Milder Becker Muscular Dystrophy Research Articles

Appendicular lean mass index changes in patients with Duchenne muscular dystrophy and Becker muscular dystrophy.

Mutations in the 79 exons of the dystrophin gene result in muscle wasting and weakness of varying clinical severity, ranging from severe/typical Duchenne muscular dystrophy (DMD) to intermediate DMD and mild Becker muscular dystrophy (BMD), depending on the frameshift of the mutation. We previously reported that males with DMD have progressively declining appendicular lean mass (ALM) and ALM index (ALMI) with age and worsening functional motor ability compared with healthy controls. These indices have not been studied in patients with intermediate DMD and BMD phenotypes and across DMD genotypes. In this study, we compared age-related trajectories of ALM and ALMI of patients who had (1) BMD without functional mobility deficits with patients who had DMD at different stages of disease and healthy controls; (2) a DMD intermediate phenotype with patients who had a typical DMD phenotype; and (3) DMD categorized by genotype. We conducted a retrospective review of ALM and ALMI data from 499 patients (ages 5-23years) with DMD (466 typical and 33 intermediate) and 46 patients (ages 5-21years) with BMD (without functional mobility deficits and functional mobility score of 1). Patients were grouped according to age reflecting disease stage (ages 5 to <7, 7 to <10, 10 to <14, and 14 to <20years) and genotype (mutations in exons 1-30, 31-44, 45-62, and 63-79). ALM and ALMI trajectories of patients with BMD paralleled those of healthy controls until adolescence, in contrast to patients with DMD. ALMI Z-scores of patients with BMD remained within ±2 SD without decline while those of patients with DMD fell below -2 SD around age 12years. Patients with BMD had increasing ALM and ALMI with age, with peak accrual between ages 10 to <14years. ALMI declined after age 14years for those with intermediate DMD compared with 10years for patients with typical DMD. Patients with mutations in exons 63-79 had a greater decline in ALMI as compared with those with other genotypes after age 10years. Age-related changes in ALMI in patients with BMD and intermediate DMD differ from those with typical DMD, reflecting their clinical phenotypes. ALM and ALMI should be further studied in patients with BMD and DMD subtypes for their potential value as surrogate markers to characterize the severity of BMD and DMD and inform clinical care decisions and clinical trial designs.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

In-Frame Deletion of Dystrophin Exons 8-50 Results in DMD Phenotype.

Mutations that prevent the production of proteins in the DMD gene cause Duchenne muscular dystrophy. Most frequently, these are deletions leading to reading-frame shift. The "reading-frame rule" states that deletions that preserve ORF result in a milder Becker muscular dystrophy. By removing several exons, new genome editing tools enable reading-frame restoration in DMD with the production of BMD-like dystrophins. However, not every truncated dystrophin with a significant internal loss functions properly. To determine the effectiveness of potential genome editing, each variant should be carefully studied in vitro or in vivo. In this study, we focused on the deletion of exons 8-50 as a potential reading-frame restoration option. Using the CRISPR-Cas9 tool, we created the novel mouse model DMDdel8-50, which has an in-frame deletion in the DMD gene. We compared DMDdel8-50 mice to C57Bl6/CBA background control mice and previously generated DMDdel8-34 KO mice. We discovered that the shortened protein was expressed and correctly localized on the sarcolemma. The truncated protein, on the other hand, was unable to function like a full-length dystrophin and prevent disease progression. On the basis of protein expression, histological examination, and physical assessment of the mice, we concluded that the deletion of exons 8-50 is an exception to the reading-frame rule.

A genome-wide association analysis of loss of ambulation in dystrophinopathy patients suggests multiple candidate modifiers of disease severity.

The major determinant of disease severity in Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether the dystrophin gene (DMD) mutation truncates the mRNA reading frame or allows expression of a partially functional protein. However, even in the complete absence of dystrophin, variability in disease severity is observed, and candidate gene studies have implicated several genes as modifiers. Here we present the largest genome-wide search to date for loci influencing severity in N = 419 DMD patients. Availability of subjects for such studies is quite limited, leading to modest sample sizes, which present a challenge for GWAS design. We have therefore taken special steps to minimize heterogeneity within our dataset at the DMD locus itself, taking a novel approach to mutation classification to effectively exclude the possibility of residual dystrophin expression, and utilized statistical methods that are well adapted to smaller sample sizes, including the use of a novel linear regression-like residual for time to ambulatory loss and the application of evidential statistics for the GWAS approach. Finally, we applied an unbiased in silico pipeline, utilizing functional genomic datasets to explore the potential impact of the best supported SNPs. In all, we obtained eight SNPs (out of 1,385,356 total) with posterior probability of trait-marker association (PPLD) ≥ 0.4, representing six distinct loci. Our analysis prioritized likely non-coding SNP regulatory effects on six genes (ETAA1, PARD6G, GALNTL6, MAN1A1, ADAMTS19, and NCALD), each with plausibility as a DMD modifier. These results support both recurrent and potentially new pathways for intervention in the dystrophinopathies.

Identification of Biallelic dystrophin gene variants during maternal carrier testing for Becker muscular dystrophy and review of the DMD exon 49-51 deletion phenotype.

Dystrophinopathies are X-linked recessive conditions caused by pathogenic variants in the dystrophin (DMD) gene. In a family that included two boys with Becker muscular dystrophy (BMD) due to a DMD deletion of exons 45-47, maternal carrier testing unexpectedly identified biallelic DMD deletions of exons 45-47 and 49-51. The patient's mild phenotype in the setting of biallelic DMD variants prompted further investigation of the exon 49-51 deletion in particular, via literature review and retrospective chart review of patients who have been evaluated in our institution's comprehensive neuromuscular center and/or diagnosed in our clinical genetic testing laboratory. To our knowledge, this is only the fifth case of confirmed biallelic DMD variants in a female. In males, the DMD exon 49-51 deletion appears to result in a mild BMD phenotype with low or normal creatine kinase levels. This deletion comprised 19% (4/21) of dystrophinopathies diagnosed by chromosomal microarray (CMA) in males during the past ten years in our clinical laboratory. Most individuals identified by chart review were diagnosed through CMA, despite the fact that microarray was genome-wide and not DMD-specific. This case raised important genetic counseling issues. The DMD exon 49-51 deletion appears to cause a variable but generally mild BMD phenotype. Its relatively frequent detection by CMA suggests it may be underdiagnosed.

High-Throughput Digital Image Analysis Reveals Distinct Patterns of Dystrophin Expression in Dystrophinopathy Patients.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.

Pre-clinical dose-escalation studies establish a therapeutic range for U7snRNA-mediated DMD exon 2 skipping

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial.

Very Low Residual Dystrophin Quantity Is Associated with Milder Dystrophinopathy.

ObjectiveThis study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD mutations.MethodsWe performed a retrospective multicentric cohort study by using molecular and clinical data from patients with DMD mutations registered in the Universal Mutation Database–DMD France database. Patients with intronic, splice site, or nonsense DMD mutations, with available muscle biopsy Western blot data, were included irrespective of whether they presented with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD). Patients were separated into 3 groups based on dystrophin protein levels. Clinical outcomes were ages at appearance of first symptoms; loss of ambulation; fall in vital capacity and left ventricular ejection fraction; interventions such as spinal fusion, tracheostomy, and noninvasive ventilation; and death.ResultsOf 3,880 patients with DMD mutations, 90 with mutations of interest were included. Forty‐two patients expressed no dystrophin (group A), and 31 of 42 (74%) developed DMD. Thirty‐four patients had dystrophin quantities < 5% (group B), and 21 of 34 (61%) developed BMD. Fourteen patients had dystrophin quantities ≥ 5% (group C), and all but 4 who lost ambulation beyond 24 years of age were ambulant. Dystrophin quantities of <5%, as low as <0.5%, were associated with milder phenotype for most of the evaluated clinical outcomes, including age at loss of ambulation (p < 0.001).InterpretationVery low residual dystrophin protein quantity can cause a shift in disease phenotype from DMD toward BMD. ANN NEUROL 2021;89:280–292

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.207 AAV.U7 technology for two mutational hotspots of the DMD gene (∼6%) results in efficient exon skipping, protein restoration and force improvement

Mutations in the DMD gene cause two disorders, the severe Duchenne muscular dystrophy (DMD), associated with no protein expression and the milder Becker muscular dystrophy (BMD) associated with the production of an internally truncated but semi-functional protein. Genotype-phenotype correlation of DMD and BMD prompted a promising therapeutic strategy named exon skipping that intends to convert a DMD to a BMD phenotype. This strategy makes use of antisense oligonucleotides (AONs) to hide exon definition. This should transform an out-of-frame DMD reading frame into an in-frame one, leading into expression of an internally truncated but functional protein. AONs are promising but have two major drawbacks: 1. stability which required repeated injection; 2. limited penetration in heart. Alternatively, an antisense sequence can be carried by a small nuclear RNA (U7snRNA) and delivered using a viral vector such as adeno-associated virus (AAV). This approach (AAV.U7) has been demonstrated by several group including ours, to overcome AON's drawbacks. In this study, we focused on skipping of exon 2 and exon 44, two mutational hotspots of the DMD gene as their skipping could be beneficial to ∼12% of patients. Our objective was to evaluate the efficiency of an AAV.U7 exon skipping strategy in these hotspot mutations both in vitro and in vivo. For in vitro studies, we used skin punches from several DMD patients and transdifferentiated them into myotubes. Using those cells, we showed that our constructs efficiently mediated exons 2 or 44 skipping (at least ≥50%) and induced expression of a slightly truncated dystrophin. For in vivo studies, we used either a new mouse model carrying an exon 2 duplication (referred to as Dup 2 mice) or more recently a new humanized DMD mouse model that is missing human exon 45 disrupting reading frame of hDMD. This new model (referred to as hDMD/mdx del45 mice) does not express mouse and human dystrophins and presents a dystrophic muscle pathology in multiple muscles. In vivo, following intramuscular or intravenous injection, we observed an efficient exon 2 or 44 skipping (at least ≥50%) and expression of a truncated dystrophin (at least ≥40%). Muscle strength using different tests was improved (by ∼50% compared to an untreated mouse). These AAV.U7- could help ∼12% of DMD patients. One of those vectors is being evaluated in a clinical trial.

DMD – BIOMARKERS & OUTCOME MEASURES: P.127 Identification of biallelic DMD variants during maternal carrier testing for Becker muscular dystrophy

Dystrophinopathies are allelic X-linked recessive muscular dystrophies caused by pathogenic variants in the dystrophin (DMD) gene. In males, out-of-frame DMD variants typically cause more severe Duchenne muscular dystrophy (DMD), while in-frame variants typically result in Becker muscular dystrophy (BMD). Females who carry a DMD variant are at increased risk for cardiomyopathy and may have skeletal muscle symptoms. We report a mother (proband) and three sons, the oldest (10-year-old) and youngest (8-year-old) of whom have a pathogenic in-frame deletion of exons 45-47 and diagnoses of Becker muscular dystrophy. Both have progressive muscle weakness, recurrent rhabdomyolysis, and elevated creatine phosphokinase (CPK) levels ranging from 2,500 – 18,000 unit/L (ref 22-250 unit/L). During confirmatory carrier testing, the mother was found to carry the familial exon 45-47 deletion in addition to an unexpected second pathogenic in-frame DMD deletion of exons 49-51, which was not present in her two previously tested sons. She reports muscle cramps and pain but no muscle weakness. Cardiac MRI shows no current evidence of cardiomyopathy. DMD testing for the family's 9-year-old son detected the exon 49-51 deletion, which supported a diagnosis of BMD for him and confirmed the two DMD changes are situated on opposite alleles in his mother. He had an existing diagnosis of Perthes disease, but had otherwise not come to clinical attention for BMD-related symptoms. The family had reported that CPK levels measured locally had been within normal limits. CPK levels measured subsequent to genetic testing were mildly elevated (up to 321 unit/L), and muscle pain and fatigue were noted during clinical visit, consistent with BMD. This case highlights unexpected outcomes of maternal dystrophinopathy carrier testing, namely the detection of a second variant not previously known in the family, and subsequent diagnosis of BMD in a son who had been presumed to be unaffected. These results underscore the importance of genetic counselling in this setting. This case also describes an additional example of mild BMD in the setting of exon 49-51 deletion, as well as a further example of biallelic DMD variants in a female, of which only a few cases have been described.

Becker muscular dystrophy caused by exon 2-truncating mutation of DMD

Nonsense and frameshift mutations of the dystrophin (DMD) gene usually cause severe Duchenne muscular dystrophy (DMD). Interestingly, however, premature stop codons in exons 1 and 2 result in relatively mild Becker muscular dystrophy (BMD). Herein, we report the clinical course of a patient with a very mild phenotype of BMD caused by a frameshift mutation, NM_004006.2: c.40_41del GA/p.(Glu14ArgfsX17), in exon 2 of the DMD gene.

P.143Correlation between dystrophin espression and clinical phenotype using high-throughput digital immunoanalysis in Duchenne and Becker muscular dystrophy patients

Dystrophin is an important endpoint in Duchenne Muscular Dystrophy (DMD) trials. Precise quantification of the variably-reduced dystrophin in biopsies from Becker muscular dystrophy (BMD) patients with different mutations in relation to the phenotype may help in understanding the correlation between dystrophin levels and clinical trial outcome. We established a novel digital script developed with Definiens software applied to digitally scanned muscle sections, generating high-throughput data for dystrophin quantification. We analysed muscle sections of 16 patients across the entire range of clinical severity, from DMD, Intermediate Muscular Dystrophy (IMD), to mild and severe BMD. We performed double immunostaining using an anti-dystrophin antibody combined with laminin-alpha2 for myofibre identification. When possible, Western blot (WB) was performed to quantify dystrophin in muscle homogenates. The mean sarcolemmal dystrophin intensity was quantified for every myofibre in the section. Total myofibre population in each section was divided into 4 groups according to sarcolemmal dystrophin coverage; the mean dystrophin intensity for each group was expressed as percentage of mean intensity of the corresponding control group. These biological outcomes were correlated with the severity grade and WB data. BMD carrying different mutations have very different patterns of dystrophin expression (% of positive fibres, individual fibre coverage and intensity) while DMD patients had a similar pattern of residual low dystrophin expression. Our results suggest that different BMD mutations give variable dystrophin coverage and sarcolemmal intensity and quantity on WB, presumably reflecting the quality of internally-deleted proteins. Analysis of a larger patient cohort will refine the correlation between dystrophin expression in BMD/IMD/DMD patients and functional outcomes.

Truncated dystrophin ameliorates the dystrophic phenotype of mdx mice by reducing sarcolipin-mediated SERCA inhibition

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45–55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45–55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

DUCHENNE MUSCULAR DYSTROPHY – IMAGING AND BIOMARKERS: P.31Serum creatinine: a promising biomarker for distinguishing Duchenne muscular dystrophy from Becker muscular dystrophy in patients aged ≤ 3 Years

Dystrophinopathy is a common muscular dystrophy, consisting of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The prognosis and treatment strategy between DMD and BMD are totally different. This study was designed to investigate correlations between the level of serum creatinine and clinical phenotypes of dystrophinopathy in young patients. We enrolled sixty-eight patients with dystrophinopathy, whose diagnosis were based on clinical manifestation, biochemical changes, and molecular analysis. The levels of serum creatinine were determined when patients were ≤ 3 years old. Each patient was followed up, and motor function and clinical phenotype were assessed when the same patients were ≥ 4 years old. The results indicated that in young patients, lower serum creatinine levels were associated with increased disease severity (p < 0.01), and that serum creatinine levels were the highest in patients exhibiting mild BMD (p < 0.001) and the lowest in patients with DMD (p < 0.01), and were significantly higher in patients carrying in-frame mutations than in patients carrying out-of-frame mutations (p < 0.001). Serum creatinine level cut-off values for identifying mild BMD [18 μmol/L; area under the curve (AUC): 0.947; p < 0.001] and DMD (17 μmol/L; AUC: 0.837; p < 0.001) were established. These results suggest that serum creatinine might be a promising biomarker for distinguishing DMD from BMD in patients aged ≤ 3 years and could assist in the selection of appropriate treatment strategies.

Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45-55 Skipping Accompanied by Rescue of Dystrophin Expression.

Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45-55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this "mutation hotspot." In addition, patients naturally harboring a dystrophin exons 45-55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45-55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45-55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.

The exon 45 skipping therapy of induced pluripotent stem cells derived cardiomyocyte from the DMD patient with exon 46-55 deletion

Background: Duchenne muscular dystrophy (DMD) is an X-linked lethal muscular disorder and is caused by a mutation in the DMD gene encoding dystrophin. It has been reported that a deletion of exon 45–55 covering a whole hot-spot mutational region of the DMD gene causes a very mild Becker muscular dystrophy or asymptomatic dystrophinopathy. In dystrophinopathy, cardiomyopathy sometimes is a critical issue; therefore, the DMD specific induced pluripotent stem cells(iPSC)-derived cardiomyocytes might be a useful for elucidation of the pathomechanism and evaluation of the therapeutic efficacy.

Functional analysis of MYOD-transduced fibroblasts and urine-derived cells from healthy individual and patients with duchenne muscular dystrophy

Background: Duchenne muscular dystrophy (DMD) is a severe muscle disorder characterized by mutations in the DMD gene that mainly disrupt the reading frame, resulting in absence of functional dystrophin. Exon-skipping using short antisense oligonucleotides is a promising therapy for DMD, which aims to convert severe DMD into mild Becker muscular dystrophy by altering pre-mRNA splicing to restore an open reading frame. We employed in vitro assay including myogenic conversion of fibroblasts by MYOD and fluorescence-activated cell sorting (FACS)-aided cell selection, for patient eligibility assessments in a first-in-human exon 53-skipping clinical trial at NCNP. However, a limitation of the in vitro assay is that it requires an invasive skin biopsy. Moreover, detailed characteristics of the MYOD-transduced fibroblasts as myogenic cells are still unclear.

Serum Creatinine Distinguishes Duchenne Muscular Dystrophy from Becker Muscular Dystrophy in Patients Aged ≤3 Years: A Retrospective Study.

Here, we investigated correlations between serum creatinine (SCRN) levels and clinical phenotypes of dystrophinopathy in young patients. Sixty-eight patients with dystrophinopathy at the Neuromuscular Clinic, The First Affiliated Hospital, Sun Yat-sen University, were selected for this study. The diagnosis of dystrophinopathy was based on clinical manifestation, biochemical changes, and molecular analysis. Some patients underwent muscle biopsies; SCRN levels were tested when patients were ≤3 years old, and reading frame changes were analyzed. Each patient was followed up, and motor function and clinical phenotype were assessed when the same patients were ≥4 years old. Our findings indicated that in young patients, lower SCRN levels were associated with increased disease severity (p < 0.01) and that SCRN levels were the highest in patients exhibiting mild Becker muscular dystrophy (BMD) (p < 0.001) and the lowest in patients with Duchenne muscular dystrophy (DMD) (p < 0.01) and were significantly higher in patients carrying in-frame mutations than in patients carrying out-of-frame mutations (p < 0.001). SCRN level cutoff values for identifying mild BMD [18 µmol/L; area under the curve (AUC): 0.947; p < 0.001] and DMD (17 µmol/L; AUC: 0.837; p < 0.001) were established. These results suggest that SCRN might be a valuable biomarker for distinguishing DMD from BMD in patients aged ≤3 years and could assist in the selection of appropriate treatment strategies.

Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G&gt;A mutation identified by NGS

Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.

Functional changes in Becker muscular dystrophy: implications for clinical trials in dystrophinopathies.

We performed a 1-year longitudinal study of Six Minute Walk Test (6MWT), North Star Ambulatory Assessment (NSAA), and timed function tests in Becker muscular dystrophy (BMD). Skeletal muscle dystrophin was quantified by immunoblot. We grouped deletions ending on exon 45 (“del 45-x”, n = 28) or 51 (“del x-51”, n = 10); isolated exon 48 deletion (“del 48”, n = 10); and other mutations (n = 21). Only patients in the “del 45-x” or “other” groups became non-ambulatory (n = 5, log-rank p = n.s.) or unable to run (n = 22, p < 0.001). All measures correlated positively with dystrophin quantity and negatively with age, and were significantly more impaired in the “del 45-x” and “other” groups. After one year, NSAA score decreased significantly (−0.9 ± 1.6, p < 0.001); in the “del 45-x” group, both NSAA (−1.3 ± 1.7, p = 0.001) and 6MWT (−12 ± 31 m, p = 0.059) decreased. We conclude that patients with “del x-51” or “del 48” mutations have mild or asymptomatic BMD, while “del 45-x” mutations cause comparatively severe weakness, and functional deterioration in 1 year. Furthermore, exon 51 skipping could be more effective than exon 45 skipping in Duchenne muscular dystrophy.