Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype.

Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner. PAPERCLIP.

T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing.

Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase.

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.

Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

An engineered N-acyltransferase-LOV2 domain fusion protein enables light-inducible allosteric control of enzymatic activity.

Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by light-oxygen-voltage 2 sensing domain. We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/∗Dbv8) and identify potential allosteric sites on the protein's surface. We insert light-oxygen-voltage 2 sensing domain at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/∗Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.

Comprehensive analysis of genomic complexity in the 5' end coding region of the DMD gene in patients of exons 1-2 duplications based on long-read sequencing.

BACKGROUND: Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic variants in the DMD gene, however, 5' end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS), are commonly used in clinics. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation. RESULTS: We used LRS technology to perform whole genome sequencing on three families and analyze the structural variations of the DMD gene, which involves the duplications of exon 1 and/or exon 2. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. In pedigree 1, the male individuals harboring duplication variant of consecutive exons 1-2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is likely benign. In pedigree 3, the patient carries complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified by LRS, enabling the classification of the variants' pathogenicity. CONCLUSIONS: Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.

Exploring lipin1 as a promising therapeutic target for the treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive and devastating muscle disease, resulting from the absence of dystrophin. This leads to cell membrane instability, susceptibility to contraction-induced muscle damage, subsequent muscle degeneration, and eventually disability and early death of patients. Currently, there is no cure for DMD. Our recent studies identified that lipin1 plays a critical role in maintaining myofiber stability and integrity. However, lipin1 gene expression levels are dramatically reduced in the skeletal muscles of DMD patients and mdx mice. METHODS: To identify whether increased lipin1 expression could prevent dystrophic pathology, we employed unique muscle-specific mdx:lipin1 transgenic (mdx:lipin1Tg/0) mice in which lipin1 was restored in the dystrophic muscle of mdx mice, intramuscular gene delivery, as well as cell culture system. RESULTS: We found that increased lipin1 expression suppressed muscle degeneration and inflammation, reduced fibrosis, strengthened membrane integrity, and resulted in improved muscle contractile and lengthening force, and muscle performance in mdx:lipin1Tg/0 compared to mdx mice. To confirm the role of lipin1 in dystrophic muscle, we then administered AAV1-lipin1 via intramuscular injection in mdx mice. Consistently, lipin1 restoration inhibited myofiber necroptosis and lessened muscle degeneration. Using a cell culture system, we further found that differentiated primary mdx myoblasts had elevated expression levels of necroptotic markers and medium creatine kinase (CK), which could be a result of sarcolemmal damage. Most importantly, increased lipin1 expression levels in differentiated myoblasts from mdx:lipin1Tg/0 mice substantially inhibited the elevation of necroptotic markers and medium CK levels. CONCLUSIONS: Overall, our data suggest that lipin1 is a promising therapeutic target for the treatment of dystrophic muscles.

Two years of newborn screening for Duchenne muscular dystrophy as a part of the statewide Early Check research program in North Carolina.

PURPOSE: Current and emerging treatments for Duchenne muscular dystrophy (DMD) position DMD as a candidate condition for newborn screening (NBS). In anticipation of the nomination of DMD for universal NBS, we conducted a prospective study under the Early Check voluntary NBS research program in North Carolina, United States. METHODS: We performed screening for creatine kinase-MM (CK-MM), a biomarker of muscle damage, on residual routine newborn dried blood spots (DBS) from participating newborns. Total creatine kinase testing and next generation sequencing of an 86-neuromuscular gene panel that included DMD were offered to parents of newborns who screened positive. Bivariate and multivariable analyses were performed to assess effects of biological and demographic predictors on CK-MM levels in DBS. RESULTS: We screened 13,354 newborns and identified 2 males with DMD. The provisional 1626 ng/mL cutoff was raised to 2032 ng/mL to improve specificity, and additional cutoffs (900 and 360 ng/mL) were implemented to improve sensitivity for older and low-birthweight newborns. CONCLUSION: Population-scale screening for elevated CK-MM in DBS is a feasible approach to identify newborns with DMD. Inclusion of birthweight- and age-specific cutoffs, repeat creatine kinase testing after 72 hours of age, and DMD sequencing improve sensitivity and specificity of screening.

Genome sequencing detects a balanced pericentric inversion with breakpoints that impact the DMD and upstream region of POU3F4 genes.

We describe a family with two maternal half-brothers both of whom presented with muscular dystrophy, autism spectrum disorder, developmental delay, and sensorineural hearing loss. The elder brother had onset of features at ~3 months of age, followed by clinical confirmation of muscular dystrophy at 3 years. Skeletal biopsy staining at 4.7 years showed an absence of dystrophin protein which prompted extensive molecular testing over 4 years that included gene panels, targeted single-gene assays, arrays, and karyotyping, all of which failed to identify a clinically significant variant in the DMD gene. At 10 years of age, clinical whole-genome sequencing (cWGS) was performed, which revealed a novel hemizygous ~50.7 Mb balanced pericentric inversion on chromosome X that disrupts the DMD gene in both siblings, consistent with the muscular dystrophy phenotype. This inversion also impacts the upstream regulatory region of POU3F4, structural rearrangements which are known to cause hearing loss. The unaffected mother is a heterozygous carrier for the pericentric inversion. This finding illustrates the ability of cWGS to detect a wide breadth of disease-causing genomic variations including large genomic rearrangements.

Flexible implementation of modulated localisation microscopy based on DMD.

Localisation microscopy of individual molecules allows one to bypass the diffraction limit, revealing cellular organisation on a nanometric scale. This method, which relies on spatial analysis of the signal emitted by molecules, is often limited to the observation of biological objects at shallow depths, or with very few aberrations. The introduction of a temporal parameter into the localisation process through a time-modulated excitation was recently proposed to address these limitations. This method, called ModLoc, is demonstrated here with an alternative flexible strategy. In this implementation, to encode the time-modulated excitation a digital micromirror device (DMD) is used in combination with a fast demodulation approach, and provides a twofold enhancement in localisation precision. Layout: Nowadays, we can use an optical microscope to observe how proteins are organised in 3D within a cell at the nanoscale. By carefully controlling the emission of molecules in both space and time, we can overcome the limitations set by the diffraction limit. This allows us to pinpoint the exact location of molecules more precisely. However, the usual spatial analysis method limits observations to shallow depths or causing low distortion of optical waves. To overcome these restrictions, a recent approach introduces a temporal element to the localisation process. This involves changing the illumination over time to enhance the precision of localisation. This method, known as ModLoc, is showcased here using a flexible and alternative strategy. In this setup, a matrix of micrometric mirrors, working together with a fast demodulation optical module, is used to encode and decode the time-modulated information. This combination results in a twofold improvement in localisation precision.

Functional abilities, respiratory and cardiac function in a large cohort of adults with Duchenne muscular dystrophy treated with glucocorticoids.

BACKGROUND AND PURPOSE: The transition to adult services, and subsequent glucocorticoid management, is critical in adults with Duchenne muscular dystrophy. This study aims (1) to describe treatment, functional abilities, respiratory and cardiac status during transition to adulthood and adult stages; and (2) to explore the association between glucocorticoid treatment after loss of ambulation (LOA) and late-stage clinical outcomes. METHODS: This was a retrospective single-centre study on individuals with Duchenne muscular dystrophy (≥16 years old) between 1986 and 2022. Logistic regression, Cox proportional hazards models and survival analyses were conducted utilizing data from clinical records. RESULTS: In all, 112 individuals were included. Mean age was 23.4 ± 5.2 years and mean follow-up was 18.5 ± 5.5 years. At last assessment, 47.2% were on glucocorticoids; the mean dose of prednisone was 0.38 ± 0.13 mg/kg/day and of deflazacort 0.43 ± 0.16 mg/kg/day. At age 16 years, motor function limitations included using a manual wheelchair (89.7%), standing (87.9%), transferring from a wheelchair (86.2%) and turning in bed (53.4%); 77.5% had a peak cough flow <270 L/min, 53.3% a forced vital capacity percentage of predicted <50% and 40.3% a left ventricular ejection fraction <50%. Glucocorticoids after LOA reduced the risk and delayed the time to difficulties balancing in the wheelchair, loss of hand to mouth function, forced vital capacity percentage of predicted <30% and forced vital capacity <1 L and were associated with lower frequency of left ventricular ejection fraction <50%, without differences between prednisone and deflazacort. Glucocorticoid dose did not differ by functional, respiratory or cardiac status. CONCLUSION: Glucocorticoids after LOA preserve late-stage functional abilities, respiratory and cardiac function. It is suggested using functional abilities, respiratory and cardiac status at transition stages for adult services planning.

Lethal immunotoxicity in high-dose systemic AAV therapy.

High-dose systemic gene therapy with adeno-associated virus (AAV) is in clinical trials to treat various inherited diseases. Despite remarkable success in spinal muscular atrophy and promising results in other diseases, fatality has been observed due to liver, kidney, heart, or lung failure. Innate and adaptive immune responses to the vector play a critical role in the toxicity. Host factors also contribute to patient death. This mini-review summarizes clinical findings and calls for concerted efforts from all stakeholders to better understand the mechanisms underlying lethality in AAV gene therapy and to develop effective strategies to prevent/treat high-dose systemic AAV-gene-therapy-induced immunotoxicity.

Three-dimensional video recordings: Accuracy, reliability, clinical and research guidelines - Reliability assessment of a 4D camera.

OBJECTIVES: In addition to studying facial anatomy, stereophotogrammetry is an efficient diagnostic tool for assessing facial expressions through 3D video recordings. Current technology produces high-quality recordings but also generates extremely excessive data. Here, we compare various recording speeds for three standardized movements using the 3dMDface camera system, to assess its accuracy and reliability. MATERIALS AND METHODS: A linear and two circular movements were performed using a 3D-printed cube mounted on a robotic arm. All movements were recorded initially at 60 fps (frames/second) and then at 30 and 15 fps. Recording accuracy was tested with best-fit superimpositions of consecutive frames of the 3D cube and calculation of the Mean Absolute Distance (MAD). The reliability of the recordings were tested with evaluation of the inter- and intra-examiner error. RESULTS: The accuracy of movement recordings was excellent at all speeds (60, 30 and 15 fps), with variability in MAD values consistently being less than 1 mm. The reliability of the camera recordings was excellent at all recording speeds. CONCLUSIONS: This study demonstrated that 3D recordings of facial expressions can be performed at 30 or even at 15 fps without significant loss of information. This considerably reduces the amount of produced data facilitating further processing and analyses.

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.

De Novo p.Asp3368Gly Variant of Dystrophin Gene Associated with X-Linked Dilated Cardiomyopathy and Skeletal Myopathy: Clinical Features and In Silico Analysis.

Dystrophin (DMD) gene mutations are associated with skeletal muscle diseases such as Duchenne and Becker Muscular Dystrophy (BMD) and X-linked dilated cardiomyopathy (XL-DCM). To investigate the molecular basis of DCM in a 37-year-old woman. Clinical and genetic investigations were performed. Genetic testing was performed with whole exome sequencing (WES) using the Illumina platform. According to the standard protocol, a variant found by WES was confirmed in all available members of the family by bi-directional capillary Sanger resequencing. The effect of the variant was investigated by using an in silico prediction of pathogenicity. The index case was a 37-year-old woman diagnosed with DCM at the age of 33. A germline heterozygous A>G transversion at nucleotide 10103 in the DMD gene, leading to an aspartic acid-glycine substitution at the amino acid 3368 of the DMD protein (c.10103A>G p.Asp3368Gly), was identified and confirmed by PCR-based Sanger sequencing of the exon 70. In silico prediction suggests that this variant could have a deleterious impact on protein structure and functionality (CADD = 30). The genetic analysis was extended to the first-degree relatives of the proband (mother, father, and sister) and because of the absence of the variant in both parents, the p.Asp3368Gly substitution was considered as occurring de novo. Then, the direct sequencing analysis of her 8-year-old son identified as hemizygous for the same variant. The young patient did not present any signs or symptoms attributable to DCM, but reported asthenia and presented with bilateral calf hypertrophy at clinical examination. Laboratory testing revealed increased levels of creatinine kinase (maximum value of 19,000 IU/L). We report an early presentation of dilated cardiomyopathy in a 33-year-old woman due to a de novo pathogenic variant of the dystrophin (DMD) gene (p.Asp3368Gly). Genetic identification of this variant allowed an early diagnosis of a skeletal muscle disease in her son.

The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

An Integrated Transcriptomics and Genomics Approach Detects an X/Autosome Translocation in a Female with Duchenne Muscular Dystrophy.

Duchenne and Becker muscular dystrophies, caused by pathogenic variants in DMD, are the most common inherited neuromuscular conditions in childhood. These diseases follow an X-linked recessive inheritance pattern, and mainly males are affected. The most prevalent pathogenic variants in the DMD gene are copy number variants (CNVs), and most patients achieve their genetic diagnosis through Multiplex Ligation-dependent Probe Amplification (MLPA) or exome sequencing. Here, we investigated a female patient presenting with muscular dystrophy who remained genetically undiagnosed after MLPA and exome sequencing. RNA sequencing (RNAseq) from the patient's muscle biopsy identified an 85% reduction in DMD expression compared to 116 muscle samples included in the cohort. A de novo balanced translocation between chromosome 17 and the X chromosome (t(X;17)(p21.1;q23.2)) disrupting the DMD and BCAS3 genes was identified through trio whole genome sequencing (WGS). The combined analysis of RNAseq and WGS played a crucial role in the detection and characterisation of the disease-causing variant in this patient, who had been undiagnosed for over two decades. This case illustrates the diagnostic odyssey of female DMD patients with complex structural variants that are not detected by current panel or exome sequencing analysis.

Is Cardiac Transplantation Still a Contraindication in Patients with Muscular Dystrophy-Related End-Stage Dilated Cardiomyopathy? A Systematic Review.

Inherited muscular diseases (MDs) are genetic degenerative disorders typically caused by mutations in a single gene that affect striated muscle and result in progressive weakness and wasting in affected individuals. Cardiac muscle can also be involved with some variability that depends on the genetic basis of the MD (Muscular Dystrophy) phenotype. Heart involvement can manifest with two main clinical pictures: left ventricular systolic dysfunction with evolution towards dilated cardiomyopathy and refractory heart failure, or the presence of conduction system defects and serious life-threatening ventricular arrhythmias. The two pictures can coexist. In these cases, heart transplantation (HTx) is considered the most appropriate option in patients who are not responders to the optimized standard therapeutic protocols. However, cardiac transplant is still considered a relative contraindication in patients with inherited muscle disorders and end-stage cardiomyopathies. High operative risk related to muscle impairment and potential graft involvement secondary to the underlying myopathy have been the two main reasons implicated in the generalized reluctance to consider cardiac transplant as a viable option. We report an overview of cardiac involvement in MDs and its possible association with the underlying molecular defect, as well as a systematic review of HTx outcomes in patients with MD-related end-stage dilated cardiomyopathy, published so far in the literature.

A case study of Duchenne muscular dystrophy caused by Alu element insertion in DMD gene and analysis of its gray-hair symptoms.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive genetic disorder caused by mutations in the DMD gene, which leads to a deficiency of the dystrophin protein. The main mutation types of this gene include exon deletions and duplications, point mutations, and insertions. These mutations disrupt the normal expression of dystrophin, ultimately leading to the disease. In this study, we reported a case of DMD caused by an insertion mutation in exon 59 (E59) of the DMD gene. The affected child exhibited significant abnormalities in related biochemical markers, early symptoms of DMD, and multiple gray hair. His mother and sister were carriers with slightly abnormal biochemical markers. The mother had mild clinical symptoms, while the sister had no clinical symptoms. Other family members were genetically and physically normal. Sequencing and sequence alignment revealed that the inserted fragment was an Alu element from the AluYa5 subfamily. This insertion produced two stop codons and a polyadenylate (polyA) tail. To understand the impact of this insertion on the DMD gene and its association with clinical symptoms, exonic splicing enhancer (ESE) prediction indicated that the insertion did not affect the splicing of E59. Therefore, we speculated that the insertion sequence would be present in the mRNA sequence of the DMD gene. The two stop codons and polyA tail likely terminate translation, preventing the production of functional dystrophin protein, which may be the mechanism leading to DMD. In addition to typical DMD symptoms, the child also exhibited premature graying of hair. This study reports, for the first time, a case of DMD caused by the insertion of an Alu element into the coding region of the DMD gene. This finding provides clues for studying gene mutations induced by Alu sequence insertion and expands the understanding of DMD gene mutations.