Severe Nemaline Myopathy Research Articles

A recurrent ACTA1 amino acid change in mosaic form causes milder asymmetric myopathy

We describe three patients with asymmetric congenital myopathy without definite nemaline bodies and one patient with severe nemaline myopathy. In all four patients, the phenotype had been caused by pathogenic missense variants in ACTA1 leading to the same amino acid change, p.(Gly247Arg). The three patients with milder myopathy were mosaic for their variants. In contrast, in the severely affected patient, the missense variant was present in a de novo, constitutional form. The grade of mosaicism in the three mosaic patients ranged between 20 % and 40 %. We speculate that the milder clinical and histological manifestations of the same ACTA1 variant in the patients with mosaicism reflect the lower abundance of mutant actin in their muscle tissue. Similarly, the asymmetry of body growth and muscle weakness may be a consequence of the affected cells being unevenly distributed. The partial improvement in muscle strength with age in patients with mosaicism might be due to an increased proportion over time of nuclei carrying and expressing two normal alleles.

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

CONGENITAL MYOPATHIES – NEMALINE MYOPATHIES: EP.38 Generation of induced pluripotent stem cell lines from a 4-month-old severe nemaline myopathy patient with a dominant ACTA1 c.553C>A (p.R183S) variant

CONGENITAL MYOPATHIES – NEMALINE MYOPATHIES: EP.38 Generation of induced pluripotent stem cell lines from a 4-month-old severe nemaline myopathy patient with a dominant ACTA1 c.553C>A (p.R183S) variant

Generation of two isogenic induced pluripotent stem cell lines from a 4-month-old severe nemaline myopathy patient with a heterozygous dominant c.553C > A (p.Arg183Ser) variant in the ACTA1 gene

Nemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of abnormal thread- or rod-like structures (nemaline bodies) in myofibres. Pathogenic variants in the skeletal muscle alpha actin gene, ACTA1, cause approximately 25% of all NM cases. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 4-month-old female with severe NM harbouring a dominant variant in ACTA1 (c.553C > A). The isogenic lines displayed characteristic iPSC morphology, expressed pluripotency markers, differentiated into cells of all three germ layers, and possessed normal karyotypes. These lines could be useful models of human ACTA1 disease.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.237Congenital fatal cap-rod myopathy due to a de novo autosomal dominant pathogenic ACTA1 variant

Cap disease is a rare structural congenital myopathy (CM) associated with hypotonia, proximal and facial muscle weakness, and frequently scoliosis and respiratory involvement. Mutations in TPM2, TPM3 and ACTA1 have been associated with cap disease, as well as nemaline myopathy. Combined caps and nemaline rods have been reported in the same patient due to a mutation in TPM3. Here, we report the first case of a severe, fatal CM with distinct and separate caps and nemaline rods identified in skeletal muscle biopsies. The patient was born at 37 weeks of gestation, with a history of polyhydramnios, little spontaneous movements at birth, generalised hypotonia, and required immediate ventilatory support. He died 20 days after birth. Ante mortem and post mortem biopsies from the quadriceps, and post mortem biopsies from the biceps and diaphragm showed diffuse fibre hypotrophy without specific structural alterations on light microscopy. Ultrastructural examination of the ante mortem quadriceps biopsy showed several classical cap lesions, and a few of these contained small nemaline rods. The post mortem quadriceps sample showed nemaline rods in several fibres. Both samples showed mitochondrial paracrystalline inclusions in a few interstitial capillary endothelial cells. Ultrastructural findings were key in directing molecular genetic testing. A next generation sequencing panel for 35 congenital myopathy genes identified a de novo ACTA1 c.739G>C p.(Gly247Arg) variant previously reported in the literature in a patient with severe nemaline myopathy, affecting a highly conserved amino acid, and predicted to affect actin function with In Silico analysis. Our case of ACTA1-related cap-rod myopathy is the most severe presentation of a CM with caps or cap-rods described till date. The case further cements the notion of caps and rods being part of the 'nemaline spectrum' and highlights the remarkable heterogeneity of lesions within the same muscle or same group of muscles.

Development and Phenotype Studies of a Slow Skeletal Troponin T E180 Nonsense Mutation Knock-In Mouse Line

A nonsense mutation in codon Glu180 of TNNT1 gene encoding the slow skeletal muscle isoform of troponin T (ssTnT) causes a recessively inherited severe nemaline myopathy (Amish nemaline myopathy, ANM). Muscle biopsies of ANM patients showed a total loss of ssTnT. The present study developed and characterized mice with the E180 nonsense mutation knocked-in the Tnnt1 gene (ssTnT-KI). Homozygotes of ssTnT-KI mice survived to adulthood. Histology studies revealed significant increases of centralized nuclei in the soleus muscle of ssTnT-KI when compared with age matched wild type controls. Immunohistochemistry staining showed atrophic slow type 1 fibers in ssTnT-KI mouse soleus muscle with severe inflammatory cells infiltration. Western blots confirmed the absence of full length ssTnT with no ssTnT fragments detectable, mimicking that seen in the muscle of ANM patients. Contractile properties of ssTnT-KI mouse soleus muscle measured in situ demonstrated a decreased fatigue resistance. Significant expression of cardiac TnT is detected in soleus muscle of ssTnT-KI mice, consistent with the presence of active muscle regeneration. Decrease of slow type isoforms and increase of fast type isoforms of other myofilament proteins with a significant hypertrophy of fast fibers in ssTnT-KI mouse soleus muscle indicate a potentially compensatory adaptation to the loss of slow fiber functions. The ssTnT-KI mouse line provides a model of ANM for pathogenesis, pathophysiology and therapeutic studies.

A Premature Stop Codon in MYO18B Is Associated with Severe Nemaline Myopathy with Cardiomyopathy (P5.067)

A Premature Stop Codon in MYO18B Is Associated with Severe Nemaline Myopathy with Cardiomyopathy (P5.067)

A Large Deletion Affecting TPM3, Causing Severe Nemaline Myopathy.

Background and Objectives:Nemaline myopathy may be caused by pathogenic variants in the TPM3 gene and is then called NEM1. All previously identified disease-causing variants are point mutations including missense, nonsense and splice-site variants. The aim of the study was to identify the disease-causing gene in this patient and verify the NM diagnosis.Methods:Mutation analysis methods include our self-designed nemaline myopathy array, The Nemaline Myopathy Comparative Genomic Hybridisation Array (NM-CGH array), whole-genome array-CGH, dHPLC, Sanger sequencing and whole-exome sequencing. The diagnostic muscle biopsy was investigated further by routine histopathological methods.Results:We present here the first large (17–21 kb) aberration in the α-tropomyosinslow gene (TPM3), identified using the NM-CGH array. This homozygous deletion removes the exons 1a and 2b as well as the promoter of the TPM3 isoform encoding Tpm3.12st. The severe phenotype included paucity of movement, proximal and axial weakness and feeding difficulties requiring nasogastric tube feeding. The infant died at the age of 17.5 months. Muscle biopsy showed variation in fibre size and rods in a population of hypotrophic muscle fibres expressing slow myosin, often with internal nuclei, and abnormal immunolabelling revealing many hybrid fibres.Conclusions:This is the only copy number variation we have identified in any NM gene other than nebulin (NEB), suggesting that large deletions or duplications in these genes are very rare, yet possible, causes of NM.

G.O.5 - A premature stop codon in MYO18B is associated with severe nemaline myopathy with cardiomyopathy

G.O.5 - A premature stop codon in MYO18B is associated with severe nemaline myopathy with cardiomyopathy

A Premature Stop Codon in MYO18B isAssociated with Severe Nemaline Myopathy with Cardiomyopathy.

Background: Nemaline myopathies (NM) are rare and severe muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Although ten genes have been implicated in the etiology of NM, an important number of patients remain without a molecular diagnosis. Objective: Here we describe the clinical and histopathological features of a sporadic case presenting with severe NM and cardiomyopathy. Using exome sequencing, we aimed to identify the causative gene. Results: We identified a homozygous nonsense mutation in the last exon of MYO18B, leading to a truncated protein lacking the most C-terminal part. MYO18B codes for an unconventional myosin protein and it is mainly expressed in skeletal and cardiac muscles, two tissues severely affected in the patient. We showed that the mutation does not impact on mRNA stability. Immunostaining and Western blot confirmed the absence of the full-length protein. Conclusion: We propose MYO18B as a novel gene associated with nemaline myopathy and cardiomyopathy.

G.P.270: A large genomic rearrangement affecting TPM3 causing severe nemaline myopathy

Using our self-designed NM-CGH microarray, which we have developed to identify large copy number variations in the currently known nine nemaline myopathy (NM) genes, we identified the first large (>20kb) aberration in the alpha-tropomyosin gene (<i>TPM3</i>). This homozygous mutation deletes the promoter as well as the muscle- specific exons 1 and 2 of <i>TPM3</i>. The promoter and exon 1 of the non-muscle isoform seem to be intact. The deletion also spans the micro-RNA-encoding <i>MIR190B</i> gene and the last two exons of the <i>C1orf189</i> gene upstream of <i>TPM3</i>. Our attempts to sequence the promoter and exon 1 of TPM3 have constantly failed, supporting the array result, showing an approximately 6kb deletion including the musclespecific exons 1 and 2 as well as regions upstream from the gene. Before analyzing the patient sample using our array, we had analyzed other NM genes including nebulin (<i>NEB</i>), in which we identified a heterozygous nonsense mutation in exon 42. No second mutation, however, was identified in <i>NEB</i> and this nonsense mutation likely has a modifying impact on the phenotype of the patient. We also performed whole-exome sequencing, but no other pathogenic mutations were revealed. The patient was born to consanguineous parents. He deceased at the age of approximately 1.5years. His biopsy confirmed the diagnosis of NM. Unfortunately, to date, no further clinical details are available. We have screened 250 samples from 185 NM and related myopathy families using the NM-CGH-array and identified nine large aberrations in <i>NEB</i> in nine families, but we have not detected such mutations in other NM genes except the one discussed here. Therefore large aberrations in known NM genes other than NEB are likely a very rare cause of NM. The NM-CGH microarray method is currently available for mutation analysis in our laboratory.

G.O.2: Mutations in LMOD3 cause severe nemaline myopathy by disrupting thin filament organisation in skeletal muscle

Nemaline myopathy (NM) is a disorder of the skeletal muscle thin filament characterised by muscle dysfunction and electron-dense protein accumulations (nemaline bodies). Pathogenic mutations have been described in nine genes to date, but the genetic basis remains unknown in many cases. We used whole exome sequencing (WES) in two families with NM and subsequent gene sequencing in over 540 additional genetically unresolved NM patients to identify and characterise a new genetic cause of NM. We developed a knock-down zebrafish model of this condition and used immunohistochemistry, western blotting, single-fibre contractility studies and recombinant protein studies to characterise the expression, localisation and biochemical functions of the new disease-related protein. We identified homozygous or compound heterozygous variants in LMOD3, which encodes leiomodin-3 (Lmod3) in 21 patients from 14 families. Affected individuals had severe generalised weakness and hypotonia, and most affected individuals died in the neonatal period. We demonstrated that Lmod3 is expressed from early muscle differentiation, localises to thin filaments with enrichment at the pointed ends, and has strong actin nucleating activity. Loss of Lmod3 in patient muscle results in shortening and disorganisation of thin filaments. Knockdown of lmod3 in the zebrafish replicates this phenotype. These findings define a new genetic subtype of congenital myopathy and demonstrate an essential, previously unrecognised role for Lmod3 in the regulation of sarcomeric thin filaments in skeletal muscle.

G.P.273: Nemaline myopathy 8 and KLHL40 in diseased and normal skeletal muscle

Mutations in the gene ( KLHL40 ), encoding Kelch-like family member 40, were recently identified in patients presenting with severe autosomal recessive nemaline myopathy (NEM8). Using exome sequencing or targeted re-sequencing of 276 known neuromuscular disease genes through a TargetSeq (Life Technologies) capture panel, we have identified two additional cases with KLHL40 mutations. Both were from consanguineous kindreds. Case 1 was diagnosed with severe SMA and congenital fractures. We identified a homozygous nonsense mutation in KLHL40 (c.46C>T, p. Gln16*). Muscle biopsy identified variation in fibre size and miliary nemaline bodies by electron microscopy seen as very fine scattered granules upon Gomori trichrome staining. Case 2 presented with severe nemaline myopathy, the biopsy showing numerous tiny nemaline bodies. We identified a homozygous missense mutation in KLHL40 (c.931C>A, p. Arg311Ser). Neither of the variants was present in 1000genomes or EVS databases. These data further suggest that muscle biopsy may be useful to inform genetic testing and that KLHL40 should be considered in patients presenting with severe miliary nemaline myopathy. Nemaline myopathy should also be considered in the differential diagnosis of severe SMA, especially in patients presenting with congenital fractures and contractures. The normal biological function of KLHL40 is largely unknown and therefore the basis of the severe muscle disease resulting from mutations of KLHL40 is also unknown. In investigations of control and diseased mouse and patient muscle, we show that KLHL40 is more abundant in the mouse soleus and diaphragm (muscles that comprise oxidative type I and IIA myofibres) than in the EDL, gastrocnemius and quadriceps muscles (type II glycolytic predominant). In addition we show that KLHL40 is increased in dystrophic mouse ( mdx ) and regenerating patient muscle (including DMD), suggesting that it may serve as a novel marker of skeletal muscle regeneration.

P17 Whole exome sequencing in patients with congenital myopathies

Congenital myopathies (CM) are a group of disorders presenting at birth or early infancy, characterised by muscle weakness and specific changes in the muscle biopsy. During the recent decade a number of genes have been discovered, however, additional novel genes are yet to be identified as genetic diagnosis cannot be currently established in many CM patients. With the aim to identify the genetic defect in 33 CM patients, in whom mutations in suspected genes have been previously excluded, we carried out whole exome sequencing (WES). From the 33 patients, 19 have been resolved and 14 patients are still being investigated as the initial WES data analysis failed to identify possible candidate genes. Among the resolved cases, two patients with muscle biopsy suggestive of core myopathy and one with centronuclear myopathy, carried heterozygous truncating TTN mutations supporting the emerging data that TTN mutations should be investigated as causative in patients with unresolved centronuclear and core myopathy. We also identified a homozygous missense mutation in STAC3 gene in a patient with King-Denborough syndrome and core-like changes on muscle biopsy, indicating a causative role of STAC3 mutations in King–Denborough syndrome. Recently, a homozygous missense mutation in STAC3 gene was identified in patients with Native American myopathy. A patient with a severe phenotype and muscle biopsy changes suggestive of nemaline myopathy, carried a homozygous missense mutation in KLHL40. Mutations in KLHL40 have been very recently identified as a frequent cause of severe autosomal-recessive nemaline myopathy. Ten of the resolved patients carried mutations in potentially causative genes which are currently under investigation.

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.

Distinct Underlying Mechanisms of Limb and Respiratory Muscle Fiber Weaknesses in Nemaline Myopathy

Nemaline myopathy is the most common congenital myopathy and is caused by mutations in various genes such as ACTA1 (encoding skeletal α-actin). It is associated with limb and respiratory muscle weakness. Despite increasing clinical and scientific interest, the molecular and cellular events leading to such weakness remain unknown, which prevents the development of specific therapeutic interventions. To unravel the potential mechanisms involved, we dissected lower limb and diaphragm muscles from a knock-in mouse model of severe nemaline myopathy expressing the ACTA1 His40Tyr actin mutation found in human patients. We then studied a broad range of structural and functional characteristics assessing single-myofiber contraction, protein expression, and electron microscopy. One of the major findings in the diaphragm was the presence of numerous noncontractile areas (including disrupted sarcomeric structures and nemaline bodies). This greatly reduced the number of functional sarcomeres, decreased the force generation capacity at the muscle fiber level, and likely would contribute to respiratory weakness. In limb muscle, by contrast, there were fewer noncontractile areas and they did not seem to have a major role in the pathogenesis of weakness. These divergent muscle-specific results provide new important insights into the pathophysiology of severe nemaline myopathy and crucial information for future development of therapeutic strategies.

C.P.12 The H40Y α-actin mutation differently affects limb and respiratory muscle contraction

Nemaline myopathy is the most common of the congenital non-dystrophic myopathies, with an estimated incidence of 1:50,000. The severe form of this disease is characterized by generalized weakness especially affecting limb and respiratory muscles. Questions that remain unanswered are (i) whether limb and respiratory muscles are affected to the same extent, and (ii) whether the underlying mechanisms are similar. The aim of the present study was to address these questions in a novel transgenic mouse model of severe nemaline myopathy. This model expresses the heterozygous amino acid substitution, H40Y, in skeletal α-actin. Hence, we dissected EDL and diaphragm muscles and evaluated the contraction mechanism. To our surprise, we observed muscle-specific defects that will be presented during the conference. These findings give valuable information for future potential therapeutic interventions.

Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression.

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy

Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary L-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that L-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.

Novel mutations in NEB cause abnormal nebulin expression and markedly impaired muscle force generation in severe nemaline myopathy

BackgroundNemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases.ResultsWe describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.ConclusionsOur findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.