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Carole L. Moncman - One of the best experts on this subject based on the ideXlab platform.
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Nebulette mutations are associated with dilated cardiomyopathy and endocardial fibroelastosis
Journal of the American College of Cardiology, 2010Co-Authors: Enkhsaikhan Purevjav, Carole L. Moncman, Jaquelin Varela, Micaela Morgado, Debra L Kearney, Hua Li, Michael D Taylor, Takuro Arimura, William J Mckenna, Ross T MurphyAbstract:Objectives Four variants (K60N, Q128R, G202R, and A592E) in the Nebulette gene were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis. We sought to determine if these mutations are cardiomyopathy causing. Background Nebulette aligns thin filaments and connects them with the myocardial Z-disk, playing a role in mechanosensation. Methods We generated transgenic mice with cardiac-restricted overexpression of human wild-type or mutant Nebulette. Chimera and transgenic mice were examined at 4, 6, and 12 months of age by echocardiography and cardiac magnetic resonance imaging. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural, and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of Nebulette underwent cyclic mechanical strain. Results We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mouse lines developed DCM with severe heart failure. An irregular localization pattern for Nebulette and impaired desmin expression were noted in the proband and chimeric Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired function with specific changes in I-band and Z-disk proteins by 6 months of age. The mutations modulated distribution of Nebulette in the sarcomere and Z-disk during stretch of H9C2 cells. Conclusions Nebulette is a new susceptibility gene for endocardial fibroelastosis and DCM. Different mutations in Nebulette trigger specific mechanisms, converging to a common pathological cascade leading to endocardial fibroelastosis and DCM.
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the Nebulette repeat domain is necessary for proper maintenance of tropomyosin with the cardiac sarcomere
Experimental Cell Research, 2008Co-Authors: Jeremy R Bonzo, Michael Esham, Andrea Norris, Carole L. MoncmanAbstract:Abstract Nebulette is a cardiac-specific isoform of the giant actin-binding protein nebulin. Nebulette, having a mass of ∼ 100 kDa, is only predicted to extend 150 nm from the edge of the Z-lines. Overexpression of the Nebulette C-terminal linker and/or SH3 domains in chicken cardiomyocytes results in a loss of endogenous Nebulette with a concomitant loss of tropomyosin (TPM) and troponin, as well as a shortening of the thin filaments. These data suggest that Nebulette's position in the sarcomere is important for the maintenance of TPM, troponin and thin filament length. To evaluate this hypothesis, N-terminal nested truncations tagged with GFP were expressed in chicken cardiomyocytes and the cells were analyzed for the distribution of myofilament proteins. Minimal effects on the myofilaments were observed with N-terminal deletions of up to 10 modules; however, deletion of 15 modules replicated the phenotype observed with expression of the C-terminal fragments. Expression of internal deletions of Nebulette verifies that a site between module 10 and 15 is important for TPM maintenance within the sarcomeric lattice. We have additionally isolated TPM cDNAs from a yeast two hybrid (Y2H) analysis. These data indicate the importance of the Nebulette–TPM interactions in the maintenance and stability of the thin filaments.
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ectopic expression of lim Nebulette lasp2 reveals roles in cell migration and spreading
Cytoskeleton, 2008Co-Authors: Xiaodi A Deng, Andrea Norris, Zivile Panaviene, Carole L. MoncmanAbstract:LIM-Nebulette (LASP2) is a small focal adhesion protein and a member of the nebulin family of actin binding proteins. This recently identified splice variant of the Nebulette locus is widely expressed and highly enriched in neuronal tissue. Other than the facts that LIM-Nebulette is a focal adhesion protein and interacts with zyxin, nothing is known about its function. Given that LIM-Nebulette has an identical modular organization and overlapping tissue distributions to that of LASP1, we have analyzed the role of LIM-Nebulette in comparison with that of LASP1. We find that LIM-Nebulette is a dynamic focal adhesion protein that increases the rate of attachment and spreading of fibroblasts on fibronectin coated surfaces. Additionally, LIM-Nebulette is recruited from the cortical cytoskeleton in non-motile cells to focal adhesions at the leading edge of stimulated cells. In confluent cultures of HeLa and NIH3T3 cells, LIM-Nebulette co-localizes with α-catenin in putative adherens junctions, whereas LASP1 is devoid of these areas. Interestingly, overexpression of LIM-Nebulette in PC6 cells inhibits neurite outgrowth in response to growth factors. Collectively, our data indicate that LIM-Nebulette and LASP1 have distinct roles in the actin cytoskeleton.
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Nebulette interacts with filamin c
Cytoskeleton, 2008Co-Authors: William B Holmes, Carole L. MoncmanAbstract:The actin-binding proteins, Nebulette, and nebulin, are comprised of a four-domain layout containing an acidic N-terminal region, a repeat domain, a serine-rich-linker region, and a Src homology-3 domain. Both proteins contain homologous N-terminal regions that are predicted to be in different environments within the sarcomere. The nebulin acidic N-terminal region is found at the distal ends of the thin filaments. Nebulette, however, is predicted to extend 150 nm from the center of the Z-line. To dissect out the functions of the N-terminal domain of Nebulette, we have performed a yeast two-hybrid screen using Nebulette residues 1–86 as bait. We have identified filamin-C, ZASP-1, and tropomyosin-1 as binding partners. Characterization of the Nebulette-filamin interaction indicates that filamin-C predominantly interacts with the modules. These data suggest that filamin-C, a known component of striated muscle Z-lines, interacts with Nebulette modules. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc.
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expression of Nebulette during early cardiac development
Cytoskeleton, 2007Co-Authors: Michael Esham, Kourtney Bryan, Jennifer Milnes, William B Holmes, Carole L. MoncmanAbstract:Nebulette, a cardiac homologue of nebulin, colocalizes with α-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of Nebulette in myofibrillogenesis, we have analyzed the expression of Nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa Nebulette in heart rudiments derived from stage 9–10 using the anti-nebulin mAb, N114, or polyclonal anti-Nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-Nebulette and anti-α-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of ∼1.0 μm indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that Nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that Nebulette may play an important role in the formation of these structures. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc.
Kuan Wang - One of the best experts on this subject based on the ideXlab platform.
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targeted disruption of Nebulette protein expression alters cardiac myofibril assembly and function
Experimental Cell Research, 2002Co-Authors: Carole L. Moncman, Kuan WangAbstract:Abstract To evaluate Nebulette's role in cardiac myofibrils, cardiomyocytes expressing green fluorescent protein (GFP)–Nebulette constructs were monitored for their ability to contract and myofilament protein distribution was analyzed. Cells expressing full-length GFP–Nebulette appear unaffected and exhibit normal beating frequencies. Expression of the GFP linker and SH3 results in loss of the endogenous Nebulette and tropomyosin; however, Z-line and thick filaments are undisturbed. Cells expressing either of these domains have dramatically reduced beating frequencies, consistent with the loss of thin filament proteins. This loss was inhibited by the addition of protease inhibitors during culturing. The GFP repeat domain disrupts both myofibrillogenesis and contraction in spreading cardiomyocytes, whereas introduction of this protein into well-spread cardiomyocytes results in localization at the Z-line and a 50% reduction in beating frequency. Ultimately, these cells form bundles containing the GFP repeat and many myofilament proteins. Interestingly, butanedione monoxime inhibition of contraction inhibited the formation of these bundles. These results show that the GFP–Nebulette domains have a dominant-negative effect on the distribution and function of the sarcomeric proteins. Taken together with the observation that Nebulette colocalizes with α-actinin in the pre-, nascent, and mature myofibrils, our data demonstrate the importance of this cardiac-specific nebulin isoform in myofibril organization and function.
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Architecture of the thin filament-Z-line junction: lessons from Nebulette and nebulin homologies
Journal of Muscle Research & Cell Motility, 2000Co-Authors: Carole L. Moncman, Kuan WangAbstract:Nebulette and nebulin are homologous proteins associated with the Z-lines of cardiac and skeletal muscle sarcomeres. Although these proteins share ∼70% sequence homology and an identical domain layout, Nebulette is one-tenth the size of nebulin. To define structurally important features of these proteins in terms of the Z-line architecture, we have analyzed the primary structure of Nebulette and nebulin from a variety of species and developmental stages. Alignment of the 35 residue nebulin-like modules from both proteins demonstrates that the individual modules share 30–90% homology across the six proteins analyzed. In addition, this analysis demonstrates a number of areas in which the identity across the six proteins is as high as 75%. These areas may be important signals for Z-line assembly and function in the striated muscles. Significantly, most of the areas of high identity also coincide with consensus phosphorylation sites. To evaluate if Nebulette, like nebulin, exhibits tissue-specific and developmental specific polymorphism, a series of immunoblot assays were performed. These data demonstrate that Nebulettes from different portions of the heart are the same size. Comparison of Nebulette from embryonic and adult cardiac muscle also demonstrates that this protein does not appear to vary in size with developmental stage. Consistent with the large number of consensus phosphorylation sites identified in the Nebulette primary structure, we find that Nebulette is phosphorylated in the cardiac muscle at serine and threonine residues. These data and sequence analyses are discussed in terms of current models for Z-line architecture.
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functional dissection of Nebulette demonstrates actin binding of nebulin like repeats and z line targeting of sh3 and linker domains
Cytoskeleton, 1999Co-Authors: Carole L. Moncman, Kuan WangAbstract:Nebulette, a 107 kDa protein associated with the I-Z-I complex of cardiac myofibrils, may play an important role in the assembly of the Z-line. Determination of the complete primary structure of 1011 residue human fetal Nebulette reveals a four-domain layout similar to skeletal muscle nebulin: a short N-terminal domain, followed by 22 nebulin-like repeats that are linked to a C-terminal Src homology 3 (SH3) domain via a short linker domain. To elucidate the mechanisms of assembly for Nebulette in the Z-line, the complete coding sequence or fusions of Nebulette domains with green fluorescent protein (GFP) were expressed in cardiomyocytes and fibroblasts. The complete protein localized to Z-lines in cardiac cells and to dense bodies in nonmuscle cells. The GFP-repeat domain forms bundles that are associated with actin filaments in both cell types and disrupts the microfilament network. In contrast, the GFP-repeat plus linker shows limited interaction with dense bodies in nonmuscle cells and the Z-lines of cardiomyocytes. Interestingly, the tagged linker or SH3 is diffusely distributed in nonmuscle cells, but localizes to the Z-lines in cardiomyocytes. Supporting the cellular localization work, recombinant Nebulette fragments bind to actin, tropomyosin, and α-actinin in in vitro binding assays. These results suggest the repeat domain contains actin binding functions and that the linker domain may target this interaction to Z-lines and dense bodies. Our data also indicate that the linker and SH3 domains can distinguish between dense bodies and Z-lines, suggesting that the ligands for their interactions are specific to these muscular substructures. Cell Motil. Cytoskeleton 44:1–22, 1999. © 1999 Wiley-Liss, Inc.
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Nebulette: A 107 kD nebulin‐like protein in cardiac muscle
Cytoskeleton, 1995Co-Authors: Carole L. Moncman, Kuan WangAbstract:A 107-kD protein has been identified in primary cultures of chicken embryonic cardiomyocytes by immunoprecipitations with certain anti-nebulin monoclonal antibodies (mAbs). These mAbs, prepared against a fragment of human skeletal muscle nebulin located near the carboxyl terminus, detect a 107-kD protein in extracts of adult chicken heart, adult mouse heart, and adult rabbit heart by immunoblot analysis. A partial cDNA corresponding to this protein has been isolated by immunological screening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380-amino acid open reading frame composed entirely of nebulin-like 35-residue modules marked by the highly conserved sequence motifs: SXXXYK and TPD. The open reading frame exhibits 60–85% homology with skeletal muscle nebulins from a variety of species. This cDNA recognizes an ˜8-kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluorescence localization of this nebulin-like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indicates that the protein is localized to the I-Z-I complex of the myofibrils, extending approximately 25% of the thin filament length. Comparisons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin-like protein becomes aligned with the nascent myofibrils early during myofibrillogenesis. To distinguish this petite nebulin-like protein from the 600–900 kD skeletal muscle nebulin, we have named it Nebulette. © 1995 Wiley-Liss, Inc.
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Nebulette a 107 kd nebulin like protein in cardiac muscle
Cytoskeleton, 1995Co-Authors: Carole L. Moncman, Kuan WangAbstract:A 107-kD protein has been identified in primary cultures of chicken embryonic cardiomyocytes by immunoprecipitations with certain anti-nebulin monoclonal antibodies (mAbs). These mAbs, prepared against a fragment of human skeletal muscle nebulin located near the carboxyl terminus, detect a 107-kD protein in extracts of adult chicken heart, adult mouse heart, and adult rabbit heart by immunoblot analysis. A partial cDNA corresponding to this protein has been isolated by immunological screening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380-amino acid open reading frame composed entirely of nebulin-like 35-residue modules marked by the highly conserved sequence motifs: SXXXYK and TPD. The open reading frame exhibits 60–85% homology with skeletal muscle nebulins from a variety of species. This cDNA recognizes an ˜8-kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluorescence localization of this nebulin-like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indicates that the protein is localized to the I-Z-I complex of the myofibrils, extending approximately 25% of the thin filament length. Comparisons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin-like protein becomes aligned with the nascent myofibrils early during myofibrillogenesis. To distinguish this petite nebulin-like protein from the 600–900 kD skeletal muscle nebulin, we have named it Nebulette. © 1995 Wiley-Liss, Inc.
Vincenzo Nigro - One of the best experts on this subject based on the ideXlab platform.
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Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation
Skeletal Muscle, 2019Co-Authors: Luciano Merlini, Patrizia Sabatelli, Manuela Antoniel, Valeria Carinci, Fabio Niro, Giuseppe Monetti, Annalaura Torella, Teresa Giugliano, Cesare Faldini, Vincenzo NigroAbstract:Background Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and Nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients.
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Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation
Skeletal Muscle, 2019Co-Authors: Luciano Merlini, Patrizia Sabatelli, Manuela Antoniel, Valeria Carinci, Fabio Niro, Giuseppe Monetti, Annalaura Torella, Teresa Giugliano, Cesare Faldini, Vincenzo NigroAbstract:Background Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and Nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients. Case presentation A consanguineous family with congenital to adult-onset muscle weakness and hanging big toe was reported. Muscle biopsy showed minimal changes with internal nuclei, type 1 fiber predominance, and ultrastructural defects of Z line. Muscle CT imaging showed marked hypodensity of the sartorius bilaterally and MRI scattered abnormal high-intensity areas in the internal tongue muscle and in the posterior cervical muscles. Cardiac involvement was demonstrated by magnetic resonance imaging and late gadolinium enhancement. Whole exome sequencing analysis identified a homozygous loss of function single nucleotide deletion in the exon 11 of the MYPN gene in two siblings. Full-length MYPN protein was undetectable on immunoblotting, and on immunofluorescence, its localization at the Z line was missed. Conclusions This report extends the phenotypic spectrum of recessive MYPN-related myopathies showing: (1) the two patients had hanging big toe and the oldest one developed spine and hand contractures, none of these signs observed in the previously reported patients, (2) specific ultrastructural changes consisting in Z line fragmentation, but (3) no nemaline or caps on muscle pathology.
Luciano Merlini - One of the best experts on this subject based on the ideXlab platform.
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Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation
Skeletal Muscle, 2019Co-Authors: Luciano Merlini, Patrizia Sabatelli, Manuela Antoniel, Valeria Carinci, Fabio Niro, Giuseppe Monetti, Annalaura Torella, Teresa Giugliano, Cesare Faldini, Vincenzo NigroAbstract:Background Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and Nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients.
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Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation
Skeletal Muscle, 2019Co-Authors: Luciano Merlini, Patrizia Sabatelli, Manuela Antoniel, Valeria Carinci, Fabio Niro, Giuseppe Monetti, Annalaura Torella, Teresa Giugliano, Cesare Faldini, Vincenzo NigroAbstract:Background Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and Nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients. Case presentation A consanguineous family with congenital to adult-onset muscle weakness and hanging big toe was reported. Muscle biopsy showed minimal changes with internal nuclei, type 1 fiber predominance, and ultrastructural defects of Z line. Muscle CT imaging showed marked hypodensity of the sartorius bilaterally and MRI scattered abnormal high-intensity areas in the internal tongue muscle and in the posterior cervical muscles. Cardiac involvement was demonstrated by magnetic resonance imaging and late gadolinium enhancement. Whole exome sequencing analysis identified a homozygous loss of function single nucleotide deletion in the exon 11 of the MYPN gene in two siblings. Full-length MYPN protein was undetectable on immunoblotting, and on immunofluorescence, its localization at the Z line was missed. Conclusions This report extends the phenotypic spectrum of recessive MYPN-related myopathies showing: (1) the two patients had hanging big toe and the oldest one developed spine and hand contractures, none of these signs observed in the previously reported patients, (2) specific ultrastructural changes consisting in Z line fragmentation, but (3) no nemaline or caps on muscle pathology.
Cesare Faldini - One of the best experts on this subject based on the ideXlab platform.
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Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation
Skeletal Muscle, 2019Co-Authors: Luciano Merlini, Patrizia Sabatelli, Manuela Antoniel, Valeria Carinci, Fabio Niro, Giuseppe Monetti, Annalaura Torella, Teresa Giugliano, Cesare Faldini, Vincenzo NigroAbstract:Background Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and Nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients.
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Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation
Skeletal Muscle, 2019Co-Authors: Luciano Merlini, Patrizia Sabatelli, Manuela Antoniel, Valeria Carinci, Fabio Niro, Giuseppe Monetti, Annalaura Torella, Teresa Giugliano, Cesare Faldini, Vincenzo NigroAbstract:Background Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and Nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients. Case presentation A consanguineous family with congenital to adult-onset muscle weakness and hanging big toe was reported. Muscle biopsy showed minimal changes with internal nuclei, type 1 fiber predominance, and ultrastructural defects of Z line. Muscle CT imaging showed marked hypodensity of the sartorius bilaterally and MRI scattered abnormal high-intensity areas in the internal tongue muscle and in the posterior cervical muscles. Cardiac involvement was demonstrated by magnetic resonance imaging and late gadolinium enhancement. Whole exome sequencing analysis identified a homozygous loss of function single nucleotide deletion in the exon 11 of the MYPN gene in two siblings. Full-length MYPN protein was undetectable on immunoblotting, and on immunofluorescence, its localization at the Z line was missed. Conclusions This report extends the phenotypic spectrum of recessive MYPN-related myopathies showing: (1) the two patients had hanging big toe and the oldest one developed spine and hand contractures, none of these signs observed in the previously reported patients, (2) specific ultrastructural changes consisting in Z line fragmentation, but (3) no nemaline or caps on muscle pathology.
