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.

Muscle weakness in TPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres.

Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.

Mutations in the TPM2 gene, which encodes ß-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of ß-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-ß-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-ß-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

Dominant mutations in KBTBD13, a member of the BTB/Kelch family, cause nemaline myopathy with cores.

We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecule's beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

Mutations in contactin-1, a neural adhesion and neuromuscular junction protein, cause a familial form of lethal congenital myopathy.

We have previously reported a group of patients with congenital onset weakness associated with a deficiency of members of the syntrophin-alpha-dystrobrevin subcomplex and have demonstrated that loss of syntrophin and dystrobrevin from the sarcolemma of skeletal muscle can also be associated with denervation. Here, we have further studied four individuals from a consanguineous Egyptian family with a lethal congenital myopathy inherited in an autosomal-recessive fashion and characterized by a secondary loss of beta2-syntrophin and alpha-dystrobrevin from the muscle sarcolemma, central nervous system involvement, and fetal akinesia. We performed homozygosity mapping and candidate gene analysis and identified a mutation that segregates with disease within CNTN1, the gene encoding for the neural immunoglobulin family adhesion molecule, contactin-1. Contactin-1 transcripts were markedly decreased on gene-expression arrays of muscle from affected family members compared to controls. We demonstrate that contactin-1 is expressed at the neuromuscular junction (NMJ) in mice and man in addition to the previously documented expression in the central and peripheral nervous system. In patients with secondary dystroglycanopathies, we show that contactin-1 is abnormally localized to the sarcolemma instead of exclusively at the NMJ. The cntn1 null mouse presents with ataxia, progressive muscle weakness, and postnatal lethality, similar to the affected members in this family. We propose that loss of contactin-1 from the NMJ impairs communication or adhesion between nerve and muscle resulting in the severe myopathic phenotype. This disorder is part of the continuum in the clinical spectrum of congenital myopathies and congenital myasthenic syndromes.

Disease severity and thin filament regulation in M9R TPM3 nemaline myopathy.

The mechanism of muscle weakness was investigated in an Australian family with an M9R mutation in TPM3 (alpha-tropomyosin(slow)). Detailed protein analyses of 5 muscle samples from 2 patients showed that nemaline bodies are restricted to atrophied Type 1 (slow) fibers in which the TPM3 gene is expressed. Developmental expression studies showed that alpha-tropomyosin(slow) is not expressed at significant levels until after birth, thereby likely explaining the childhood (rather than congenital) disease onset in TPM3 nemaline myopathy. Isoelectric focusing demonstrated that alpha-tropomyosin(slow) dimers, composed of equal ratios of wild-type and M9R-alpha-tropomyosin(slow), are the dominant tropomyosin species in 3 separate muscle groups from an affected patient. These findings suggest that myopathy-related slow fiber predominance likely contributes to the severity of weakness in TPM3 nemaline myopathy because of increased proportions of fibers that express the mutant protein. Using recombinant proteins and far Western blot, we demonstrated a higher affinity of tropomodulin for alpha-tropomyosin(slow) compared with beta-tropomyosin; the M9R substitution within alpha-tropomyosin(slow) greatly reduced this interaction. Finally, transfection of the M9R mutated and wild-type alpha-tropomyosin(slow) into myoblasts revealed reduced incorporation into stress fibers and disruption of the filamentous actin network by the mutant protein. Collectively, these results provide insights into the clinical features and pathogenesis of M9R-TPM3 nemaline myopathy.

Intranuclear rod myopathy: molecular pathogenesis and mechanisms of weakness.

OBJECTIVE: Mutations in the alpha-skeletal actin gene (ACTA1) result in a variety of inherited muscle disorders characterized by different pathologies and variable clinical phenotypes. Mutations at Val163 in ACTA1 result in pure intranuclear rod myopathy; however, the molecular mechanisms by which mutations at Val163 lead to intranuclear rod formation and muscle weakness are unknown. METHODS AND RESULTS: We investigated the effects of the Val163Met mutation in ACTA1 in tissue culture and Drosophila models, and in patient muscle. In cultured cells, the mutant actin tends to aggregate rather than incorporate into cytoplasmic microfilaments, and it affects the dynamics of wild-type actin, causing it to accumulate with the mutant actin in the nucleus. In Drosophila, the Val163Met mutation severely disrupts the structure of the muscle sarcomere. The intranuclear aggregates in patient muscle biopsies impact on nuclear structure and sequester normal Z-disc-associated proteins within the nucleus; however, the sarcomeric structure is relatively well preserved, with evidence of active regeneration. By mass spectrometry, the levels of mutant protein are markedly reduced in patient muscle compared with control. INTERPRETATION: Data from our tissue culture and Drosophila models show that the Val163Met mutation in alpha-skeletal actin can affect the dynamics of other actin isoforms and severely disrupt sarcomeric structure, processes that can contribute to muscle weakness. However, in human muscle, there is evidence of regeneration, and the mutant protein tends to aggregate rather than incorporate into cytoplasmic microfilaments in cells. These are likely compensatory processes that ameliorate the effects of the mutant actin and contribute to the milder clinical and pathological disease phenotype.

Mechanisms underlying intranuclear rod formation.

Specific mutations within the alpha-skeletal actin gene (ACTA1) result in intranuclear rod myopathy (IRM), characterized by rod-like aggregates containing actin and alpha-actinin-2 inside the nucleus of muscle cells. The mechanism leading to formation of intranuclear aggregates containing sarcomeric proteins and their impact on cell function and contribution to disease pathogenesis is unknown. In this study, we transfected muscle and non-muscle cells with mutants of alpha-skeletal actin (Val163Leu, Val163Met) associated with intranuclear rod myopathy. By live-cell imaging we demonstrate that nuclear aggregates of actin form within the nuclear compartment, rather than entering the nucleus after formation in the cytoplasm, and are highly motile and dynamic structures. Thus, the nuclear environment supports the polymerization of actin and the movement and coalescence of the polymerized actin into larger structures. We show that the organization of actin within these aggregates is influenced by the binding of alpha-actinin, and that alpha-actinin is normally present in the nucleus of muscle and non-muscle cells. Furthermore, we demonstrate that, under conditions of cell stress (cytoskeletal disruption and ATP depletion), WT skeletal actin forms aggregates within the nucleus that are similar in morphology to those formed by the mutant actin, suggesting a common pathogenic mechanism for aggregate formation. Finally, we show that the presence of intranuclear actin aggregates significantly decreases the mitotic index and hence impacts on the function of the cell. Intranuclear aggregates thus likely contribute to the pathogenesis of muscle weakness in intranuclear rod myopathy.

Investigations into the pathobiology of thin-filament myopathies.

Mutations in actin and tropomyosin, identified in patients with myopathic disease have been used in tissue culture models and functional studies with a view to understand how these mutations impact on skeletal muscle structure and function and result in muscle weakness. The likely mode of pathogenesis in these disorders is via a dominant negative effect i.e., the production of 'poison' proteins that interfere with the normal function of the native protein. Tissue culture models and in vitro binding studies highlight the defects of different actin mutants including abnormal folding, aggregation and altered polymerization which would likely impact on skeletal muscle structure and function. The most widely studied mutation in tropomyosin is the M9R substitution identified in a large Australian family with nemaline myopathy. The M9R mutant protein has a reduced affinity for actin, does not bind to tropomodulin in a model peptide and results in reduced sensitivity of isometric force to activating calcium in cardiac myocytes. The pathological consequences of mutations identified in troponin, nebulin, and cofilin are also discussed. Although mutations in alpha-actinin have not been associated with NM, tissue culture models using tagged constructs of different regions of the alpha-actinin gene suggest that this protein plays a role in nemaline body formation.

Autosomal dominant nemaline myopathy with intranuclear rods due to mutation of the skeletal muscle ACTA1 gene: clinical and pathological variability within a kindred.

Nemaline Myopathy with Intranuclear Rods is a rare variant of nemaline myopathy, due in almost all instances to mutation of ACTA1, the gene encoding skeletal muscle alpha-actin. We describe the novel autosomal dominant occurrence in a three-generation kindred, and review previously reported cases. Onset of myopathic symptoms in our kindred was in infancy or early childhood. Beyond infancy, limb muscle weakness was non-disabling and minimally progressive. A tall thin face and facial myopathy were prominent features in the affected adults. By light microscopy, muscle biopsies ranged from almost normal, to chronic myopathy with sarcoplasmic and intranuclear rods. A heterozygous GTG-ATG mutation (Val163Met) was found in exon 4 of ACTA1 in affected individuals. Actin is normally present within the nucleus in only trace amounts. Mutation at postion 163 may result in intranuclear rods by virtue of its close proximity to a nuclear export signal within the actin molecule.

Defining alpha-skeletal and alpha-cardiac actin expression in human heart and skeletal muscle explains the absence of cardiac involvement in ACTA1 nemaline myopathy.

Mutations in alpha-skeletal actin (ACTA1) underlie several congenital muscle disorders including nemaline myopathy (NM). Almost all ACTA1-NM patients have normal cardiac function, and, even lethally affected congenital NM patients exhibit an unremarkable gestation with decreased foetal movement just prior to birth. Although alpha-skeletal actin is thought to be the predominant sarcomeric actin in human heart (Boheler KR, Carrier L, de la Bastie D, et al. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest 1991;88:323-30 ), ACTA1-NM patients almost never exhibit a cardiac phenotype. In this study, we define the relative expression of skeletal and cardiac actin proteins in human heart and skeletal muscle. We show that alpha-cardiac actin is the predominant sarcomeric isoform in human donor hearts and in early foetal skeletal muscle development. Skeletal actin is the predominant isoform from 25 to 27 weeks gestation and is the exclusive isoform expressed in muscle from infancy through to adulthood. These findings are consistent with clinical observations of NM patients and assist us to better understand the pathogenesis of inherited myopathies and cardiomyopathies with mutations in actin.

Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine.

Variants in ACTA1, which encodes α-skeletal actin, cause several congenital myopathies, most commonly nemaline myopathy. Autosomal recessive variants comprise approximately 10% of ACTA1 myopathy. All recessive variants reported to date have resulted in loss of skeletal α-actin expression from muscle and severe weakness from birth. Targeted next-generation sequencing in two brothers with congenital muscular dystrophy with rigid spine revealed homozygous missense variants in ACTA1. Skeletal α-actin expression was preserved in these patients. This report expands the clinical and histological phenotype of ACTA1 disease to include congenital muscular dystrophy with rigid spine and dystrophic features on muscle biopsy. This represents a new class of recessive ACTA1 variants, which do not abolish protein expression.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

In vitro analysis of rod composition and actin dynamics in inherited myopathies.

Rods are the pathological hallmark of nemaline myopathy, but they can also occur as a secondary phenomenon in other disorders, including mitochondrial myopathies such as complex I deficiency. The mechanisms of rod formation are not well understood, particularly when rods occur in diverse disorders with very different structural and metabolic defects. We compared the characteristics of rods associated with abnormalities in structural components of skeletal muscle thin filament (3 mutations in the skeletal actin gene ACTA1) with those of rods induced by the metabolic cell stress of adenosine triphosphate depletion. C2C12 and NIH/3T3 cell culture models and immunocytochemistry were used to study rod composition and conformation. Fluorescent recovery after photobleaching was used to measure actin dynamics inside the rods. We demonstrate that not all rods are the same. Rods formed under different conditions contain a unique fingerprint of actin-binding proteins (cofilin and alpha-actinin) and display differences in actin dynamics that are specific to the mutation, to the cellular location of the rods (intranuclear vs cytoplasmic), and/or to the underlying pathological process (i.e. mutant actin or adenosine triphosphate depletion). Thus, rods likely represent a common morphological end point of a variety of different pathological processes, either structural or metabolic.

Expanding the clinical, pathological and MRI phenotype of DNM2-related centronuclear myopathy.

Mutations in dynamin-2 (DNM2) cause autosomal dominant centronuclear myopathy (CNM). We report a series of 12 patients from eight families with CNM in whom we have identified a number of novel features that expand the reported clinicopathological phenotype. We identified two novel and five recurrent missense mutations in DNM2. Early clues to the diagnosis include relative weakness of neck flexors, external ophthalmoplegia and ptosis, although these are not present in all patients. Pes cavus was present in two patients, and in another two members of one family there was mild slowing of nerve conduction velocities. Whole-body MRI examination in two children and one adult revealed a similar pattern of involvement of selective muscles in head (lateral pterygoids), neck (extensors), trunk (paraspinal) and upper limbs (deep muscles of forearm). Findings in lower limbs and pelvic region were similar to that previously reported in adults with DNM2 mutations. Two patients presented with dystrophic changes as the predominant pathological feature on muscle biopsies; one of whom had a moderately raised creatine kinase, and both patients were initially diagnosed as congenital muscular dystrophy. DNM2 mutation analysis should be considered in patients with a suggestive clinical phenotype despite atypical histopathology, and MRI findings can be used to guide genetic testing. Subtle neuropathic features in some patients suggest an overlap with the DNM2 neuropathy phenotype. Missense mutations in the C-terminal region of the PH domain appear to be associated with a more severe clinical phenotype evident from infancy.

Tropomyosin 4 defines novel filaments in skeletal muscle associated with muscle remodelling/regeneration in normal and diseased muscle.

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.

The pathogenesis of ACTA1-related congenital fiber type disproportion.

OBJECTIVE: Mutations in ACTA1 have been associated with a variety of changes in muscle histology that likely result from fundamental differences in the way that ACTA1 mutations disrupt muscle function. Recently, we reported three patients with congenital fiber type disproportion (CFTD) caused by novel heterozygous missense mutations in ACTA1 (D292V, L221P, P332S) with marked type 1 fiber hypotrophy as the only pathological finding on muscle biopsy. We have investigated the basis for the histological differences between these CFTD patients and patients with ACTA1 nemaline myopathy (NM). METHODS AND RESULTS: Mass spectrometry and two-dimensional gel electrophoresis demonstrate that mutant actin accounts for 25 and 50% of alpha-skeletal actin in the skeletal muscle of patients with the P332S and D292V mutations, respectively, consistent with a dominant-negative disease mechanism. In vitro motility studies indicate that abnormal interactions between actin and tropomyosin are the likely principal cause of muscle weakness for D292V, with tropomyosin stabilized in the "switched off" position. Both the D292V and P322S CFTD mutations are associated with normal sarcomeric structure on electron microscopy, which is atypical for severe NM. In contrast, we found no clear difference between ACTA1 mutations associated with NM and CFTD in tendency to polymerize or aggregate in C2C12 expression models. INTERPRETATION: These data suggest that ACTA1 CFTD mutations cause weakness by disrupting sarcomere function rather than structure. We raise the possibility that the presence or absence of structural disorganization when mutant actin incorporates into sarcomeres may be an important determinant of whether the histological patterns of CFTD or NM develop in ACTA1 myopathy.

Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms.

We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle alpha-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased gamma-filamin, myotilin, desmin and alpha-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actin(EGFP) constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actin(EGFP) constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganization.