) is an ongoing, multicenter registry providing real-world evidence regarding ataluren use in patients with nonsense mutation Duchenne muscular dystrophy (nmDMD). We examined the effectiveness of ataluren + standard of care (SoC) in the registry versus SoC alone in the Cooperative International Neuromuscular Research Group (CINRG) Duchenne Natural History Study (DNHS), DMD genotype-phenotype/-ataluren benefit correlations and ataluren safety. Patients & methods: Propensity score matching was performed to identify STRIDE and CINRG DNHS patients who were comparable in established disease progression predictors (registry cut-off date, 9 July 2018). Results & conclusion: Kaplan-Meier analyses demonstrated that ataluren + SoC significantly delayed age at loss of ambulation and age at worsening performance in timed function tests versus SoC alone (p ≤ 0.05). There were no DMD genotype-phenotype/ataluren benefit correlations. Ataluren was well tolerated. These results indicate that ataluren + SoC delays functional milestones of DMD progression in patients with nmDMD in routine clinical practice. ClinicalTrials.gov identifier: NCT02369731. ClinicalTrials.gov identifier: NCT02369731.

OBJECTIVE: To study vamorolone, a first-in-class steroidal anti-inflammatory drug, in Duchenne muscular dystrophy (DMD). METHODS: An open-label, multiple-ascending-dose study of vamorolone was conducted in 48 boys with DMD (age 4-

Extensive biomarker discoveries for DMD have occurred in the past 7 years, and a vast array of these biomarkers were confirmed in independent cohorts and across different laboratories. In these previous studies, glucocorticoids and age were two major confounding variables. In this new study, using SomaScan technology and focusing on a subset of young DMD patients who were not yet treated with glucocorticoids, we identified 108 elevated and 70 decreased proteins in DMD relative to age matched healthy controls (p value

BACKGROUND: Duchenne muscular dystrophy (DMD) patients experience skeletal muscle degeneration, including respiratory muscles. Respiratory decline in glucocorticoid-treated DMD patients, measured by percent predicted forced vital capacity (FVC% p), is typically 5% annually in patients aged 10 to 18 years. OBJECTIVE: Evaluate the effects of eteplirsen on FVC% p annual change in 3 trials versus matched Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG DNHS) controls. METHODS: Eteplirsen studies 201/202 evaluated eligible ambulatory DMD patients for at least 4 years, study 204 evaluated primarily non-ambulatory DMD patients for 2 years, and ongoing study 301 is evaluating ambulatory DMD patients for 2 years (interim analysis is included). Eteplirsen-treated patients (n = 74) were amenable to exon 51 skipping and were receiving glucocorticoids. Three CINRG DNHS cohorts included: glucocorticoid-treated patients amenable to exon 51 skipping (Exon 51 CINRG DNHS; n = 20), all glucocorticoid-treated CINRG patients (All CINRG DNHS; n = 172), and all glucocorticoid-treated genotyped CINRG DNHS patients (Genotyped CINRG DNHS; n = 148). FVC% p assessments between ages 10 and

PurposePediatric diffuse midline glioma (DMG) are highly malignant tumors with poor clinical outcomes. Over 70% of patients with DMG harbor the histone 3 p.K27M (H3K27M) mutation, which correlates with a poorer clinical outcome, and is also used as a criterion for enrollment in clinical trials. Because complete surgical resection of DMG is not an option, biopsy at presentation is feasible, but rebiopsy at time of progression is rare. While imaging and clinical-based disease monitoring is the standard of care, molecular-based longitudinal characterization of these tumors is almost nonexistent. To overcome these hurdles, we examined whether liquid biopsy allows measurement of disease response to precision therapy.Experimental designWe established a sensitive and specific methodology that detects major driver mutations associated with pediatric DMGs using droplet digital PCR (n = 48 subjects, n = 110 specimens). Quantification of circulating tumor DNA (ctDNA) for H3K27M was used for longitudinal assessment of disease response compared with centrally reviewed MRI data.ResultsH3K27M was identified in cerebrospinal fluid (CSF) and plasma in 88% of patients with DMG, with CSF being the most enriched for ctDNA. We demonstrated the feasibility of multiplexing for detection of H3K27M, and additional driver mutations in patient's tumor and matched CSF, maximizing the utility of a single source of liquid biome. A significant decrease in H3K27M plasma ctDNA agreed with MRI assessment of tumor response to radiotherapy in 83% (10/12) of patients.ConclusionsOur liquid biopsy approach provides a molecularly based tool for tumor characterization, and is the first to indicate clinical utility of ctDNA for longitudinal surveillance of DMGs.

Introduction: During aging, sarcopenia and decline in physiological processes lead to partial loss of muscle strength, atrophy, and increased fatigability. Muscle changes may be related to a reduced intake of essential amino acids playing a role in proteostasis. We have recently shown that branched-chain amino acid (BCAA) supplements improve atrophy and weakness in models of muscle disuse and aging. Considering the key roles that the alteration of Ca2+-related homeostasis and store-operated calcium entry (SOCE) play in several muscle dysfunctions, this study has been aimed at gaining insight into the potential ability of BCAAbased dietary formulations in aged mice on various players of Ca2+ dyshomeostasis. Methods: Seventeen-month-old male C57BL/6J mice received a 12-week supplementation with BCAAs alone or boosted with two equivalents of L-alanine (2-Ala) or with dipeptide L-alanyl-L-alanine (Di-Ala) in drinking water. Outcomes were evaluated on ex vivo skeletal muscles indices vs. adult 3-month-old male C57BL/6J mice. Results: Ca2+ imaging confirmed a decrease in SOCE and an increase of resting Ca2+ concentration in aged vs. adult mice without alteration in the canonical components of SOCE. Aged muscles vs. adult muscles were characterized by a decrease in the expression of ryanodine receptor 1 (RyR1), the SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) pump, and sarcalumenin together with an alteration of the expression of mitsugumin 29 and mitsugumin 53, two recently recognized players in the SOCE mechanism. BCAAs, particularly the formulation BCAAs+2-Ala, were able to ameliorate all these alterations. Discussion: These results provide evidence that Ca2+ homeostasis dysfunction plays a role in the functional deficit observed in aged muscle and supports the interest of dietary BCAA supplementation in counteracting sarcopenia-related SOCE dysregulation. [ABSTRACT FROM AUTHOR]

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor β [TGFβ]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFβ pathways. The minor allele at rs1883832, in the 5-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Corticosteroids are extensively used in pediatrics, yet the burden of side effects is significant. Availability of a simple, fast, and reliable biochemical read out of steroidal drug pharmacodynamics could enable a rapid and objective assessment of safety and efficacy of corticosteroids and aid development of corticosteroid replacement drugs. To identify potential corticosteroid responsive biomarkers we performed proteome profiling of serum samples from DMD and IBD patients with and without corticosteroid treatment using SOMAscan aptamer panel testing 1,129 proteins in

OBJECTIVE: To correlate time to loss of ambulation (LoA) and different truncating DMD gene mutations in a large, prospective natural history study of Duchenne muscular dystrophy (DMD), with particular attention to mutations amenable to emerging molecular treatments. METHODS: We analyzed data from the Cooperative International Neuromuscular Research Group Duchenne Natural History Study for participants with DMD single- or multi-exon deletions or duplications with defined exon boundaries (n = 186), or small mutations identified by sequencing (n = 26, including 16 nonsense point mutations). We performed a time-to-event analysis of LoA, a strong indicator of overall disease severity, adjusting for glucocorticoid treatment and genetic modifiers. RESULTS: Participants with deletions amenable to skipping of exon 44 had later LoA (median 14.8 years, hazard ratio 0.31, 95% confidence interval 0.14-0.69, p = 0.004). Age at LoA did not differ significantly in participants with deletions amenable to exon 45, 51, and 53 skipping, duplications, and small rearrangements. Nonsense mutation DMD also showed a typical median age at LoA (11.1 years), with a few outliers (ambulatory around or after 16 years of age) carrying stop codons within in-frame exons, more often situated in the rod domain. CONCLUSIONS: As exon 44 skipping-amenable DMD has a later LoA, mutation-specific randomization and selection of placebo groups are essential for the success of clinical trials.

ObjectiveWe aimed to perform an observational study of age at loss of independent ambulation (LoA) and side-effect profiles associated with different glucocorticoid corticosteroid (GC) regimens in Duchenne muscular dystrophy (DMD).MethodsWe studied 340 participants in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). LoA was defined as continuous wheelchair use. Effects of prednisone or prednisolone (PRED)/deflazacort (DFZ), administration frequency, and dose were analyzed by time-varying Cox regression. Side-effect frequencies were compared using χ(2) test.ResultsParticipants treated ≥1 year while ambulatory (n = 252/340) showed a 3-year median delay in LoA (p < 0.001). Fourteen different regimens were observed. Nondaily treatment was common for PRED (37%) and rare for DFZ (3%). DFZ was associated with later LoA than PRED (hazard ratio 0.294 ± 0.053 vs 0.490 ± 0.08, p = 0.003; 2-year difference in median LoA with daily administration, p < 0.001). Average dose was lower for daily PRED (0.56 mg/kg/d, 75% of recommended) than daily DFZ (0.75 mg/kg/d, 83% of recommended, p < 0.001). DFZ showed higher frequencies of growth delay (p < 0.001), cushingoid appearance (p = 0.002), and cataracts (p < 0.001), but not weight gain.ConclusionsUse of DFZ was associated with later LoA and increased frequency of side effects. Differences in standards of care and dosing complicate interpretation of this finding, but stratification by PRED/DFZ might be considered in clinical trials. This study emphasizes the necessity of a randomized, blinded trial of GC regimens in DMD.Classification of evidenceThis study provides Class IV evidence that GCs are effective in delaying LoA in patients with DMD.

BACKGROUND: Cardiac disease is a major cause of death in patients with muscular dystrophies. The use of feasible and reproducible echocardiographic measures of cardiac function is critical to advance the field of therapeutics for dystrophic cardiomyopathy. METHODS: Participants aged 8 to 18 years with genetically confirmed Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, or limb-girdle muscular dystrophy were enrolled at five centers, and standardized echocardiographic examinations were performed. Measures of systolic and diastolic function and speckle-tracking echocardiography-derived cardiac strain were reviewed independently by two central readers. Furthermore, echocardiographic measures from participants with DMD were compared with those from retrospective age-matched control subjects from a single site to assess measures of myocardial function. RESULTS: Forty-eight participants (mean age, 13.3 ± 2.7 years) were enrolled. Shortening fraction had a greater interobserver correlation (intraclass correlation coefficient [ICC] = 0.63) compared with ejection fraction (ICC = 0.49). One reader could measure ejection fraction in only 53% of participants. Myocardial performance index measured by pulse-wave Doppler and Doppler tissue imaging showed similar ICCs (0.55 and 0.54). Speckle-tracking echocardiography showed a high ICC (0.96). Focusing on participants with DMD (n = 33), significantly increased mitral A-wave velocities, lower E/A ratios, and lower Doppler tissue imaging mitral lateral E velocities were observed compared with age-matched control subjects. Speckle-tracking echocardiography demonstrated subclinical myocardial dysfunction with decreased average circumferential and longitudinal strain in three distinct subgroups: participants with DMD with normal shortening fractions, participants with DMD aged < 13 years, and participants with DMD with myocardial performance index scores < 0.40 compared with control subjects. CONCLUSIONS: In a muscular dystrophy cohort, assessment of cardiac function is feasible and reproducible using shortening fraction, diastolic measures, and myocardial performance index. Cardiac strain measures identified early myocardial disease in patients with DMD.

Serum biomarkers in Duchenne muscular dystrophy (DMD) may provide deeper insights into disease pathogenesis, suggest new therapeutic approaches, serve as acute read-outs of drug effects, and be useful as surrogate outcome measures to predict later clinical benefit. In this study a large-scale biomarker discovery was performed on serum samples from patients with DMD and age-matched healthy volunteers using a modified aptamer-based proteomics technology. Levels of 1,125 proteins were quantified in serum samples from two independent DMD cohorts: cohort 1 (The Parent Project Muscular Dystrophy-Cincinnati Childrens Hospital Medical Center), 42 patients with DMD and 28 age-matched normal volunteers; and cohort 2 (The Cooperative International Neuromuscular Research Group, Duchenne Natural History Study), 51 patients with DMD and 17 age-matched normal volunteers. Forty-four proteins showed significant differences that were consistent in both cohorts when comparing DMD patients and healthy volunteers at a 1% false-discovery rate, a large number of significant protein changes for such a small study. These biomarkers can be classified by known cellular processes and by age-dependent changes in protein concentration. Our findings demonstrate both the utility of this unbiased biomarker discovery approach and suggest potential new diagnostic and therapeutic avenues for ameliorating the burden of DMD and, we hope, other rare and devastating diseases.

ObjectiveWe studied the effects of LTBP4 and SPP1 polymorphisms on age at loss of ambulation (LoA) in a multiethnic Duchenne muscular dystrophy (DMD) cohort.MethodsWe genotyped SPP1 rs28357094 and LTBP4 haplotype in 283 of 340 participants in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). Median ages at LoA were compared by Kaplan-Meier analysis and log-rank test. We controlled polymorphism analyses for concurrent effects of glucocorticoid corticosteroid (GC) treatment (time-varying Cox regression) and for population stratification (multidimensional scaling of genome-wide markers).ResultsHispanic and South Asian participants (n = 18, 41) lost ambulation 2.7 and 2 years earlier than Caucasian subjects (p = 0.003,

It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials.

IntroductionCardiomyopathy is a common cause of morbidity and death in patients with Duchenne muscular dystrophy (DMD).MethodsThis investigation was a cross-sectional cross-sectional analysis of clinical data from the multi-institutional Cooperative International Neuromuscular Research Group (CINRG) DMD Natural History Study of 340 DMD patients aged 2-28 years. Cardiomyopathy was defined as shortening fraction (SF)  0.68). In patients with cardiomyopathy, 57% (27 of 47) reported not taking any cardiac medications. Cardiac medications were used in 12% (15 of 127) of patients without cardiomyopathy.ConclusionsWe found that echocardiograms were underutilized, and cardiomyopathy was undertreated in this DMD natural history cohort.