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• Novel clinical finding of muscle hypertonia in tropomyosin-caused congenital myopathy. • Unusual symptom of trismus. • Unspecific histological findings. Patients with myopathies caused by pathogenic variants in tropomyosin genes TPM2 and TPM3 usually have muscle hypotonia and weakness, their muscle biopsies often showing fibre size disproportion and nemaline bodies. Here, we describe a series of patients with hypercontractile molecular phenotypes, high muscle tone, and mostly non-specific myopathic biopsy findings without nemaline bodies. Three of the patients had trismus, whilst in one patient, the distal joints of her fingers flexed on extension of the wrists. In one biopsy from a patient with a rare TPM3 pathogenic variant, cores and minicores were observed, an unusual finding in TPM3 -caused myopathy. The variants alter conserved contact sites between tropomyosin and actin. [ABSTRACT FROM AUTHOR]

• NEM type 10 is caused by bi-allelic mutations in the LMOD3 gene. • NEM type 10 is usually characterized by a severe congenital nemaline myopathy. • We report two adult patients with mild NM, due to a new missense variant in LMOD3. Nemaline myopathy (NEM) type 10, caused by biallelic mutations in LMOD3 , is a severe congenital myopathy clinically characterized by generalized hypotonia and muscle weakness, respiratory insufficiency, joint contractures, and bulbar weakness. Here, we describe a family with two adult patients presenting mild nemaline myopathy due to a novel homozygous missense variant in LMOD3. Both patients presented mild delayed motor milestones, frequent falls during infancy, prominent facial weakness and mild muscle weakness in the four limbs. Muscle biopsy showed mild myopathic changes and small nemaline bodies in a few fibers. A neuromuscular gene panel revealed a homozygous missense variant in LMOD3 that co-segregated with the disease in the family (NM_198271.4: c.1030C>T; p.Arg344Trp). The patients described here provide evidence of the phenotype-genotype correlation, suggesting that non-truncating variants in LMOD3 lead to milder phenotypes of NEM type 10. [ABSTRACT FROM AUTHOR]

LAMA2-related dystrophy (LAMA2-RD) is one of the most common forms of congenital muscular dystrophy (CMD). The severe phenotype, which manifests congenitally with muscle weakness and hypotonia, accounts for one-third of all CMD cases. Establishing disease-relevant biomarkers that can help monitor disease severity and progression beginning in infancy is crucial for clinical trial readiness. While muscle imaging remains a potential biomarker in CMDs, muscle MRI is not ideal given the requirement of sedation in infants and young children and its associated risks and discomfort. Muscle ultrasound (MUS) offers better sensitivity than MRI in capturing early muscle changes, does not require sedation, and can be performed conveniently at the bedside. However, MUS may reach a ceiling in reflecting fibroadipose muscle replacement relatively early in dystrophic muscle disorders. Here, we present findings from 27 muscle ultrasound studies performed in a cohort of 17 patients with LAMA2-RD (ages 5-71 months; males: 13; females: 4, 186 images total). The mean age at which examined muscle groups reached maximum echogenicity on the modified Heckmatt scale (grade 3, severely increased muscle echogenicity with loss of bone echo) was 34 months of age. Maximum echogenicity was demonstrated as early as 8 months for the hip adductors, hamstrings, and gastrocnemius, while certain proximal muscles of the upper extremities (triceps and deltoid) showed a persistence of bone echo in the majority of patients through 71 months of age. These results justify further investigation into MUS as a potential imaging biomarker for LAMA2-RD, particularly for the muscles of the proximal upper extremities. Overall, MUS remains an accessible, non-invasive way to monitor disease progression and may serve as an imaging biomarker to evaluate the potential efficacy of future therapies in very young patients with LAMA2-RD. [ABSTRACT FROM AUTHOR]

Pathogenic variants in RYR1 , which encodes the type 1 ryanodine receptor, are the most frequently diagnosed cause of congenital myopathy (CM). CM is a genetic disorder characterized by weakness and muscle hypotonia. There are many RYR1 variants of unknown significance (VUS) for which the contribution to disease (i.e., pathogenicity) is unclear. Model organisms are valuable tools for exploring how VUS may contribute to pathology. Advantages of zebrafish (Danio rerio) over other vertebrate model systems include their potential for high-throughput screening and transparent larvae which enable muscle function characterization. Collectively, these features make zebrafish an ideal model in which to study CM. Prior studies have demonstrated that zebrafish lacking ryr1, stac3 or cacna1s recapitulate human skeletal muscle pathology, however these knockout lines are of limited clinical relevance. To understand how clinically relevant RYR1 variants contribute to disease, we are using a three-step approach: (1) employ base editing to generate variants of known and unknown significance in the RyR1 cytosolic shell and channel domains, (2) cross these variants into muscle reporter zebrafish lines to analyze calcium flux and mitochondria abundance, redox state, and calcium levels and, (3) observe how selected variants affect muscle function in behavioral assays. These approaches allow us to (1) compare the impact of known pathogenic variants against other model systems, (2) functionally characterize VUS, and (3) generate data supporting/refuting pathogenicity. In support of zebrafish as a model to study CM, our preliminary results indicate that generating pathogenic RYR1 variant I4898T in zebrafish results in impaired muscle function. Once validated, these models will provide a scalable system to investigate underlying disease mechanism(s) and screen compounds for drug repurposing. [ABSTRACT FROM AUTHOR]

RYR1 gene, encoding the ryanodine receptor 1, contains 106 exons. RYR1 variants are linked to autosomal dominant (AD) or recessive (AR) congenital myopathy and to malignant hyperthermia. The gene's length with multitude of identified variants, variable phenotype and mixed inheritance patterns pose diagnostic challenges in RYR1-related disorders. This study presents 18 patients from 15 families, revealing nine with AR and eight with AD RYR1-related myopathies and one with AD susceptibility to malignant hyperthermia without myopathic features. Malignant hyperthermia occurred in one AR-RYR1 myopathy patient and two AD-RYR1 myopathy patients. Additionally, one patient with AR-RYR1 myopathy and two patients with AD-RYR1 myopathies experienced an attack of malignant hyperthermia. 12 causal variants for AR-RYR1 myopathies, five of which were previously unpublished, and eight for AD-RYR1 myopathies, two of which were unpublished, were identified, including a rare large deletion variant in the RYR1 gene. The clinical symptoms of myopathy manifested in infancy in all patients, with 16 experiencing hypotonia, 12 having feeding difficulties, and nine presenting respiratory insufficiency. In two patients, initial symptoms included arthrogryposis and multiple skeletal deformities, with development of myopathic symptoms over time. Motor development delays were observed in 17 patients, yet 13 patients achieved independent walking. All 18 patients displayed proximal muscle weakness and hypotonia with hyperlaxity. Specifically, four AR-RYR1 myopathy patients presented with external ophthalmoplegia. Nine patients had scoliosis, four had spine rigidity, and six exhibited hip subluxations. Two patients required invasive ventilation, two needed non-invasive ventilation, and two were fed via PEG. This study contributes to diagnosing RYR1-related disorders and highlighting the importance of detecting large deletions and performing CNV detection in cases of unidentified causal variants. [ABSTRACT FROM AUTHOR]

• We describe an infant with neutral lipid storage disease with myopathy (NLSDM). • Lipid accumulation was observed within the muscle fibers and liver. • Jordan's anomaly was observed in a blood smear. • NLSDM may manifest as a mildly symptomatic myopathy in infants. • This is the first report to demonstrate hepatic involvement in a pediatric patient. Neutral lipid storage disease with myopathy is an ultra-rare, inherited autosomal recessive neuromuscular metabolic disorder caused by pathogenic variants in PNPLA2. It typically presents in adults as a progressive myopathy and is associated with myocardiopathy, hepatic involvement, and high creatine kinase levels. Only three children and adolescents with neutral lipid storage disease with myopathy have been reported. We report a female infant with congenital hypotonia born to consanguineous parents, whose mother presented with polyhydramnios during pregnancy. She demonstrated delayed acquisition of motor milestones, hepatomegaly, and elevated creatine kinase levels. Homozygous pathogenic variants in PNPLA2 were identified. Lipid accumulation was observed within the muscle fibers and Jordans' anomaly was observed in a blood smear. This is the first report to describe an infant with mildly symptomatic neutral lipid storage disease with myopathy and demonstrate hepatic involvement in a pediatric patient. Despite her mild symptoms, her ancillary test results were markedly abnormal. [ABSTRACT FROM AUTHOR]

Highlights • All children with congenital myopathies should have regular respiratory muscle tests. • Respiratory muscle tests should privilege natural maneuvers such as sniff and cough. • Respiratory muscle tests should be adapted to the “respiratory muscle phenotype”. • Respiratory muscle function should be assessed on a combination of complementary tests. Abstract Congenital muscular dystrophies and congenital myopathies represent a heterogeneous group of disorders of the muscle characterized by an early onset of hypotonia and muscle weakness and consequently, a high respiratory morbidity and mortality. The diagnosis and characterization of the weakness of the respiratory muscles is crucial for clinical management of patients and the evaluation of innovative therapies. Routine respiratory evaluation is based on noninvasive volitional tests, such as the measurement of lung volumes, spirometry, and maximal static pressures, which may be difficult or impossible to obtain in young children. Tests using natural maneuvers such as a sniff, a cough or a whistle, are easier to perform and may be more informative in young children. The combination of multiple tests of respiratory muscle function is essential and both increases diagnosis accuracy and the strength of the data in case of clinical trials assessing new therapies for these diseases. [ABSTRACT FROM AUTHOR]

Highlights • Two new patients are described with FAD synthase deficiency (FLAD1 mutations). • Patients partially responded to riboflavin despite frameshift mutations in exon 2. • Infantile hypotonia resulted in early death despite partial response to riboflavin. • Dysmorphic auricular helix and hypospadias may be novel phenotypic features. Abstract Inherited defects of vitamin B 2 (riboflavin) metabolism may cause different phenotypes with common biochemical markers of multiple acyl-CoA dehydrogenase deficiency (MADD). Most recently, mutations in FLAD1 , which encodes flavin adenine dinucleotide (FAD) synthase, has been implicated in MADD with combined respiratory chain deficiency in nine patients. Here, we describe two siblings with FAD synthase deficiency, who were diagnosed post-mortem upon suspicion of this newly-described disease. Hypotonia was evident at two months of age in both infants, followed by feeding difficulties, respiratory distress and death in six months despite partial response to riboflavin. The older sibling had documented lipid storage myopathy and biochemical markers of MADD. Our observations support the previous reports of unexpected riboflavin-responsiveness in frameshift mutations in the second exon of FLAD1 and suggest dysmorphic auricular helix and hypospadias as possible additional clinical features. More reports and studies are needed to better describe and treat FAD synthase deficiency. [ABSTRACT FROM AUTHOR]

Fukuyama congenital muscular dystrophy (FCMD) is the second most common muscular dystrophy in Japan. FCMD is an autosomal recessive disorder caused by mutations in the fukutin gene. The main features of FCMD are a combination of infantile-onset hypotonia, generalized muscle weakness, eye abnormalities, and mental retardation associated with cortical migration defects, and most patients are never able to walk. To date, the development of a quantitative motor scale for FMCD has been difficult due to the moderate-to-severe intellectual impairment that accompanies FCMD. Gross motor function measure (GMFM), originally developed as a quantitative motor scale for cerebral palsy, can precisely and quantitatively assess motor function without complicated instructions, and was recently reported to be useful in the assessment of Down syndrome and spinal muscular atrophy. To confirm the validity of GMFM for the assessment of FCMD, 41 FCMD patients (age range: 0.6–24.4 years) were recruited for this study. The GMFM scores correlated significantly with those of two previously used motor scales, and the time-dependent change in GMFM scores was consistent with the natural course of FCMD. The inter-rater reliability, based on determinations made by four physiotherapists blinded to each other's assessment results, was excellent. We concluded GMFM to be a useful and valid measure of motor function in FCMD patients. [ABSTRACT FROM AUTHOR]

Nemaline myopathy is among the most common non-dystrophic congenital myopathies, and is characterized by the presence of nemaline rods in skeletal muscles fibers, general muscle weakness, and hypotonia. Although respiratory failure is the main cause of death in nemaline myopathy, only little is known regarding the contractile strength of the diaphragm, the main muscle of inspiration. To investigate diaphragm contractility, in the present study we took advantage of a mouse model for nebulin-based nemaline myopathy that we recently developed. In this mouse model, exon 55 of Neb is deleted ( Neb ΔExon55 ), a mutation frequently found in patients. Diaphragm contractility was determined in permeabilized muscle fibers and was compared to the contractility of permeabilized fibers from three peripheral skeletal muscles: soleus, extensor digitorum longus, and gastrocnemius. The force generating capacity of diaphragm muscle fibers of Neb ΔExon55 mice was reduced to 25% of wildtype levels, indicating severe contractile weakness. The contractile weakness of diaphragm fibers was more pronounced than that observed in soleus muscle, but not more pronounced than that observed in extensor digitorum longus and gastrocnemius muscles. The reduced muscle contractility was at least partly caused by changes in cross-bridge cycling kinetics which reduced the number of bound cross-bridges. The severe diaphragm weakness likely contributes to the development of respiratory failure in Neb ΔExon55 mice and might explain their early, postnatal death. [ABSTRACT FROM AUTHOR]

Autosomal dominantly inherited mutations of BICD2 are associated with congenital-onset spinal muscular atrophy characterised by lower limb predominance. A few cases have also showed upper motor neuron pathology, including presenting with features resembling hereditary spastic paraplegia. The age-of-onset for the published families is usually at birth but also included cases with childhood- and adult-onset disease. In this report we described two isolated probands that presented in utero with features associated with reduced fetal movements. Both cases were diagnosed at birth with arthrogryposis multiplex congenita (AMC) and hypotonia. Other variable features included congenital fractures, hip dislocation, micrognathia, respiratory insufficiency, microcephaly and bilateral perisylvian polymicrogyria. Patient 1 is 4 years of age and stable, but shows significant motor developmental delay and delayed speech. Patient 2 passed away at 7 weeks of age. Through next generation sequencing we identified the same missense substitution in BICD2 (p.Arg694Cys) in both probands. Sanger sequencing showed that in both cases the mutation arose de novo . The in utero onset in both cases suggests that the p.Arg694Cys substitution may have a more deleterious effect on BICD2 function than previously described mutations. Our results broaden the phenotypes associated with BICD2 mutations to include AMC and cortical malformations and therefore to a similar phenotypic spectrum to that associated with its binding partner DYNC1H1. [ABSTRACT FROM AUTHOR]

Nemaline myopathies are clinically and genetically heterogeneous muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Mutations in the KLHL40 (kelch-like family member 40) gene (NEM 8) are common cause of severe/lethal nemaline myopathy. We report an 8-year-old girl born to consanguineous Moroccan parents, who presented with hypotonia and poor sucking at birth, delayed motor development, and further mild difficulties in walking and fatigability. A muscle biopsy revealed the presence of nemaline bodies. KLHL40 gene Sanger sequencing disclosed a never before reported pathogenic homozygous mutation which resulted in absent KLHL40 protein expression in the muscle. This further expands the phenotypical spectrum of KLHL40 related nemaline myopathy. [ABSTRACT FROM AUTHOR]

Nemaline Myopathy is a genetically and clinically heterogeneous group of neuromuscular disorder most commonly presenting with congenital/childhood-onset hypotonia and muscle weakness. Despite the substantial progress in uncovering the genetics of these disorders, there is still no specific treatment available due to a lack of understanding of the origin and progression of pathophysiological processes contributing to the disease state. Mutations in Kelch proteins encoding genes are associated with recessive and dominant rare forms of nemaline myopathy (Kelch-NM). We have recently created zebrafish and mouse models of NM causing Kelch genes that recapitulate the clinical and pathological defects associated with NM patients. Using in vivo proteomics and imaging approaches, we have identified critical roles of NM causing Kelch genes in sarcomere development, maintenance, and cellular trafficking. These studies reveal the mechanism of muscle abnormalities in nemaline myopathy and provide insights into new pathways regulating normal and disease muscle. Finally, using gene targeting and small molecules-based screening approaches in Kelch-NM animal models, we aim to develop therapeutics for NM and related muscle disorders. [ABSTRACT FROM AUTHOR]

Infantile X-linked spinal muscular atrophy (SMAX2) is a rare form of spinal muscular atrophy manifesting as severe hypotonia, areflexia, arthrogryposis, facial weakness and cryptorchidism, and frequently accompanied by bone fractures. We present a male patient with SMAX2 who presented with typical symptoms at birth, preceded by reduced fetal movements in the second and third trimesters of pregnancy. Clinical examination revealed a myopathic face with a characteristic tent-shaped open mouth, tongue fibrillations, profound muscle weakness, areflexia, multiple contractures, mild skeletal abnormalities and cryptorchidism. In the first days of the patient's life, fractures of the right femur and right humerus were found; however, calcium–phosphate metabolism and densitometric examination were normal. Molecular analysis revealed a de novo c.1731C>T substitution in the UBA1 gene, which was localized in exon 15, the specific hot spot for mutation. [ABSTRACT FROM AUTHOR]

Mutations in ACTA1 have been associated with different pathologic findings including nemaline myopathy, intranuclear rod myopathy, actin myopathy, cap myopathy, congenital fiber type disproportion, and core myopathy. Myofibrillar myopathies are morphologically distinct but genetically heterogeneous muscular dystrophies arising from mutations in Z-disk related proteins. We report a 26-month-old boy with significantly delayed motor development requiring mechanical ventilation and tube-feeding since birth. The muscle biopsy displayed typical features of myofibrillar myopathy with abnormal expression of multiple proteins. Whole exome sequencing revealed two-amino-acid duplication in ACTA1 . In cell culture system, mutant actin was expressed at ~11% of wild-type, and mutant actin formed pleomorphic cytoplasmic aggregates whereas wild-type actin appeared in filamentous structures. We conclude that mutations in ACTA1 can cause pathologic features consistent with myofibrillar myopathy, and mutations in ACTA1 should be considered in patients with severe congenital hypotonia associated with muscle weakness and features of myofibrillar myopathy. [ABSTRACT FROM AUTHOR]

Congenital myopathies are a clinically and genetically heterogeneous group of disorders characterized by early onset hypotonia, weakness and characteristic, but not pathognomonic, structural abnormalities in muscle fibres. The clinical features overlap with muscular dystrophies, myofibrillar myopathies, neurogenic conditions and congenital myasthenic syndromes. We describe a case of cap myopathy with myasthenic features due to a mutation in the TPM2 gene that responded to anticholinesterase therapy. We also review other published cases of congenital myopathies with neuromuscular transmission abnormalities. This report expands the spectrum of congenital myopathies with secondary neuromuscular transmission defects. The recognition of these cases is important since these conditions can benefit from treatment with drugs enhancing neuromuscular transmission. [ABSTRACT FROM AUTHOR]

Abstract: Over the past decade there have been major advances in defining the genetic basis of the majority of congenital myopathy subtypes. However the relationship between each congenital myopathy, defined on histological grounds, and the genetic cause is complex. Many of the congenital myopathies are due to mutations in more than one gene, and mutations in the same gene can cause different muscle pathologies. The International Standard of Care Committee for Congenital Myopathies performed a literature review and consulted a group of experts in the field to develop a summary of (1) the key features common to all forms of congenital myopathy and (2) the specific features that help to discriminate between the different genetic subtypes. The consensus statement was refined by two rounds of on-line survey, and a three-day workshop. This consensus statement provides guidelines to the physician assessing the infant or child with hypotonia and weakness. We summarise the clinical features that are most suggestive of a congenital myopathy, the major differential diagnoses and the features on clinical examination, investigations, muscle pathology and muscle imaging that are suggestive of a specific genetic diagnosis to assist in prioritisation of genetic testing of known genes. As next generation sequencing becomes increasingly used as a diagnostic tool in clinical practise, these guidelines will assist in determining which sequence variations are likely to be pathogenic. [Copyright &y& Elsevier]

Abstract: Infantile-onset X-linked spinal muscular atrophy (SMAX2) is a rare lethal disorder linked to mutations in the UBA1 (previously UBE1) gene, encoding ubiquitin-activating enzyme 1 that has an important role in the ubiquitin–proteasome pathway. Published pathological reports are scarce. Here we report a male infant who presented from birth with predominantly truncal hypotonia following an antenatal history of reduced fetal movements. He had a myopathic face, profound weakness, multiple contractures and areflexia. Creatine kinase was moderately raised. Brain MRI showed non-specific symmetrical periventricular white matter changes. Neurophysiology revealed evidence of motor and sensory involvement and muscle biopsy showed marked inflammatory changes with subtle features suggestive of acute denervation. UBA1 sequencing revealed a novel hemizygous missense mutation (c.1670A>T; p.Glu557Val). He died from progressive respiratory failure at 4months. On post mortem assessment, in addition to severe ventral motor neuron pathology, there was widespread involvement of the sensory system, as well as developmental and degenerative cerebellar abnormalities. In contrast to typical SMN1-associated SMA, the thalamus was unaffected. These findings indicate that SMAX2 is more accurately classified as a motor sensory neuronopathy rather than a pure anterior horn cell disorder. Ubiquitin–proteasome pathway defects may not only cause neurodegeneration but also affect normal neuronal development. [Copyright &y& Elsevier]

Abstract: Severe forms of myotubular myopathy (MTM) and congenital myotonic dystrophy type 1 (CDM), both present as floppy infants with hypotonia, respiratory failure and bulbar insufficiency. Muscle biopsy is often performed as part of the diagnostic process, but these two disorders share very similar histopathological features. It is well documented that CDM muscle has nuclear foci that contain muscleblind-like 1 (MBNL1) protein. In muscle biopsies from eight neonates showing central nuclei, MBNL1 immunolocalisation identified discrete, intensely stained foci in three cases that were subsequently confirmed as CDM by DNA analysis. In the five remaining non-CDM patients and two controls, MBNL1 staining was heterogeneous in nuclei, not as foci. MBNL1 staining patterns in CDM were easily distinguishable from MTM. We suggest that in cases of hypotonia with suspected CDM or MTM, when biopsy has been taken, sections should additionally be stained for MBNL1 to provide a rapid indication of a CDM diagnosis. [Copyright &y& Elsevier]

Abstract: This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient’s biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy. [Copyright &y& Elsevier]

Abstract: The fatal infantile neuromuscular presentation of branching enzyme deficiency (glycogen storage disease type IV) due to mutations in the gene encoding the glycogen branching enzyme, is a rare but probably underdiagnosed cause of congenital hypotonia. We report an infant girl with severe generalized hypotonia, born at 33weeks gestation who required ventilatory assistance since birth. She had bilateral ptosis, mild knee and foot contractures and echocardiographic evidence of cardiomyopathy. A muscle biopsy at 1month of age showed typical polyglucosan storage. The autopsy at 3.5months of age showed frontal cortex polymicrogyria and polyglucosan bodies in neurons of basal ganglia, thalamus, substantia innominata, brain stem, and myenteric plexus, as well as liver involvement. Glycogen branching enzyme activity in muscle was virtually undetectable. Sequencing of the GBE1 gene revealed a homozygous 28 base pair deletion and a single base insertion at the same site in exon 5. This case confirms previous observations that GBE deficiency ought to be included in the differential diagnosis of congenital hypotonia and that the phenotype correlates with the ‘molecular severity’ of the mutation. [ABSTRACT FROM AUTHOR]

Abstract: X-linked centronuclear myopathy (XLMTM), also called myotubular myopathy, is a severe congenital myopathy characterized by generalized hypotonia and weakness at birth and the typical histological finding of centralization of myo-nuclei. It is caused by mutations in the MTM1 gene encoding the 3-phosphoinositides phosphatase myotubularin. Mutations in dynamin 2 and amphiphysin 2 genes lead to autosomal forms of centronuclear myopathy (CNM). While XLMTM is the most frequent and severe form of CNM, no mutations are found in about 30% of patients by sequencing all MTM1 exons. Moreover, the impact of MTM1 sequence variants is sometimes difficult to assess. It is thus important to devise a complete molecular diagnostic strategy that includes analysis of the myotubularin transcript and protein expression. We therefore developed novel antibodies against human myotubularin and showed that they are able to detect the endogenous protein by direct Western blot from muscle samples and from cultured cells. In conjunction with RT-PCR analysis we validated the consequences of missense and splice mutations on transcript integrity and protein level. We also detected and characterized a novel deep intronic mutation consisting of a single nucleotide change that induces exonisation of a conserved intronic sequence. Patients with centronuclear myopathy and no molecular diagnosis should be investigated for MTM1 defects at the cDNA and protein level. [Copyright &y& Elsevier]

Abstract: Dynamin 2 (DNM2)-related dominant centronuclear myopathy is usually a mild disorder, but more severe variants have been associated with mutations affecting the pleckstrin homology (PH) domain of the protein, mainly implicated in different forms of Charcot–Marie–Tooth Disease (CMT). Whilst DNM2-related CMT may feature non-neurological findings including cataracts, this has not been reported in DNM2-related centronuclear myopathy. We report a girl presenting from birth with hypotonia, respiratory and feeding difficulties. Motor development was delayed and at 9years she lost the ability to walk. She had ptosis, external ophthalmoplegia and bilateral cataracts. Muscle biopsy showed increase in central nuclei with type 1 hypotrophy and fibrosis. DNM2 screening revealed a novel heterozygous substitution (c.1862T>C; p.Leu621Pro) affecting the PH domain of the protein. Her further course was progressive and at 14years she died from respiratory failure. Our findings expand the phenotypical spectrum associated with DNM2 mutations and provide a new clinical indicator for involvement of this gene in patients with centronuclear myopathy. [Copyright &y& Elsevier]

Abstract: Muscle–eye–brain disease (MEB) is characterised by congenital muscular dystrophy, structural brain malformations and eye abnormalities. We report a MEB case whose presenting sign was congenital blindness. She was investigated primarily for eye abnormalities at onset. She had bilateral retinal detachment and microphthalmia. Mild axial hypotonia and motor retardation were attributed to cerebral disorder in another center. Muscle biopsy showed mild myopathic changes and significant α-dystroglycan deficiency. Analysis of the POMGnT1 showed a novel homozygous mutation 1814G>C, causing p.Arg605Pro change. This case expands the clinical spectrum of MEB with unusually severe eye abnormalities compared to mild skeletal muscle and brain involvement. [Copyright &y& Elsevier]

Abstract: Neonatal hypotonia is frequently observed with a highly variable clinical presentation. Congenital myopathies that are classically characterized by the presence of structural changes of the muscle fibres such as cores, rods and aggregates have been reported to be occasionally associated with this presentation. However, the identification of the causing defect can be a challenging task in severe neonatal forms of the disease since specific structural changes might not always be present in affected newborn’s muscles. The RYR1 gene encodes the skeletal muscle isoform of a calcium channel, the ryanodine receptor, and has been involved in both dominant and recessive congenital myopathies associated with structural changes and presenting with various degree of severity. Here we report the case of a child presenting at birth with a lethal form of neonatal hypotonia associated with an atypical congenital myopathy. Molecular investigations showed that the disease was caused by two novel RYR1 mutations. One of the mutations was a large-sized genomic deletion. This is the first genomic rearrangement identified into the RYR1 gene to our knowledge. This new class of mutation of the RYR1 gene will clearly have consequences for the molecular investigation of RYR1-related diseases. [Copyright &y& Elsevier]

Abstract: Nemaline myopathy is among the most common congenital myopathies. We describe for the first time a novel double de novo mutation in two adjacent codons resulting in two amino acid changes E74D and H75Y in the ACTA1 gene. The hypotonic male infant was the first son of healthy unrelated parents with no family history of neuromuscular disorders. Pregnancy was complicated: decreased fetal movements were noted on the 25th week of gestation, premature labour pains were present from the 29th week onwards and because of breech presentation a Caesarian section was carried out in the 39th week. The patient presented with multiple congenital fractures and joint contractures. He was dependent on ventilatory support until his death at 2months. Muscle biopsy revealed severely atrophic and rounded muscle fibers with considerable variation in diameter and pronounced disorganization of the myofibers. Electron microscopy indicated a distinct disturbance of the myofibrillar architecture and nemaline rods. In view of previously described cases carrying different single missense mutations of the amino acid residues E74 or H75, we suggest that the particular genotype E74D/H75Y is compatible with the severity of the patient’s phenotype. The possibility of germ cell mosaicism should be taken into account in genetic counseling. [Copyright &y& Elsevier]

Abstract: Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy. [Copyright &y& Elsevier]

Abstract: Anti-MuSK antibodies have been reported in about 40–50% of patients with seronegative myasthenia gravis. Curiously, this condition has never been reported in association with fetal or transient neonatal myasthenia gravis, despite a known female predominance. We report the case of a 22-year-old woman who developed seronegative, mild steroid-responsive myasthenia gravis. When aged 26, she gave birth to a baby boy with neonatal myasthenia gravis characterized by hypotonia, stridor and sucking difficulties. Intubation was required for a few weeks. Anti-MuSK antibodies were assessed and found positive in both patients. Progressive hydramnios during the last trimester, with a decrease in spontaneous fetal mobility in the last weeks, long-lasting stridor, ptosis and occasional difficulties in swallowing liquids till two years of age, despite anti-MuSK antibodies becoming negative, suggest a fetal onset. The possible pathophysiology of this disorder, based on recent findings on the expression and function of MuSK protein, is reviewed. [Copyright &y& Elsevier]

Abstract: We report a family where a predominantly proximal myopathy has become increasingly severe with successive generations of the maternal lineage. This pure myopathy has been caused by a mutation (m.5650G>A) in the mt-tRNAAla gene that has been reported only once previously in a patient with CADASIL where the phenotype was dominated by neurological complications. This report is therefore the first description of the phenotype associated solely with this mutation and confirms its pathogenicity. [Copyright &y& Elsevier]

Abstract: We report a large family with a mild form of autosomal dominant nemaline myopathy and a new phenotype. Onset of symptoms was in infancy with hypotonia and motor delay. Weakness involved neck flexors, abdominal and proximal limb muscles. There was no bulbar, respiratory or foot dorsiflexion weakness and no slowness in movement. Patients had remarkably good physical endurance and no limitation in daily activities, but were slow runners since childhood. Nemaline rods were seen in less than 5% of muscle fibres. No linkage to the five known nemaline myopathy genes (α-tropomyosin-3, nebulin, α-actin, troponin T1 and β-tropomyosin), to the ryanodine receptor gene (associated with core-rod myopathy) or to the 15q21–23 locus was found. [Copyright &y& Elsevier]

Abstract: X-linked myotubular myopathy (XLMTM) is a congenital muscle disorder caused by mutations in the MTM1 gene. Affected males usually present at birth with severe hypotonia and respiratory insufficiency, and most of them die within the first few years of life. We report here on a 68-year-old patient with a very mild form of the disease who was diagnosed after his grandson showed muscular weakness and respiratory problems at birth. The E404K mutation in the MTM1 gene was found in both patients. To our knowledge, this grandfather is one of the oldest and most mildly affected known patients with an MTM1 mutation to date. Thus, this family represents a remarkable phenotypic variation of XLMTM ranging from a congenital to a mild adult form. [Copyright &y& Elsevier]

Abstract: We report a 3-year-old female with type I spinal muscular atrophy (SMA) born to a young and non-consanguineous couple. The child presented at two months of life with intense muscle weakness affecting predominantly proximal portions of the limbs, especially the legs, muscle hypotonia, fasciculation of the tongue, and severe respiratory muscle involvement. She remained in an intensive care unit with an assisted ventilation system from the fourth month of life. She died at 3years of age from pulmonary infection. Molecular analysis confirmed the diagnosis of SMA but revealed that only the father was an asymptomatic carrier. Because SMN1 is mapped in a complex region containing repetitive elements due to an inverted duplication of approximately 500kb, we carry out an SNP array and detected a 1.3Mb deletion including the SMN1 and SMN2 genes that explain the disease. [Copyright &y& Elsevier]

Congenital myopathies are a heterogeneous group of diseases that typically present in childhood with hypotonia and weakness and are commonly defined by characteristic changes observed on muscle biopsy. Currently, no treatments or disease-modifying therapies have been identified. Therapy development is hindered because approximately 40% of congenital myopathies are genetically unresolved. An overarching goal of our laboratory is to identify and characterize new causes of congenital myopathy. To this end, using whole exome sequencing we recently identified a splice-acceptor variant in the coiled-coil domain containing 78 gene (CCDC78) in a family with autosomal dominant core myopathy. CCDC78 is a previously uncharacterized gene with no known function that is highly enriched in skeletal muscle and localized to the perinuclear compartment and to the excitation contraction coupling apparatus. The specific goals of this study are to determine how mutation in CCDC78 results in muscle disease, establish the relationship between CCDC78 and other congenital myopathies, and to elucidate the normal function of CCDC78. To accomplish these goals we have created both morpholino based and stable genetic models of CCDC78 mutation in the zebrafish, including characterization of a newly developed loss of function CCDC78 mutant (sa3254). We have combined analysis of these zebrafish models with cell culture studies and examination of a range of human muscle biopsies. Current data suggests that CCDC78 participates in both the regulation of excitation-contraction coupling and in the regulation of autophagy. Additional ongoing experimentation will examine expression of CCDC78 in other congenital muscle diseases, will determine interacting partners for CCDC78, and will establish the normal function for CCDC78 in skeletal muscle development and homeostasis. In all, we present the first extensive characterization of this newly identified congenital myopathy gene. [Copyright &y& Elsevier]

Nemaline myopathy (NM) is the most common congenital myopathy with considerable clinical and genetic heterogeneity. It is characterized by muscle weakness and hypotonia at birth. Respiratory and feeding problems are common features especially in neonatal period and many of the patients complain of running difficulty during childhood. We report a family of autosomal dominant (AD) myopathy with marked intrafamilial phenotypic variability. A 12month old female neonate presented with hypotonia, developmental delay and failure to thrive. The patient had lip cyanosis followed by cardiac arrest. After treatment, she was unable to wean the ventilator, and currently shows hypotonia, with slight improvement. The laboratory findings and brain imaging were unremarkable. Genetic tests for common inherited neuromuscular disorders returned normal. The muscle biopsy showed intranuclear rods with selective type 1 fiber atrophy. Electron microscopy demonstrated severe loss of myofibrillary structure with nemaline rod accumulation within atrophic fibers. The familial history revealed that the mother, grandfather, as well as younger sister and mother’s cousin experienced developmental delay. While patient‘s mother showed muscle atrophy in limb muscles on muscle computed tomography, none of them experienced any severe respiratory difficulties like the proband. On mutational analysis of ACTA1 and TPM3, a heterozygous missense mutation (c.32T>A) was identified in exon 1 of TPM3 from affected members of the family. The mode of inheritance of our patient is compatible with AD trait. Mutations in ACTA1, TPM3 and TPM2 are reported to cause AD NM. ACTA1 is known to be the most common mutation that accounts for 15–26% while TPM3 accounts for about 3%. There are few reports on intrafamilial phenotypic variability in TPM3 mutations, but not as phenotypically variable as in our family. We report a novel mutation in exon 1 of TPM3 which is the hot spot for most of the mutations found. [Copyright &y& Elsevier]

Abstract: Prader–Willi Syndrome (PWS) is a multi-system disorder caused by absent expression of the paternal region of chromosome 15q11–13. Newborns with PWS are severely hypotonic and inactive, with subtle dysmorphic features (almond shaped eyes, small hands and feet and hypogonadism). Ability to suck is reduced and feeding problems are common, however respiratory distress is rare. Characteristically, within weeks, these infants become more responsive and develop more spontaneous movement. The aetiology of the extreme hypotonia is unknown, however both central factors and primary muscle pathology have been proposed. We describe five infants with severe hypotonia, feeding difficulties and varying degrees of respiratory distress. The diagnosis of PWS was initially not clinically apparent therefore they underwent neurophysiological assessment, including stimulated single fibre EMG (SSFEMG) of orbicularis oculi. SSFEMG was considered abnormal if the grand average of the mean consecutive difference (MCD), of the intervals between the stimulus and the potential (the jitter), was greater than 26μs, or if more than 10% of the individual measurements were greater than 34μs. All five infants showed significant abnormalities in SSFEMG, suggestive of a neuromuscular transmission disorder. Jitter measurements ranged from 34.6 to 68.7. One infant was initially diagnosed with congenital myasthenic syndrome and responded well to treatment with pyridostigmine. Cytogenetic studies subsequently confirmed the diagnosis of PWS in all five cases. This is the first report of abnormal SSFEMG findings in infants with PWS. We suggest that these infants may have an abnormality in neuromuscular junction transmission contributing to their early hypotonia and inactivity, which improves with maturation. PWS should therefore be considered in the differential diagnosis of hypotonic infants with neuromuscular junction abnormalities diagnosed by SSFEMG. [Copyright &y& Elsevier]