Progressive cardiomyopathy with intercalated disc disorganization in a rat model of Becker dystrophy.

Becker muscular dystrophy (BMD) is an X-linked disorder due to in-frame mutations in the DMD gene, leading to a less abundant and truncated dystrophin. BMD is less common and severe than Duchenne muscular dystrophy (DMD) as well as less investigated. To accelerate the search for innovative treatments, we developed a rat model of BMD by deleting the exons 45-47 of the Dmd gene. Here, we report a functional and histopathological evaluation of these rats during their first year of life, compared to DMD and control littermates. BMD rats exhibit moderate damage to locomotor and diaphragmatic muscles but suffer from a progressive cardiomyopathy. Single nuclei RNA-seq analysis of cardiac samples revealed shared transcriptomic abnormalities in BMD and DMD rats and highlighted an altered end-addressing of TMEM65 and Connexin-43 at the intercalated disc, along with electrocardiographic abnormalities. Our study documents the natural history of a translational preclinical model of BMD and reports a cellular mechanism for the cardiac dysfunction in BMD and DMD offering opportunities to further investigate the organization role of dystrophin in intercellular communication.

Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Myotonia congenita in a Greek cohort: Genotype spectrum and impact of the CLCN1:c.501C > G variant as a genetic modifier.

INTRODUCTION/AIMS: Myotonia congenita (MC) is the most common hereditary channelopathy in humans. Characterized by muscle stiffness, MC may be transmitted as either an autosomal dominant (Thomsen) or a recessive (Becker) disorder. MC is caused by variants in the voltage-gated chloride channel 1 (CLCN1) gene, important for the normal repolarization of the muscle action potential. More than 250 disease-causing variants in the CLCN1 gene have been reported. This study provides an MC genotype-phenotype spectrum in a large cohort of Greek patients and focuses on novel variants and disease epidemiology, including additional insights for the variant CLCN1:c.501C > G. METHODS: Sanger sequencing for the entire coding region of the CLCN1 gene was performed. Targeted segregation analysis of likely candidate variants in additional family members was performed. Variant classification was based on American College of Medical Genetics (ACMG) guidelines. RESULTS: Sixty-one patients from 47 unrelated families were identified, consisting of 51 probands with Becker MC (84%) and 10 with Thomsen MC (16%). Among the different variants detected, 11 were novel and 16 were previously reported. The three most prevalent variants were c.501C > G, c.2680C > T, and c.1649C > G. Additionally, c.501C > G was detected in seven Becker cases in-cis with the c.1649C > G. DISCUSSION: The large number of patients in whom a diagnosis was established allowed the characterization of genotype-phenotype correlations with respect to both previously reported and novel findings. For the c.501C > G (p.Phe167Leu) variant a likely nonpathogenic property is suggested, as it only seems to act as an aggravating modifying factor in cases in which a pathogenic variant triggers phenotypic expression.

Aß-protein polymerization in Alzheimer disease: Optimal control for nucleation parameter estimation.

In this paper, we are interested in the modeling of Alzheimer's disease from the angle of the amyloid cascade hypothesis, where the pathogenic agent is the Aß protein in its oligomeric form. The formation dynamics of this pathogenic form is modeled by a Becker-Doring type model where APP (Amyloid Precursor Protein) protein, after cleavage by specific enzymes, forms monomeric Aß proteins, which, by polymerization and/or nucleation, lead to the formation of oligomeric Aß. We propose an optimal control problem to estimate the rate of nucleation which seems to be at the base of this amyloid cascade hypothesis and for which no (experimental) measurement exists for the moment.

Cognitive abnormalities in Becker muscular dystrophy: a mysterious link between dystrophin deficiency and executive functions.

BACKGROUND: Distrophinopathies are a heterogeneous group of neuromuscular disorders due to mutations in the DMD gene. Different isoforms of dystrophin are also expressed in the cerebral cortex and Purkinje cells. Despite cognitive abnormalities in Duchenne muscular dystrophy subjects that have been described in the literature, little is known about a comprehensive cognitive profile in Becker muscular dystrophy patients. AIM: The aim of this study was to assess cognitive functioning in Becker muscular dystrophy patients by using an extensive neuropsychological battery. Our hypothesis is that the most impaired functions are the highly intentional and conscious ones, such as working memory functions, which require a prolonged state of cellular activation. METHODS: We performed an extensive neuropsychological assessment on 28 Becker muscular dystrophy patients from 18 to 65 years old. As control subjects, we selected 20 patients with limb-girdle muscular dystrophy, whose clinical picture was similar except for cognitive integrity. The evaluation, although extended to all areas, was focused on prefrontal control skills, with a distinction between inhibitory processes of selective attention and activating processes of working memory. RESULTS AND CONCLUSIONS: Significant underperformances were found exclusively in the Dual Task and PASAT tests, to demonstrate a selective impairment of working memory that, while not causing intellectual disability, reduces the intellectual potential of patients with Becker muscular dystrophy.

Social cognition in two brothers with Becker muscular dystrophy: an exploratory study revealing divergent behavioral phenotypes.

BACKGROUND AND OBJECTIVE: Only few studies investigated social cognition in Becker muscular dystrophy (BMD). However, brain dystrophin deficiency could be a neural substrate for cognitive, emotional, and neuropsychological features in BMD. METHODS: We compared interoceptive accuracy and interpersonal comfort distance in two brothers with BMD presenting with the same genetic deletion and a healthy control. When possible, we collected neuropsychological and psychopathological assessments. RESULTS: Our BMD patients were significantly different in interoceptive accuracy, with patient 1 being extremely accurate and patient 2 being significantly less accurate than his brother but more accurate than the control. Interestingly, they presented opposite patterns of interpersonal distance. Patient 1 was comfortable with very short interpersonal distance (≤50 cm from the confederate/object) vs the control and patient 2. By contrast, patient 2 preferred larger distance vs the control and patient 1. Patient 1 also presented difficulties in social and emotional skills on the psychopathological assessment. CONCLUSIONS: We are aware this is a small sample; nonetheless, this is also the first description of such aspects in BMD and the first report ever of such divergent behavioral pattern. As impaired social cognition affects the quality of life and social relationship, further studies are needed for a closer understanding of involved mechanisms.

A retrospective cohort study and review of the literature about germline mosaicism in Duchenne/Becker muscular dystrophy prenatal counseling: How to estimate the recurrence risk in clinical settings?

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common inherited neuromuscular diseases. Following the identification of a pathogenic causative variant in the DMD gene of a proband, potential carriers can be informed of their risk of having offspring with the disease. Germline mosaicism is a variant that is confined to the gonads that can be transmitted to offspring and is usually reported when a non-carrier of a DMD pathogenic variant has two or more offspring carrying the variant in question. On average, one third of cases are the result of a de novo variant, and as DMD and BMD are prone to germline mosaicism, its inclusion in genetic counseling is mandatory. In this retrospective cohort study, we presented clinical data from an unpublished DMD/BMD cohort of 332 families with incidence of germline mosaicism in families with de novo transmission of 8.1%. This is also the first systematic literature review searching PubMed to provide an accurate assessment of the current literature on germline mosaicism in DMD and BMD, including 17 case reports and 20 original studies. The incidence of documented germline mosaicism in de novo event families ranged from 6.0 to 40%, with a mean of 8.3%. The estimated recurrence risk for mothers of a patient with a proven de novo causal variant ranged from 4.3 to 11%, with a mean of 5.8% for a male fetus. By providing an up-to-date and comprehensive overview of the literature, this review aims to improve our understanding of germline mosaicism in DMD and to promote the development of effective strategies and reliable data for occurrence risk assessment in genetic counseling of de novo event families.

Genetic counseling for the dystrophinopathies-Practice resource of the National Society of Genetic Counselors.

The dystrophinopathies encompass the phenotypically variable forms of muscular dystrophy caused by pathogenic variants in the DMD gene. The dystrophinopathies include the most common inherited muscular dystrophy among 46,XY individuals, Duchenne muscular dystrophy, as well as Becker muscular dystrophy and other less common phenotypic variants. With increased access to and utilization of genetic testing in the diagnostic and carrier setting, genetic counselors and clinicians in diverse specialty areas may care for individuals with and carriers of dystrophinopathy. This practice resource was developed as a tool for genetic counselors and other health care professionals to support counseling regarding dystrophinopathies, including diagnosis, health risks and management, psychosocial needs, reproductive options, clinical trials, and treatment. Genetic testing efforts have enabled genotype/phenotype correlation in the dystrophinopathies, but have also revealed unexpected findings, further complicating genetic counseling for this group of conditions. Additionally, the therapeutic landscape for dystrophinopathies has dramatically changed with several FDA-approved therapeutics, an expansive research pathway, and numerous clinical trials. Genotype-phenotype correlations are especially complex and genetic counselors' unique skill sets are useful in exploring and explaining this to families. Given the recent advances in diagnostic testing and therapeutics related to dystrophinopathies, this practice resource is a timely update for genetic counselors and other healthcare professionals involved in the diagnosis and care of individuals with dystrophinopathies.

Follicular Becker's nevi: A case series and review of literature.

The follicular variant of Becker's nevus is an under-reported entity. We present the rare occurrence of follicular Becker's nevus in 7 patients, confirmed through dermoscopy and histopathological examination. Dermoscopy shows perifollicular hypopigmentation surrounded by a well-defined net-like pigmentation corresponding clinically to the presence of folliculocentric macules. Histology shows prominent basal and suprabasal melanization surrounding the follicle, corresponding to well-defined net-like pigmentation seen on dermoscopy. However, the melanization does not extend along the entire length of the follicular epithelium leading to perifollicular hypopigmentation on dermoscopy. Though biopsy is confirmatory, it is not usually necessary.

The role of fibrosis in the pathophysiology of muscular dystrophy.

Muscular dystrophy exerts significant and dramatic impacts on affected patients, including progressive muscle wasting leading to lung and heart failure, and results in severely curtailed lifespan. Although the focus for many years has been on the dysfunction induced by the loss of function of dystrophin or related components of the striated muscle costamere, recent studies have demonstrated that accompanying pathologies, particularly muscle fibrosis, also contribute adversely to patient outcomes. A significant body of research has now shown that therapeutically targeting these accompanying pathologies via their underlying molecular mechanisms may provide novel approaches to patient management that can complement the current standard of care. In this review, we discuss the interplay between muscle fibrosis and muscular dystrophy pathology. A better understanding of these processes will contribute to improved patient care options, restoration of muscle function, and reduced patient morbidity and mortality.

Proteomic Comparison of Three Wild-Type Pseudorabies Virus Strains and the Attenuated Bartha Strain Reveals Reduced Incorporation of Several Tegument Proteins in Bartha Virions.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky's disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha-an attenuated strain created by serial passaging-represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.

Combination 1550 nm non-ablative fractional resurfacing and laser hair removal for treatment of Becker's nevi in skin types III-VI.

OBJECTIVES: Becker's nevus is a cosmetically bothersome benign hamartoma typically associated with basal layer hyperpigmentation and hypertrichosis. We herein present the largest case series characterizing treatment parameters and clinical outcomes of combined 1550 nm non-ablative fractional resurfacing and laser hair removal (long-pulsed neodymium-doped yttrium aluminum garnet or alexandrite) in the treatment of Becker's nevus. This is also the largest case series of laser treatment of Becker's nevus in Fitzpatrick skin types V and VI. METHODS: We performed a retrospective review of patients treated between 2016 and 2021. Clinical photographs were graded by three independent physicians using a 5-point visual analog scale. RESULTS: Twelve patients (mean age: 24.8 years, Fitzpatrick skin types III-VI) were treated for Becker's nevus on the face (4) or the trunk and/or extremities (8). Four patients were Fitzpatrick skin types V or VI. On average, patients received 5.3 treatments in 1-4-month intervals. Ten of the 12 patients had concomitant laser hair removal preceding same-day non-ablative fractional resurfacing (n = 7 with long-pulsed 1064 nm neodymium-doped yttrium aluminum garnet and n = 3 with long-pulsed 755 nm alexandrite). The number of treatments with each modality was determined by patient satisfaction with improvement in hyperpigmentation and hypertrichosis. At follow-up, which ranged from 6 to 40 weeks (mean 10.5 weeks), patients were given a mean improvement score of 51%-75%. No long-term adverse events were encountered in either group. Limitations include a small sample size and a lack of long-term follow-up. CONCLUSION: Combination 1550 nm non-ablative fractional resurfacing and laser hair removal is safe and efficacious in the cosmetic reduction of hyperpigmentation and hypertrichosis associated with Becker's nevus, including those with Fitzpatrick skin types V and VI.

Efficacy of non-ablative 1927-nm fractional thulium fiber laser for the treatment of Becker's nevus: Intra- and interobserver agreement study.

OBJECTIVE: To assess the efficacy and safety of a 1927-nm fractional thulium fiber laser treatment for Becker's nevus. METHODS: A retrospective analysis of patients with Becker's nevus who were treated with a 1927-nm fractional thulium fiber laser was conducted. Grading for lightening was set quarterly by physician global assessment of the pre- and postphotographs (I: <25%; II: 25%-50%; III: 51%-75%; and IV: >75% for marked lightening). Grading validation was exercised using intra- and interobserver agreement analysis among 10 dermatologists to determine the percent of agreement and the intraclass correlation coefficient. Grades I and II were classified as treatment failure. RESULTS: A total of 21 patients were recruited for analysis. The average number of treatment sessions was 2.1 per patient. A total of 13 patients achieved successful outcomes (grades III and IV) while 8 patients experienced treatment failure (grades I and II). Partial recurrence and transient hypopigmentation on adjacent skin were the main adverse effects. Only one patient reported worsening of the nevus. Overall agreement among the raters for the outcomes was graded from "very good" to "excellent," and the intraclass correlation coefficient, kappa, was 0.8. CONCLUSION: A 1927-nm fractional thulium fiber laser may be effective to ameliorate the pigment of Becker's nevus and achieve acceptable cosmetic outcomes. However, further studies are required in this area to improve and optimize the results.

Diversity of mutations in the dystrophin gene and details of muscular lesions in porcine dystrophinopathies.

During meat inspections in pigs, dystrophinopathies are among the muscle lesions targeted for disposal. In this study, the authors examined the lesions and the distribution of dystrophin expression in 25 pigs with dystrophinopathy. In addition, complementary deoxyribonucleic acid (cDNA) sequencing and western blotting were performed in 6 of the 25 cases, all of which were characterized by degeneration, necrosis, and fat replacement of muscle fibers. Comparing the results of immunohistochemistry with anti-dystrophin antibodies that recognized at different sites in the protein, the authors noted that the loss of dystrophin expression was most pronounced in the C-terminus-recognizing antibody (19/25 cases). The authors detected 5 missense mutations and 3 types of shortened transcripts generated by the skipping of exons in the cDNA, which were associated with the pathogenesis. One missense mutation had been reported previously, whereas the remaining mutations identified had not been previously documented in pigs. In the cases with shortened transcripts, normal-sized transcripts were detected together with the defective transcripts, suggesting that these mutations were caused by splicing abnormalities. In addition, they were in-frame mutations, all of which have similar pathogeneses of Becker muscular dystrophy in humans. These cases were 6 months of age and exhibited macroscopic discoloration, fatty replacement, and muscle degeneration, suggesting that the effect of these mutations on skeletal muscle was significant.

Patterns of cardiac involvement in different muscular dystrophies assessed by magnetic resonance imaging.

BACKGROUND: In muscular dystrophies, it is not only skeletal muscles that can be affected, but also the myocardium. This cardiac involvement can represent a major cause of morbidity and mortality. PURPOSE: To investigate cardiac involvement in Duchenne (DMD), Becker (BMD), and limb girdle muscular dystrophy (LGMD) patients, and carriers of DMD/BMD by cardiac magnetic resonance (CMR) imaging and to search for differences in the pattern of cardiac involvement. MATERIAL AND METHODS: All patients with genetically or histologically proven DMD, BMD, and LGMD, or confirmed carriers of DMD/BMD who had undergone CMR at our clinic between January 2008 and November 2018 were retrospectively included and re-evaluated for regional and global left ventricular function, increased trabecularization, and late enhancement. RESULTS: A total of 26 DMD, 10 BMD, 11 LGMD, and seven DMD/BMD carriers were included. Only one carrier of DMD presented with normal CMR results; all other participants showed cardiac abnormalities. Regional wall motion abnormalities (RWMA; prevalence in LGMD patients: 55%) and late enhancement (prevalence in LGMD patients: 82%) were frequent. RWMA were accentuated basal inferolateral in DMD/BMD carriers, while in LGMD they were accentuated apical. In all groups late enhancement was located mainly subepicardial/midmyocardial with a basal inferolateral accentuation. Apart from the different RWMA distribution, no further group-specific differences were found. CONCLUSION: We found a high rate of cardiac involvement not only in DMD/BMD, but also in LGMD and DMD/BMD carriers with a different RWMA accentuation (apical in LGMD and basal inferolateral in DMD/BMD) as a single group-specific difference.

Direct to implant reconstruction with adjustable permanent Becker implant expanders with ADM following de-epithelised skin reducing mastectomy in high-risk overweight and obese patients with severe ptosis.

Overweight and obese patients with severe ptosis undergoing mastectomy and reconstruction present a challenge to the oncoplastic breast surgeon. This paper investigates early-to medium-term outcome of high-risk overweight and obese women with severe ptosis undergoing de-epithelised skin reducing mastectomy and direct to implant reconstruction with Becker implant expanders. From Nov 2016 and April 2021, 20 Wise pattern skin reducing mastectomies (SRM) were performed, 18 with Adjustable Permanent Becker Implant expanders (APBI) and 2 with fixed volume implants. Median age was 48 years (27-73), and median FU was 21 months (4-49). There were 8 smokers and 7 patients needed radiation treatment. In the ABPI group, there was one infection, 3 patients had threatened wounds which needed revision, and one patient lost her implant. There was no locoregional recurrence or distant metastasis. There was 1 week delay in adjuvant chemotherapy for a patient with wound issues following reconstruction. One patient is awaiting autologous reconstruction following deformity subsequent to radiation treatment. In conclusion the use of APBI following de-epithelised skin reduction surgery in this high-risk group is an option with acceptable early-to medium-term outcome.

Multidimensional Biomechanics-Based Score to Assess Disease Progression in Duchenne Muscular Dystrophy.

(1) Background: Duchenne (DMD) is a rare neuromuscular disease that progressively weakens muscles, which severely impairs gait capacity. The Six Minute-Walk Test (6MWT), which is commonly used to evaluate and monitor the disease's evolution, presents significant variability due to extrinsic factors such as patient motivation, fatigue, and learning effects. Therefore, there is a clear need for the establishment of precise clinical endpoints to measure patient mobility. (2) Methods: A novel score (6M+ and 2M+) is proposed, which is derived from the use of a new portable monitoring system capable of carrying out a complete gait analysis. The system includes several biomechanical sensors: a heart rate band, inertial measurement units, electromyography shorts, and plantar pressure insoles. The scores were obtained by processing the sensor signals and via gaussian-mixture clustering. (3) Results: The 6M+ and 2M+ scores were evaluated against the North Star Ambulatory Assessment (NSAA), the gold-standard for measuring DMD, and six- and two-minute distances. The 6M+ and 2M+ tests led to superior distances when tested against the NSAA. The 6M+ test and the 2M+ test in particular were the most correlated with age, suggesting that these scores better characterize the gait regressions in DMD. Additionally, the 2M+ test demonstrated an accuracy and stability similar to the 6M+ test. (4) Conclusions: The novel monitoring system described herein exhibited good usability with respect to functional testing in a clinical environment and demonstrated an improvement in the objectivity and reliability of monitoring the evolution of neuromuscular diseases.

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).

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.