Dystrophy Gene Research Articles

Exome sequencing reveals neurodevelopmental genes in simplex consanguineous Iranian families with syndromic autism

Background and objectiveAutosomal recessive genetic disorders pose significant health challenges in regions where consanguineous marriages are prevalent. The utilization of exome sequencing as a frequently employed methodology has enabled a clear delineation of diagnostic efficacy and mode of inheritance within multiplex consanguineous families. However, these aspects remain less elucidated within simplex families.MethodsIn this study involving 12 unrelated simplex Iranian families presenting syndromic autism, we conducted singleton exome sequencing. The identified genetic variants were validated using Sanger sequencing, and for the missense variants in FOXG1 and DMD, 3D protein structure modeling was carried out to substantiate their pathogenicity. To examine the expression patterns of the candidate genes in the fetal brain, adult brain, and muscle, RT-qPCR was employed.ResultsIn four families, we detected an autosomal dominant gene (FOXG1), an autosomal recessive gene (CHKB), and two X-linked autism genes (IQSEC2 and DMD), indicating diverse inheritance patterns. In the remaining eight families, we were unable to identify any disease-associated genes. As a result, our variant detection rate stood at 33.3% (4/12), surpassing rates reported in similar studies of smaller cohorts. Among the four newly identified coding variants, three are de novo (heterozygous variant p.Trp546Ter in IQSEC2, heterozygous variant p.Ala188Glu in FOXG1, and hemizygous variant p.Leu211Met in DMD), while the homozygous variant p.Glu128Ter in CHKB was inherited from both healthy heterozygous parents. 3D protein structure modeling was carried out for the missense variants in FOXG1 and DMD, which predicted steric hindrance and spatial inhibition, respectively, supporting the pathogenicity of these human mutants. Additionally, the nonsense variant in CHKB is anticipated to influence its dimerization – crucial for choline kinase function – and the nonsense variant in IQSEC2 is predicted to eliminate three functional domains. Consequently, these distinct variants found in four unrelated individuals with autism are likely indicative of loss-of-function mutations.ConclusionsIn our two syndromic autism families, we discovered variants in two muscular dystrophy genes, DMD and CHKB. Given that DMD and CHKB are recognized for their participation in the non-cognitive manifestations of muscular dystrophy, it indicates that some genes transcend the boundary of apparently unrelated clinical categories, thereby establishing a novel connection between ASD and muscular dystrophy. Our findings also shed light on the complex inheritance patterns observed in Iranian consanguineous simplex families and emphasize the connection between autism spectrum disorder and muscular dystrophy. This underscores a likely genetic convergence between neurodevelopmental and neuromuscular disorders.

Confined placental mosaicism is a diagnostic pitfall in dystrophinopathies: a clinical report.

Single-gene copy number variants (CNVs) limited to placenta although rarely identified may have clinical implications. We describe a pregnant woman referred for chorionic villus sampling due to increased fetal nuchal translucency. Incident intragenic deletion of Duchenne muscular dystrophy (DMD) gene, affecting exons 56 and 57, was identified in a male fetus in ~23-30% of placental cells by chromosomal microarray and confirmed using multiplex ligation-dependent probe amplification (MLPA). Rapid aneuploidy testing showed normal results and the deletion was not detected in the mother. Subsequent analyses on amniotic cells yielded a normal DMD gene result, corroborating the confined placental nature of the mosaicism. Hence, this report emphasizes the importance of conducting amniocentesis following detection of mosaicism for single gene CNVs on chorionic villi, in order to preclude confined placental mosaicism (CPM). As far as we know, this report marks only the second documented situation of CPM involving an intragenic DMD deletion.

Boy Dosed with Pfizer's Duchenne Muscular Dystrophy Gene Therapy Dies a Year After Phase II Trial.

Boy Dosed with Pfizer's Duchenne Muscular Dystrophy Gene Therapy Dies a Year After Phase II Trial.

Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

System-level analysis of genes mutated in muscular dystrophies reveals a functional pattern associated with muscle weakness distribution

Muscular dystrophies (MDs) are inherited genetic diseases causing weakness and degeneration of muscles. The distribution of muscle weakness differs between MDs, involving distal muscles or proximal muscles. While the mutations in most of the MD-associated genes lead to either distal or proximal onset, there are also genes whose mutations can cause both types of onsets. We hypothesized that the genes associated with different MD onsets code proteins with distinct cellular functions. To investigate this, we collected the MD-associated genes and assigned them to three onset groups: genes mutated only in distal onset dystrophies, genes mutated only in proximal onset dystrophies, and genes mutated in both types of onsets. We then systematically evaluated the cellular functions of these gene sets with computational strategies based on functional enrichment analysis and biological network analysis. Our analyses demonstrate that genes mutated in either distal or proximal onset MDs code proteins linked with two distinct sets of cellular processes. Interestingly, these two sets of cellular processes are relevant for the genes that are associated with both onsets. Moreover, the genes associated with both onsets display high centrality and connectivity in the network of muscular dystrophy genes. Our findings support the hypothesis that the proteins associated with distal or proximal onsets have distinct functional characteristics, whereas the proteins associated with both onsets are multifunctional.

Differential expression of DMD transcripts as a novel prognostic biomarker in histologically diverse mesotheliomas.

The identification of prognostic biomarkers is crucial for guiding treatment strategies in mesothelioma patients. The Duchenne muscular dystrophy (DMD) gene and its specific transcripts have been associated with patient survival in various tumours. In this study, we aimed to investigate the prognostic potential of DMD gene expression and its transcripts in mesothelioma patients. We analysed The Cancer Genome Atlas (TCGA) mesothelioma RNAseq, mutation, and clinical data to assess the association between DMD gene expression and its transcripts (Dp427, Dp71 splice variants) and mesothelioma survival. We also evaluated the specific Dp71 transcript as a unique prognostic biomarker across mesothelioma subtypes. Additionally, we performed differential gene expression analysis between high and low DMD gene/transcript expression groups. The analysis included 57 epithelioid, 23 biphasic, two sarcomatoid, and five not otherwise specified (NOS) histological subtypes of mesothelioma samples. Univariate analysis revealed that high expression of the DMD gene and its Dp71 transcript was significantly associated with shorter survival in mesothelioma patients (P=0.003 and P<0.001, respectively). In a multivariate analysis, the association between Dp71 expression and survival remained significant [hazard ratio (HR) 2.29, 95% confidence interval (CI): 1.24-4.23, P=0.008] across all mesothelioma patients, and also among patients with mesotheliomas without deep CDKN2A deletions (HR 3.58, 95% CI: 1.31-9.80, P=0.01). Pathway analysis revealed enrichment of cell cycle (P=3.01×10-4) and homologous recombination (P=0.01) pathways in differentially expressed genes (DEGs) between high and low Dp71 groups. Furthermore, there were correlations between Dp71 transcript expression and tumour microenvironment (TME) cells, including a weak positive correlation with macrophages (R=0.32, P=0.002) specifically M2 macrophages (R=0.34, P=0.001). Our findings indicate that the differential expression of specific DMD transcripts is associated with poor survival in mesothelioma patients. The specific Dp71 transcript can serve as a potential biomarker for predicting patient survival in diverse histological subtypes of mesothelioma. Further studies are needed to understand the role of specific dystrophin transcripts in cancer and TME cells, and their implications in the pathogenesis and progression of mesothelioma. Identifying patients at risk of poor survival based on DMD transcript expression can guide treatment strategies in mesothelioma, informing decisions regarding treatment intensity, follow-up schedules, eligibility for clinical trials, and ultimately, end-of-life care planning.

Clinical and genetic characterisation of a large Indian congenital myasthenic syndrome cohort.

Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and β2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014-19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1-56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0-49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects.

Myocardial native T1 mapping and extracellular volume quantification in asymptomatic female carriers of Duchenne muscular dystrophy gene mutations

BackgroundFemale carriers of dystrophin gene mutations (DMD-FC) were previously considered non-manifesting, but in recent decades, cardiomyopathy associated with muscular dystrophy and myocardial fibrosis has been described. Our study aimed to assess prospectively myocardial fibrosis in asymptomatic DMD-FC compared to a sex-matched control group (CG) with similar age distribution using native T1 mapping and extracellular volume (ECV) quantification by cardiovascular magnetic resonance (CMR) imaging.Materials and methods38 DMD-FC with verified genetic mutation and 22 healthy volunteers were included. Using CMR, native T1 relaxation time and ECV quantification were determined in each group. Late gadolinium enhancement (LGE) was assessed in all cases.ResultsThere were 38 DMD-FC (mean age 39.1 ± 8.8 years) and 22 healthy volunteers (mean age 39.9 ± 12.6 years) imagined by CMR. The mean global native T1 relaxation time was similar for DMD-FC and CG (1005.1 ± 26.3 ms vs. 1003.5 ± 25.0 ms; p-value = 0.81). Likewise, the mean global ECV value was also similar between the groups (27.92 ± 2.02% vs. 27.10 ± 2.89%; p-value = 0.20). The segmental analysis of mean ECV values according to the American Heart Association classification did not show any differences between DMD-FC and CG. There was a non-significant trend towards higher mean ECV values of DMD-FC in the inferior and inferolateral segments of the myocardium (p-value = 0.075 and 0.070 respectively).ConclusionThere were no statistically significant differences in the mean global and segmental native T1 relaxation times and the mean global or segmental ECV values. There was a trend towards higher segmental mean ECV values of DMD-FC in the inferior and inferolateral walls of the myocardium.

Advances in Dystrophinopathy Diagnosis and Therapy.

Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the Dystrophin gene, including Duchenne and Becker muscular dystrophy, and dilated cardiomyopathy. However, Duchenne muscular dystrophy interconnects with bone loss and osteoporosis, which are exacerbated by glucocorticoids therapy. Procedures for diagnosing dystrophinopathies include creatine kinase assay, haplotype analysis, Southern blot analysis, immunological analysis, multiplex PCR, multiplex ligation-dependent probe amplification, Sanger DNA sequencing, and next generation DNA sequencing. Pharmacological therapy for dystrophinopathies comprises glucocorticoids (prednisone, prednisolone, and deflazacort), vamorolone, and ataluren. However, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and β-blockers are the first-line to prevent dilated cardiomyopathy in dystrophinopathy patients. Duchenne muscular dystrophy gene therapy strategies involve gene transfer, exon skipping, exon reframing, and CRISPR gene editing. Eteplirsen, an antisense-oligonucleotide drug for skipping exon 51 from the Dystrophin gene, is available on the market, which may help up to 14% of Duchenne muscular dystrophy patients. There are various FDA-approved exon skipping drugs including ExonDys-51 for exon 51, VyonDys-53 and Viltolarsen for exon 53 and AmonDys-45 for exon 45 skipping. Other antisense oligonucleotide drugs in the pipeline include casimersen for exon 45, suvodirsen for exon 51, and golodirsen for exon 53 skipping. Advances in the diagnosis and therapy of dystrophinopathies offer new perspectives for their early discovery and care.

Sildenafil increases AAV9 transduction after a systemic administration and enhances AAV9-dystrophin therapeutic effect in mdx mice.

Adeno-associated virus (AAV) vectors have been successfully used to deliver genes for treating rare diseases. However, the systemic administration of high AAV vector doses triggers several adverse effects, including immune response, the asymptomatic elevation of liver transaminase levels, and complement activation. Thus, improving AAV transduction and reducing AAV dosage for treatment is necessary. Recently, we found that a phosphodiesterase-5 inhibitor significantly promoted AAV9 transduction in vitro by regulating the caveolae and macropinocytosis pathways. When AAV9-Gaussian luciferase (AAV9-Gluc) and AAV9-green fluorescent protein (AAV9-GFP) were injected intravenously into mice pre-treated with sildenafil, the expressions of Gluc in the plasma and GFP in muscle tissues significantly increased (P < 0.05). Sildenafil also improved Evans blue permeation in tissues. Additionally, we found that sildenafil promoted Treg proliferation, inhibited B-cell activation, and decreased anti-AAV9 IgG levels (P < 0.05). Furthermore, sildenafil significantly promoted Duchenne muscular dystrophy gene therapy efficacy using AAV9 in mdx mice; it increased micro-dystrophin gene expression, forelimb grip strength, and time spent on the rotarod test, decreased serum creatine kinase levels, and ameliorated histopathology by improving muscle cell morphology and reducing fibrosis (P < 0.05). These results show that sildenafil significantly improved AAV transduction, suppressed the levels of anti-AAV9 IgG, and enhanced the efficacy of gene therapy.

Food and Drug Administration Sets Stage for Approval of First Duchenne Muscular Dystrophy Gene Therapy.

Human Gene TherapyVol. 34, No. 11-12 Gene Therapy BriefsFood and Drug Administration Sets Stage for Approval of First Duchenne Muscular Dystrophy Gene TherapyAlex PhilippidisAlex Philippidis*Correspondence: E-mail Address: aphilippidis@liebertpub.comMary Ann Liebert, Inc., Publishers, New Rochelle, New York, USA.Search for more papers by this authorPublished Online:16 Jun 2023https://doi.org/10.1089/hum.2023.29243.bfsAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 34Issue 11-12Jun 2023 InformationCopyright 2023, by Mary Ann Liebert, Inc., publishersTo cite this article:Alex Philippidis.Food and Drug Administration Sets Stage for Approval of First Duchenne Muscular Dystrophy Gene Therapy.Human Gene Therapy.Jun 2023.477-480.http://doi.org/10.1089/hum.2023.29243.bfsPublished in Volume: 34 Issue 11-12: June 16, 2023PDF download

Duchenne Muscular Dystrophy Gene Therapy in 2023: Status, Perspective, and Beyond.

Duchenne muscular dystrophy (DMD) was named more than 150 years ago. About four decades ago, the DMD gene was discovered, and the reading frame shift was determined as the genetic underpinning. These pivotal findings significantly changed the landscape of DMD therapy development. Restoration of dystrophin expression with gene therapy became a primary focus. Investment in gene therapy has led to the approval of exon skipping by regulatory agencies, multiple clinical trials of systemic microdystrophin therapy using adeno-associated virus vectors, and revolutionary genome editing therapy using the CRISPR technology. However, many important issues surfaced during the clinical translation of DMD gene therapy (such as low efficiency of exon skipping, immune toxicity-induced serious adverse events, and patient death). In this issue of Human Gene Therapy, several research articles highlighted some of the latest developments in DMD gene therapy. Importantly, a collection of articles from experts in the field reviewed the progress, major challenges, and future directions of DMD gene therapy. These insightful discussions have significant implications for gene therapy of other neuromuscular diseases.

Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

A Deletion in Duchenne Muscular Dystrophy Gene Found Through Whole Exome Sequencing in Iran.

Duchenne muscular dystrophy (DMD) is a severe progressive X-linked neuromuscular illness that affects movement through mutations in dystrophin gene. The mutation leads to insufficient, lack of, or dysfunction of dystrophin. The cause of DMD was determined in an Iranian family. Exome sequencing was carried out along with a complete physical examination of the family. In silico methods were applied to find the alteration in the protein structure. The homozygous variant in DMD gene (NM-004006.2) was defined as c.2732-2733delTT (p.Phe911CysfsX8) in exon 21. In addition, phylogenetic conservation study of the human dystrophin protein sequence revealed that phenylalanine 911 is one of the evolutionarily conserved amino acids. In conclusion, our study indicated a new deletion in the DMD gene in the affected family. This deletion with an X-linked inheritance pattern is new in Iran. These findings could facilitate genetic counseling for this family and other patients in the future.

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.

Uncovering the Gene Regulatory Network of Endothelial Cells in Mouse Duchenne Muscular Dystrophy: Insights from Single-Nuclei RNA Sequencing Analysis.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by mutations in the dystrophin gene, which leads to heart and respiratory failure. Despite the critical impact of DMD on endothelial cells (ECs), there is limited understanding of its effect on the endothelial gene network. The aim of this study was to investigate the impact of DMD on the gene regulatory network of ECs. To gain insights into the role of the dystrophin muscular dystrophy gene (DMD) in ECs from Duchenne muscular dystrophy; the study utilized single-nuclei RNA sequencing (snRNA-seq) to evaluate the transcriptomic profile of ECs from skeletal muscles in DMD mutant mice (DMDmut) and wild-type control mice. The analysis showed that the DMD mutation resulted in the suppression of several genes, including SPTBN1 and the upregulation of multiple long noncoding RNAs (lncRNAs). GM48099, GM19951, and GM15564 were consistently upregulated in ECs and skeletal muscle cells from DMDmut, indicating that these dysregulated lncRNAs are conserved across different cell types. Gene ontology (GO) enrichment analysis revealed that the DMD mutation activated the following four pathways in ECs: fibrillary collagen trimer, banded collagen fibril, complex of collagen trimers, and purine nucleotide metabolism. The study also found that the metabolic pathway activity of ECs was altered. Oxidative phosphorylation (OXPHOS), fatty acid degradation, glycolysis, and pyruvate metabolism were decreased while purine metabolism, pyrimidine metabolism, and one carbon pool by folate were increased. Moreover, the study investigated the impact of the DMD mutation on ECs from skeletal muscles and found a significant decrease in their overall number, but no change in their proliferation. Overall, this study provides new insights into the gene regulatory program in ECs in DMD and highlights the importance of further research in this area.

Variants in the Sequence of the Probe Hybridization Site May Affect MLPA Performance in Patients with Duchenne/Becker Muscular Dystrophy.

The MLPA (multiplex ligation dependent probe amplification) technique is currently the test most widely used to detect mutations in the Duchenne/Becker muscular dystrophy (DMD) gene in the initial assessment. However, several studies have suggested that MLPA results require implementing other detection methods due to false duplication. Our aim was to evaluate variables that could alter the peak ratio in MLPA in individuals with Duchenne/Becker muscular dystrophy (DMD/BMD) who present sequence variants at the probe hybridization site, such as the location of sequence variants (SVs), melting temperature of the probe, and the type of variant. We analyzed patients with clinical suspicion of DMD/BMD through the MLPA technique. The DMD gene was sequenced in patients with normal results in MLPA. Of 111 patients, 72 had an abnormal MLPA result, of which 10 had a single exon abnormal peak, and 39 had a normal peak ratio. Out of 10 patients, 4 (40%) with a single exon abnormal peak ratio had SV at the hybridization site of the probe. In the other 6, the deletion was confirmed. Out of 39 patients with a normal peak ratio, 11 presented SVs at the hybridization site of the probe, and DMD/BMD was confirmed. In cases of abnormal peak ratio results of MLPA in a single exon, it would be valuable to sequence the DMD gene to assess whether variants in the probe hybridization site might result in a false positive that could be interpreted as an exon deletion.

P558: Two distinct rearrangements in one family within the Duchenne Muscular Dystrophy gene

P558: Two distinct rearrangements in one family within the Duchenne Muscular Dystrophy gene

FRG1 is a direct transcriptional regulator of nonsense-mediated mRNA decay genes

FRG1 is the primary candidate gene for Fascioscapulohumeral Muscular Dystrophy. So far, its role has been reported in muscle development, vasculogenesis, angiogenesis, and tumorigenesis. Mechanistically studies suggest FRG1's role in RNA biogenesis which may have implications in multiple physiological processes and diseases, including tumorigenesis. Its probable role as hnRNP and association with NMD-related genes prompted us to look into FRG1's effect on NMD gene expression and the mechanism. Using microarray profiling in cell lines, we found that FRG1 altered the mRNA surveillance pathway and associated pathways, such as RNA transport and spliceosome machinery molecules. Multiple sequence alignment of core factors, namely, UPF1, UPF3B, and SMG1, showed conserved stretches of nucleotide sequence ‘CTGGG’. Structural modeling followed by EMSA, ChIP-qPCR, and luciferase reporter assays showed ‘CTGGG’ as a FRG1 binding site. Analysis of the publicly available datasets showed that the expression of FRG1 correlates with NMD genes in different tissue types. We validated the effect of FRG1 on NMD gene transcription by qRT-PCR. Overall, FRG1 might be a transcriptional regulator of NMD genes.

Dp71 Point Mutations Induce Protein Aggregation, Loss of Nuclear Lamina Integrity and Impaired Braf35 and Ibraf Function in Neuronal Cells.

Dystrophin Dp71 is the most abundant product of the Duchenne muscular dystrophy gene in the nervous system, and mutations impairing its function have been associated with the neurodevelopmental symptoms present in a third of DMD patients. Dp71 is required for the clustering of neurotransmitter receptors and the neuronal differentiation of cultured cells; nonetheless, its precise role in neuronal cells remains to be poorly understood. In this study, we analyzed the effect of two pathogenic DMD gene point mutations on the Dp71 function in neurons. We engineered C272Y and E299del mutations to express GFP-tagged Dp71 protein variants in N1E-115 and SH-SY5Y neuronal cells. Unexpectedly, the ectopic expression of Dp71 mutants resulted in protein aggregation, which may be mechanistically caused by the effect of the mutations on Dp71 structure, as predicted by protein modeling and molecular dynamics simulations. Interestingly, Dp71 mutant variants acquired a dominant negative function that, in turn, dramatically impaired the distribution of different Dp71 protein partners, including β-dystroglycan, nuclear lamins A/C and B1, the high-mobility group (HMG)-containing protein (BRAF35) and the BRAF35-family-member inhibitor of BRAF35 (iBRAF). Further analysis of Dp71 mutants provided evidence showing a role for Dp71 in modulating both heterochromatin marker H3K9me2 organization and the neuronal genes’ expression, via its interaction with iBRAF and BRAF5.