A novel deep intronic variant introduce dystrophin pseudoexon in Becker muscular dystrophy: A case report.

Most pathogenic DMD variants are detectable and interpretable by standard genetic testing for dystrophinopthies. However, approximately 1∼3% of dystrophinopthies patients still do not have a detectable DMD variant after standard genetic testing, most likely due to structural chromosome rearrangements and/or deep intronic pseudoexon-activating variants. Here, we report on a boy with a suspected diagnosis of Becker muscular dystrophy (BMD) who remained without a detectable DMD variant after exonic DNA-based standard genetic testing. Dystrophin mRNA studies and genomic Sanger sequencing were performed in the boy, followed by in silico splicing analyses. We successfully detected a novel deep intronic disease-causing variant in the DMD gene (c.2380 + 3317A > T), which consequently resulting in a new dystrophin pseudoexon activation through the enhancement of a cryptic donor splice site. The patient was therefore genetically diagnosed with BMD. Our case report further emphasizes the significant role of disease-causing splicing variants within deep intronic regions in genetically undiagnosed dystrophinopathies.

The Discharge Behavior and Mechanism of Polyimide Aerogel under Electron Irradiation.

The response and mechanism of polyimide aerogel under electron irradiations were investigated. The experimental results indicated that electron irradiation could not damage the skeleton polyimide in the aerogel due to its high stability, but could result in a discharge within. The morphology of the discharge shows some dendritic discharge patterns, and the material surrounding the discharge channels was carbonized. The numerical simulation results indicated that the incident electrons, and also large amount induced secondary electrons, would be deposited inhomogeneously within the nano-porous polyimide aerogel. This would result in forming an ultra-high electrical potential of up to about 8.5 × 1010 V/m (which is far higher than the breakdown strength (2 × 108 V/m) of bulk polyimide materials) in a local region. This may be the leading cause of the obvious discharge in the materials. Furthermore, it was found that the actual reason for the discharge is related to the residual gas within the nano-porous structure; namely, the more internal residual gas (as a shorter-time vacuum pumping in the irradiated chamber), the more serious the discharge phenomenon. Correspondingly, the phenomenon may largely consist of both residual-gas discharge and surface flashover due to ultra-high local potentials induced by unevenly deposited charges in the porous aerogel.

Clinical and genetic interpretation of uncertain DMD missense variants: evidence from mRNA and protein studies.

BACKGROUND: Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their pathogenicity interpretation remains complicated. We aimed to investigate whether DMD missense variants would cause aberrant splicing and re-interpret their pathogenicity based on mRNA and protein studies. METHODS: Nine unrelated patients who had an elevated serum creatine kinase level with or without muscle weakness were enrolled. They underwent a detailed clinical, imaging, and pathological assessment. Routine genetic testing and muscle-derived mRNA and protein studies of dystrophin and sarcoglycan genes were performed in them. RESULTS: Three of the 9 patients presented with a Duchenne muscular dystrophy (DMD) phenotype and the remaining 6 patients had a suspected diagnosis of Becker muscular dystrophy (BMD) or sarcoglycanopathy based on their clinical and pathological characteristics. Routine genetic testing detected only 9 predicted DMD missense variants in them, of which 6 were novel and interpreted as uncertain significance. Muscle-derived mRNA studies of sarcoglycan genes didn't reveal any aberrant transcripts in them. Dystrophin mRNA studies confirmed that 3 predicted DMD missense variants (c.2380G > C, c.4977C > G, and c.5444A > G) were in fact splicing and frameshift variants due to aberrant splicing. The 9 DMD variants were re-interpreted as pathogenic or likely pathogenic based on mRNA and protein studies. Therefore, 3 patients with DMD splicing variants and 6 patients with confirmed DMD missense variants were diagnosed with DMD and BMD, respectively. CONCLUSION: Our study highlights the importance of muscle biopsy and aberrant splicing for clinical and genetic interpretation of uncertain DMD missense variants.

A new pseudoexon activation due to ultrarare branch point formation in Duchenne muscular dystrophy.

Deep-intronic variants that create or enhance a splice site are increasingly reported as a significant cause of monogenic diseases. However, deep-intronic variants that activate pseudoexons by affecting a branch point are extremely rare in monogenic diseases. Here, we describe a novel deep-intronic DMD variant that created a branch point in a Duchenne muscular dystrophy (DMD) patient. A 7.0-year-old boy was enrolled because he was suspected of DMD based on his clinical, muscle imaging, and pathological features. Routine genetic testing did not discover a pathogenic DMD variant. We then performed muscle-derived dystrophin mRNA analysis and detected an aberrant pseudoexon-containing transcript. Further genomic Sanger sequencing and bioinformatic analyses revealed a novel deep-intronic splicing variant in DMD (NM_004006.2:c.5325+1759G>T), which created a new branch point sequence and thus activated a new dystrophin pseudoexon (NM_004006.2:r.5325_5326ins5325+1779_5325+1855). Our study highlights the significant role of branch point alterations in the pathogenesis of monogenic diseases.

Comparing adverse events of tenecteplase and alteplase: a real-world analysis of the FDA adverse event reporting system (FAERS).

OBJECTIVES: The aim of this study is to monitor, identify, and compare the adverse events (AEs) related to tenecteplase and alteplase, with the objective of exploring the potential safety of tenecteplase for acute ischemic stroke (AIS) and guiding its use to enhance patient safety. METHODS: In order to evaluate the disproportionality of AEs associated with tenecteplase and alteplase in real-world data, four algorithms (ROR, PRR, BCPNN, EBGM) were utilized as measures to detect signals of AEs related to both drugs. Subsequently, Breslow-Day statistical analysis was applied to compare the RORs of the main system organ classes (SOCs) and key preferred terms (PTs) between tenecteplase and alteplase. RESULTS: A statistical analysis was performed utilizing data gleaned from the Food and Drug Administration Adverse Event Reporting System (FAERS) database, encompassing 19,514,140 case reports from 2004Q1 to 2023Q1. There were 1,004 cases where tenecteplase was reported as the primary suspected (PS) and 2,363 tenecteplase-related adverse drug reactions (ADRs) at the PTs level were identified, the two data of alteplase were 10,945 and 25,266, respectively. The occurrence of drug-induced ADRs was analyzed across 27 organ systems, The analysis revealed several expected ADRs, such as Haemorrhage, Hypersensitivity which were consistent with the two drug-labels. It is of note that the signal strengths of 'death,' 'ventricular fibrillation,' 'cardiogenic shock' and 'pneumonia aspiration' at the PT level were markedly higher for tenecteplase than for alteplase, whereas the signal strength of 'angioedema' at the PT level was significantly higher for alteplase in comparison to tenecteplase. Additionally, unexpected significant ADRs associated with ocular adverse reactions and pneumonia aspiration at the PT level were identified, indicating potential AEs not currently mentioned in the drug instructions. CONCLUSION: This study identified and compared signals of ADRs associated with tenecteplase and alteplase, although tenecteplase is as effective as alteplase and has advantages such as ease of use and affordability, it cannot replace alteplase in the treatment of AIS until its safety profile is fully recognized. Additionally, previously unreported ocular ADRs and pneumonia were identified, providing valuable insights into the relationship between ADRs and the use of these thrombolytic drugs. These findings underscore the importance of continuous monitoring and effective detection of AEs to ultimately enhance the safety of AIS patients undergoing thrombolytic therapy.

A novel biomarker of fibrofatty replacement in dystrophinopathies identified by integrating transcriptome, magnetic resonance imaging, and pathology data.

BACKGROUND: We aimed to analyse genome-wide transcriptome differences between Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients and identify biomarkers that correlate well with muscle magnetic resonance imaging (MRI) and histological fibrofatty replacement in both patients, which have not been reported. METHODS: One hundred and one male patients with dystrophinopathies (55 DMD and 46 BMD) were enrolled. Muscle-derived genome-wide RNA-sequencing was performed in 31 DMD patients, 29 BMD patients, and 11 normal controls. Fibrofatty replacement was scored on muscle MRI and histological levels in all patients. A unique pipeline, single-sample gene set enrichment analysis combined with Spearman's rank correlations (ssGSEA-Cor) was developed to identify the most correlated gene signature for fibrofatty replacement. Quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, and single-nucleus RNA-sequencing (snRNA-seq) were performed in the remaining patients to validate the most correlated gene signature. RESULTS: Comparative transcriptomic analysis revealed that 31 DMD muscles were characterized by a significant increase of inflammation/immune response and extracellular matrix remodelling compared with 29 BMD muscles (P < 0.05). The ssGSEA-Cor pipeline revealed that the gene set of CDKN2A and CDKN2B was the most correlated gene signature for fibrofatty replacement (histological rs  = 0.744, P < 0.001; MRI rs  = 0.718, P < 0.001). Muscle qRT-PCR confirmed that CDKN2A mRNA expression in both 15 DMD (median = 25.007, P < 0.001) and 12 BMD (median = 5.654, P < 0.001) patients were significantly higher than that in controls (median = 1.101), while no significant difference in CDKN2B mRNA expression was found among DMD, BMD, and control groups. In the 27 patients, muscle CDKN2A mRNA expression respectively correlated with muscle MRI (rs  = 0.883, P < 0.001) and histological fibrofatty replacement (rs  = 0.804, P < 0.001) and disease duration (rs  = 0.645, P < 0.001) and North Star Ambulatory Assessment total scores (rs  = -0.698, P < 0.001). Muscle western blot analysis confirmed that both four DMD (median = 2.958, P < 0.05) and four BMD (median = 1.959, P < 0.01) patients had a significantly higher level of CDKN2A protein expression than controls (median = 1.068). The snRNA-seq analysis of two DMD muscles revealed that CDKN2A was mainly expressed in fibro-adipogenic progenitors, satellite cells, and myoblasts. CONCLUSIONS: We identify CDKN2A expression as a novel biomarker of fibrofatty replacement, which might be a new target for antifibrotic therapy in dystrophinopathies.

Key phonon modes to determine the phase transition of two dimensional Janus transition metal dichalcogenides: a DFT and tight-binding study.

Phase stability and the phase transition of Janus transition metal chalcogenides (TMDs) have become interesting issues that have not been fully resolved since their successful synthesis. By fitting the results from first principles calculations, a tight-binding dynamics matrix of the 1T' phase is constructed and the eigenvectors are also obtained. We propose a method to project the atomic motion causing the phase transition from 2H to 1T' onto these eigenvectors, and identify four key phonon modes which are the major factors to trigger phase transition. Temperature excitation is used to excite the key modes and the free energy criterion is used to determine the phase stability. The relatively large enthalpy difference between the 2H and 1T' phases favours the 2H one as the stable phase at low temperature. While the 1T' phase has a quick increase in vibrational free energy with rising temperature, especially for 1T' Janus TMDs which have a quicker increase in the total free energy than that of 1T' non-Janus TMDs, making them show a lower phase transition temperature. Our work will deepen our understanding of the phase transition behavior of 2D Janus TMDs, and the tight-binding dynamics matrix and the method to obtain the key modes will be a useful tool for further study of the phase transitions of 2D Janus TMDs and other related materials.

Cryptic exon activation caused by a novel deep-intronic splice-altering variant in Becker muscular dystrophy.

BACKGROUND: An accurate genetic diagnosis of Becker muscular dystrophy (BMD) can be sometimes challenging due to deep intronic DMD variants. Here, we report on the genetic diagnosis of a BMD patient with a novel deep-intronic splice-altering variant in DMD. METHODS: The index case was a 3.8-year-old boy who was suspected of having a diagnosis of BMD based on his clinical, muscle imaging, and pathological features. Routine genomic detection approaches did not detect any disease-causing variants in him. Muscle-derived DMD mRNA studies, followed by genomic Sanger sequencing and in silico bioinformatic analyses, were performed in the patient. RESULTS: DMD mRNA studies detected a cryptic exon-containing transcript and normally spliced DMD transcript in the patient. The cryptic exon-containing transcript encoded a frameshift and premature termination codon (NP_003997.1:p.[=,Asp2740Valfs*52]). Further genomic Sanger sequencing and bioinformatic analysis identified a novel deep-intronic splice-altering variant in DMD (c.8217 + 23338A > G). The novel variant strengthened a cryptic donor splice site and activated a cryptic acceptor splice site in the deep-intronic region of DMD intron 55, resulting in the activation of a new dystrophin cryptic exon found in the patient. CONCLUSION: Our case report expands the genetic spectrum of BMD and highlights the essential role of deep-intronic cryptic exon-activating variants in genetically unsolved BMD patients.

Pathogenic PSAT1 Variants and Autosomal Recessive Axonal Charcot-Marie-Tooth Disease With Ichthyosis.

BACKGROUND: Biallelic pathogenic phosphoserine aminotransferase 1 (PSAT1) variants generally cause a severe phenotype predominantly involving the central nervous system. Here, for the first time, we report two patients harboring pathogenic PSAT1 variants only manifested as polyneuropathy and ichthyosis. METHODS: Two patients from unrelated families presenting with polyneuropathy and ichthyosis were enrolled. Whole exome sequencing was performed to identify possible disease-causing variants. Their clinical, electrophysiological, imaging, biochemical, and pathologic changes were in detail assessed and investigated. RESULTS: Homozygous variant c.43G>C and compound heterozygous variants c.112A>C and c.43G>C in PSAT1 were identified in patients 1 and 2, respectively. Nerve conduction studies revealed preserved or mild slowing motor nerve conduction velocities of the median nerves in the two patients, whereas the compound motor action potential in patient 1 was severely decreased. Brain magnetic resonance imaging of the two patients found no abnormalities. Median nerve enlargement was observed on ultrasound in patient 1. Both patients had normal level of serine and glycine in plasma and cerebrospinal fluid. Sural nerve biopsy found severe loss of myelinated fibers. Electron microscopy revealed neurofilament accumulation and mitochondrial aggregation in axons. Both variants in PSAT1 were classified as likely pathogenic or pathogenic variants according to the standard guidelines. CONCLUSIONS: Our study confirms that pathogenic PSAT1 variants can cause a mild phenotype, predominantly as autosomal recessive axonal Charcot-Marie-Tooth disease.

An in-frame pseudoexon activation caused by a novel deep-intronic variant in the dysferlin gene.

The precise detection and interpretation of pathogenic DYSF variants are sometimes challenging, largely due to rare deep-intronic splice-altering variants. Here, we report on the genetic diagnosis of a male patient with dysferlinopathy. He remained genetically unsolved after routine exonic detection approaches that only detected a novel heterozygous frameshift variant (c.407dup, p.Thr137Tyrfs*11) in DYSF exon 5. Via muscle-derived DYSF mRNA studies, we identified a novel deep-intronic DYSF variant in the other allele (c.1397 + 649C > T), which causing in-frame alterations in DYSF mRNA and protein structure and confirmed his genetic diagnosis of dysferlinopathy. Our study emphasizes the potential role of undetected deep-intronic splice-altering variants in monogenic diseases.

Clinical, muscle imaging, and genetic characteristics of dystrophinopathies with deep-intronic DMD variants.

BACKGROUND: Phenotypic heterogeneity within or between families with a same deep-intronic splice-altering variant in the DMD gene has never been systematically analyzed. This study aimed to determine the phenotypic and genetic characteristics of patients with deep-intronic DMD variants. METHODS: Of 1338 male patients with a suspected dystrophinopathy, 38 were confirmed to have atypical pathogenic DMD variants via our comprehensive genetic testing approach. Of the 38 patients, 30 patients from 22 unrelated families with deep-intronic DMD variants underwent a detailed clinical and imaging assessment. RESULTS: Nineteen different deep-intronic DMD variants were identified in the 30 patients, including 15 with Duchenne muscular dystrophy (DMD), 14 with Becker muscular dystrophy (BMD), and one with X-linked dilated cardiomyopathy. Of the 19 variants, 15 were single-nucleotide variants, 2 were structural variants (SVs), and 2 were pure-intronic large-scale SVs causing aberrant inclusion of other protein-coding genes sequences into the mature DMD transcripts. The trefoil with single fruit sign was observed in 18 patients and the concentric fatty infiltration pattern was observed in 2 patients. Remarkable phenotypic heterogeneity was observed not only in skeletal but also cardiac muscle involvement in 2 families harboring a same deep-intronic variant. Different skeletal muscle involvement between families with a same variant was observed in 4 families. High inter-individual phenotypic heterogeneity was observed within two BMD families and one DMD family. CONCLUSIONS: Our study first highlights the variable phenotypic expressivity of deep-intronic DMD variants and demonstrates a new class of deep-intronic DMD variants, i.e., pure-intronic SVs involving other protein-coding genes.

Simulation of the Particle Transport Behaviors in Nanoporous Matter.

The transport behaviors of proton into nanoporous materials were investigated using different Monte Carlo simulation codes such as GEANT4, Deeper and SRIM. The results indicated that porous structure could enhance the proton scattering effects due to a higher specific surface area and more boundaries. The existence of voids can deepen and widen the proton distribution in the targets due to relatively lower apparent density. Thus, the incident protons would transport deeper and form a wider Bragg peak in the end of the range, as the target materials are in a higher porosity state and/or have a larger pore size. The existence of voids also causes the local inhomogeneity of proton/energy distribution in micro/nano scales. As compared, the commonly used SRIM code can only be used to estimate roughly the incident proton range in nanoporous materials, based on a homogeneous apparent density equivalence rule. Moreover, the estimated errors of the proton range tend to increase with the porosity. The Deeper code (designed for evaluation of radiation effects of nuclear materials) can be used to simulate the transport behaviors of protons or heavy ions in a real porous material with porosity smaller than 52.3% due to its modeling difficulty, while the GEANT4 code has shown advantages in that it is suitable and has been proven to simulate proton transportation in nanoporous materials with porosity in its full range of 0~100%. The GEANT4 simulation results are proved consistent with the experimental data, implying compatibility to deal with ion transportation into homogeneously nanoporous materials.

Exonization of a deep intronic long interspersed nuclear element in Becker muscular dystrophy.

The precise identification of pathogenic DMD variants is sometimes rather difficult, mainly due to complex structural variants (SVs) and deep intronic splice-altering variants. We performed genomic long-read whole DMD gene sequencing in a boy with asymptomatic hyper-creatine kinase-emia who remained genetically undiagnosed after standard genetic testing, dystrophin protein and DMD mRNA studies, and genomic short-read whole DMD gene sequencing. We successfully identified a novel pathogenic SV in DMD intron 1 via long-read sequencing. The deep intronic SV consists of a long interspersed nuclear element-1 (LINE-1) insertion/non-tandem duplication rearrangement causing partial exonization of the LINE-1, establishing a genetic diagnosis of Becker muscular dystrophy. Our study expands the genetic spectrum of dystrophinopathies and highlights the significant role of disease-causing LINE-1 insertions in monogenic diseases.

First Identification of Rare Exonic and Deep Intronic Splice-Altering Variants in Patients With Beta-Sarcoglycanopathy.

Background: The precise genetic diagnosis of a sarcoglycanopathy or dystrophinopathy is sometimes extremely challenging, as pathogenic non-coding variants and/or complex structural variants do exist in DMD or sarcoglycan genes. This study aimed to determine the genetic diagnosis of three patients from two unrelated families with a suspected sarcoglycanopathy or dystrophinopathy based on their clinical, radiological, and pathological features, for whom routine genomic detection approaches failed to yield a definite genetic diagnosis. Methods: Muscle-derived reverse transcription-polymerase chain reaction analysis and/or TA cloning of DMD, SGCA, SGCB, SGCD, and SGCG mRNA were performed to identify aberrant transcripts. Genomic Sanger sequencing around the aberrant transcripts was performed to detect possible splice-altering variants. Bioinformatic and segregation studies of the detected genomic variants were performed in both families. Results: In patients F1-II1 and F1-II2, we identified two novel pathogenic compound heterozygous variants in SGCB. One is a deep intronic splice-altering variant (DISV), c.243 + 1558C > T in intron 2 causing the activation of an 87-base pair (bp) pseudoexon, and the other one is a non-canonical splicing site variant, c.243 + 6T > A leading to the partial intron inclusion of 10-bp sequence. A novel DISV, c.243 + 1576C > G causing a 106-bp pseudoexon activation, and a nonsense variant in SGCB were identified in compound heterozygous state in patient F2-II1. Unexpectedly, the predicted nonsense variant, c.334C > T in exon 3, created a new donor splice site in exon 3 that was stronger than the natural one, resulting in a 97-bp deletion of exon 3 (r.333_429del). Conclusion: This is the first identification of rare exonic and DISVs in the SGCB gene.

MnIn2S4 nanosheets growing on rods-like ß-MnO2 via covalent bonds as high-performance photocatalyst for boosting Cr(VI) photocatalytic reduction under visible light irradiation: Behavior and mechanism study.

It is an urgent and onerous task to develop catalysts for photocatalytic reduction of Cr(VI) in wastewater under wide pH range. In this work, a novel hierarchical Z-scheme MnO2/MnIn2S4 (MISO) heterojunction photocatalyst with MnIn2S4 nanosheets growing on the surface of ß-MnO2 nanorods is constructed for efficient photocatalytic reduction of Cr(VI). The optimized 2.0-MISO photocatalyst exhibits the almost 100% reduction efficiency in the pH range of 2.1-5.6 under visible light irradiation, and the apparent rate constant is 0.05814 min-1, which is 29.96 and 3.27 times higher than the pure ß-MnO2 and MnIn2S4, respectively. A efficient photocatalytic reduction of Cr(VI) to Cr(III) species on 2.0-MISO photocatalyst in actual industry wastewater (286.7 mg/L) up to 99.8% is achieved. Under natural light, the 2.0-MISO photocatalyst also shows rapid reduction of Cr(VI) species. The photocorrosion of MnIn2S4 was significantly hindered by the construction of heterojunction. And the O2- and e- species are the main active species during the Cr(VI) photoreduction process. The connection mode between MnIn2S4 and ß-MnO2 is verified by DFT calculations and a possible photocatalytic mechanism is also proposed.

Efficient CO2 Electroreduction via Au-Complex Derived Carbon Nanotube Supported Au Nanoclusters.

The production of value-added chemicals from CO2 electroreduction, using renewable energy, provides an appealing route to achieve the goal of carbon neutrality. Challenges remain in designing and understanding of high-performance catalysts with restructuring behavior under electrochemical conditions. Here, the intrinsic performance enhancement of an Au-complex derived carbon nanotube-supported Au nanoclusters catalyst was demonstrated for CO2 reduction. This catalyst exhibited impressive activity for yielding CO in both H-cell and flow cell reactors. Experimental results revealed that the synthesis procedure via metal complex reconstructing on proper support induced charge transfer between Au nanoclusters and carbon nanotubes, forming a rather electron-rich state for Au active sites, which greatly contributed to the CO2 activation pathway.

Practical approach to the genetic diagnosis of unsolved dystrophinopathies: a stepwise strategy in the genomic era.

OBJECTIVE: To investigate the diagnostic value of implementing a stepwise genetic testing strategy (SGTS) in genetically unsolved cases with dystrophinopathies. METHODS: After routine genetic testing in 872 male patients with highly suspected dystrophinopathies, we identified 715 patients with a pathogenic DMD variant. Of the 157 patients who had no pathogenic DMD variants and underwent a muscle biopsy, 142 patients were confirmed to have other myopathies, and 15 suspected dystrophinopathies remained genetically undiagnosed. These 15 patients underwent a more comprehensive evaluation as part of the SGTS pipeline, which included the stepwise analysis of dystrophin mRNA, short-read whole-gene DMD sequencing, long-read whole-gene DMD sequencing and in silico bioinformatic analyses. RESULTS: SGTS successfully yielded a molecular diagnosis of dystrophinopathy in 11 of the 15 genetically unsolved cases. We identified 8 intronic and 2 complex structural variants (SVs) leading to aberrant splicing in 10 of 11 patients, of which 9 variants were novel. In one case, a molecular defect was detected on mRNA and protein level only. Aberrant splicing mechanisms included 6 pseudoexon inclusions and 4 alterations of splice sites and splicing regulatory elements. We showed for the first time the exonisation of a MER48 element as a novel pathogenic mechanism in dystrophinopathies. CONCLUSION: Our study highlights the high diagnostic utility of implementing a SGTS pipeline in dystrophinopathies with intronic variants and complex SVs.

Splicing Characteristics of Dystrophin Pseudoexons and Identification of a Novel Pathogenic Intronic Variant in the DMD Gene.

Pseudoexon (PE) inclusion has been implicated in various dystrophinopathies; however, its splicing characteristics have not been fully investigated. This study aims to analyze the splicing characteristics of dystrophin PEs and compare them with those of dystrophin canonical exons (CEs). Forty-two reported dystrophin PEs were divided into a splice site (ss) group and a splicing regulatory element (SRE) group. Five dystrophin PEs with characteristics of poison exons were identified and categorized as the possible poison exon group. The comparative analysis of each essential splicing signal among different groups of dystrophin PEs and dystrophin CEs revealed that the possible poison exon group had a stronger 3' ss compared to any other group. As for auxiliary SREs, different groups of dystrophin PEs were found to have a smaller density of diverse types of exonic splicing enhancers and a higher density of several types of exonic splicing silencers compared to dystrophin CEs. In addition, the possible poison exon group had a smaller density of 3' ss intronic splicing silencers compared to dystrophin CEs. To our knowledge, our findings indicate for the first time that poison exons might exist in DMD (the dystrophin gene) and present with different splicing characteristics than other dystrophin PEs and CEs.

Long-read whole-genome sequencing for the genetic diagnosis of dystrophinopathies.

The precise genetic diagnosis of dystrophinopathies can be challenging, largely due to rare deep intronic variants and more complex structural variants (SVs). We report on the genetic characterization of a dystrophinopathy patient. He remained without a genetic diagnosis after routine genetic testing, dystrophin protein and mRNA analysis, and short- and long-read whole DMD gene sequencing. We finally identified a novel complex SV in DMD via long-read whole-genome sequencing. The variant consists of a large-scale (~1Mb) inversion/deletion-insertion rearrangement mediated by LINE-1s. Our study shows that long-read whole-genome sequencing can serve as a clinical diagnostic tool for genetically unsolved dystrophinopathies.