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Background: Anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)-myopathy is a usually rapidly progressive form of immune-mediated necrotizing myopathy (IMNM). Rarer clinical courses show slow progression and resemble the phenotype of limb-girdle dystrophy (LGMD). Objective: We demonstrate the difficulties in differentiating LGMD versus anti-HMGCR-myopathy. Methods: We report on a 48-year-old patient with slowly progressive tetraparesis and hyperCKemia for more than 20 years. Results: Due to myopathic changes in initial and second muscle biopsy and typical clinical presentation, the patient was diagnosed with LGMD 20 years ago; despite comprehensive genetic testing including exome diagnostics, the genetic cause of disease could not be identified. Finally, HMG-CoA reductase antibodies were detected, confirming the diagnosis of anti-HMGCR-myopathy. By re-work-up of a second muscle biopsy specimen from year 2009, the diagnosis of a IMNM was made in retrospect. Seven cycles of high-dose immunoglobulins were administered; patient reported outcome measures have mildly improved. Conclusion: Patients with clinical LGMD phenotype, degenerative changes in muscle biopsy but without genetic confirmation of the disease should be tested for HMG-CoA-myopathy, thereby allowing for an early start of treatment.

Background and ObjectivesFacioscapulohumeral muscular dystrophy type 2 (FSHD2) and arhinia are 2 distinct disorders caused by pathogenic variants in the same gene: SMCHD1. The mechanism underlying this phenotypic divergence remains unclear. In this study, we characterize the neuromuscular phenotype of individuals with arhinia caused by SMCHD1 variants and analyze their complex genetic and epigenetic criteria to assess their risk for FSHD2.MethodsEleven individuals with congenital nasal anomalies, including arhinia, nasal hypoplasia, or anosmia, underwent a neuromuscular examination, genetic testing, muscle ultrasound, and muscle MRI. Risk for FSHD2 was determined by combined genetic and epigenetic analysis of 4q35 haplotype, D4Z4 repeat length, and methylation profile. We also compared expression levels of pathogenic DUX4 mRNA in primary myoblasts or dermal fibroblasts (upon myogenic differentiation or epigenetic transdifferentiation, respectively) in these individuals vs those with confirmed FSHD2.ResultsAmong the 11 individuals with rare, pathogenic, heterozygous missense variants in exons 3–11 of SMCHD1, only a subset (n = 3/11; 1 male, 2 female; age 25–51 years) met the strict genetic and epigenetic criteria for FSHD2 (D4Z4 repeat unit length cis with a 4qA haplotype and D4Z4 methylation DUX4 expression in dermal fibroblasts under the epigenetic de-repression by drug treatment and in the primary myoblasts undergoing myogenic differentiation.DiscussionIn this cross-sectional study, we identified patients with arhinia who meet the full genetic and epigenetic criteria for FSHD2 and display the molecular hallmark of FSHD—DUX4 de-repression and expression in vitro—but who do not manifest with the typical clinicopathologic phenotype of FSHD2. The distinct dichotomy between FSHD2 and arhinia phenotypes despite an otherwise poised DUX4 locus implies the presence of novel disease-modifying factors that seem to operate as a switch, resulting in one phenotype and not the other. Identification and further understanding of these disease-modifying factors will provide valuable insight with therapeutic implications for both diseases.

Objective To define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI‐related muscular dystrophy (COL6‐RD). Methods COL6‐RD patient muscle biopsies were stratified into three groups based on the overall level of pathologic severity considering degrees of fibrosis, muscle fiber atrophy, and fatty replacement of muscle tissue. Using microarray and RNA‐Seq, we then performed global gene expression profiling on the same muscle biopsies and compared their transcriptome with age‐ and sex‐matched controls. Results COL6‐RD muscle biopsy transcriptomes as a group revealed prominent upregulation of muscle extracellular matrix component genes and the downregulation of skeletal muscle and mitochondrion‐specific genes. Upregulation of the TGFβ pathway was the most conspicuous change across all biopsies and was fully evident even in the mildest/earliest histological group. There was no difference in the overall transcriptional signature between the different histologic groups but polyserial analysis identified relative changes along with COL6‐RD histological severity. Interpretation Overall, our study establishes the prominent dysregulation of extracellular matrix genes, TGFβ signaling, and its downstream cellular pathways at the transcriptomic level in COL6‐RD muscle.

SPTLC1-related disorder is a late onset sensory-autonomic neuropathy associated with perturbed sphingolipid homeostasis which can be improved by supplementation with the serine palmitoyl-CoA transferase (SPT) substrate, l-serine. Recently, a juvenile form of motor neuron disease has been linked to SPTLC1 variants. Variants affecting the p.S331 residue of SPTLC1 cause a distinct phenotype, whose pathogenic basis has not been established. This study aims to define the neuropathological and biochemical consequences of the SPTLC1 p.S331 variant, and test response to l-serine in this specific genotype.We report clinical and neurophysiological characterisation of two unrelated children carrying distinct p.S331 SPTLC1 variants. The neuropathology was investigated by analysis of sural nerve and skin innervation. To clarify the biochemical consequences of the p.S331 variant, we performed sphingolipidomic profiling of serum and skin fibroblasts. We also tested the effect of l-serine supplementation in skin fibroblasts of patients with p.S331 mutations.In both patients, we recognised an early onset phenotype with prevalent progressive motor neuron disease. Neuropathology showed severe damage to the sensory and autonomic systems. Sphingolipidomic analysis showed the coexistence of neurotoxic deoxy-sphingolipids with an excess of canonical products of the SPT enzyme. l-serine supplementation in patient fibroblasts reduced production of toxic 1-deoxysphingolipids but further increased the overproduction of sphingolipids.Our findings suggest that p.S331 SPTLC1 variants lead to an overlap phenotype combining features of sensory and motor neuropathies, thus proposing a continuum in the spectrum of SPTLC1-related disorders. l-serine supplementation in these patients may be detrimental.

JAG2 encodes the Notch ligand Jagged2. The conserved Notch signaling pathway contributes to the development and homeostasis of multiple tissues, including skeletal muscle. We studied an international cohort of 23 individuals with genetically unsolved muscular dystrophy from 13 unrelated families. Whole-exome sequencing identified rare homozygous or compound heterozygous JAG2 variants in all 13 families. The identified bi-allelic variants include 10 missense variants that disrupt highly conserved amino acids, a nonsense variant, two frameshift variants, an in-frame deletion, and a microdeletion encompassing JAG2. Onset of muscle weakness occurred from infancy to young adulthood. Serum creatine kinase (CK) levels were normal or mildly elevated. Muscle histology was primarily dystrophic. MRI of the lower extremities revealed a distinct, slightly asymmetric pattern of muscle involvement with cores of preserved and affected muscles in quadriceps and tibialis anterior, in some cases resembling patterns seen in POGLUT1-associated muscular dystrophy. Transcriptome analysis of muscle tissue from two participants suggested misregulation of genes involved in myogenesis, including PAX7. In complementary studies, Jag2 downregulation in murine myoblasts led to downregulation of multiple components of the Notch pathway, including Megf10. Investigations in Drosophila suggested an interaction between Serrate and Drpr, the fly orthologs of JAG1/JAG2 and MEGF10, respectively. In silico analysis predicted that many Jagged2 missense variants are associated with structural changes and protein misfolding. In summary, we describe a muscular dystrophy associated with pathogenic variants in JAG2 and evidence suggests a disease mechanism related to Notch pathway dysfunction.

Genes 13(5), 893 (2022). doi:10.3390/genes13050893 special issue: "Special Issue "Genetics of Motor Neuron Diseases" / Special Issue Editor: Christopher Grunseich, Guest Editor", Published by MDPI, Basel

SummaryAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Mutations in the SPTLC1 subunit of serine-palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased sphingolipid synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause the peripheral sensory neuropathy HSAN1 due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

Patients with bi-allelic loss-of-function mutations in the gene ANO5 most commonly present with muscular dystrophy. In some studies, patients with ANO5-related dystrophy (ANO5-RD) had evidence of mild cardiac abnormalities; however, cardiac magnetic resonance imaging (MRI) has not been used for myocardial characterization. Ten patients with genetically confirmed ANO5-RD were enrolled in a phenotyping study to better characterize cardiac involvement. Evaluations included medical history, neurological examination and cardiac evaluations (electrocardiogram, echocardiogram and cardiac MRI). All patients were clinically asymptomatic from a cardiac perspective. Muscle MRI was consistent with previous studies of ANO5-RD with increased T1 signal in the posterior and medial compartments of the upper leg and the posterior compartment of the lower leg. Cardiac studies using echocardiography and cardiac MRI revealed dilation of the aortic root and thickening of the aortic valve without significant stenosis in 3/10 patients. There was evidence of abnormal late gadolinium enhancement (LGE) on cardiac MRI in 2/10 patients. In ANO5-RD, the development of cardiac fibrosis, edema or inflammation as demonstrated by LGE has not yet been reported. Cardiac MRI can characterize cardiac tissue and may detect subtle changes before they appear on echocardiography, with potential prognostic implications.

BACKGROUND AND OBJECTIVES: Facioscapulohumeral muscular dystrophy type 2 (FSHD2) and arhinia are 2 distinct disorders caused by pathogenic variants in the same gene: SMCHD1. The mechanism underlying this phenotypic divergence remains unclear. In this study, we characterize the neuromuscular phenotype of individuals with arhinia caused by SMCHD1 variants and analyze their complex genetic and epigenetic criteria to assess their risk for FSHD2. METHODS: Eleven individuals with congenital nasal anomalies, including arhinia, nasal hypoplasia, or anosmia, underwent a neuromuscular examination, genetic testing, muscle ultrasound, and muscle MRI. Risk for FSHD2 was determined by combined genetic and epigenetic analysis of 4q35 haplotype, D4Z4 repeat length, and methylation profile. We also compared expression levels of pathogenic DUX4 mRNA in primary myoblasts or dermal fibroblasts (upon myogenic differentiation or epigenetic transdifferentiation, respectively) in these individuals vs those with confirmed FSHD2. RESULTS: Among the 11 individuals with rare, pathogenic, heterozygous missense variants in exons 3–11 of SMCHD1, only a subset (n = 3/11; 1 male, 2 female; age 25–51 years) met the strict genetic and epigenetic criteria for FSHD2 (D4Z4 repeat unit length

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.

Objective To characterize the natural history and clinical features of myopathies caused by mono‐allelic, dominantly acting pathogenic variants in COL12A1. Methods Patients with dominant COL12A1‐related myopathies were characterized by history and clinical examination, muscle imaging, and genetic analysis. Pathogenicity of the variants was assessed by immunostaining patient‐derived dermal fibroblast cultures for collagen XII. Results Four independent families with childhood‐onset weakness due to novel, dominantly acting pathogenic variants in COL12A1 were identified. Adult patients exhibited distal‐predominant weakness. Three families carried dominantly acting glycine missense variants, and one family had a heterozygous, intragenic, in‐frame deletion of exon 52 of COL12A1. All pathogenic variants resulted in increased intracellular retention of collagen XII in patient‐derived fibroblasts as well as loss of extracellular, fibrillar collagen XII deposition. Since haploinsufficiency for COL12A1 is largely clinically asymptomatic, we designed and evaluated small interfering RNAs (siRNAs) that specifically target the mutant allele containing the exon 52 deletion. Immunostaining of the patient fibroblasts treated with the siRNA showed a near complete correction of collagen XII staining patterns. Interpretation This study characterizes a distal myopathy phenotype in adults with dominant COL12A1 pathogenic variants, further defining the phenotypic spectrum and natural history of COL12A1‐related myopathies. This work also provides proof of concept of a precision medicine treatment approach by proposing and validating allele‐specific knockdown using siRNAs specifically designed to target a patient’s dominant COL12A1 disease allele.

SummaryRNA-binding proteins (RBPs) are essential for post-transcriptional regulation and processing of RNAs. Pathogenic missense variants in RBPs underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, inclusion body myopathy, distal myopathy, and Paget’s disease of the bone. Here, we present ten independent families with a severe, progressive, early-onset muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD), caused by heterozygous frameshift variants in the prion-like domain of hnRNPA2B1. We found that in contrast with the previously reported missense variants, the frameshift hnRNPA2B1 variants do not promote, but rather decelerate the fibrillization of the protein. Importantly, the frameshift variants harbor altered nuclear-localization sequences and exhibit reduced affinity for the nuclear-import receptor, Karyopherin-β2, which promotes their cytoplasmic accumulation in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with hnRNPA2B1 to include a severe, early-onset disease reminiscent of OPMD, caused by a distinct class of frameshift variants that alter its nucleocytoplasmic transport dynamics.

Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca(2+) to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca(2+) and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients’ biopsies revealed a markedly reduced force response of the sarcomeres to [Ca(2+)]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients’ myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.

Type VI collagen is well known for its role in muscular disorders, however its function in bone is still not well understood. To examine its role in bone we analyzed femoral and vertebral bone mass by micro-computed tomography analysis, which showed lower bone volume/total volume and trabecular number in Col6α2-KO mice compared with WT. Dynamic histomorphometry showed no differences in trabecular bone formation between WT and Col6α2-KO mice based on the mineral appositional rate, bone formation rate, and mineralizing perimeter. Femoral sections were assessed for the abundance of Tartrate Resistant Acid Phosphatase-positive osteoclasts, which revealed that mutant mice had more osteoclasts compared with WT mice, indicating that the primary effect of Col6a2 deficiency is on osteoclastogenesis. When bone marrow stromal cells (BMSCs) from WT and Col6α2-KO mice were treated with rmTNFα protein, the Col6α2-KO cells expressed higher levels of TNFα mRNA compared with WT cells. This was accompanied by higher levels of p-p65, a down-stream target of TNFα, suggesting that BMSCs from Col6α2-KO mice are highly sensitive to TNFα signaling. Taken together, our data imply that Col6a2 deficiency causes trabecular bone loss by enhancing osteoclast differentiation through enhanced TNFα signaling.

Background Only a few small studies have previously reported episodes of hypoglycemia in children with neuromuscular diseases; however, there has been no broader investigation into the occurrence of hypoglycemia in children with congenital muscle disease (CMD). Methods Pediatric patients enrolled in the CMD International Registry (CMDIR) with a history of hypoglycemia were included in this retrospective review. Hypoglycemic episodes and associated clinical and biochemical characteristics were characterized. Results Ten patients with CMD (5 with LAMA2-related muscular dystrophy) reported at least one episode of hypoglycemia beginning at an average age of 3.5 years. Predominant symptoms included altered mental status and nausea/vomiting, and laboratory studies demonstrated metabolic acidosis and ketonuria, consistent with ketotic hypoglycemia. Conclusion Patients with CMD may have an increased risk of hypoglycemia during fasting, illness, or stress due to their relatively low muscle mass and hence, paucity of gluconeogenic substrate. Clinicians should therefore maintain a high index of suspicion for hypoglycemia in this high-risk patient population and caregivers should routinely be trained to recognize and treat hypoglycemia.

Contains fulltext : 229372.pdf (Publisher’s version ) (Closed access) Synaptotagmins are integral synaptic vesicle membrane proteins that function as calcium sensors and regulate neurotransmitter release at the presynaptic nerve terminal. Synaptotagmin-2 (SYT2), is the major isoform expressed at the neuromuscular junction. Recently, dominant missense variants in SYT2 have been reported as a rare cause of distal motor neuropathy and myasthenic syndrome, manifesting with stable or slowly progressive distal weakness of variable severity along with presynaptic NMJ impairment. These variants are thought to have a dominant-negative effect on synaptic vesicle exocytosis, although the precise pathomechanism remains to be elucidated. Here we report seven patients of five families, with biallelic loss of function variants in SYT2, clinically manifesting with a remarkably consistent phenotype of severe congenital onset hypotonia and weakness, with variable degrees of respiratory involvement. Electrodiagnostic findings were consistent with a presynaptic congenital myasthenic syndrome (CMS) in some. Treatment with an acetylcholinesterase inhibitor pursued in three patients showed clinical improvement with increased strength and function. This series further establishes SYT2 as a CMS-disease gene and expands its clinical and genetic spectrum to include recessive loss-of-function variants, manifesting as a severe congenital onset presynaptic CMS with potential treatment implications.

Skeletal muscle function relies on the myofibrillar apparatus inside myofibers as well as an intact extracellular matrix surrounding each myofiber. Muscle extracellular matrix (ECM) plays several roles including but not limited to force transmission, regulation of growth factors and inflammatory responses, and influencing muscle stem cell (i.e. satellite cell) proliferation and differentiation. In most myopathies, muscle ECM undergoes remodeling and fibrotic changes that may be maladaptive for normal muscle function and recovery. In addition, mutations in skeletal muscle ECM and basement proteins can cause muscle disease. In this review, we summarize the clinical features of two of the most common congenital muscular dystrophies, COL6-related dystrophies and LAMA2-related dystrophies, which are caused by mutations in muscle ECM and basement membrane proteins. The study of clinical features of these diseases has helped to inform basic research and understanding of the biology of muscle ECM. In return, basic studies of muscle ECM have provided the conceptual framework to develop therapeutic interventions for these and other similar disorders of muscle.

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype–phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease. Electronic supplementary material The online version of this article (10.1007/s00401-019-02059-z) contains supplementary material, which is available to authorized users.

Anti-HMGCR myopathy was first recognized and characterized in patients with a history of statin exposure and immune-mediated necrotizing myopathy. After the discovery of anti-HMGCR autoantibodies, several international groups identified and characterized more patients, expanding the phenotypic spectrum of this disease to include pediatric patients and young adults without statin exposure and those with a chronic myopathy resembling limb-girdle muscular dystrophy. We provide a summary of clinical findings, pathologic features, muscle imaging, and immunogenetic risk factors of the disease. We also discuss the current treatment strategies and approaches to monitoring the therapeutic response. Lastly, we briefly summarize the current understanding of the pathophysiology of the disease and postulate a model for autoimmunity initiation and propagation in this disease.

Mutations in ACTA1 cause a group of myopathies with expanding clinical and histopathological heterogeneity. We describe three patients with severe ACTA1-related myopathy who have muscle fiber cytoplasmic bodies but no classic nemaline rods. Patient 1 is a five-year-old boy who presented at birth with severe weakness and respiratory failure, requiring mechanical ventilation. Whole exome sequencing identified a heterozygous c.282C>A (p.Asn94Lys) ACTA1 mutation. Patients 2 and 3 were twin boys with hypotonia, severe weakness, and respiratory insufficiency at birth requiring mechanical ventilation. Both died at 6 months of age. The same heterozygous c.282C>A (p.Asn94Lys) ACTA1 mutation was identified by whole exome sequencing. We conclude that clinically severe ACTA1-related myopathy can present with muscle morphological findings suggestive of cytoplasmic body myopathy in the absence of definite nemaline rods. The Asn94Lys mutation in skeletal muscle sarcomeric α-actin may be linked to this histological appearance. These novel ACTA1 cases also illustrate the successful application of whole exome sequencing in directly arriving at a candidate genetic diagnosis in patients with unexpected phenotypic and histologic features for a known neuromuscular gene.

Introduction Immune-mediated necrotizing myopathies (IMNMs) are characterized by progressive weakness, elevated serum creatine kinase levels, and necrotizing myopathic features on muscle biopsy. Presence of highly specific autoantibodies against signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR) can aid in recognition and confirmation of IMNMs. Methods In this study we describe a boy with HMGCR-positive necrotizing myopathy and highlight the clinical features of the patient. Results In contrast to most adults, the patient described had a more indolent disease course, reminiscent of a muscular dystrophy. Intravenous immunoglobulin monotherapy resulted in a dramatic clinical response with return to normal strength. Conclusions Systematic consideration of IMNMs and testing for relevant autoantibodies in children with suspected but genetically unconfirmed muscular dystrophy may help improve diagnostic accuracy and allow timely treatment with potentially highly effective immunotherapies. Muscle Nerve 56: 175–179, 2017

Collagen prolyl 4-hydroxylases (C-P4Hs) play a central role in the formation and stabilization of the triple helical domain of collagens. P4HA1 encodes the catalytic α(I) subunit of the main C-P4H isoenzyme (C-P4H-I). We now report human bi-allelic P4HA1 mutations in a family with a congenital-onset disorder of connective tissue, manifesting as early-onset joint hypermobility, joint contractures, muscle weakness and bone dysplasia as well as high myopia, with evidence of clinical improvement of motor function over time in the surviving patient. Similar to P4ha1 null mice, which die prenatally, the muscle tissue from P1 and P2 was found to have reduced collagen IV immunoreactivity at the muscle basement membrane. Patients were compound heterozygous for frameshift and splice site mutations leading to reduced, but not absent, P4HA1 protein level and C-P4H activity in dermal fibroblasts compared to age-matched control samples. Differential scanning calorimetry revealed reduced thermal stability of collagen in patient-derived dermal fibroblasts versus age-matched control samples. Mutations affecting the family of C-P4Hs, and in particular C-P4H-I, should be considered in patients presenting with congenital connective tissue/myopathy overlap disorders with joint hypermobility, contractures, mild skeletal dysplasia and high myopia.

A statistical model to find disease genes Genetic variation is high among individuals, which makes it difficult to identify any one specific pathogenetic variant in patients with idiopathic disease, especially those that are in noncoding regions of the genome. Examining tissue-specific and population-level RNA sequencing data, Mohammadi et al. developed a statistical test, analysis of expression variation (ANEVA), that can quantify how one individual's gene expression fits in the context of the variation within the general population. By applying ANEVA to a dosage outlier test, the authors identified pathogenic gene transcripts in patients with Mendelian muscle dystrophy. Science , this issue p. 351

Transcriptome data holds substantial promise for better interpretation of rare genetic variants in basic research and clinical settings. Here, we introduce ANalysis of Expression VAriation (ANEVA) to quantify genetic variation in gene dosage from allelic expression (AE) data in a population. Application to GTEx data showed that this variance estimate is robust across datasets and is correlated with selective constraint in a gene. We next used ANEVA variance estimates in a Dosage Outlier Test (ANEVA-DOT) to identify genes in an individual that are affected by a rare regulatory variant with an unusually strong effect. Applying ANEVA-DOT to AE data form 70 Mendelian muscular disease patients showed high accuracy in detecting genes with pathogenic variants in previously resolved cases, and lead to one confirmed and several potential new diagnoses in cases previously unresolved. Using our reference estimates from GTEx data, ANEVA-DOT can be readily incorporated in rare disease diagnostic pipelines to better utilize RNA-seq data.One Sentence SummaryNew statistical framework for modelling allelic expression characterizes genetic regulatory variation in populations and informs diagnosis in rare disease patients

Calpainopathy, also known as limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) or LGMD R1 Calpain3-related, is one of the most common genetically characterized forms of limb-girdle muscular dystrophy with a wide range of phenotypic severity. We evaluated a consanguineous family with a clinical phenotype consistent with calpainopathy in whom conventional sequencing did not detect any mutations in the CAPN3 gene. Using whole exome sequencing paired with haplotype analysis, we identified a homozygous deep intronic single base pair deletion in CAPN3 (c.946-29delT). Familial segregation studies were consistent with recessive inheritance. Immunoblotting of muscle tissue from the patient showed complete absence of calpain 3. In silico analysis predicted the deletion to disrupt the branch point and subsequently alter splicing of exon 7. Studies of patient fibroblasts and muscle tissue confirmed altered splicing, resulting in an inclusion of a 389-bp intronic sequence upstream of exon 7, originating from a cryptic splice acceptor site in intron 6. This out-of-frame insertion results in a premature stop codon, leading to an apparent absence of protein likely due to degradation of the transcript via nonsense-mediated decay. We then designed phosphorodiamidate morpholino oligomers (PMOs) as splice modulators to block the new splice acceptor site. This approach successfully prevented the aberrant splicing - reverting the majority of the splice to the wildtype transcript. These results confirm the pathogenicity of this novel deep intronic mutation and provide a mutation-specific therapeutic strategy. Thus, deep intronic mutations in CAPN3 may be pathogenic and should be considered in the appropriate clinical setting. The identification of mutations which may be missed by traditional Sanger sequencing is essential as they may be excellent targets for individualized therapeutic strategies using RNA-directed splice modulation.

ObjectiveTo identify the rate of change of clinical outcome measures in children with 2 types of congenital muscular dystrophy (CMD), COL6-related dystrophies (COL6-RDs) and LAMA2-related dystrophies (LAMA2-RDs).MethodsOver the course of 4 years, 47 individuals (23 with COL6-RD and 24 with LAMA2-RD) 4 to 22 years of age were evaluated. Assessments included the Motor Function Measure 32 (MFM32), myometry (knee flexors and extensors, elbow flexors and extensors), goniometry (knee and elbow extension), pulmonary function tests, and quality-of-life measures. Separate linear mixed-effects models were fitted for each outcome measurement, with subject-specific random intercepts.ResultsTotal MFM32 scores for COL6-RDs and LAMA2-RDs decreased at a rate of 4.01 and 2.60 points, respectively, each year (p < 0.01). All muscle groups except elbow flexors for individuals with COL6-RDs decreased in strength between 1.70% (p < 0.05) and 2.55% (p < 0.01). Range-of-motion measurements decreased by 3.21° (p < 0.05) at the left elbow each year in individuals with LAMA2-RDs and 2.35° (p < 0.01) in right knee extension each year in individuals with COL6-RDs. Pulmonary function demonstrated a yearly decline in sitting forced vital capacity percent predicted of 3.03% (p < 0.01) in individuals with COL6-RDs. There was no significant change in quality-of-life measures analyzed.ConclusionResults of this study describe the rate of change of motor function as measured by the MFM32, muscle strength, range of motion, and pulmonary function in individuals with COL6-RDs and LAMA2-RDs.

ObjectiveTo determine the prevalence and clinical features of anti-HMGCR myopathy among patients with presumed limb-girdle muscular dystrophy (LGMD) in whom genetic testing has failed to elucidate causative mutations.MethodsPatients with presumed LGMD and unrevealing genetic testing were selected based on a few clinico-pathologic features and tested for anti-HMGCR autoantibodies (n = 11). These clinico-pathologic features are peak creatine kinase (CK) greater than 1,000 IU/L and at least 3 of the following features: (1) limb-girdle pattern of weakness, (2) selective involvement of posterior thigh on clinical examination or muscle imaging, (3) dystrophic changes on muscle biopsy, and (4) no family history of muscular dystrophy.ResultsSix patients tested positive for anti-HMGCR autoantibodies. In 4, there was a presymptomatic phase, lasting as long as 10 years, characterized by elevated CK levels without weakness. Muscle biopsies revealed variable degrees of a dystrophic pathology without prominent inflammation. In an independent cohort of patients with anti-HMGCR myopathy, 17 of 51 (∼33%) patients were initially presumed to have a form of LGMD based on clinico-pathologic features but were ultimately found to have anti-HMGCR myopathy. Most of these patients responded favorably to immunomodulatory therapies, evidenced by reduction of CK levels and improved strength.ConclusionsAnti-HMGCR myopathy can resemble LGMD. Diagnosis of patients with a LGMD-like presentation of anti-HMGCR myopathy is critical because these patients may respond favorably to immunotherapy, especially those with shorter disease duration.

International audience; Mutations in RYR1 give rise to diverse skeletal muscle phenotypes, ranging from classical central core disease to susceptibility to malignant hyperthermia. Next-generation sequencing has recently shown that RYR1 is implicated in a wide variety of additional myopathies, including centronuclear myopathy. In this work, we established an international cohort of 21 patients from 18 families with autosomal recessive RYR1-related centronuclear myopathy, to better define the clinical, imaging, and histological spectrum of this disorder. Early onset of symptoms with hypotonia, motor developmental delay, proximal muscle weakness, and a stable course were common clinical features in the cohort. Ptosis and/or ophthalmoparesis, facial weakness, thoracic deformities, and spinal involvement were also frequent but variable. A common imaging pattern consisted of selective involvement of the vastus lateralis, adductor magnus, and biceps brachii in Comparison to adjacent muscles. In addition to a variable prominence of central nuclei, muscle biopsy from 20 patients showed type 1 fiber predominance and a wide range of intermyofibrillary architecture abnormalities. All families harbored compound heterozygous mutations, most commonly a truncating mutation combined with a missense mutation. This work expands the phenotypic characterization of patients with recessive RYR1-related centronuclear myopathy by highlighting common and variable clinical, histological, and imaging findings in these patients. (C) 2017 Elsevier B.V. All rights reserved.

Objective Autoantibodies against transcription intermediary factor 1γ (TIF1γ) are found in many patients with dermatomyositis (DM). Although TIF1γ is known to play a role in the differentiation of other tissues, its functional role in muscle regeneration has not been elucidated. This study was undertaken to explore the regulation and functional role of this protein during muscle differentiation and regeneration. Methods TIF1γ expression was analyzed in human muscle biopsy specimens using immunofluorescence microscopy. Immunofluorescence microscopy and immunoblotting analyses were used to study TIF1γ expression in a mouse model of muscle injury and repair. The effect of premature TIF1γ silencing on muscle differentiation was studied in cultured mouse myoblasts. Results In muscle biopsy specimens from DM patients, TIF1γ was expressed at low levels in the nuclei of histologically normal muscle cells but at high levels in the centralized nuclei of atrophic, perifascicular myofibers expressing markers of regeneration. TIF1γ levels were also increased in regenerating myonuclei following muscle injury in mice. Premature silencing of TIF1γ in vitro using small interfering RNA did not accelerate the expression of myogenin, a transcription factor that plays a central role in regulating relatively early stages of muscle differentiation. However, premature silencing of TIF1γ did accelerate myotube fusion and the expression of myosin heavy chain (MyHC), a later marker of muscle differentiation. Conclusion The DM autoantigen TIF1γ is markedly up-regulated during muscle regeneration in human and mouse muscle cells. Premature silencing of this protein in cultured myoblasts accelerates MyHC expression and myoblast fusion. However, TIF1γ may function independently of, or downstream from, myogenin.

Background The causes of intellectual disability (ID) are diverse and de novo mutations are increasingly recognised to account for a significant proportion of ID. Methods and results In this study, we performed whole exome sequencing on a large cohort of patients with ID or neurodevelopmental delay and identified four novel de novo predicted deleterious missense variants in HECW2 in six probands with ID/developmental delay and hypotonia. Other common features include seizures, strabismus, nystagmus, cortical visual impairment and dysmorphic facial features. HECW2 is an ubiquitin ligase that stabilises p73, a crucial mediator of neurodevelopment and neurogenesis. Conclusion This study implicates pathogenic genetic variants in HECW2 as potential causes of neurodevelopmental disorders in humans.