Becker Muscular Dystrophy Research Articles

Sleep apnoea and hypoventilation in patients with five major types of muscular dystrophy

BackgroundThe characteristics of and relationship between sleep apnoea and hypoventilation in patients with muscular dystrophy (MD) remain to be fully understood.MethodsWe analysed 104 in-laboratory sleep studies of 73 patients with...

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).

Change in the spectrum of detected mutations in the <I>DMD</I> gene depending on the methodological capabilities of the laboratory

Background. Duchenne muscular dystrophy (DMD) is a severe, progressive form of muscular dystrophy that occurs in children between one and three years of age. The disease is mainly characterized by weakness of the proximal muscles, which leads to difficulty in movement, and ultimately to complete disability. Becker muscular dystrophy (BMD) is a milder allelic form of the disorder characterized by late onset and slow progression. The cause of the development of DMD/BMD is mutations in the DMD gene, leading to a deficiency in the production of various isoforms of the dystrophin protein family. The most common mutations in case of DMD/BMD are gross deletions (55–65 %) and duplications (6–11 %) of one or several exons The remaining cases of DMD/BMD are due to small mutations (approximately 20–30 %). Depending on the methodological capabilities of the laboratory, the idea of the spectrum of mutations in the DMD gene changed, which is important in genetic counseling of patients and planning the therapy available today.Aim. To analyze the spectrum of mutations in the DMD gene, including three time slices, depending on the methodological capabilities of the laboratory.Materials and methods. We analyzed the spectrum of mutations in the DMD gene for a sample of 2957 patients admitted to the laboratory of DNA diagnostics of the Research Centre for Medical Genetics with a referral diagnosis of DMD/BMD. Depending on the time of treatment and the capabilities of the laboratory, patients were divided into three groups: 2008–2015, 2016–2018, 2019–2022.Results. As a result of the study, the full range of mutations in the DMD gene was analyzed over three-time intervals, which makes it possible to get an idea of the distribution of mutation types in the sample among Russian patients. Regardless of the methodological capabilities of the laboratory, the spectrum of mutations in the DMD gene remains biased relative to world data. At the moment, there is a significant decrease in the proportion of extended deletions (50.7–59.6 %), while the proportion of extended duplications (11.8–17.2 %) and small mutations (23.2–35.0 %) increased. We assume that the main reason for such features of the spectrum is ethnic and population differences.Conclusion. Duchenne/Becker muscular dystrophy (DMD/BMD) is the most common form of muscular dystrophy, accounting for more than 50 % of all cases. Determination of the spectrum of mutations provides an understanding of their frequencies, which in the future may help patients in the appointment of therapy specific to a particular type of mutation.

Sources of variation in estimates of Duchenne and Becker muscular dystrophy prevalence in the United States

BackgroundDirect estimates of rare disease prevalence from public health surveillance may only be available in a few catchment areas. Understanding variation among observed prevalence can inform estimates of prevalence in other locations. The Muscular Dystrophy Surveillance, Tracking, and Research Network (MD STARnet) conducts population-based surveillance of major muscular dystrophies in selected areas of the United States. We identified sources of variation in prevalence estimates of Duchenne and Becker muscular dystrophy (DBMD) within MD STARnet from published literature and a survey of MD STARnet investigators, then developed a logic model of the relationships between the sources of variation and estimated prevalence.ResultsThe 17 identified sources of variability fell into four categories: (1) inherent in surveillance systems, (2) particular to rare diseases, (3) particular to medical-records-based surveillance, and (4) resulting from extrapolation. For the sources of uncertainty measured by MD STARnet, we estimated each source’s contribution to the total variance in DBMD prevalence. Based on the logic model we fit a multivariable Poisson regression model to 96 age–site–race/ethnicity strata. Age accounted for 74% of the variation between strata, surveillance site for 6%, race/ethnicity for 3%, and 17% remained unexplained.ConclusionVariation in estimates derived from a non-random sample of states or counties may not be explained by demographic differences alone. Applying these estimates to other populations requires caution.

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.

Dystrophin Short Product, Dp71, Interacts with AQP4 and Kir4.1 Channels in the Mouse Cerebellar Glial Cells in Contrast to Dp427 at Inhibitory Postsynapses in the Purkinje Neurons.

Dystrophin is the causative gene for Duchenne and Becker muscular dystrophy (DMD/BMD), and it produces full-length and short dystrophin, Dp427 and Dp71, respectively, in the brain. The existence of the different dystrophin molecular complexes has been known for a quarter century, so it is necessary to derive precise expression profiles of the molecular complexes in the brain to elucidate the mechanism of cognitive symptoms in DMD/BMD patients. In order to investigate the Dp71 expression profile in cerebellum, we employed Dp71-specific tag-insertion mice, which allowed for the specific detection of endogenous Dp71 in the immunohistochemical analysis and found its expressions in the glial cells, Bergmann glial (BG) cells, and astrocytes, whereas Dp427 was exclusively expressed in the inhibitory postsynapses within cerebellar Purkinje cells (PCs). Interestingly, we found different cell-type dependent dystrophin molecular complexes; i.e., glia-associated Dp71 was co-expressed with dystroglycan (DG) and dystrobrevinα, whereas synapse-associated Dp427 was co-expressed with DG and dystrobrevinβ. Furthermore, we investigated the molecular relationship of Dp71 to the AQP4 water channel and the Kir4.1 potassium channel, and found biochemical associations of Dp71 with AQP4 and Kir4.1 in both the cerebellum and cerebrum. Immunohistochemical and cytochemical investigations revealed partial co-localizations of Dp71 with AQP4 and Kir4.1 in the glial cells, indicating Dp71 interactions with the channels in the BG cells and astrocytes. Taken together, different cell-types, glial cells and Purkinje neurons, in the cerebellum express different dystrophin molecular complexes, which may contribute to pathological and physiological processes through the regulation of the water/ion channel and inhibitory postsynapses.

Dystrophin Genotype and Risk of Neuropsychiatric Disorders in Dystrophinopathies: A Systematic Review and Meta-Analysis.

Dystrophinopathies are associated with neuropsychiatric disorders due to alterations in dystrophin/DMD expression. The objective was to estimate the association of developmental disorders, autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), depression, anxiety disorders, and obsessive-compulsive disorder with the dystrophin/DMD genotype in population with dystrophinopathies. Systematic searches of Medline, Scopus, Web of Science, and Cochrane Library were performed from inception to September 2022. We included observational studies in the population with Becker or Duchenne muscular dystrophies (BMD, DMD) that estimated the prevalence of these disorders according to Dp140 and/or Dp71 genotype. Meta-analysis of the prevalence ratio (PR) of genotype comparisons was conducted for each disorder. Ten studies were included in the systematic review. In BMD, Dp140+ vs. Dp140- and Dp71+ vs. Dp71- were associated with developmental disorders with a PR of 0.11 (0.04, 0.34) and 0.22 (0.07, 0.67), respectively. In DMD, Dp140+/Dp71+ vs. Dp140- /Dp71- had a PR of 0.40 (0.28, 0.57), and Dp71+ vs. Dp71- had a PR of 0.47 (0.36, 0.63) for ADHD. However, there was no association of genotype with ASD, only a trend was observed for Dp71+ vs. Dp71-, with a PR of 0.61 (0.35, 1.06). Moreover, the data showed no association of these isoforms with emotional-related disorders. In BMD, Dp140 and Dp71 could be associated with developmental disorders, while ADHD might be associated with the Dp71 genotype in DMD. Further research is needed regarding Dp140 and Dp71, especially in DMD for ASD.

Next-generation sequencing approach to molecular diagnosis of Iranian patients with Duchenne/Becker muscular dystrophy: Several novel variants identified.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) constitute the second most prevalent muscular dystrophy, with large deletions or duplications accounting for 66% of cases. No effective treatment exists for DMD/BMD. At present, genetic diagnosis serves as the foundation for gene therapy treatments. In this study, a comprehensive molecular investigation was conducted. The subjects diagnosed with DMD/BMD were initially examined using multiplex ligation-dependent probe amplification (MLPA) technology. The negative MLPA results were analyzed further using next-generation sequencing (NGS) technology. The MLPA detected 201 deletions (65.9%) and 20 duplications (6.6%) along the dystrophin gene among the 305 Iranian patients examined. The deletion of exon 52 in the amenable skipping subgroup was associated with an earlier onset age and a more severe phenotype. Twenty-one of the small mutations found in 58 MLPA-negative patients were novel. The most prevalent variants were nonsense variants (46.5%), frameshift variants (31%), splicing variants (6.9%), missense variants (10.4%), and synonymous mutations (5.1%). Our results demonstrate that MLPA and NGS can be effective diagnostic tools for very young patients with a single exon deletion.

Subclinical cardiac involvement present as electrocardiographic abnormalities in various neuromuscular diseases

BackgroundCardiac involvement is commonly present in various neuromuscular diseases which may develop life-threatening consequences. The early manifestation is often asymptomatic which however has been insufficiently studied. ObjectivesWe aim to characterize electrocardiographic (ECG) changes in neuromuscular diseases without cardiac symptoms. MethodsAdults having genetically and/or pathologically confirmed type 1 myotonic dystrophy (DM1), Becker muscular dystrophy (BMD), limb girdle muscular dystrophies (LGMDs) and mitochondrial diseases (MtDs) but without history of heart diseases and cardiovascular symptoms were enrolled. The 12-lead ECG characteristics and other test results at diagnosis were retrieved and analyzed. Results196 patients with neuromuscular diseases (44 DM1, 25 BMD, 82 LGMDs, 45 MtDs) were consecutively enrolled. ECG abnormalities were identified in 107 (54.6%) patients with a prevalence of 59.1% in DM1, 76.0% in BMD, 40.2% in LGMDs and 64.4% in MtDs. Conduction block was more commonly present in DM1 than the other groups (P < 0.01), which had a longest PR interval and QRS duration of 186.1 ± 38.3 ms and 104.2 [90.0–108.0]ms, respectively. QT prolongation was most frequently seen in DM1 (P < 0.001). Left ventricular hypertrophy features were found in BMD, LGMDs and MtDs (P < 0.05) without intergroup difference, while a significantly higher right ventricular amplitude is observed in BMD than in other groups (P < 0.001). ConclusionsSubclinical cardiac involvement is commonly present as ECG abnormalities in multiple adult neuromuscular diseases before associated symptoms occur and show diversity in different groups.

Modeling Reduced Contractility and Stiffness Using iPSC-Derived Cardiomyocytes Generated From Female Becker Muscular Dystrophy Carrier

Study investigators encountered a female Becker muscular dystrophy (BMD) carrier with advanced heart failure (HF) and identified a stop-gain variant in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) as a potential second-hit variant. Isogenic induced pluripotent stem cells (iPSCs) with dominant expression of WT-DMD, Δ45-48-DMD, or Δ45-48-DMD with corrected PLOD3 variant were established. Microforce testing using 3-dimensional self-organized tissue rings (SOTRs) generated from iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated that correction of the heterozygous PLOD3 variant did not improve the reduced force, but it significantly recovered the reduced stiffness in Δ45-48-DMD SOTRs. Correction of the PLOD3 variant restored collagen synthesis in iPSC-CMs. Our findings revealed the pathogenesis underlying advanced HF in a female BMD carrier.

Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods: This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results: Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion: This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Givinostat for Becker muscular dystrophy: A randomized, placebo-controlled, double-blind study.

No treatments are approved for Becker muscular dystrophy (BMD). This study investigated the efficacy and safety of givinostat, a histone deacetylase pan-inhibitor, in adults with BMD. Males aged 18-65 years with a diagnosis of BMD confirmed by genetic testing were randomized 2:1 to 12 months treatment with givinostat or placebo. The primary objective was to demonstrate statistical superiority of givinostat over placebo for mean change from baseline in total fibrosis after 12 months. Secondary efficacy endpoints included other histological parameters, magnetic resonance imaging and spectroscopy (MRI and MRS) measures, and functional evaluations. Of 51 patients enrolled, 44 completed treatment. At baseline, there was greater disease involvement in the placebo group than givinostat, based on total fibrosis (mean 30.8 vs. 22.8%) and functional endpoints. Mean total fibrosis did not change from baseline in either group, and the two groups did not differ at Month 12 (least squares mean [LSM] difference 1.04%; p = 0.8282). Secondary histology parameters, MRS, and functional evaluations were consistent with the primary. MRI fat fraction in whole thigh and quadriceps did not change from baseline in the givinostat group, but values increased with placebo, with LSM givinostat-placebo differences at Month 12 of -1.35% (p = 0.0149) and -1.96% (p = 0.0022), respectively. Adverse events, most mild or moderate, were reported by 88.2% and 52.9% patients receiving givinostat and placebo. The study failed to achieve the primary endpoint. However, there was a potential signal from the MRI assessments suggesting givinostat could prevent (or slow down) BMD disease progression.

How Important are Cardiac Electrical Abnormalities in Duchenne and Becker Muscular Dystrophy?

How Important are Cardiac Electrical Abnormalities in Duchenne and Becker Muscular Dystrophy?

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.

P2 Receptor Signaling in Motor Units in Muscular Dystrophy.

The purine signaling system is represented by purine and pyrimidine nucleotides and nucleosides that exert their effects through the adenosine, P2X and P2Y receptor families. It is known that, under physiological conditions, P2 receptors play only a minor role in modulating the functions of cells and systems; however, their role significantly increases under some pathophysiological conditions, such as stress, ischemia or hypothermia, when they can play a dominant role as a signaling molecule. The diversity of P2 receptors and their wide distribution in the body make them very attractive as a target for the pharmacological action of drugs with a new mechanism of action. The review is devoted to the involvement of P2 signaling in the development of pathologies associated with a loss of muscle mass. The contribution of adenosine triphosphate (ATP) as a signal molecule in the pathogenesis of a number of muscular dystrophies (Duchenne, Becker and limb girdle muscular dystrophy 2B) is considered. To understand the processes involving the purinergic system, the role of the ATP and P2 receptors in several models associated with skeletal muscle degradation is also discussed.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy(DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P<0.0001), wire hang (P=0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P=0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P<0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P=0.0017) and centrally located nuclei indicating degeneration/regeneration (P<0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P<0.0001), collagen deposition (+1.4-fold, P=0.0217), and sarcolemmal damage measured by intracellular IgM (P=0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

The efficacy and safety of kinesytherapy in Becker muscular dystrophy

The efficacy and safety of kinesytherapy in Becker muscular dystrophy

Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA)signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3–0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics.

The importance of direct genetic testing for determining female carriers of the mutation in dystrophinopathies

Background/Aim. Duchenne muscular dystrophy (MD) and Becker MD are caused by mutations in the gene for dystrophin (DMD). They are X chromosome-linked recessive diseases where males are affected, and females are healthy carriers of the mutation in most cases. It is estimated that 2/3 of mothers of Duchenne MD probands are carriers, while 1/3 of probands have de novo mutations. The aim of the study was to confirm the carrier status of female members of the families of Duchenne MD/Becker MD probands using direct genetic testing methods. Methods. The study included 38 females from 31 families of Duchenne MD/Becker MD probands with deletion/duplication in the DMD gene. Moreover, 4 cases of prenatal diagnosis of Duchenne MD/Becker MD were included. The methods of polymerase chain reaction - PCR and the multiplex ligation-dependent probe amplification - MLPA were applied for detecting deletions, i.e., deletion/duplication mutations in the DMD gene. Results. In the total of 31 Duchenne MD/Becker MD probands, 87.1% of deletions and 12.9% of duplications of one or more exons in the DMD gene were detected. Of the 29 tested mothers, mutations were found in 17 of them (14 deletions and 3 duplications). Mutations were detected in 11 (57.9%) out of 19 mothers of probands with the Duchenne MD phenotype and 6 (60%) out of 10 mothers of Becker MD probands. Furthermore, 14 (56%) out of 25 mothers were carriers in probands with deletions, and 3 (75%) out of 4 mothers were carriers in probands with duplications. In the remaining 9 other female relatives of the patients, mutations were found in 4. In prenatal diagnosis, we identified a deletion in one male and one female fetus of one single mother who was confirmed as a carrier. Conclusion. The study showed that mothers were carriers in almost 60% of sporadic cases of Duchenne MD/Becker MD with deletions and duplications. In addition, the carrier frequency tended to be higher in mothers of the probands with duplications (75%) compared to mothers of probands with deletions (56%).

Proximal myopathy: causes and associated conditions.

Proximal myopathy presents as generalized muscle weakness commonly involving the muscles of upper and/or lower limbs. Toxins, long-term use of statins, corticosteroids, alcohol, SGLT2 inhibitors, COVID-19 vaccination, and antimalarials have been attributed to its development. In endocrine and metabolic disorders, adrenal dysfunction including both overproduction and insufficiency of the adrenal gland hormones has been reported to cause myopathy. Moreover, parathyroid and thyroid disorders along with pituitary gland disorders can also directly or indirectly contribute to this condition. In idiopathic inflammatory myopathies including polymyositis, dermatomyositis, inclusion body myositis (IBM), and Systemic Lupus Erythematosus (SLE), Sjögren's Syndrome, and overlap syndromes, moderate to severe muscle weakness has been observed. IBM has been reported to be the most prevalent acquired myopathy above the age of 50. Hereditary or congenital myopathies include limb girdle muscular dystrophies, facioscapulohumeral muscular dystrophy, Duchenne and Becker muscular dystrophy, and proximal myotonic myopathy. In addition to these, glycogen storage diseases such as the McArdle disease can also cause fast exhaustion, myalgia, and cramping in working muscles. It is pertinent to mention here that a class of hereditary metabolic myopathies, referred to as "lipid deposition myopathy" causes lipids to accumulate in skeletal muscle fibers, leading to lesions and degeneration. Among viral causes, HIV, dengue virus, influenza virus, hepatitis B virus, hepatitis C virus, SARS-CoV2 are also associated with muscle weakness. Sarcoidosis, an inflammatory disease, can also manifest as muscle weakness and myalgia. Owing to this complicated pathophysiology of proximal myopathy, this review aims to summarize the existing literature on conditions associated with this phenomenon and other recent developments that have been made regarding events leading to development of generalized muscle weakness. To the authors' knowledge this is the first narrative review that discusses causes and conditions associated with proximal myopathy in thorough detail.