Comprehensive analysis of m6A regulators characterized by the immune microenvironment in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked, incurable, degenerative neuromuscular disease that is exacerbated by secondary inflammation. N6-methyladenosine (m6A), the most common base modification of RNA, has pleiotropic immunomodulatory effects in many diseases. However, the role of m6A modification in the immune microenvironment of DMD remains elusive. METHODS: Our study retrospectively analyzed the expression data of 56 muscle tissues from DMD patients and 26 from non-muscular dystrophy individuals. Based on single sample gene set enrichment analysis, immune cells infiltration was identified and the result was validated by flow cytometry analysis and immunohistochemical staining. Then, we described the features of genetic variation in 26 m6A regulators and explored their relationship with the immune mircoenvironment of DMD patients through a series of bioinformatical analysis. At last, we determined subtypes of DMD patients by unsupervised clustering analysis and characterized the molecular and immune characteristics in different subgroups. RESULTS: DMD patients have a sophisticated immune microenvironment that is significantly different from non-DMD controls. Numerous m6A regulators were aberrantly expressed in the muscle tissues of DMD and inversely related to most muscle-infiltrating immune cell types and immune response-related signaling pathways. A diagnostic model involving seven m6A regulators was established using LASSO. Furthermore, we determined three m6A modification patterns (cluster A/B/C) with distinct immune microenvironmental characteristics. CONCLUSION: In summary, our study demonstrated that m6A regulators are intimately linked to the immune microenvironment of muscle tissues in DMD. These findings may facilitate a better understanding of the immunomodulatory mechanisms in DMD and provide novel strategies for the treatment.

NOTCH2NLC-related oculopharyngodistal myopathy type 3 complicated with focal segmental glomerular sclerosis: a case report.

BACKGROUND: Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ocular, facial, pharyngeal, and distal limb muscle involvement. Recent research showed that GGC repeat expansions in the NOTCH2NLC gene were observed in a proportion of OPDM patients, and these patients were designated as having OPDM type 3 (OPDM3). Heterogeneous neuromuscular manifestations have been described previously in studies of OPDM3; however, kidney involvement in this disease has rarely been reported. CASE PRESENTATION: Here, we report the case of a 22-year-old Chinese patient with typical manifestations of OPDM complicated with focal segmental glomerular sclerosis (FSGS). This patient with sporadic FSGS exhibited distal motor neuropathy and rimmed vacuolar myopathy in clinical and pathological examinations. An expansion of 122 CGG repeats located in the 5' untranslated region (UTR) of the NOTCH2NLC gene was identified as the causative mutation in this patient. The clinical and histopathological findings fully met the criteria for the diagnosis of OPDM3. In addition, intranuclear inclusions were detected in the renal tubule epithelial cells of this patient, indicating that the kidney may also be impaired in NOTCH2NLC-related GGC repeat expansion disorders (NREDs). CONCLUSIONS: Our case report demonstrated the clinicopathological cooccurrence of sporadic FSGS and OPDM3 in a patient, which highlighted that the kidney may show inclusion depositions in OPDM3, thus expanding the clinical spectrum of NREDs.

The clinical, myopathological, and molecular characteristics of 26 Chinese patients with dysferlinopathy: a high proportion of misdiagnosis and novel variants.

BACKGROUND: Dysferlinopathy is an autosomal recessive muscular dystrophy caused by pathogenic variants in the dysferlin (DYSF) gene. This disease shows heterogeneous clinical phenotypes and genetic characteristics. METHODS: We reviewed the clinical and pathological data as well as the molecular characteristics of 26 Chinese patients with dysferlinopathy screened by immunohistochemistry staining and pathogenic variants in DYSF genes. RESULTS: Among 26 patients with dysferlinopathy, 18 patients (69.2%) presented as Limb-girdle Muscular Dystrophy Type R2 (LGMD R2), 4 (15.4%) had a phenotype of Miyoshi myopathy (MM), and 4 (15.4%) presented as asymptomatic hyperCKemia. Fifteen patients (57.7%) were originally misdiagnosed as inflammatory myopathy or other diseases. Fifteen novel variants were identified among the 40 variant sites identified in this cohort. CONCLUSION: Dysferlinopathy is a clinically and genetically heterogeneous group of disorders with various phenotypes, a high proportion of novel variants, and a high rate of misdiagnosis before immunohistochemistry staining and genetic analysis.

The clinical, pathological, and genetic characteristics of lipid storage myopathy in northern China.

BACKGROUND: The lipid storage myopathy (LSM) diagnosis is based on the patient's clinical manifestations and muscle pathology. However, when genetic testing is lacking, there is a high rate of misdiagnosis of the disease. This study aimed to investigate the clinical and pathological features of genetically diagnosed LSM in northern China, analyze genetic mutations' characteristics, and improve the LSM diagnostic rate. METHODS: Twenty patients with LSM diagnosed were collected; meanwhile, the clinical data, muscle samples, and routine pathological staining of muscle specimens were collected. The morphological changes of muscle fibers were observed under an optical microscope. RESULTS: Among the included patients, 18 cases had ETFDH (HGNC ID: 3483) mutations, and two had PNPLA2 mutations. Family pedigree verification was performed on three patients with heterozygous mutations in the ETFDH gene complex. Histopathological staining showed that all patients had fine vacuoles in the muscle fibers, and some of them merged to form fissures, and the lipid droplets increased in cells. After therapy, 18 patients were associated with a favorable prognosis, and two patients were ineffective with the treatment of neutral lipid storage myopathy (NLSDM) caused by PNPLA2 mutation. DISCUSSION: The clinical manifestations of LSM are complex and diverse, mainly manifested by proximal muscle weakness and exercise intolerance in the extremities. The pathological images of LSM muscles are abnormal storage of lipid droplets in muscle fibers, primarily involving type I fibers. The LSM patients were mainly multiple acyl-CoA dehydrogenase deficiency (MADD) caused by the ETFDH gene mutation. It is necessary to perform an accurate typing diagnosis of LSM.

Golgi apparatus fragmentation participates in oxidized low-density lipoprotein-induced endothelial cell injury.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial injury plays crucial roles in the development of arteriosclerosis (AS). Golgi apparatus (GA) fragmentation is involved in various pathological processes, including endothelial injury. However, the role of GA fragmentation in ox-LDL-induced endothelial injury has not been determined. In this study, human umbilical vein endothelial cells (HUVECs) subjected to ox-LDL were used as an in vitro AS model. Herein, we showed that ox-LDL restrained proliferation and induced apoptosis and GA fragmentation of HUVECs. Moreover, overexpression of GRASP65 significantly prevented ox-LDL-induced GA fragmentation and endothelial cell injury by enhancing cell viability, nitric oxide production, and endothelial NOS expression and reducing apoptosis. Mechanistically, ox-LDL resulted in the activation of the extracellular signal-regulated kinase (ERK) pathway in HUVECs. Inactivation of the ERK pathway by U0126 suppressed the phosphorylation of GRASP65, GA fragmentation, and endothelial cell injury induced by ox-LDL. In conclusion, ox-LDL triggers GA fragmentation in HUVECs via activating the ERK signaling pathway, which participates in endothelial injury during the development of AS.

Adolescent Hyperuricemia with Lipid Storage Myopathy: A Clinical Study.

BACKGROUND In this study, we investigated the clinical and pathological features of patients with lipid storage myopathy (LSM) complicated with hyperuricemia, to improve clinicians' understanding of metabolic multi-muscular disorder with metabolic disorders, and to reduce the risk of missed diagnosis of LSM. MATERIAL AND METHODS From January 2005 to December 2017, 8 patients underwent muscle biopsy and diagnosed by muscle pathology and genetic testing in our hospital. All 8 patients were in compliance with LSM diagnosis. We collected data on the patient's clinical performance, adjuvant examination, treatment, and outcomes to provide a comprehensive report and description of LSM patients with hyperuricemia. RESULTS All patients were diagnosed as having ETFDH gene mutations. The main clinical manifestations of patients were chronic limb and trunk weakness, limb numbness, and muscle pain. The serum creatine kinase (CK) values in all patients were higher than normal values. Electromyography showed 3 cases of simple myogenic damage and 3 cases of neurogenic injury. Hematuria metabolic screening showed that 2 patients had elevated glutaric aciduria, and 1 patient had elevated fatty acyl carnitine in the blood. All patients were given riboflavin treatment, and the clinical symptoms were significantly improved, and 3 patients returned to normal uric acid levels after treatment. Pathological staining showed an abnormal deposition of lipid droplets in muscle fibers. CONCLUSIONS If an adolescent hyperuricemia patient has abnormal limb weakness, exercise intolerance, and elevated serum CK values, clinicians need to be highly alert to the possibility of LSM. Early diagnosis and treatment of LSM should improve the clinical symptoms and quality of life and reduce complications.

Identification of a novel SGCA missense mutation in a case of limb-girdle muscular dystrophy 2D with the absence of four sarcoglycan proteins.

Limb-girdle muscular dystrophy 2D (LGMD2D) is caused by mutations in the α-sarcoglycan gene (SGCA). Due to lack of specificity, it is impossible to identify LGMD2D only by clinical symptoms and conventional immunohistochemical staining. The loss of any protein (α-, ß-, γ-, δ-sarcoglycan) that represent sarcoglycanopathy may cause reduction or absence of the other three proteins. Here, we report a patient with a complete loss of all the four proteins. Next generation sequencing (NGS) results showed a missense mutation (C.218 C > T) and a partial heterozygous deletion containing exons 7 and 8 of SGCA, which led to the final diagnosis of the patient. The discovery of this new mutation could broaden the spectrum of SGCA mutations, which may be associated with putative LGMD2D, especially when all the four proteins are completely missing.

A novel mutation in the DYSF gene in a patient with a presumed inflammatory myopathy.

Dysferlinopathy, a progressive muscular dystrophy, results from mutations in the Dysferlin gene (DYSF, MIM*603009). Traditional diagnosis relies on the reduction or absence of dysferlin. However, altered dysferlin has been observed in other myopathies, leading to a precise diagnosis through molecular genetics. In this study, we report a patient who was previously misdiagnosed as inflammatory myopathy based on routine clinicopathological examinations alone. However, muscle biopsy specimens were analyzed further by immunohistochemistry of muscular dystrophy-related proteins, and gene-targeted next generation sequencing (NGS) was used to correctly identify muscular dystrophy. DNA was sequenced with NGS and the detected mutation was verified by Sanger sequencing. Our targeted NGS found a novel missense mutation (c.5392G > A) in the DYSF gene, allowing correct diagnosis of LGMD2B in our patient. We discovered of a novel missense mutation in the DYSF gene and have broadened the DYSF mutation spectrum, which may be correlated in patients with presumed dysferlinopathy, especially when lymphocytic infiltration is observed.

Case Report: A Novel Splice-Site Mutation in DNAJB6 Associated With Juvenile-Onset Proximal-Distal Myopathy in a Chinese Patient.

DNAJB6 was identified as the causative gene of limb-girdle muscular dystrophy type 1D. In recent years, the phenotypic and molecular spectrum of DNAJB6-myopathy has been expanded, and several mutations of DNAJB6 have been identified in Europe, North America, and Asia. Interestingly, almost all identified mutations in previous reports were point mutations, and most of them were clustered in exon 5, which encodes the G/F domain of DNAJB6. The so-far unique splice site mutation eliminating the entire G/F domain was reported to cause a severe, early-onset phenotype. Here, we report a juvenile-onset Chinese patient who presented with proximal-distal myopathy as well as esotropia and facial weakness. Muscle pathology showed rimmed vacuolation and myofibrillar disarrangement. A novel splice-site mutation NM_058246:c.236-1_240delGGTGGA of the DNAJB6 gene was identified by targeted exome sequencing, which results in a severe defect of the G/F domain. This rare mutation type expands the molecular spectrum of DNAJB6-myopathy and further underlines the importance of the G/F region.

Identification of Auxiliary Biomarkers and Description of the Immune Microenvironmental Characteristics in Duchenne Muscular Dystrophy by Bioinformatical Analysis and Experiment.

Background: Duchenne muscular dystrophy (DMD) is a genetic muscle disorder characterized by progressive muscle wasting associated with persistent inflammation. In this study, we aimed to identify auxiliary biomarkers and further characterize the immune microenvironment in DMD. Methods: Differentially expressed genes (DEGs) were identified between DMD and normal muscle tissues based on Gene Expression Omnibus (GEO) datasets. Bioinformatical analysis was used to screen and identify potential diagnostic signatures of DMD which were further validated by real-time quantitative reverse transcription PCR (RT-qPCR). We also performed single-sample gene-set enrichment analysis (ssGSEA) to characterize the proportion of tissue-infiltrating immune cells to determine the inflammatory state of DMD. Results: In total, 182 downregulated genes and 263 upregulated genes were identified in DMD. C3, SPP1, TMSB10, TYROBP were regarded as adjunct biomarkers and successfully validated by RT-qPCR. The infiltration of macrophages, CD4+, and CD8+ T cells was significantly higher (p < 0.05) in DMD compared with normal muscle tissues, while the infiltration of activated B cells, CD56dim natural killer cells, and type 17 T helper (Th17) cells was lower. In addition, the four biomarkers (C3, SPP1, TMSB10, TYROBP) were strongly associated with immune cells and immune-related pathways in DMD muscle tissues. Conclusion: Analyses demonstrated C3, SPP1, TMSB10, and TYROBP may serve as biomarkers and enhance our understanding of immune responses in DMD. The infiltration of immune cells into the muscle microenvironment might exert a critical impact on the development and occurrence of DMD.

Effect of AAV9-hIGF-1 on inflammatory reaction in mdx mice and its mechanism.

This study aimed to the role of insulin-like growth factor 1 (IGF-1) in Duchenne muscular dystrophy (DMD), the inflammatory response and the potential mechanism of the effect hIGF1 exerted in muscle inflammation were also been explored. In this study, AAV9, a carrier of the human IGF-1 gene, was injected into mdx mice to observe the role of IGF-1 in DMD. Routine histopathological staining, immunofluorescence and western blot were used to detect the inflammatory response. In addition, we also explored the potential mechanism of the role of hIGF1 in muscle inflammation. The expression of AAV9 in myocardium and muscle tissue of AAV9-GFP group was detected by GFP method. GFP was expressed in different tissues of mdx mice, especially in anterior tibial muscle, triceps muscle and other tissues. The percentage of anterior tibial muscle inflammation area in CD68 and AAV9-hIGF-1 group was lower than that in AAV-GFP group, and the percentage of anterior tibial muscle inflammation area in AAV9-hIGF-1 group (1.78 ± 0.47%) was significantly lower than that in AAV GFP group (3.4 ± 1.22%) (P < 0.05). Western-blot showed that AAV-hIGF-1 group (0.45 + 0.07%) was lower than that of AAV-GFP group (0.76 + 0.13%), higher than the normal group (0.38 + 0.06%). The difference was statistically significant (P < 0.05). In conclusion, this study confirmed that hIGF-1 can reduce the inflammatory response and macrophage infiltration in mdx mice, and further proved that hIGF-1 can down regulate the expression of NF-κB signal pathway, which has anti-inflammatory effect.

Activation of the Notch Signaling Pathway and Cellular Localization of Notch Signaling Molecules in the Spinal Cord of SOD1-G93A ALS Model Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron loss and gliosis in the spinal cord, brain stem and cortex. The Notch signaling pathway has been reported to be dysfunctional in neurodegenerative diseases, including ALS. However, the exact mechanism is still unclear. Here, we detected Notch signaling activation in proliferating glial cells, Notch inactivation in motor neurons in the spinal cord of the SOD1-G93A model, and dramatic changes of cellular relocalization of Notch pathway signaling molecules, including activated Notch intracellular domain (NICD), Notch ligands (Jagged1 and DLL4) and the target gene Hes1. We found that Notch activation was universal in proliferating astrocytes and that the Notch ligand Jagged1 was uniquely upregulated in proliferating microglia, while DLL4 expression was increased in both activated astrocytes and degenerating oligodendrocytes. Our results indicate that microglia may play an important role in the intercellular receptor-ligand interaction of the Notch signaling pathway and contribute to the pathogenesis of motor neuron loss in ALS mice. Further experiments are required to clarify the exact mechanism responsible for Notch dysfunction in ALS.

A novel DMD splicing mutation found in a family responsible for X-linked dilated cardiomyopathy with hyper-CKemia.

This study was aimed to detect a new mutation responsible for X-linked dilated cardiomyopathy with hyper-CKemia.We studied a proband who presented with cardiac symptoms with hyper-CKemia, but no clinical skeletal involvement in physical examination, laboratory tests, electromyography, echocardiography, and magnetic resonance imaging (MRI) of cardiac muscles. Muscle biopsy for histopathology and immunohistochemistry for accessing sarcolemma changes. The next-generation sequencing and bioinformatics analysis were performed on the patient and Sanger sequencing was confirmed on the other 6 unaffected families.The clinic investigations illustrated a dilated cardiomyopathy. Histopathology and immunohistochemistry showed dystrophic changes and an obvious reduction of dystrophin-N and δ-sarcoglycan, respectively. One hemizygous splicing pathogenic mutation c.31 + 1G > C of exon 1 in the DMD gene (chrX33229398, NM_00 4006) was finally identified in the patient and his nephew, but it was carried in his mother and sister.A novel small mutation was identified at the first exon-intron boundary splicing site by next-generation sequencing and bioinformatics analysis.

Adeno-associated virus serotype 9 mediated vascular endothelial growth factor gene overexpression in mdx mice.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by the absence of dystrophin. Vascular endothelial growth factor (VEGF) is a heparin-binding dimeric glycoprotein and principal angiogenic factor stimulating the migration, proliferation and expression of various genes in endothelial cells. Recently, VEGF was demonstrated to exhibit an antiapoptotic and direct myogenic effect, as well as to enhance muscle force restoration subsequent to traumatic injury. Therefore, the present study attempted to assess the muscle damage of VEGF overexpression in mdx mice. Adeno-associated virus serotype 9 (AAV9)-VEGF was administered intravenously to mdx mice. At 4 weeks after injection, VEGF was observed to be upregulated in the tibialis anterior muscle. In addition, the serum creatine kinase levels were significantly reduced and fatigue was slowed down, whereas the limb grip strength and weight of mice were markedly increased compared with the saline-treated mdx mice. Furthermore, significantly reduced inflammation and necrosis areas were observed in the muscle tissues of mice in the AAV9-VEGF group. These results suggested that AAV9-mediated VEGF gene overexpression was able to improve the muscle damage in mdx mice.

Ultrastructural diversity of inclusions and aggregations in the lumbar spinal cord of SOD1-G93A transgenic mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron death. We report the characteristics of ultrastructural pathological changes of inclusions and aggregations in the neuronal axons, glial cells and ventral roots of lumbar spinal cord in SOD1-G93A transgenic mice using light and electron transmission microscope at different stages of disease. The most noteworthy is that mutant SOD1 accumulations in the cytoplasm of motor neurons precede the numerous inclusions. Inclusions manifested differently according to the specified locations. This study provided further information to the previous reports about pathological changes of ALS.

Reduced Nrf2 and Phase II enzymes expression in immune-mediated spinal cord motor neuron injury.

The transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a major regulator of genes encoding Phase II detoxifying enzymes and antioxidant proteins, is important for protecting cells against oxidative damage. In this work, we report that in the immune-mediated motor neuron injury animal model, expression of Nrf2 and antioxidative enzymes including glutathione S-transferase, nicotinamide adenine dinucleotide phosphate (reduced)-quinone oxidoreductase 1 and heme oxygenase 1 were greatly reduced in motor neurons of spinal cord anterior horn in paralyzed guinea pigs, whereas the antioxidant enzymes in the dorsal horn of paralyzed guinea pigs were generally preserved. Our findings suggest that declined antioxidative capacity may contribute to the damage to motor neurons in the process of immune-mediated motor neuron injury. Although the exact mechanism of immune reactivity and Nrf2-antioxidant response element pathway inactivation remains to be elucidated, inducers of Phase II detoxification enzymes may be an attractive therapeutic target for immune-mediated motor neuron degeneration.