Loss of C2orf69 defines a fatal autoinflammatory syndrome in humans and zebrafish that evokes a glycogen-storage-associated mitochondriopathy.

Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.

Alterations in insulin-like growth factor system in spinal muscular atrophy.

INTRODUCTION/AIMS: Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by survival motor neuron (SMN) protein deficiency. Insulin-like growth factor-I (IGF-I) is a myotrophic and neurotrophic factor that has been reported to be dysregulated in in vivo SMA model systems. However, detailed analyses of the IGF-I system in SMA patients are missing. In this study, we analyzed the components of the IGF-I system in serum and archived skeletal muscle biopsies of SMA patients. METHODS: Serum IGF-I, IGF binding protein (IGFBP)-3, and IGFBP-5 levels were analyzed in 11 SMA patients and 13 healthy children by immunoradiometric and enzyme-linked immunosorbent assays. The expression of IGF-I, IGF-I receptor, and IGFBP-5 proteins was investigated by immunofluorescence analysis in the archived skeletal muscle biopsies of nine SMA patients, six patients with non-SMA-related neuromuscular disease and atrophic fibers in muscle biopsy, and four controls. RESULTS: A significant decrease in IGF-I levels (mean ± SD: -1.39 ± 1.46 vs. 0.017 ± 0.83, p = .02) and increase in IGFBP-5 levels (mean ± SD: 2358.5 ± 1617.4 ng/mL vs. 1003.4 ± 274.3 ng/mL, p = .03) were detected in serum samples of SMA patients compared to healthy controls. Increased expression of IGF-I, IGF-I receptor, and IGFBP-5 was detected in skeletal muscle biopsies of SMA patients and non-SMA neuromuscular diseases, indicating atrophy-specific alterations in the pathway. DISCUSSION: Our findings suggested that the components of the IGF-I system are altered in SMA patients at both the systemic and tissue-specific levels.

A Rare Pediatric Case of Severe Rhabdomyolysis Owing to Dual Infection.

AIM: We aimed to report a severe and rare pediatric rhabdomyolysis case associated with a dual viral infection. CASE: A 13 year-old, healthy girl presented with the complaints of fever, abdominal pain, weakness and dark-colored urine. She was diagnosed with rhabdomyolysis based on clinical signs and laboratory findings. The diagnosis was confirmed by serological tests and real-time polymerase chain reaction for Epstein-Barr virus (EBV) and cytomegalovirus (CMV), respectively. Other potential genetic, metabolic and infectious causes were evaluated meticulously but no evidence was found. This case is also important as it is the first reported case to our knowledge on rhabdomyolysis associated with EBV and CMV co-infection in children. CONCLUSION: The presented case experienced tetraplegia due to the severe muscular damage and muscle power returned to normal range after 3 months. This suggests that EBV and CMV may have exert synergistic effects leading to more severe inflammation and degeneration.

The Common miRNA Signatures Associated with Mitochondrial Dysfunction in Different Muscular Dystrophies.

Secondary mitochondrial damage in skeletal muscles is a common feature of different neuromuscular disorders, which fall outside the mitochondrial cytopathies. The common cause of mitochondrial dysfunction and structural changes in skeletal muscle tissue remains to be discovered. Although they are associated with different clinical, genetic, and pathologic backgrounds, the pathomechanisms underlying neuromuscular disorders might be attributed to the complex interaction and cross talk between mitochondria and the associated miRNAs. This study aimed to identify the common miRNA signatures that are associated with mitochondrial damage in different muscular dystrophies (MDs; Duchenne muscular dystrophy, megaconial congenital muscular dystrophy, Ullrich congenital muscular dystrophy, and α-dystroglycanopathy). The miRNome profiles of skeletal muscle biopsies acquired from four different MD groups and control individuals were analyzed by miRNA microarray. We identified 17 common up-regulated miRNAs in all of the tested MD groups. A specific bioinformatics approach identified 10 of these miRNAs to be specifically related to the mitochondrial pathways. Six miRNAs, miR-134-5p, miR-199a-5p, miR-382-5p, miR-409-3p, miR-497-5p, and miR-708-5p, were associated with the top four mitochondrial pathways and were thus selected as priority candidates for further validation by quantitative real-time PCR analysis. We demonstrate, for the first time, common up-regulated miRNAs that are associated with mitochondrial damage in different MD groups, therefore contributing to the pathophysiology. Our findings may open a new gate toward therapeutics.

Clinical and genetic characterization of PYROXD1-related myopathy patients from Turkey.

Congenital myopathies (CMs) are a heterogeneous group of inherited muscle disorders characterized by muscle weakness at birth, while limb-girdle muscular dystrophies (LGMD) have a later onset and slower disease progression. Thus, detailed clinical phenotyping of genetically defined disease entities are required for the full understanding of genotype-phenotype correlations. A recently defined myopathic genetic disease entity is caused by bi-allelic variants in a gene coding for pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1) with unknown substrates. Here, we present three patients from two consanguineous Turkish families with mild LGMD, facial weakness, normal CK levels, and slow progress. Genomic analyses revealed a homozygous known pathogenic missense variant (c.464A>G, p.Asn155Ser) in family 1 with two affected females. In the affected male of family 2, we found this variant in a compound heterozygous state together with a novel frameshift variant (c.329_332delTCTG, p.Leu112Valfs*8), which is the second frameshift variant known so far in PYROXD1. We have been able to define a large homozygous region in family 1 sharing a common haplotype with family 2 in the critical region. Our data suggest that c.464A>G is a Turkish founder mutation. To gain deeper insights, we performed a systematic review of all published PYROXD1-related myopathy cases. Our analysis showed that the c.464A > G variant was found in 87% (20/23) of the patients and that it may cause either a childhood- or adult-onset phenotype, irrespective of its presence in a homozygous or compound heterozygous state. Interestingly, only four patients had elevated CK levels (up to 1000 U/L), and cardiac involvement was found in few compound heterozygous cases.

Validation of the EULAR/ACR 2017 idiopathic inflammatory myopathy classification criteria in juvenile dermatomyositis patients.

OBJECTIVES: In 2017, a new set of criteria was proposed by EULAR/ACR to classify idiopathic inflammatory myopathies. Our aim was to validate the EULAR/ACR 2017 classification criteria in juvenile dermatomyositis (JDM) patients. METHODS: This study was carried out at Hacettepe University Children's Hospital Department of Paediatrics, Divisions of Rheumatology, Neurology and Paediatric Pathology Unit. Control patients included inborn errors of metabolism presenting with myopathy and/or rhabdomyolysis, idiopathic rhabdomyolysis, dystrophinopathies, neuromyotonia and systemic rheumatic disorders. Patients' data were collected retrospectively from patient files. RESULTS: Fifty-eight JDM patients (60.3% female) and 40 controls (32.5% female) were included in this study. When the probability cut-off was set at 55% as recommended, the sensitivity/specificity of the new criteria to diagnose JDM were 96.5%/85% in the total cohort, 95.8%/84.6% without the muscle biopsy data and 97%/85.7% with biopsy data. With the ROC curve analysis, the optimal probability cut-off for the whole cohort was found to be >62%; providing a sensitivity/specificity of 96.6% (95% CI: 88.1% to 99.6)/90% (95% CI: 76.3% to 97.2%), and >68.5% for the patients with muscle biopsy providing sensitivity/specificity of 97% (84.7-99.9%)/100% (76.8-100%), respectively. The new EULAR/ACR criteria were the most sensitive, however, the least specific compared to the Tanimoto criteria (sensitivity/specificity 64%/97.5%) and Bohan-Peter criteria (sensitivity/ specificity 74.1%/92.5%). CONCLUSIONS: The new EULAR/ACR criteria performed favourably in our JDM cohort especially with the probability cut-off of >62%.

Diagnostic yield of muscle biopsy in infants: Retrospective analysis of clinical and histopathologic findings.

The aim was to define the clinical and histopathologic findings of infants who underwent muscle biopsy and identify the diagnostic yield of muscle biopsy in this cohort. Infants who underwent muscle biopsy from January 2010 to March 2017 at a tertiary hospital were included in the study (N = 87; 64 boys (73.6%), 23 girls (26.4%); age range 0 - 2 years; mean age 9.73 ± 7.04 months). Clinical and histopathologic data were obtained from medical records. Developmental delay (64.4%) and hypotonia (59.8%) were the most frequent clinical findings, and mitochondrial disease (61%) was the most frequent clinical diagnosis, followed by muscular dystrophy (15.9%) and congenital myopathy (11.5%). Creatine kinase level was normal in 65.9% and > 1,000 U/L in 17.1%. Specific pathologic findings were identified from 38 biopsies (43.7%). The most frequent pathologic findings were features compatible with mitochondrial/metabolic myopathy (14 patients, 16.1%) and muscular dystrophy (12 patients, 13.8%). Myopathic changes were present in 7 biopsy samples (8.0%) and neurogenic changes in 5 (5.7%). The clinical and pathologic diagnoses were compatible in 24 patients (63.2%). The diagnostic yield of muscle biopsy remains significant, especially in this age group. Mitochondrial disease is a major diagnostic challenge, and muscle biopsy helps to support the clinical diagnosis and guide further studies.

Inflammatory milieu of muscle biopsies in juvenile dermatomyositis.

Juvenile dermatomyositis (JDM) is an inflammatory myopathy which causes severe morbidity and high mortality if untreated. In this study, we aimed to define the T-helper cell profile in the muscle biopsies of JDM patients. Muscle biopsies of twenty-six patients (50% female) were included in the study. Immunohistochemical expression of CD3, CD20, CD138, CD68, IL-17, Foxp3, IFN-É£, IFN-alpha and IL-4 was studied and muscle biopsies were scored using the JDM muscle biopsy scoring tool. Inflammatory cells were in small clusters in perimysium and perivascular area or scattered throughout the endomysium in most biopsies; however in 2 biopsies, lymphoid follicle-like big clusters were observed, and in one, there was a very dense and diffuse inflammatory infiltration nearly destroying all the muscle architecture. Seventy-three per cent of the biopsies had T cells, 88% had B cells, 57% had plasma cells, and all had macrophages. As for T-helper cell subtypes, 80% of the biopsies were Th1 positive, 92% Th17 positive and 30% Treg positive. No IL-4 positive inflammatory cell was detected, and only 2 biopsies showed IFN-alpha positivity. The mean JDM biopsy score was 17.6, meaning moderate to severe muscular involvement. Visual analogue score of the pathologist was strongly correlated with histopathological features. B cells, macrophages, plasma cells and T cells constitute the inflammatory milieu of the JDM muscle biopsies. As for T cells, JDM is a disease mainly related with Th1 and Th17 T-helper cell subtypes and to some extend Treg. Th2 cells are not involved in the pathogenesis.

Bi-allelic mutations in MYL1 cause a severe congenital myopathy.

Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, ∼50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain (ELC) in both probands. A homozygous essential splice acceptor variant (c.479-2A > G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modelling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin ELC is critical for myofibre development and function. Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with severe congenital myopathy.

Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization.

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

LARGE expression in different types of muscular dystrophies other than dystroglycanopathy.

BACKGROUND: Alpha-dystroglycan (αDG) is an extracellular peripheral glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin globular domains and certain arenaviruses. An important enzyme, known as Like-acetylglucosaminyltransferase (LARGE), has been shown to transfer repeating units of -glucuronic acid-ß1,3-xylose-α1,3- (matriglycan) to αDG that is required for functional receptor as an extracellular matrix protein scaffold. The reduction in the amount of LARGE-dependent matriglycan result in heterogeneous forms of dystroglycanopathy that is associated with hypoglycosylation of αDG and a consequent lack of ligand-binding activity. Our aim was to investigate whether LARGE expression showed correlation with glycosylation of αDG and histopathological parameters in different types of muscular dystrophies, except for dystroglycanopathies. METHODS: The expression level of LARGE and glycosylation status of αDG were examined in skeletal muscle biopsies from 26 patients with various forms of muscular dystrophy [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), sarcoglycanopathy, dysferlinopathy, calpainopathy, and merosin and collagen VI deficient congenital muscular dystrophies (CMDs)] and correlation of results with different histopathological features was investigated. RESULTS: Despite the fact that these diseases are not caused by defects of glycosyltransferases, decreased expression of LARGE was detected in many patient samples, partly correlating with the type of muscular dystrophy. Although immunolabelling of fully glycosylated αDG with VIA4-1 was reduced in dystrophinopathy patients, no significant relationship between reduction of LARGE expression and αDG hypoglycosylation was detected. Also, Merosin deficient CMD patients showed normal immunostaining with αDG despite severe reduction of LARGE expression. CONCLUSIONS: Our data shows that it is not always possible to correlate LARGE expression and αDG glycosylation in different types of muscular dystrophies and suggests that there might be differences in αDG processing by LARGE which could be regulated under different pathological conditions.

Prenatal Diagnosis of Merosin-Deficient Muscular Dystrophy.

GOAL: We evaluated the potential for prenatal diagnosis of merosin-negative muscular dystrophies by immunohistochemistry. MATERIALS AND METHODS: This is a retrospective study of 12 pregnancies with merosin-negative muscular dystrophy in a prior child. Chorionic villus sampling (CVS) was performed between 11th to 13th gestational weeks. Merosin immunohistochemical studies were performed on trophoblastic cells. RESULTS: Two of 12 were "merosin-negative," both were from the same family. Fetal ultrasonographies were evaluated as normal in these pregnancies. Eight of the 10 merosin-positive cases delivered healthy babies. Two were lost to follow-up. CONCLUSION: Prenatal diagnosis of merosin-negative muscular dystrophies can be accomplished by immunohistochemical analysis.

SPEG interacts with myotubularin, and its deficiency causes centronuclear myopathy with dilated cardiomyopathy.

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.

Recessive PIEZO2 stop mutation causes distal arthrogryposis with distal muscle weakness, scoliosis and proprioception defects.

The genetic work-up of arthrogryposis is challenging due to the diverse clinical and molecular etiologies. We report a-183/12-year-old boy, from a 2nd degree consanguineous family, who presented at 36/12 years with hypotonia, distal laxity, contractures, feeding difficulties at birth. He required surgery for progressive scoliosis at 16 years of age, and walked independently since then with an unstable gait and coordination defects. His latest examination at 18 years of age revealed a proprioceptive defect and loss-of-joint position sense in the upper limbs. Somatosensory evoked potentials supported bilateral involvement of dorsal column-medial lemniscal sensory pathways and nerve conduction studies revealed a mild axonal neuropathy. Muscle biopsy showed myopathic changes with neonatal myosin expression. Mendeliome sequencing led to the discovery of a recessive stop mutation in piezo-type mechanosensitive ion channel component 2 (PIEZO2, NM_022068, c.1384C>T, p.R462*). PIEZO2 is a nonselective cation channel, expressed in sensory endings of proprioceptors innervating muscle spindles and Golgi tendon organs. Dominant PIEZO2 mutations were described in patients with distal arthrogryposis type 5 and Marden-Walker syndrome. Sensory ataxia and proprioception defect with dorsal column involvement together with arthrogryposis, myopathy, scoliosis and progressive respiratory failure may represent a distinct clinical phenotype, and indicate recessive mutations in PIEZO2.

Recessive TTN truncating mutations define novel forms of core myopathy with heart disease.

Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Clinical characteristics of megaconial congenital muscular dystrophy due to choline kinase beta gene defects in a series of 15 patients.

A new form of congenital muscular dystrophy (CMD) with multisystem involvement and characteristic mitochondrial structural changes, due to choline kinase beta (CHKB) gene defects has been characterized by intellectual disability, autistic features, ichthyosis-like skin changes, and dilated cardiomyopathy. We define the clinical characteristics in 15 patients, from 14 unrelated families with so-called 'megaconial CMD', all having mutations in CHKB. Core clinical phenotype included global developmental delay prominent in gross-motor and language domains, severe intellectual disability (ID), and/or muscle weakness in all cases. Muscle biopsies were equivocally 'megaconial' in all. Other peculiarities were: ichthyosis-like skin changes (n = 11), increased serum CK levels (n = 12), microcephaly (n = 6), dysmorphic facial features (n = 7), neonatal hypotonia (n = 3), seizures (n = 3), epileptiform activity without clinically overt seizures (n = 2), dilated cardiomyopathy (n = 2), decreased left ventricular systolic function (n = 2), congenital heart defects (n = 3), sensorineural (n = 1), and conductive hearing loss (n = 1). Ten patients had cranial neuroimaging (MRI-MRS) study, which was notably normal in all, other than one patient having a decreased choline: creatine peak. Intra-familial variability in clinical expression of the disease is noted in four families. Two siblings from the same family, one presenting with global developmental delay and dilated cardiomyopathy, and the other with ichthyosis, ID and proximal weakness without cardiomyopathy died at the ages of 2 years 1 month, and 7 years 4 months respectively. Evolution was progressive (n = 13) and static (n = 2).