Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.

Update on RYR1-related myopathies.

PURPOSE OF REVIEW: RYR1-related myopathy (RYR1-RM) is a group of myopathies caused by mutations in the RYR1 gene, which encodes the ryanodine receptor 1 (RYR1). This review discusses recent advances in the clinical features, pathology, pathogenesis, and therapeutics of RYR1-RM. RECENT FINDINGS: Although treatments such as salbutamol, pyridostigmine, and N-acetylcysteine have been explored as potential therapies for RYR1-RM, none have been conclusively proven to be effective. However, recent clinical trials of Rycal ARM210 in patients with RYR1-RM have shown promising results, including reduced fatigue and improved proximal muscle strength.Recent advances in three-dimensional structural analysis of RYR1 channels, facilitated by cryo-electron microscopy (cryo-EM), have elucidated the distinct molecular mechanisms underlying RYR1 functionality. Additionally, high-throughput screening methods, including FRET-based and endoplasmic reticulum Ca2+-based assays, have been successful in identifying potential candidates for the treatment of RYR1-RM. SUMMARY: Recent advances in clinical and pathological understanding have provided new insights into RYR1-RM. Novel pathomechanisms elucidated by cryo-EM and rapid screening methods have led to the identification of several promising drug candidates. We are hopeful about the potential of Rycal, other new drugs, and gene therapy, offering a promising outlook for the future.

Best practice guidelines on genetic diagnostics of facioscapulohumeral muscular dystrophy: Update of the 2012 guidelines.

The gold standard for facioscapulohumeral muscular dystrophy (FSHD) genetic diagnostic procedures was published in 2012. With the increasing complexity of the genetics of FSHD1 and 2, the increase of genetic testing centers, and the start of clinical trials for FSHD, it is crucial to provide an update on our knowledge of the genetic features of the FSHD loci and renew the international consensus on the molecular testing recommendations. To this end, members of the FSHD European Trial Network summarized the evidence presented during the 2022 ENMC meeting on Genetic diagnosis, clinical outcome measures, and biomarkers. The working group additionally invited genetic and clinical experts from the USA, India, Japan, Australia, South-Africa, and Brazil to provide a global perspective. Six virtual meetings were organized to reach consensus on the minimal requirements for genetic confirmation of FSHD1 and FSHD2. Here, we present the clinical and genetic features of FSHD, specific features of FSHD1 and FSHD2, pros and cons of established and new technologies (Southern blot in combination with either linear or pulsed-field gel electrophoresis, molecular combing, optical genome mapping, FSHD2 methylation analysis and FSHD2 genotyping), the possibilities and challenges of prenatal testing, including pre-implantation genetic testing, and the minimal requirements and recommendations for genetic confirmation of FSHD1 and FSHD2. This consensus is expected to contribute to current clinical management and trial-readiness for FSHD.

Comprehensive Analysis of a Japanese Pedigree with Biallelic ACAGG Expansions in RFC1 Manifesting Motor Neuronopathy with Painful Muscle Cramps.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive multisystem neurologic disorder caused by biallelic intronic repeats in RFC1. Although the phenotype of CANVAS has been expanding via diagnostic case accumulation, there are scant pedigree analyses to reveal disease penetrance, intergenerational fluctuations in repeat length, or clinical phenomena (including heterozygous carriers). We identified biallelic RFC1 ACAGG expansions of 1000 ~ repeats in three affected siblings having sensorimotor neuronopathy with spinocerebellar atrophy initially presenting with painful muscle cramps and paroxysmal dry cough. They exhibit almost homogeneous clinical and histopathological features, indicating motor neuronopathy. Over 10 years of follow-up, painful intractable muscle cramps ascended from legs to trunks and hands, followed by amyotrophy and subsequent leg pyramidal signs. The disease course combined with the electrophysical and imagery data suggest initial and prolonged hyperexcitability and the ensuing spinal motor neuron loss, which may progress from the lumbar to the rostral anterior horns and later expand to the corticospinal tract. Genetically, heterozygous ACAGG expansions of similar length were transmitted in unaffected family members of three successive generations, and some of them experienced muscle cramps. Leukocyte telomere length assays revealed comparatively shorter telomeres in affected individuals. This comprehensive pedigree analysis demonstrated a non-anticipating ACAGG transmission and high penetrance of manifestations with a biallelic state, especially motor neuronopathy in which muscle cramps serve as a prodromal and disease progress marker. CANVAS and RFC1 spectrum disorder should be considered when diagnosing lower dominant motor neuron disease, idiopathic muscle cramps, or neuromuscular hyperexcitability syndromes.

Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

[Current and Future Status of Muscle Pathology: The Position of Muscle Pathology Diagnosis in the Future].

Many muscle disease names are mostly based on muscle pathology findings. Naturally, muscle pathology is important in the diagnosis of muscle diseases. Moreover, in recent years, extensive genetic analysis and autoantibody testing for myositis have been applied clinically, although muscle biopsies are less performed. However, muscle pathology should be proactively considered when a single gene presents multiple phenotypes, when variants of unknown pathological significance are detected, or in cases of autoimmune myositis that may be misdiagnosed as muscular dystrophy.

Splicing Switching of Alternative Last Exons Due to a Deletion Including Canonical Polyadenylation Site in COL6A2 Gene Causes Recessive UCMD.

Objectives: Collagen VI-related myopathy spans a clinical continuum from severe Ullrich congenital muscular dystrophy to milder Bethlem myopathy, caused by genetic variants in COL6A1, COL6A2, and COL6A3 genes. Our objective was to report a newly identified patient with the pathogenic variants restricted to a polyadenylation signal in the 3'-untranslated region, which have not been reported in hereditary muscle disease. Methods: We performed clinicopathologic diagnosis and analysis using whole-genome and RNA sequencing. Results: We report Ullrich congenital muscular dystrophy caused by a homozygous deletion, c.*198_*466del, which includes a polyadenylation signal in the canonical last exon of the COL6A2 gene. The parents were consanguineously married and had the heterozygous variant, but they were completely asymptomatic. In the patient's muscles, collagen VI was deficient in the sarcolemma, but present in the interstitium, showing the pattern of sarcolemma-specific collagen VI deficiency rather than a pattern of complete deficiency despite the lack of a polyadenylation signal. The RNA sequencing of the patient's muscle showed that alternative last exons were raised in COL6A2 transcript. Discussion: Our case provides a valuable example of the mechanism of alternative splicing switches for polyadenylation selection.

[Diagnosis of anti-melanoma differentiation-associated gene 5 antibody-positive dermatomyositis led by sarcoplasmic myxovirus resistance protein A expression on muscle pathology].

A 44-year-old woman with autism spectrum disorder developed bulbar symptoms and generalized muscle weakness 7 months before referral. Six months before, she was administered glucocorticoid for liver involvement. During the course, while she presented alopecia, skin ulcers, and poikiloderma, hyperCKemia was observed only twice. Due to complications including cardiac involvement and hearing loss as well, we suspected mitochondrial disease and performed a muscle biopsy. The muscle pathology showed sarcoplasmic myxovirus resistance A (MxA) expression with scattered pattern. Since anti-melanoma differentiation-associated gene 5 (MDA5) antibody was detected, we diagnosed the patient with anti-MDA5 antibody-positive dermatomyositis (DM). We reinforced immunosuppressive therapy, and her clinical symptoms and liver involvement were improved. When we diagnose a case of anti-MDA5 antibody-positive DM who is difficult to make clinical diagnosis, it may be valuable to evaluate sarcoplasmic MxA expression on muscle pathology.

Impact of sex, age at onset, and anti-cN1A antibodies on sporadic inclusion body myositis.

BACKGROUND: Inclusion body myositis (IBM) is a progressive myopathy occurring in patients over 45 years of age, with heterogeneous and variable clinical features. This study aimed to determine the influence of autoantibodies, gender, and age of onset on the clinical features of IBM. METHODS: Medical records and muscle histology findings of 570 participants with suspected IBM were reviewed. Various characteristics of patients who met the 2011 ENMC IBM diagnostic criteria were compared based on the presence of anti-cytosolic 5'-nucleotidase 1 A (cN1A) autoantibodies, gender, age of onset, and disease duration. RESULTS: Of the 353 patients who met the criteria, 41.6% were female. The mean age at onset was 64.6 ± 9.3 years, and the mean duration from onset to diagnosis was 5.7 ± 4.7 years. 196 of the 353 patients (55.5%) were positive for anti-cN1A autoantibodies and 157 were negative. Logistic regression showed that patients with anti-cN1A autoantibodies had a higher frequency of finger flexion weakness. Multiple regression showed that patients with later age of onset had shorter disease duration, lower BMI, and lower serum CK levels. Male patients had a higher frequency of onset with finger weakness and female patients had a lower BMI. CONCLUSION: Autoantibodies, gender, age of onset, and disease duration may influence the clinical presentation of IBM, highlighting the need for a precision medicine approach that considers these factors along with the underlying mechanisms of the disease.

X-linked Myotubular Myopathy Manifesting Carrier with Central and Peripheral Nervous System Involvement.

X-linked myotubular myopathy (XLMTM) is a rare genetic disorder caused by X-linked mutations in the MTM1 gene. Although heterozygous females are typically asymptomatic, affected cases have recently been reported. We herein report a case of XLMTM manifesting carrier of the pathogenic c.206dupG mutation in MTM1 with uncommon extramuscular symptoms. She developed gaze nystagmus and cognitive impairment in addition to muscle weakness. Electrophysiological studies and brain magnetic resonance imaging indicated the involvement of the central and peripheral nervous systems. XLMTM manifesting carriers may have a wider spectrum of clinical phenotypes than currently assumed. Appropriate follow-up of extramuscular and conventional muscular manifestations is important in such cases.

Sporadic Late-onset Nemaline Myopathy Associated with Sjögren's Syndrome: A Case Report.

We report the case of a 46-year-old female patient who developed a subacute progression of axial and proximal muscle weakness. Laboratory findings revealed mildly elevated serum creatine kinase levels. No monoclonal gammopathy was detected. A muscle biopsy revealed that she had nemaline myopathy. Serological tests and a lip biopsy revealed Sjögren's syndrome (SjS). We diagnosed her as having sporadic late-onset nemaline myopathy without monoclonal gammopathy of undetermined significance associated with SjS. Her symptoms improved after methylprednisolone pulse therapy followed by intravenous immunoglobulin therapy. A good response to immunotherapy demonstrates the necessity of making a correct diagnosis, for which a muscle biopsy is required.

[Juvenile-onset anti-nuclear matrix protein 2 (NXP-2) antibody-positive dermatomyositis with joint contractures before manifestation of myositis: a case report].

A 23-year-old man was admitted to our hospital with a one-year history of muscle weakness and atrophy. He had noticed contractures of the fingers of both hands from the age of 18. Examination revealed a skin rash including heliotrope rash and Gottron's sign, joint contractures in the extremities, dysphagia, extensive muscle weakness and marked muscle atrophy. The serum creatine kinase level was 272 |IU/l and muscle biopsy showed typical perifascicular atrophy but little lymphocyte invasion. There was no interstitial pneumonia or malignancy, but muscle tendons showed elevated CT values suggesting calcification or fibrosis. Anti-nuclear matrix protein 2 (NXP-2) antibody-positive dermatomyositis was diagnosed on the basis of the serum antibody level. Methylprednisolone pulse therapy ameliorated the skin rash and bulbar palsy, but muscle weakness, atrophy and joint contractures were resistant to the treatment. There have been no previous reports of young adults with anti-NXP-2 antibody-positive dermatomyositis in whom joint contracture became evident as early as 4 years beforehand, which is a important feature for differential diagnosis of dermatomyositis.

Large phenotypic diversity by genotype in patients with GNE myopathy: 10 years after the establishment of a national registry in Japan.

BACKGROUND: GNE myopathy is an ultra-rare autosomal recessive distal myopathy caused by pathogenic variants of the GNE gene, which encodes a key enzyme in sialic acid biosynthesis. The present study aimed to examine the long-term progression of GNE myopathy, genotype-phenotype correlations, and complications to provide useful information for predicting patient progression and designing clinical trials using a large collection of registry data over a 10-year period. METHODS: We analyzed 220 Japanese patients with GNE myopathy from a national registry in Japan. Diagnoses were confirmed by genetic curators based on genetic analysis reports. We analyzed registration sheets and annually updated items completed by attending physicians. RESULTS: In total, 197 of 220 participants (89.5%) carried p.D207V or p.V603L in at least one allele. The median disease duration to loss of ambulation was estimated to be 10 years in p.V603L homozygotes (n = 48), whereas more than 90% of p.D207V/p.V603L compound heterozygotes were estimated to be ambulatory even 20 years after disease onset according to Kaplan-Meier analysis (p < 0.001). Moreover, participants with a younger age of onset lost ambulation earlier regardless of genotype. A decline in respiratory function was observed as the disease progressed, particularly in p.V603L homozygotes, whereas none of the p.D207V/p.V603L compound heterozygotes showed a decline. CONCLUSIONS: The present study demonstrated large differences in disease progression and respiratory function between genotypes. Moreover, age of onset was found to be an indicator of disease severity regardless of genotype in GNE myopathy patients. These results may help stratify patients in clinical trials and predict disease progression.

A Case of a Patient with Calpainopathy Carrying Compound Heterozygous Mutations of a de novo Pathogenic Variant of c.1333G>A and a Novel Variant of c.1331C>T in CAPN3.

Calpainopathy is primarily an autosomal recessive inherited myopathy; however, dominantly inherited cases with a pathogenic variant of c.1333G>A have been reported. A 13-year-old Japanese girl presented with toe walking and elevated serum creatine kinase levels. Genetic panel testing revealed compound heterozygosity for c.1333G>A and a novel variant of c.1331C>T in CAPN3, leading to a diagnosis of calpainopathy. A genetic analysis of her parents revealed the possibility that c.1333G>A was de novo. In this patient, the onset age was earlier than that of the reported autosomal dominant cases, suggesting the influence of the novel variant in the contralateral allele.

Possible correlation between serum interleukin-8 levels and the activity of myositis in anti-NXP2 antibody-positive dermatomyositis.

Anti-nuclear matrix protein 2 (NXP2) antibody-positive dermatomyositis (DM) is characterized by extensive and severe myositis. In this study, we evaluated which cytokines/chemokines involved with the activity of the myositis. We performed quantitative immunoassays using the MILLIPLEX® Multiplex Assays Using Luminex to evaluate serum levels of interferon-γ, interleukin (IL)-1ß, IL-6, IL-8, IL-12p40, and tumor necrosis factor-α in samples collected over time from a 9-year-old female with anti-NXP2 antibody-positive DM. In our case, the serum level of IL-8 was elevated when the myositis worsened, and decreased in accordance with the improvement of myositis, suggesting that the serum IL-8 levels were correlated with the myositis activity. Serum levels of IL-8 in samples from five patients with anti-NXP2 antibody-positive DM and five patients with anti-transcriptional intermediary factor 1γ (TIF1γ) antibody-positive DM without both interstitial lung disease (ILD) and malignancy before starting treatments, along with five healthy controls, were also evaluate by an enzyme-linked immunosorbent assay. Serum IL-8 levels were significantly elevated in anti-NXP2 or anti-TIF1γ antibody-positive DM patients with myositis but not ILD, than healthy controls. It was suggested that serum levels of IL-8 correlate with the activity of myositis in DM including anti-NXP2 antibody-positive DM.

Pathologic Features of Anti-Ku Myositis.

OBJECTIVE: Characteristics of myositis with anti-Ku antibodies are poorly understood. The purpose of this study was to elucidate the pathologic features of myositis associated with anti-Ku antibodies, compared with immune-mediated necrotizing myopathy (IMNM) with anti-signal recognition particle (SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies, in muscle biopsy-oriented registration cohorts in Japan and Germany. METHODS: We performed a retrospective pathology review of patients with anti-Ku myositis samples diagnosed in the Japanese and German cohorts. We evaluated histologic features and performed HLA phenotyping. RESULTS: Fifty biopsied muscle samples in the Japanese cohort and 10 in the German cohort were obtained. After exclusion of myositis-specific autoantibodies or other autoimmune connective tissue diseases, 26 samples (43%) of anti-Ku antibody-positive myositis were analyzed. All the samples shared some common features with IMNM, whereas they showed expression of MHC class II and clusters of perivascular inflammatory cells more frequently than the anti-SRP/HMGCR IMNM samples (71% vs 7%/16%; p < 0.005/<0.005; 64% vs 0%/0%; p < 0.005/<0.005). Anti-Ku myositis biopsies could be divided into 2 subgroups based on the extent of necrosis and regeneration. The group with more abundant necrosis and regeneration showed a higher frequency of MHC class II expression and perivascular inflammatory cell clusters. HLA phenotyping in the 44 available patients showed possible associations of HLA-DRB1*03:01, HLA-DRB1*11:01, and HLA-DQB1*03:01 (p = 0.0045, 0.019, and 0.027; odds ratio [OR] 50.2, 4.6, and 2.8; 95% CI 2.6-2942.1, 1.1-14.5, and 1.0-7.0) in the group with less conspicuous necrosis and regeneration. On the contrary, in the group of more abundant necrosis and regeneration, the allele frequencies of HLA-A*24:02, HLA-B*52:01, HLA-C*12:02, and HLA-DRB1*15:02 were lower than those of healthy controls (p = 0.0036, 0.027, 0.016, and 0.026; OR = 0.27, 0, 0, and 0; 95% CI 0.1-0.7, 0-0.8, 0-0.8, and 0-0.8). However, these HLA associations did not remain significant after statistical correction for multiple testing. DISCUSSION: While anti-Ku myositis shows necrotizing myopathy features, they can be distinguished from anti-SRP/HMGCR IMNM by their MHC class II expression and clusters of perivascular inflammatory cells. The HLA analyses suggest that anti-Ku myositis may have different subsets associated with myopathologic subgroups.

A Case of Vesiculobullous Dermatomyositis with Anti-NXP-2 Antibody without Malignancy.

Vesiculobullous dermatomyositis (VD) is a rare manifestation of dermatomyositis (DM) and has been suggested to be associated with malignancy. Although the myositis-specific autoantibodies are associated with distinct clinical presentations of DM, those associated with VD remain unclear. Here, we present the case of a 54-year-old man with VD who tested positive for anti-nuclear matrix protein 2 (NXP-2) antibody, one of the DM-specific autoantibodies. Serological and histopathological findings did not support autoimmune blistering disease. Physical and histological findings suggested that the severe edema in combination with the interface dermatitis of DM contributed to blister formation. Although a systemic examination was performed, no evidence of malignancy was found. Following initiation of immunosuppressive therapy, the patient showed significant improvement in both skin lesions and myositis. This case represents the first report of anti-NXP-2-positive VD without malignancy or autoimmune blistering disease. Subcutaneous edema, a characteristic feature of anti-NXP-2-positive DM, could be related to the formation of VD.