BMD Patients Research Articles

Trabecular bone mineral density as measured by thoracic vertebrae predicts incident hip and vertebral fractures: the multi-ethnic study of atherosclerosis.

Osteoporosis affects an enormous number of patients, of all races and both sexes, and its prevalence increases as the population ages. Few studies have evaluated the association between the vertebral trabecular bone mineral density(vBMD) and osteoporosis-related hip fracture in a multiethnic population, and no studies have demonstrated the predictive value of vBMD for fractures. We sought to determine the predictive value of QCT-based trabecular vBMD of thoracic vertebrae derived from coronary artery calcium scan for hip fractures in the Multi-Ethnic Study of Atherosclerosis(MESA), a nationwide multicenter cohort included 6814 people from six medical centers across the USA and assess if low bone density by QCT can predict future fractures. Measures were done using trabecular bone measures, adjusted for individual patients, from three consecutive thoracic vertebrae (BDI Inc, Manhattan Beach CA, USA) from non-contrast cardiac CT scans. Six thousand eight hundred fourteen MESA baseline participants were included with a mean age of 62.2 ± 10.2years, and 52.8% were women. The mean thoracic BMD is 162.6 ± 46.8mg/cm3 (95% CI 161.5, 163.7), and 27.6% of participants (n = 1883) had osteoporosis (T-score 2.5 or lower). Over a median follow-up of 17.4years, Caucasians have a higher rate of vertebral fractures (6.9%), followed by Blacks (4.4%), Hispanics (3.7%), and Chinese (3.0%). Hip fracture patients had a lower baseline vBMD as measured by QCT than the non-hip fracture group by 13.6mg/cm3 [P < 0.001]. The same pattern was seen in the vertebral fracture population, where the mean BMD was substantially lower 18.3mg/cm3 [P < 0.001] than in the non-vertebral fracture population. Notably, the above substantial relationship was unaffected by age, gender, race, BMI, hypertension, current smoking, medication use, or activity. Patients with low trabecular BMD of thoracic vertebrae showed a 1.57-fold greater risk of first hip fracture (HR 1.57, 95% CI 1.38-1.95) and a nearly threefold increased risk of first vertebral fracture (HR 2.93, 95% CI 1.87-4.59) compared to normal BMD patients. There is significant correlation between thoracic trabecular BMD and the incidence of future hip and vertebral fracture. This study demonstrates that thoracic vertebrae BMD, as measured on cardiac CT (QCT), can predict both hip and vertebral fractures without additional radiation, scanning, or patient burden. Osteopenia and osteoporosis are markedly underdiagnosed. Finding occult disease affords the opportunity to treat the millions of people undergoing CT scans every year for other indications.

Imaging mass cytometry analysis of Becker muscular dystrophy muscle samples reveals different stages of muscle degeneration

Becker muscular dystrophy (BMD) is characterised by fiber loss and expansion of fibrotic and adipose tissue. Several cells interact locally in what is known as the degenerative niche. We analysed muscle biopsies of controls and BMD patients at early, moderate and advanced stages of progression using Hyperion imaging mass cytometry (IMC) by labelling single sections with 17 markers identifying different components of the muscle. We developed a software for analysing IMC images and studied changes in the muscle composition and spatial correlations between markers across disease progression. We found a strong correlation between collagen-I and the area of stroma, collagen-VI, adipose tissue, and M2-macrophages number. There was a negative correlation between the area of collagen-I and the number of satellite cells (SCs), fibres and blood vessels. The comparison between fibrotic and non-fibrotic areas allowed to study the disease process in detail. We found structural differences among non-fibrotic areas from control and patients, being these latter characterized by increase in CTGF and in M2-macrophages and decrease in fibers and blood vessels. IMC enables to study of changes in tissue structure along disease progression, spatio-temporal correlations and opening the door to better understand new potential pathogenic pathways in human samples.

Innovative Computerized Dystrophin Quantification Method Based on Spectral Confocal Microscopy

Several clinical trials are working on drug development for Duchenne and Becker muscular dystrophy (DMD and BMD) treatment, and, since the expected increase in dystrophin is relatively subtle, high-sensitivity quantification methods are necessary. There is also a need to quantify dystrophin to reach a definitive diagnosis in individuals with mild BMD, and in female carriers. We developed a method for the quantification of dystrophin in DMD and BMD patients using spectral confocal microscopy. It offers the possibility to capture the whole emission spectrum for any antibody, ensuring the selection of the emission peak and allowing the detection of fluorescent emissions of very low intensities. Fluorescence was evaluated first on manually selected regions of interest (ROIs), proving the usefulness of the methodology. Later, ROI selection was automated to make it operator-independent. The proposed methodology correctly classified patients according to their diagnosis, detected even minimal traces of dystrophin, and the results obtained automatically were statistically comparable to the manual ones. Thus, spectral imaging could be implemented to measure dystrophin expression and it could pave the way for detailed analysis of how its expression relates to the clinical course. Studies could be further expanded to better understand the expression of dystrophin-associated protein complexes (DAPCs).

Low Bone Density and High Sclerostin in Non-Functioning Pituitary Adenoma

Purpose: Sclerostin reduces bone formation by inhibiting the Wnt signaling pathway in bone tissue. This study evaluated the serum sclerostin level in non-functioning pituitary adenoma (NFPA) patients and analyzed its relationship with bone metabolism. Method: The data of the patients who applied to the Dicle University Endocrinology, diagnosed with non-functioning pituitary adenoma, and the control group consisting of healthy individuals were included in the study. Serum sclerostin levels and DXA analysis parameters were evaluated and compared with healthy control groups. Results: The study consisted of 39 patients (F / M: 27/12) with NFPA (patient group) and 43 control groups (F / M: 26/17). There was no difference in terms of gender, age, height, weight and serum calcium, phosphorus, creatinine, 25-OH vitamin D, parathyroid hormone levels. Serum sclerostin levels (32.31 ± 1.53 ng / ml) in the patient group was found to be significantly higher than the control group (22.45 ± 8.9 ng / ml) (p < 0.001). BMD (Patients groups vs control group); total lumbar BMD (0.951-1.56 gr / cm2) (p < 0.001), femoral neck BMD (0.752-1.15 g / cm2) (p < 0.001), femoral total BMD (0.995- 1.63 gr / cm2) (p < 0.001), were found to be statistically significantly lower. Conclusion: This study provides the first evidence that serum sclerostin levels were increased in non-functioning pituitary adenomas, which showed that bone parameters were negatively affected.

Spectrum of Genetic Variants in the Dystrophin Gene: A Single Centre Retrospective Analysis of 750 Duchenne and Becker Patients from Southern Italy.

Dystrophinopathies are X-linked recessive muscle disorders caused by mutations in the dystrophin (DMD) gene that include deletions, duplications, and point mutations. Correct diagnosis is important for providing adequate patient care and family planning, especially at this time when mutation-specific therapies are available. We report a large single-centre study on the spectrum of DMD gene variants observed in 750 patients analyzed for suspected Duchenne (DMD) or Becker (BMD) muscular dystrophy, over the past 30 years, at the Cardiomyology and Medical Genetics of the University of Campania. We found 534 (71.21%) large deletions, 73 (9.73%) large duplications, and 112 (14.93%) point mutations, of which 44 (5.9%) were small ins/del causing frame-shifts, 57 (7.6%) nonsense mutations, 8 (1.1%) splice site and 3 (0.4%) intronic mutations, and 31 (4.13%) non mutations. Moreover, we report the prevalence of the different types of mutations in patients with DMD and BMD according to their decade of birth, from 1930 to 2020, and correlate the data to the different techniques used over the years. In the most recent decades, we observed an apparent increase in the prevalence of point mutations, probably due to the use of Next-Generation Sequencing (NGS). In conclusion, in southern Italy, deletions are the most frequent variation observed in DMD and BMD patients followed by point mutations and duplications, as elsewhere in the world. NGS was useful to identify point mutations in cases of strong suspicion of DMD/BMD negative on deletions/duplications analyses. In the era of personalized medicine and availability of new causative therapies, a collective effort is necessary to enable DMD and BMD patients to have timely genetic diagnoses and avoid late implementation of standard of care and late initiation of appropriate treatment.

1099 ERGOREFLEX SENSITIVITY IS ASSOCIATED WITH CARDIAC INVOLVEMENT IN BECKER MUSCULAR DYSTROPHY

Abstract Background and aims Becker muscular dystrophy (BMD) is a X-linked recessive inherited skeletal myopathy due to dystrophin deficiency ensuing in a variable phenotype characterized by slowly progressing muscle weakness and, often, the development of dilated cardiomyopathy. The relationship between muscle and cardiac involvement in BMD is yet to be clarified. We aimed to assess the association between ergoreflex sensitivity, a marker of skeletal muscle work regulating ventilatory and autonomic responses to exercise, and cardio-muscular involvement in BMD. Methods Fifteen male BMD patients (median age 37 years [25-42]) underwent a comprehensive evaluation including ECG, biohumoral assessment (high sensitivity troponin T/I [hs-TnT/I], natriuretic peptides, soluble suppression of tumorigenesis 2 [sST2], catecholamines, creatine phosphokinase [CPK], myoglobin), cardiac magnetic resonance (CMR), and ergoreflex determination. Muscular phenotype was distinguished into “classic” (proximal weakness affecting the shoulders and pelvis with waddling gait) or “mild”. Cardiac involvement was defined based on the presence of late gadolinium enhancement (LGE) at CMR. Ergoreflex sensitivity was calculated as the percentage of the ventilatory response to a handgrip exercise maintained by circulatory occlusion through the inflation of a forearm tourniquet (clamp) during the third minute of recovery compared with the third minute of recovery without clamp: [(Rec∕Ex) clamp − (Rec∕Ex) no clamp]∗100, where Ex is the mean ventilation during the last 30 s of exercise, Rec the mean ventilation during the last 30 s of the third minute of recovery. Results Ten patients (67%) had a “classic” muscular phenotype. Nine patients (60%) had LGE at CMR (median number of segments 9 [6-12]). The percentage of patients with cardiac involvement was not different between those with “classic” or mild muscular phenotype. Patients with cardiac involvement had higher ergoreflex sensitivity (24% [8-38] vs. 4% [0-24], p=0.036), hs-TnI (9.8 ng/L [5.9-14.5] vs. 2.1 ng/L [1.7-4.5], p=0.018), and hs-TnT (24 ng/L [20-35] vs. 11 ng/L [5-20], p=0.012), but did not show significant differences in terms of cardiac chamber size or systolic function compared to those without LGE. Ergoreflex sensitivity correlated with biomarkers of both cardiac (hs-TnI, rho=0.654, p=0.008; hs-TnT, rho=0.529, p=0.043; sST2, rho=0.654, p=0.009) and muscular damage (CPK, rho=0.645, p=0.009), as well as with the number of LGE segments (rho=0.583, p=0.022). No difference in ergoreflex sensitivity was detected in patients with “classic” versus mild muscular phenotype. Conclusions In BMD, there is no relationship between cardiac involvement and the muscular phenotype. Nonetheless, cardiac involvement in BMD is characterized by enhanced ergoreflex sensitivity, which is associated with biomarkers of both cardiac and muscular damage.

Assessment of left atrial function and diastolic dysfunction in patients with Becker muscular dystrophy

Abstract Background Becker muscular dystrophy (BMD) is frequently characterized by myocardial involvement1 but little is known about the prevalence of left ventricular (LV) diastolic dysfunction (DD) in these patients, particularly when using more recently proposed measure of left atrial (LA) function. Purpose Purpose of our study was to assess LVDD in patients with BMD using the currently recommended echocardiographic multiparametric approach and adding LA reservoir strain as advanced measure of LVDD. Methods A total of 33 BMD patients (38±13 years) were analyzed including standard and advanced echocardiography at the time of their first visit and at 24 months follow-up. A control group consisted of 20 age- and gender-matched healthy subjects. Results 18% of BMD patients showed an e'lateral &amp;lt;10, 0% an E/e' &amp;gt;14, 22% a LAVI &amp;gt;34ml/m2 and 11% a tricuspid velocity &amp;gt;2.8m/s. When applying the currently recommended multiparametric approach, 83% of BMD patients showed normal DD and 17% showed indeterminate LV diastolic dysfunction (50% positive parameters); no BMD patient had &amp;gt;50% positive DD parameters for confirmed DD. LA reservoir strain was significantly lower in BMD patients as compared to controls (28±10% vs. 42±11%; p&amp;lt;0.001) (Figure), while LA indexed volume (LAVI) was not (26±19 ml/m2 vs. 21±6 ml/m2; p=0.142). When using the reported median value of LA reservoir strain (47%) for normal subjects with comparable age2, 31 (94%) BMD patients had impaired LA strain, but when using the cut-off value of &amp;lt;19% for increased LV filling pressure, 6 (18%) patients showed affected LA reservoir strain. Patients with more impaired six minute walk test (6MWT), defined as in the 1st tertile (1st tertile 0–309m; 2nd tertile 310–523m; 3rd tertile &amp;gt;523m) had significantly lower LA reservoir strain (1st tertile, 22±6% vs. 2nd tertile, 29±14% vs. 3rd tertile, 33±5%; p=0.022). LA reservoir strain tended to deteriorate at 1 year follow-up but not significantly (from 29±10% to 26±12%; p=0.200). Conclusions LVDD is not highly prevalent in BMD patients but LA dysfunction as assessed by LA reservoir strain is reduced and may improve detection of myocardial involvement in these patients, also over time. Funding Acknowledgement Type of funding sources: None.

FP.04 Three-year natural history study in Becker muscular dystrophy in The Netherlands

The slow and highly variable disease progression observed in BMD stresses the need to develop outcome measures for clinical trials. We evaluated the responsiveness of different assessments in a cohort of adult BMD patients followed for 3 years. We included 36 genetically confirmed BMD patients (median age at baseline 42.3 years, range 18.6-67.3) of whom 28 were ambulant at baseline (78%). We conducted ambulatory assessments (6WMT, NSAA, 10-meter walk velocity (TMWv), time to rise from floor velocity (TRFv)), PUL1.2 and bilateral maximal voluntary isometric contraction (grip, knee/hip/elbow flexion and extension). The median change and the standard response mean (SRM) at 1- and 3-year follow-up were calculated. Sample sizes (SS) were calculated assuming 50% reduction in disease progression over a 3 year clinical trial with 1:1 randomization. We observed a large variability across all measures, for example the median at baseline for the 6MWT was 439.10m (range 84.30 to 650.00), median change at 1 year follow-up -8.15 (-151.90 to 182.22) and at 3-years -14.50(-151.90 to 158.72). After 1-year follow-up, the SRM was below 0.8 for all considered outcomes. At 3-year follow-up, TMWv and TRFv showed a responsiveness with SRM above 0.8 (1.91, SS 17 and -0.90, SS 79 respectively). Three strength measurements showed SRM >0.8: right elbow flexion (SRM = -1.01, SS 62), left elbow flexion (SRM = -0.85, SS 88) and left hip flexion (SRM = -0.81, SS 96). Of the 29 patients who completed the PUL1.2 at 3rd year follow-up visit, 20 (69%) obtained the maximum score of 74 points. Only one patient lost ambulation during the study. BMD is hallmarked by large inter-individual variability at baseline and follow-up. A relatively stable disease course is common even with 3 years. The ceiling effect of the PUL1.2 illustrates the need for adequate outcome measures in non-ambulant patients.

The neurocognitive profile of adults with Becker muscular dystrophy in the Netherlands.

Background:In Becker muscular dystrophy evidence for neurocognitive and behavioral comorbidity is evolving. More insight into the extend of these problems is of great importance for early detection and remediation in clinical practice.Objective:In this study we aimed to describe the neurocognitive and behavioral features of a Dutch adult cohort of BMD patients, and to evaluate correlations to motor function outcomes.Methods:28 adult BMD patients were included. Intelligence, executive functioning, verbal memory and reaction times were assessed cross-sectionally. Additionally, patients completed questionnaires on behavioral and emotional symptoms, psychosocial and executive functions. Results were compared to normative data and correlated with disease severity as measured by the 10-meter run/walk test and Performance of the Upper Limb version 1.2.Results:15 patients (53.6%) had a high educational level despite frequent grade repeating (48.3%) during primary or secondary school. Neuropsychological testing revealed that intellectual abilities, verbal memory, processing speed and executive functioning were statistically significant below average, but still within normal range. Regarding outcomes of the behavioral questionnaires, no significant differences were reported compared to the norm population. No relevant correlations with disease severity were found.Conclusions:This cohort of adult BMD patients exhibits minor cognitive impairments and no significant behavioral problems. The lower outcomes on processing speed and verbal memory, combined with the relatively high prevalence of grade repeating during primary and secondary school, implies that these minor impairments played a role in childhood. However, the on average high educational levels suggests that they grow out of their cognitive impairments with ageing.

Serum cardiac troponin I is a candidate biomarker for cardiomyopathy in Duchenne and Becker muscular dystrophies.

Serum cardiac troponin I (cTnI), its relation to cardiomyopathy, and the contribution of the ACTN3 genotype to serum levels of cTnI in Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) remain unknown. In this study we aimed to reveal the characteristics of cTnI, assess whether cTnI is a biomarker for cardiomyopathy in these dystrophinopathies, and evaluate the contribution of the ACTN3 genotype to the serum levels of cTnI in DMD patients. Serum cTnI values obtained from 127 DMD and 47 BMD patients were analyzed retrospectively. The relationship between cTnI and echocardiography data or the ACTN3 XX genotype was assessed. The cTnI levels and proportion of patients with abnormal cTnI levels were significantly higher among DMD patients than BMD, especially in the second decade of life. In DMD, the cTnI level reached a maximum at 13 years, and left ventricular ejection fraction (LVEF) became abnormal approximately 1year subsequently. In BMD, the cTnI level peaked at the age of 14 years, and LVEF became abnormal 3 years later. Decreased LVEF was observed after cTnI elevation in both populations. cTnI levels by age in DMD patients with the ACTN3 XX genotype tended to increase significantly and early. Myocardial injury indicated by cTnI elevation was more common and severe in DMD patients. cTnI elevation preceding cardiac dysfunction may represent an early phase of cardiomyopathy progression and may be a biomarker for early detection of cardiomyopathy in these dystrophinopathies. The ACTN3 XX genotype may be a risk factor for early myocardial injury.

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin.

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Clinical and genetic profile of duchenne and becker muscular dystrophy patients in the far west of Algeria

Duchenne's (DMD) and Becker's (BMD) muscular dystrophy are the principals causes of myopathy in male children. It is linked to a mutation in the dystrophygene and isinheritedin an X-linked mode. The aims of our study are to Describe the clinical and genetic phenotype of BMD and DMD patients in the far west of Algeria.

Muscle architecture is associated with muscle fat replacement in Duchenne and Becker muscular dystrophies.

Introduction/AimsDuchenne and Becker muscular dystrophies (DMD and BMD, respectively) are characterized by fat replacement of different skeletal muscles in a specific temporal order. Given the structural role of dystrophin in skeletal muscle mechanics, muscle architecture could be important in the progressive pathophysiology of muscle degeneration. Therefore, the aim of this study was to assess the role of muscle architecture in the progression of fat replacement in DMD and BMD.MethodsWe assessed the association between literature‐based leg muscle architectural characteristics and muscle fat fraction from 22 DMD and 24 BMD patients. Dixon‐based magnetic resonance imaging estimates of fat fractions at baseline and 12 (only DMD) and 24 months were related to fiber length and physiological cross‐sectional area (PCSA) using age‐controlled linear mixed modeling.ResultsDMD and BMD muscles with long fibers and BMD muscles with large PCSAs were associated with increased fat fraction. The effect of fiber length was stronger in muscles with larger PCSA.DiscussionMuscle architecture may explain the pathophysiology of muscle degeneration in dystrophinopathies, in which proximal muscles with a larger mass (fiber length × PCSA) are more susceptible, confirming the clinical observation of a temporal proximal‐to‐distal progression. These results give more insight into the mechanical role in the pathophysiology of muscular dystrophies. Ultimately, this new information can be used to help support the selection of current and the development of future therapies.

Elevation of fast but not slow troponin I in the circulation of patients with Becker and Duchenne muscular dystrophy.

IntroductionOne of the hallmarks of injured skeletal muscle is the appearance of elevated skeletal muscle proteins in circulation. Human skeletal muscle generally consists of a mosaic of slow (type I) and fast (type IIa, IIx/d) fibers, defined by their myosin isoform expression. Recently, measurement of circulating fiber‐type specific isoforms of troponin I has been used as a biomarker to suggest that muscle injury in healthy volunteers (HV) results in the appearance of muscle proteins from fast but not slow fibers. We sought to understand if this is also the case in severe myopathy patients with Becker and Duchenne muscular dystrophy (BMD, DMD).MethodsAn enzyme‐linked immunosorbent assay (ELISA) that selectively measures fast and slow skeletal troponin I (TNNI2 and TNNI1) was used to measure a cross‐section of patient plasma samples from HV (N = 50), BMD (N = 49), and DMD (N = 132) patients. Creatine kinase (CK) activity was also measured from the same samples for comparison.ResultsTNNI2 was elevated in BMD and DMD and correlated with the injury biomarker, CK. In contrast, TNNI1 levels were indistinguishable from levels in HV. There was an inverse relationship between CK and TNNI2 levels and age, but no relationship for TNNI1.DiscussionWe define a surprising discrepancy between TNNI1 and TNNI2 in patient plasma that may have implications for the interpretation of elevated muscle protein levels in dystrophinopathies.

Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Dystrophinopathies

Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked muscular disorders produced by mutations in the DMD gene which encodes the protein dystrophin. Both diseases are characterized by progressive involvement of skeletal, cardiac, and respiratory muscles. As new treatment strategies become available, reliable biomarkers and outcome measures that can monitor disease progression are needed for clinical trials.Methods: We collected clinical and functional data and blood samples from 19 DMD patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the serum concentration of 4 growth factors involved in the process of muscle fibrosis. We correlated the serum concentration of these growth factors with several muscle function tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI.Results: We found significant differences in the serum concentration of Platelet Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, and in and Transforming Growth Factor- β1 (TGF-β1) between BMD and DYSF patients. PDGF-AA showed a good correlation with several muscle function tests for both DMD and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies.Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases.

Utrophin modulator drugs as potential therapies for Duchenne and Becker muscular dystrophies.

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin‐deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co‐localise in human foetal muscle, in the dystrophin‐competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.

The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

Small mutations in Duchenne/Becker muscular dystrophy in 164 unrelated Polish patients.

In the 164 patients with Duchenne/Becker muscular dystrophy, we found 142 different small mutations including 51 novel mutations not listed in the LOVD, the UMD-DMD, the ClinVar, and the HGMD databases. Among all mutations, nonsense mutations occurred in 45.7%, frameshift mutations in 32.9%, and splicing mutations in 19.5%. Small mutations were distributed throughout the whole dystrophin gene. Splicing mutations were twice more common in BMD patients than in DMD patients. Eighty-two percent of mothers of the males affected with DMD/BMD were found to be carriers of small mutations.

DMD – BIOMARKERS & OUTCOME MEASURES: P.118 Elevation of fast, but not slow TnI in plasma from Duchenne and Becker muscular dystrophy patients suggests differential fiber response to disease

Duchenne and Becker muscular dystrophy (DMD/BMD) are marked by an absence (DMD) or truncation (BMD) of the dystrophin protein. Dystrophin provides a structural link between the contractile elements of the sarcomere and the basement membrane of the myofiber. When absent, contraction of muscle leads to muscle stress, the opening of membrane stress channels, and myofiber degeneration. Associated with this breakdown is the leak of a number of muscle proteins into the circulation, including creatine kinase (CK), lactate dehydrogenase (LDH), myoglobin and fast skeletal troponin I. Human skeletal muscle consists of both fast (type IIa, IIx) and slow (type I) myofibers defined by their myosin isoform. Most human skeletal muscles are composed of approximately 50% slow and 50% fast fibers in a mosaic pattern. In DMD, fast fibers appear to be the first to degenerate as young patients exhibit higher levels of embryonic myosin in fast fibers (a marker of recent regeneration) and early loss of type IIx fibers. Slow fibers are commonly affected later in the disease course. Troponin I (TnI) has three different isoforms, depending on the striated muscle of origin, TNNI1 (slow skeletal), TNNI2 (fast skeletal) and TNNI3 (cardiac). We took advantage of these different isoforms to develop selective fast/slow skeletal muscle TnI ELISAs to explore the fiber-specific source of muscle proteins in circulation. Cross-sectional collections of DMD (N=132), BMD (N=52) and healthy volunteer plasma samples (N=20) were examined using these ELISAs and TnI levels compared to circulating CK. As expected, DMD patient samples exhibited the highest CK with BMD intermediate and healthy samples in the normal range. Surprisingly, only fast skeletal TNNI2 was elevated in the circulation of both BMD and DMD patients with highest levels in DMD samples and a good correlation between TNNI2 and CK. TNNI1 levels were not significantly different from healthy volunteer samples, were not different between DMD and BMD patients, and did not correlate with CK levels. This biomarker evidence suggests that different fiber types respond differently to myopathy with a marked elevation of secreted biomarker associated with fast but not slow skeletal muscle fibers. The data update our understanding of muscle biomarker levels in patients and their interpretation.

DMD – BIOMARKERS & OUTCOME MEASURES: P.120 Phenotype and genotype characterization as predictors of DMD 45 to 55 multi-exon skipping therapy

Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy (DCM). As described in literature, 63% of DMD patients are eligible for a multi-exon skipping therapy, thus becoming BMD patients with exons 45 to 55 deletion (BMDdel45-55). Interestingly, new regulatory actors as long non-coding RNA (lncRNA) are localized in introns 44 and 56 (Bovolenta et al., 2012). Here we present a population of 54 ``skip-equivalent'' BMDdel45-55 patients. The objective was to study the phenotype variability in these patients by performing a detailed clinical characterization, assessing a whole genome sequencing analysis (WGS) and investigated lncRNA sequences boarding patient's deletion. The phenotype characterization underlined that 100% patients have BMD phenotype. The patients with available cardiac investigation have a DCM in 50%. Based on the clinical characterization we assessed a new skeletal muscle clinical severity scale in 27 patients and in 54% we have identified an intermediate severity degree presentation. The WGS data in 19 patients allowed us to i) analyze the association of variants in DCM pathogenic genes with the cardiac presentation in our patients and ii) identify deletion breakpoints with specific sequence for all analyzed patients. We established the profile of lncRNA presence in 38/54 patients at genomic level and the expression profile of this lncRNA in healthy controls and DMD 45-52 exons deletion patient immortalized myoblasts. We underline the difference of the expression of these lncRNA during the myogenesis. This study presents the phenotype description in the largest reported cohort of BMDdel45-55 patients. The new clinical severity scale and genomic data profiling in this particular population would have a favorable contribution in the design of clinical trials and the new therapeutic approaches.