Hepatic veno-occlusive disease accompanied by thrombotic microangiopathy developed during treatment of juvenile dermatomyositis and macrophage activation syndrome: A case report.

Hepatic veno-occlusive disease (VOD) is a complication of haematopoietic stem cell transplantation. VOD is associated with the occurrence of thrombotic microangiopathy (TMA). In haematopoietic stem cell transplantation, VOD and TMA are endothelial syndromes resulting from endothelial cell activation and dysfunction. In rheumatic disease, while TMA is not rare, there are few reports of VOD. In idiopathic myositis, only one case with VOD and TMA complications has been reported, and there are no published cases in juvenile dermatomyositis (JDM). We report a case of JDM manifesting VOD and TMA complications during the treatment for myositis and macrophage activation syndrome (MAS). A 5-year-old boy diagnosed as anti-nuclear matrix protein 2 antibody-positive JDM was complicated by MAS. He received pulsed methylprednisolone, prednisolone, and tacrolimus, but JDM and MAS progressed. He was then treated with intravenous cyclophosphamide and cyclosporine A, with improvement in myositis symptoms and MAS. After initiation of cyclophosphamide and cyclosporine A, he developed haemolysis, painful hepatomegaly, liver damage, and ascites. He was diagnosed with VOD and TMA. Cyclophosphamide and cyclosporine A were discontinued, with recovery from VOD and TMA. The patient remained well on treatment with methotrexate, without any relapse of JDM and MAS to date. The presence of vasculopathy and hypercytokinaemia because of JDM and MAS exacerbated endothelial cell damage. In the present case, we suggest that the main cause of VOD was medication with CY and CsA, which had been used to treat acute exacerbation of MAS and JDM.

RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy.

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.

Becker Muscular Dystrophy Accompanied by Anti-HMGCR Antibody-positive Immune-mediated Necrotizing Myopathy.

Becker muscular dystrophy (BMD) is an X-linked neuromuscular disease characterized by progressive muscle weakness that currently has no cure. Immune-mediated necrotizing myopathy (IMNM) is a type of autoimmune inflammatory myopathy characterized by proximal muscle weakness that is treated with immunosuppressive therapy. We herein report a patient diagnosed with BMD complicated with IMNM by a pathological analysis. Notably, the patient had an elevated serum anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibody level. Oral glucocorticoid and methotrexate treatment partially improved the muscle weakness with decreased levels of serum creatine kinase. An accurate diagnosis is important for therapeutic decisions in these complicated cases.

Multidimensional analyses of the pathomechanism caused by the non-catalytic GNE variant, c.620A>T, in patients with GNE myopathy.

GNE myopathy is a distal myopathy caused by biallelic variants in GNE, which encodes a protein involved in sialic acid biosynthesis. Compound heterozygosity of the second most frequent variant among Japanese GNE myopathy patients, GNE c.620A>T encoding p.D207V, occurs in the expected number of patients; however, homozygotes for this variant are rare; three patients identified while 238 homozygotes are estimated to exist in Japan. The aim of this study was to elucidate the pathomechanism caused by c.620A>T. Identity-by-descent mapping indicated two distinct c.620A>T haplotypes, which were not correlated with age onset or development of myopathy. Patients homozygous for c.620A>T had mildly decreased sialylation, and no additional pathogenic variants in GNE or abnormalities in transcript structure or expression of other genes related to sialic acid biosynthesis in skeletal muscle. Structural modeling of full-length GNE dimers revealed that the variant amino acid localized close to the monomer interface, but far from catalytic sites, suggesting functions in enzymatic product transfer between the epimerase and kinase domains on GNE oligomerization. In conclusion, homozygotes for c.620A>T rarely develop myopathy, while symptoms occur in compound heterozygotes, probably because of mildly decreased sialylation, due to partial defects in oligomerization and product trafficking by the mutated GNE protein.

[Development of Deep Convolutional Neural Network-Based Algorithm for Muscle Biopsy Diagnosis].

To diagnose muscle disease, histopathologic evaluation of muscle biopsy is essential. In addition, since myositis has a well-established treatment, an accurate diagnosis is required. However, distinguishing myositis from other muscle diseases is challenging for pathologists. Thus, artificial intelligence is expected to improve medical productivity. Therefore, we developed an algorithm based on deep convolutional neural networks to make the algorithm for muscle biopsy diagnosis. We used 1,400 hematoxylin-and-eosin-stained pathology slides for training and testing. Our trained algorithm achieved better sensitivity and specificity than the diagnoses made by physicians.

A Japanese Patient with Hereditary Myopathy with Early Respiratory Failure Due to the p.P31732L Mutation of Titin.

Hereditary myopathy with early respiratory failure (HMERF) is caused by titin A-band mutations in exon 344 and is considered quite rare. Respiratory insufficiency can be the sole symptom in the disease course. We herein report the first Japanese HMERF patient with a p.P31732L mutation in titin. The patient manifested respiratory failure and mild weakness of the neck flexor muscle at 69 years old and showed fatty replacement of the bilateral semitendinosus muscles on muscle imaging. Our case indicates that HMERF with a heterozygous p.P31732L mutation should be included in the differential diagnosis of muscular diseases presenting with early respiratory failure.

Deep convolutional neural network-based algorithm for muscle biopsy diagnosis.

Histopathologic evaluation of muscle biopsy samples is essential for classifying and diagnosing muscle diseases. However, the numbers of experienced specialists and pathologists are limited. Although new technologies such as artificial intelligence are expected to improve medical reach, their use with rare diseases, such as muscle diseases, is challenging because of the limited availability of training datasets. To address this gap, we developed an algorithm based on deep convolutional neural networks (CNNs) and collected 4041 microscopic images of 1400 hematoxylin-and-eosin-stained pathology slides stored in the National Center of Neurology and Psychiatry for training CNNs. Our trained algorithm differentiated idiopathic inflammatory myopathies (mostly treatable) from hereditary muscle diseases (mostly non-treatable) with an area under the curve (AUC) of 0.996 and achieved better sensitivity and specificity than the diagnoses done by nine physicians under limited diseases and conditions. Furthermore, it successfully and accurately classified four subtypes of the idiopathic inflammatory myopathies with an average AUC of 0.958 and classified seven subtypes of hereditary muscle disease with an average AUC of 0.936. We also established a method to validate the similarity between the predictions made by the algorithm and the seven physicians using visualization technology and clarified the validity of the predictions. These results support the reliability of the algorithm and suggest that our algorithm has the potential to be used straightforwardly in a clinical setting.

TNNI1 Mutated in Autosomal Dominant Proximal Arthrogryposis.

OBJECTIVES: The main objective of this case report is to identify a gene associated with a Japanese family with autosomal dominant arthrogryposis. METHODS: We performed clinicopathologic diagnosis and genomic analysis using trio-based exome sequencing. RESULTS: A 14-year-old boy had contractures in the proximal joints, and the serum creatine kinase level was elevated. Muscle biopsy demonstrated a moth-eaten appearance in some type 1 fibers, and electron microscopic analysis revealed that type 1 fibers had Z disk streaming. We identified a heterozygous nonsense variant, c.523A>T (p.K175*), in TNNI1 in the family. DISCUSSION: The altered amino acid residue is within the tropomyosin-binding site near the C-terminus, in a region homologous to the variational hotspot of Troponin I2 (TNNI2), which is associated with distal arthrogryposis type 1 and 2b. Compared with patients with TNNI2 variants, our patient had a milder phenotype and proximal arthrogryposis. We report here a case of proximal arthrogryposis associated with a TNNI1 nonsense variant, which expands the genetic and clinical spectrum of this disease. Further functional and genetic studies are required to clarify the role of TNNI1 in the disease.

Imaging-based evaluation of pathogenicity by novel DNM2 variants associated with centronuclear myopathy.

A centronuclear myopathy (CNM) is a group of inherited congenital diseases showing clinically progressive muscle weakness associated with the presence of centralized myonuclei, diagnosed by genetic testing and muscle biopsy. The gene encoding dynamin 2, DNM2, has been identified as a causative gene for an autosomal dominant form of CNM. However, the information of a DNM2 variant alone is not always sufficient to gain a definitive diagnosis as the pathogenicity of many gene variants is currently unknown. In this study, we identified five novel DNM2 variants in our cohort. To establish the pathogenicity of these variants without using clinicopathological information, we used a simple in cellulo imaging-based assay for T-tubule-like structures to provide quantitative data that enable objective determination of pathogenicity by novel DNM2 variants. With this assay, we demonstrated that the phenotypes induced by mutant dynamin 2 in cellulo are well correlated with biochemical gain-of-function features of mutant dynamin 2 as well as the clinicopathological phenotypes of each patient. Our approach of combining an in cellulo assay with clinical information of the patients also explains the course of a disease progression by the pathogenesis of each variant in DNM2-associated CNM.

Mild form of Danon disease: two case reports.

Danon disease is typically lethal by the mid-twenties in male patients due to cardiomyopathy. This report aims to describe two unrelated male patients showing mild manifestations of the disease. A 39-year-old man presented with a 10-year history of elevated serum creatine kinase levels with slowly progressive muscle weakness. Muscle pathology showed autophagic vacuoles with sarcolemmal features. Genetic testing revealed a hemizygous mutation in exon 9b, an alternatively spliced exon, of lysosome-associated membrane protein-2 (LAMP-2) (c.1097_1098delAA). Cardiac testing showed asymptomatic mild left ventricular hypertrophy. He had borderline intelligence. Early stage of retinopathy was detected. Another male patient, currently 53-year-old, had asymptomatic supraventricular extrasystole and muscle weakness but no intellectual disability, harboring the same mutation. He also had retinopathy. The present patients commonly carry a mutation in exon 9b of LAMP-2, suggesting that mutations in the exon are associated with a mild form of Danon disease.

Whole genome sequencing of 45 Japanese patients with intellectual disability.

Intellectual disability (ID) is characterized by significant limitations in both intellectual functioning and adaptive behaviors, originating before the age of 18 years. However, the genetic etiologies of ID are still incompletely elucidated due to the wide range of clinical and genetic heterogeneity. Whole genome sequencing (WGS) has been applied as a single-step clinical diagnostic tool for ID because it detects genetic variations with a wide range of resolution from single nucleotide variants (SNVs) to structural variants (SVs). To explore the causative genes for ID, we employed WGS in 45 patients from 44 unrelated Japanese families and performed a stepwise screening approach focusing on the coding variants in the genes. Here, we report 12 pathogenic and likely pathogenic variants: seven heterozygous variants of ADNP, SATB2, ANKRD11, PTEN, TCF4, SPAST, and KCNA2, three hemizygous variants of SMS, SLC6A8, and IQSEC2, and one homozygous variant in AGTPBP1. Of these, four were considered novel. Furthermore, a novel 76 kb deletion containing exons 1 and 2 in DYRK1A was identified. We confirmed the clinical and genetic heterogeneity and high frequency of de novo causative variants (8/12, 66.7%). This is the first report of WGS analysis in Japanese patients with ID. Our results would provide insight into the correlation between novel variants and expanded phenotypes of the disease.

Mutant BIN1-Dynamin 2 complexes dysregulate membrane remodeling in the pathogenesis of centronuclear myopathy.

Membrane remodeling is required for dynamic cellular processes such as cell division, polarization, and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis and how mutations in these molecules cause CNM to develop. Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild-type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation, thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related CNM.

Duchenne muscular dystrophy-like phenotype in an LGMD2I patient with novel FKRP gene variants.

A 32-year-old man initially received a diagnosis of Duchenne muscular dystrophy (DMD). Genetic analysis revealed two novel heterozygous FKRP variants: c.169G>A (p.Glu57Lys) and c.692G>A (p.Trp231*). These results indicated that the patient had limb-girdle muscular dystrophy type 2I (LGMD2I) caused by recessive FKRP variants. Patients with LGMD2I and DMD have many overlapping phenotypes. LGMD2I should be considered in patients who have a DMD phenotype but not a DMD pathogenic variant.

Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.

Association of inattention with slow-spindle density in sleep EEG of children with attention deficit-hyperactivity disorder.

OBJECTIVE: We evaluated the power of slow sleep spindles during sleep stage 2 to clarify their relationship with executive function, especially with attention, in children with attention deficit-hyperactivity disorder (ADHD). METHODS: Subjects were 21 children with ADHD and 18 aged-matched, typically developing children (TDC). ADHD subjects were divided into groups of only ADHD and ADHD + autism spectrum disorder (ASD). We employed the Continuous Performance Test (CPT) to measure attention. We focused on sleep spindle frequencies (12-14 Hz) in sleep stage 2 and performed a power spectral analysis using fast Fourier transform techniques and compared sleep spindles with the variability of reaction time in CPT. RESULTS: In the CPT, reaction variabilities in ADHD and ADHD + ASD significantly differed from those in TDC. Twelve-hertz spindles were mainly distributed in the frontal pole and frontal area and 14-Hz spindles in the central area. The ratio of 12-Hz frontal spindle power was higher in ADHD than in TDC, especially in ADHD + ASD. Significant correlation between the ratio of 12-Hz spindles and reaction time variability was observed. CONCLUSIONS: Twelve-hertz frontal spindle EEG activity may have positive associations with sustained attention function. Slow frontal spindles may be useful as a biomarker of inattention in children with ADHD.

Three novel MTM1 pathogenic variants identified in Japanese patients with X-linked myotubular myopathy.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a form of the severest congenital muscle diseases characterized by marked muscle weakness, hypotonia, and feeding and breathing difficulties in male infants. It is caused by mutations in the myotubularin gene (MTM1). METHODS: Evaluation of clinical history and examination of muscle pathology of three patients and comprehensive genome analysis on our original targeted gene panel system for muscular diseases. RESULTS: We report three patients, each of whom presents distinct muscle pathological features. The three patients have novel hemizygous MTM1 variants, including c.527A>G (p.Gln176Arg), c.595C>G (p.Pro199Ala), or c.688T>C (p.Trp230Arg). CONCLUSIONS: All variants were assessed as "Class 4 (likely pathogenic)" on the basis of the guideline of American College of Medical Genetics and Genomics. These distinct pathological features among the patients with variants in the second cluster of PTP domain in MTM1 provides an insight into microheterogeneities in disease phenotypes in XLMTM.

RNA sequencing solved the most common but unrecognized NEB pathogenic variant in Japanese nemaline myopathy.

PURPOSE: The diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20-40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants. METHODS: Aberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES. RESULTS: We identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases. CONCLUSION: These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.