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.

Nemaline myopathies: a current view.

Nemaline myopathies are a heterogenous group of congenital myopathies caused by de novo, dominantly or recessively inherited mutations in at least twelve genes. The genes encoding skeletal α-actin (ACTA1) and nebulin (NEB) are the commonest genetic cause. Most patients have congenital onset characterized by muscle weakness and hypotonia, but the spectrum of clinical phenotypes is broad, ranging from severe neonatal presentations to onset of a milder disorder in childhood. Most patients with adult onset have an autoimmune-related myopathy with a progressive course. The wide application of massively parallel sequencing methods is increasing the number of known causative genes and broadening the range of clinical phenotypes. Nemaline myopathies are identified by the presence of structures that are rod-like or ovoid in shape with electron microscopy, and with light microscopy stain red with the modified Gömöri trichrome technique. These rods or nemaline bodies are derived from Z lines (also known as Z discs or Z disks) and have a similar lattice structure and protein content. Their shape in patients with mutations in KLHL40 and LMOD3 is distinctive and can be useful for diagnosis. The number and distribution of nemaline bodies varies between fibres and different muscles but does not correlate with severity or prognosis. Additional pathological features such as caps, cores and fibre type disproportion are associated with the same genes as those known to cause the presence of rods. Animal models are advancing the understanding of the effects of various mutations in different genes and paving the way for the development of therapies, which at present only manage symptoms and are aimed at maintaining muscle strength, joint mobility, ambulation, respiration and independence in the activities of daily living.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy.

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

Specific localization of nesprin-1-α2, the short isoform of nesprin-1 with a KASH domain, in developing, fetal and regenerating muscle, using a new monoclonal antibody.

BACKGROUND: Nesprin-1-giant (1008kD) is a protein of the outer nuclear membrane that links nuclei to the actin cytoskeleton via amino-terminal calponin homology domains. The short nesprin-1 isoform, nesprin-1-α2, is present only in skeletal and cardiac muscle and several pathogenic mutations occur within it, but the functions of this short isoform without calponin homology domains are unclear. The aim of this study was to determine mRNA levels and protein localization of nesprin-1-α2 at different stages of muscle development in order to shed light on its functions. RESULTS: mRNA levels of all known nesprin-1 isoforms with a KASH domain were determined by quantitative PCR. The mRNA for the 111 kD muscle-specific short isoform, nesprin-1-α2, was not detected in pre-differentiation human myoblasts but was present at significant levels in multinucleate myotubes. We developed a monoclonal antibody against the unique amino-terminal sequence of nesprin-1-α2, enabling specific immunolocalization for the first time. Nesprin-1-α2 protein was undetectable in pre-differentiation myoblasts but appeared at the nuclear rim in post-mitotic, multinucleate myotubes and reached its highest levels in fetal muscle. In muscle from a Duchenne muscular dystrophy biopsy, nesprin-1-α2 protein was detected mainly in regenerating fibres expressing neonatal myosin. Nesprin-1-giant was present at all developmental stages, but was also highest in fetal and regenerating fibres. In fetal muscle, both isoforms were present in the cytoplasm, as well as at the nuclear rim. A pathogenic early stop codon (E7854X) in nesprin-1 caused reduced mRNA levels and loss of protein levels of both nesprin-1-giant and (unexpectedly) nesprin-1-α2, but did not affect myogenesis in vitro. CONCLUSIONS: Nesprin-1-α2 mRNA and protein expression is switched on during myogenesis, alongside other known markers of muscle differentiation. The results show that nesprin-1-α2 is dynamically controlled and may be involved in some process occurring during early myofibre formation, such as re-positioning of nuclei.

Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of α-dystroglycan.

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a ß-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.

Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy.

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

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.

Phenotypic and molecular insights into spinal muscular atrophy due to mutations in BICD2.

Spinal muscular atrophy is a disorder of lower motor neurons, most commonly caused by recessive mutations in SMN1 on chromosome 5q. Cases without SMN1 mutations are subclassified according to phenotype. Spinal muscular atrophy, lower extremity-predominant, is characterized by lower limb muscle weakness and wasting, associated with reduced numbers of lumbar motor neurons and is caused by mutations in DYNC1H1, which encodes a microtubule motor protein in the dynein-dynactin complex and one of its cargo adaptors, BICD2. We have now identified 32 patients with BICD2 mutations from nine different families, providing detailed insights into the clinical phenotype and natural history of BICD2 disease. BICD2 spinal muscular atrophy, lower extremity predominant most commonly presents with delayed motor milestones and ankle contractures. Additional features at presentation include arthrogryposis and congenital dislocation of the hips. In all affected individuals, weakness and wasting is lower-limb predominant, and typically involves both proximal and distal muscle groups. There is no evidence of sensory nerve involvement. Upper motor neuron signs are a prominent feature in a subset of individuals, including one family with exclusively adult-onset upper motor neuron features, consistent with a diagnosis of hereditary spastic paraplegia. In all cohort members, lower motor neuron features were static or only slowly progressive, and the majority remained ambulant throughout life. Muscle MRI in six individuals showed a common pattern of muscle involvement with fat deposition in most thigh muscles, but sparing of the adductors and semitendinosus. Muscle pathology findings were highly variable and included pseudomyopathic features, neuropathic features, and minimal change. The six causative mutations, including one not previously reported, result in amino acid changes within all three coiled-coil domains of the BICD2 protein, and include a possible 'hot spot' mutation, p.Ser107Leu present in four families. We used the recently solved crystal structure of a highly conserved region of the Drosophila orthologue of BICD2 to further-explore how the p.Glu774Gly substitution inhibits the binding of BICD2 to Rab6. Overall, the features of BICD2 spinal muscular atrophy, lower extremity predominant are consistent with a pathological process that preferentially affects lumbar lower motor neurons, with or without additional upper motor neuron involvement. Defining the phenotypic features in this, the largest BICD2 disease cohort reported to date, will facilitate focused genetic testing and filtering of next generation sequencing-derived variants in cases with similar features.

ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies.

Dystroglycanopathies are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe of the congenital muscular dystrophies, Walker-Warburg syndrome, to mild forms of adult-onset limb-girdle muscular dystrophy. Their hallmark is a reduction in the functional glycosylation of α-dystroglycan, which can be detected in muscle biopsies. An important part of this glycosylation is a unique O-mannosylation, essential for the interaction of α-dystroglycan with extracellular matrix proteins such as laminin-α2. Mutations in eight genes coding for proteins in the glycosylation pathway are responsible for ∼50% of dystroglycanopathy cases. Despite multiple efforts using traditional positional cloning, the causative genes for unsolved dystroglycanopathy cases have escaped discovery for several years. In a recent collaborative study, we discovered that loss-of-function recessive mutations in a novel gene, called isoprenoid synthase domain containing (ISPD), are a relatively common cause of Walker-Warburg syndrome. In this article, we report the involvement of the ISPD gene in milder dystroglycanopathy phenotypes ranging from congenital muscular dystrophy to limb-girdle muscular dystrophy and identified allelic ISPD variants in nine cases belonging to seven families. In two ambulant cases, there was evidence of structural brain involvement, whereas in seven, the clinical manifestation was restricted to a dystrophic skeletal muscle phenotype. Although the function of ISPD in mammals is not yet known, mutations in this gene clearly lead to a reduction in the functional glycosylation of α-dystroglycan, which not only causes the severe Walker-Warburg syndrome but is also a common cause of the milder forms of dystroglycanopathy.

Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Troponin-T type 1 (TNNT1)-related nemaline myopathy: unique respiratory phenotype and muscle pathology findings.

Biallelic pathogenic variants in the troponin T type 1 (TNNT1) gene cause a severe form of congenital nemaline myopathy. Typical features include severe motor delay, proximal contractures and weakness, pectus carinatum, chest wall rigidity and tremor. If left untreated, respiratory failure leads to early death at a median age of 18 months. Here we report on three non-Amish, unrelated patients harbouring novel TNNT1 variants. The peculiar combination of respiratory muscle weakness and chest wall stiffness caused early severe hypoventilation warranting the use of high pressures on BiPAP ventilator, with subsequent rapid escalation of pressures delivered with limited efficacy secondary to the extreme rib cage stiffness. Severe respiratory impairment occurred despite a relatively milder motor involvement in one patient. Muscle biopsies from two individuals showed predominant involvement of type 1 fibres, abundant nemaline bodies, marked fibrosis and loss of TNNT1 protein. We aim to increase the awareness of the challenges of managing respiratory support in patients with this unique respiratory phenotype.

Nesprins, but not sun proteins, switch isoforms at the nuclear envelope during muscle development.

Nesprins are a family of nuclear transmembrane proteins anchored via Sun proteins to the nuclear membrane. Analysis of nesprins during human muscle development revealed an increase in nesprin-1-giant during early myogenesis in vitro. During the transition from immature to mature muscle fibres in vivo, nesprin-2 partly replaced nesprin-1 at the nuclear envelope and short nesprin isoforms became dominant. Sun1 and Sun2 proteins remained unchanged during this fibre maturation. In emerin-negative skin fibroblasts, nesprin-2-giant was relocated from the nuclear envelope to the cytoplasm, not to the endoplasmic reticulum, while nesprin-1 remained at the nuclear envelope. In emerin-negative keratinocytes lacking nesprin-1, nesprin-2 remained at the nuclear envelope. HeLa cell nuclear envelopes lacked nesprin-1, which was the dominant form in myoblasts, while a novel 130-kD nesprin-2 isoform dominated Ntera-2 cells. The results suggest the possibility of isoform-specific and tissue-specific roles for nesprins in nuclear positioning.

High-Throughput Digital Image Analysis Reveals Distinct Patterns of Dystrophin Expression in Dystrophinopathy Patients.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.

Muscleblind-like proteins: similarities and differences in normal and myotonic dystrophy muscle.

In myotonic dystrophy, muscleblind-like protein 1 (MBNL1) protein binds specifically to expanded CUG or CCUG repeats, which accumulate as discrete nuclear foci, and this is thought to prevent its function in the regulation of alternative splicing of pre-mRNAs. There is strong evidence for the role of the MBNL1 gene in disease pathology, but the roles of two related genes, MBNL2 and MBNL3, are less clear. Using new monoclonal antibodies specific for each of the three gene products, we found that MBNL2 decreased during human fetal development and myoblast culture, while MBNL1 was unchanged. In Duchenne muscular dystrophy muscle, MBNL2 was elevated in immature, regenerating fibres compared with mature fibres, supporting some developmental role for MBNL2. MBNL3 was found only in C2C12 mouse myoblasts. Both MBNL1 and MBNL2 were partially sequestered by nuclear foci of expanded repeats in adult muscle and cultured cells from myotonic dystrophy patients. In adult muscle nucleoplasm, both proteins were reduced in myotonic dystrophy type 1 compared with an age-matched control. In normal human myoblast cultures, MBNL1 and MBNL2 always co-distributed but their distribution could change rapidly from nucleoplasmic to cytoplasmic. Functional differences between MBNL1 and MBNL2 have not yet been found and may prove quite subtle. The dominance of MBNL1 in mature, striated muscle would explain why ablation of the mouse mbnl1 gene alone is sufficient to cause a myotonic dystrophy.

Muscular dystrophies: an update on pathology and diagnosis.

Muscular dystrophies are clinically, genetically, and molecularly a heterogeneous group of neuromuscular disorders. Considerable advances have been made in recent years in the identification of causative genes, the differentiation of the different forms and in broadening the understanding of pathogenesis. Muscle pathology has an important role in these aspects, but correlation of the pathology with clinical phenotype is essential. Immunohistochemistry has a major role in differential diagnosis, particularly in recessive forms where an absence or reduction in protein expression can be detected. Several muscular dystrophies are caused by defects in genes encoding sarcolemmal proteins, several of which are known to interact. Others are caused by defects in nuclear membrane proteins or enzymes. Assessment of both primary and secondary abnormalities in protein expression is useful, in particular the hypoglycosylation of alpha-dystroglycan. In dominantly inherited muscular dystrophies it is rarely possible to detect a change in the expression of the primary defective protein; an exception to this is caveolin-3.

De novo LMNA mutations cause a new form of congenital muscular dystrophy.

OBJECTIVE: To describe a new entity of congenital muscular dystrophies caused by de novo LMNA mutations. METHODS: Fifteen patients presenting with a myopathy of onset in the first year of life were subjected to neurological and genetic evaluation. Histopathological and immunohistochemical analyses were performed for all patients. RESULTS: The 15 patients presented with muscle weakness in the first year of life, and all had de novo heterozygous LMNA mutations. Three of them had severe early-onset disease, no motor development, and the rest experienced development of a "dropped head" syndrome phenotype. Despite variable severity, there was a consistent clinical pattern. Patients typically presented with selective axial weakness and wasting of the cervicoaxial muscles. Limb involvement was predominantly proximal in upper extremities and distal in lower extremities. Talipes feet and a rigid spine with thoracic lordosis developed early. Proximal contractures appeared later, most often in lower limbs, sparing the elbows. Ten children required ventilatory support, three continuously through tracheotomy. Cardiac arrhythmias were observed in four of the oldest patients but were symptomatic only in one. Creatine kinase levels were mild to moderately increased. Muscle biopsies showed dystrophic changes in nine children and nonspecific myopathic changes in the remaining. Markedly atrophic fibers were common, most often type 1, and a few patients showed positive inflammatory markers. INTERPRETATION: The LMNA mutations identified appear to correlate with a relatively severe phenotype. Our results further broaden the spectrum of laminopathies and define a new disease entity that we suggest is best classified as a congenital muscular dystrophy (LMNA-related congenital muscular dystrophy, or L-CMD).