Limb-girdle muscular dystrophy with severe heart failure overlapping with lipodystrophy in a patient with LMNA mutation p.Ser334del.

Laminopathies, a group of heterogeneous disorders associated with lamin A/C gene (LMNA) mutations, encompass a wide spectrum of clinical phenotypes, which may present as separate disease or as overlapping syndromes. We describe a 35-year-old female in whom a novel sporadic heterozygous mutation c.1001_1003delGCC (p.Ser334del) of the LMNA gene was found. The patient presented with overlapping syndrome of heart failure secondary to dilated cardiomyopathy, limb-girdle dystrophy and partial lipodystrophy. Endomyocardial biopsy revealed strong up-regulation of HLA classes I and II antigens on microvessels and induction of the class I antigens on cardiomyocytes. On muscle biopsy, a wide range of fiber sizes and small clusters of inflammatory infiltrations were found. In the rapid progression of heart failure with arrhythmias or conduction defect, accompanied with muscle atrophy and lipodystrophy, the genetic disease should be taken into consideration. In addition, undefined inflammatory response and fibrosis in the heart or skeletal muscle might further justify screening of the lamin A/C gene.

The Frequency of c.550delA Mutation of the CANP3 Gene in the Polish LGMD2A Population.

BACKGROUND: Limb girdle muscular dystrophy 2A (LGMD2A) is the most frequent LGMD variant in the European population, representing about 40% of LGMD. The c.550delA mutation in the CANP3 (calcium activated neutral protease 3) gene is the most commonly reported mutation in LGMD2A. Prevalence of this mutation in the Polish population has not been previously investigated. The aim of this study was to identify and estimate the frequency of the c.550delA mutation in Polish LGMD2A patients. METHODS: Polymerase chain reaction-sequencing analysis, restriction fragment length polymorphism polymerase chain reaction method. RESULTS: We analyzed 76 families affected with LGMD and identified 62 probands with mutations in the CANP3 gene. C.550delA was the most common mutation identified, being found in 78% of the LGMD2A families. The remaining mutations observed multiple times were as follows: c.598-612del15ntd; c.2242C>T; c.418dupC; c.1356insT, listed in terms of decreasing frequency. Two novel variants in the CANP3 gene, that is, c.700G>A Gly234Arg and c.661G>A Gly221Ser were also characterized. Overall, mutations in the LGMD2A gene were estimated to be present in 81% of patients with the LGMD phenotype who were without sarcoglycans and dysferlin deficiency on immunocytochemical analysis. The frequency of the heterozygous c.550delA mutation in the healthy Polish population was estimated at 1/124. CONCLUSIONS: The c.550delA is the most frequent CANP3 mutation in the Polish population, thus sequencing of exon 4 of this gene could identify the majority of LGMD2A patients in Poland.

Does aberrant architecture of nuclear LINC complex stop muscle cell development?

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a structure that spans the entire nuclear envelope (NE) and integrates the nuclear interior with the cytoskeleton. Lamin A/C together with nesprins that mainly reside along the inner nuclear membrane (INM) and outer nuclear membrane (ONM) are core components of the LINC complex. Integrity of this specific nuclear structure is critical for muscle cell maturation and function. In the present study, we analyzed the ultrastructure of the LINC complex observed in two neonates with severe hypotonia and respiratory distress. Disruption of the LINC complex manifests in a wide separation of the ONM from the INM; the loss of perinuclear space (PNS) and delayed muscle cell maturation were predominating findings. This nuclear phenomenon has never been reported and provides further support for the appearance of a neonatal form of laminopathy.

Severe phenotypes of SMARD1 associated with novel mutations of the IGHMBP2 gene and nuclear degeneration of muscle and Schwann cells.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a very rare autosomal recessive form of spinal muscular atrophy manifested in low birth weight, diaphragmatic palsy and distal muscular atrophy. Caused by a mutation in the IGHMBP2 gene, the disease is addressed here by reference to five Polish patients in which SMARD1 has been confirmed genetically. All presented a severe form of the disease and had evident symptoms during the second month of life; with four displaying weak cries, feeding difficulties and hypotonia from birth. Two were afflicted by severe dysfunction of the autonomic nervous system. Ultrastructural analysis of a muscle biopsy revealed progressive degeneration within the nuclei of the muscle cells and Schwann cells. Neuromuscular junctions were also defective. It proved possible to identify in our patients 6 novel IGHMBP2 mutations: three missense (c.595G>C, c.1682T>C and c.1794C>A), two nonsense (c.94C>T and c.1336C>T) and one in-frame deletion (c.1615_1623del). One nonsense mutation (c.429C>T) that had been described previously was also identified. Observation of our patients makes it clear that clinical picture is still the most important factor suggesting diagnosis of SMARD1, though further investigations concerning some of the symptoms are required. As the IGHMBP2 gene is characterized by significant heterogeneity, genetic counseling of affected families is rendered more complex. IGHMBP2 protein deficiency can lead to the degeneration of nuclei, in both muscle and Schwann cells.

Is mutation p.Arg168Gly in TPM3 gene responsible for Type 1 fiber hypoplasia and cap structure formation?

Congenital fiber type disproportion with delayed fiber type maturation and the appearance of cap structures were analyzed in a child with p.Arg168Gly mutation in TPM3 gene. Very narrow myotube-like Type 1 fibers with single nuclei decorated by cap structures seem to be a result of a failure in fusion process and mature fiber formation. Repeated mutations in exon 5 of TPM3 gene giving cap structures may be a different consequence of the loss of specific isoform normally operating in the fusion process and sarcomer formation.

Might prepatterned acetylcholine-receptor clusters on surface myotubes be a sign of neuromuscular-junction maturation failure?

INTRODUCTION: During human myogenesis and synaptogenesis, the first contact between multiaxonal nerve terminals and the primary myotube occurs at an early stage of gestation, then monoaxonal nerve terminals form and postsynaptic clusters of acetylcholine-receptor are modified and redistributed to the site of muscle-nerve contact. The aim of this study is to present the ultrastructural features of muscle and motor-junction immaturity severe enough to lead to death in the first months of life. MATERIAL AND METHODS: Ultrastructural-level analysis was carried out on the quadriceps femoris muscle of an infant born at full term with severe respiratory distress but with normal SMN1 and IGHMBP2 genes. RESULTS: Arrested muscle maturation was manifested in the presence of primary and mature myotubes, prepatterned acetylcholine-receptor clusters devoid of terminal axons, lack of synapses and multiaxonal unmyelinated intramuscular nerves. CONCLUSION: The "naked" prepatterned clusters observed on the surface of myotubes normally never observed in neonates might be a sign of a new genetic defect in innervation.

[Cardiac involvement in neuronal ceroid lipofuscinosis]. / Zajecie serca w przebiegu neuronalnej lipofuscynozy ceroidowej.

We present a case of a 58-year-old female with neuropsychiatric symptoms, followed by recurrent episodes of atrial flagellation and symptoms of heart failure. Based on intraoperative myocardial biopsy, neuronal ceroid lipofuscinosis was diagnosed.

LMNA mutations in Polish patients with dilated cardiomyopathy: prevalence, clinical characteristics, and in vitro studies.

BACKGROUND: LMNA mutations are most frequently involved in the pathogenesis of dilated cardiomyopathy with conduction disease. The goal of this study was to identify LMNA mutations, estimate their frequency among Polish dilated cardiomyopathy patients and characterize their effect both in vivo and in vitro. METHODS: Between January, 2008 and June, 2012 two patient populations were screened for the presence of LMNA mutations by direct sequencing: 66 dilated cardiomyopathy patients including 27 heart transplant recipients and 39 dilated cardiomyopathy patients with heart failure referred for heart transplantation evaluation, and 44 consecutive dilated cardiomyopathy patients, referred for a family evaluation and mutation screening. RESULTS: We detected nine non-synonymous mutations including three novel mutations: p.Ser431*, p.Val256Gly and p.Gly400Argfs*11 deletion. There were 25 carriers altogether in nine families. The carriers were mostly characterized by dilated cardiomyopathy and heart failure with conduction system disease and/or complex ventricular arrhythmia, although five were asymptomatic. Among the LMNA mutation carriers, six underwent heart transplantation, fourteen ICD implantation and eight had pacemaker. In addition, we obtained ultrastructural images of cardiomyocytes from the patient carrying p.Thr510Tyrfs*42. Furthermore, because the novel p.Val256Gly mutation was found in a sporadic case, we verified its pathogenicity by expressing the mutation in a cellular model. CONCLUSIONS: In conclusion, in the two referral centre populations, the screening revealed five mutations among 66 heart transplant recipients or patients referred for heart transplantation (7.6%) and four mutations among 44 consecutive dilated cardiomyopathy patients referred for familial evaluation (9.1%). Dilated cardiomyopathy patients with LMNA mutations have poor prognosis, however considerable clinical variability is present among family members.

Main steps of skeletal muscle development in the human: morphological analysis and ultrastructural characteristics of developing human muscle.

During embryogenesis, paraxial mesoderm undergoes segmentation into somites, progressing from head to tail. Somites differentiate into dermomyotomes, then into dermatomes and myotomes. Some head muscles derive from the anterior paraxial and precordal mesoderm. Between 10 and 13 weeks of gestation, the fusion of myoblasts generates primary myotubes with central nuclei, and the latter form the second generation of myotubes which requires active innervation. Nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase appears before succinate dehydrogenase, and ATPase shows an intermediary activity. ß-Sarcoglycan appears by 7 gestational weeks and α-sarcoglycan by 10-12 weeks. ß-Spectrin, dystrophin, and utrophin appear by 9 weeks, vimentin and desmin appear by 10 weeks and stain strongly between 10 and 15 weeks. Slow, embryonic myosin heavy chain (MHC) isoenzymes appear before 15 weeks, whereas fetal fast MCH occur later (15-18 weeks). Myotubes become myofibers with peripheral nuclei between 15 and 18 weeks. Large muscle fibers (Wohlfart B) are visible by 20-21 weeks, Wohlfart A by 21-25 weeks. Perimysium surrounds compacted and grouped fibers by 24 weeks, and utrophin disappears, whereas dystrophin stains intensely. At 29 weeks, type I fibers are visible, and by 31-33 weeks they are mature for ATPase staining. Three types of type II fibers can be seen. Vimentin disappears between 15 and 30 weeks, while desmin remains weakly positive at birth.

Morphologic and clinical aspects of Danon disease in a patient with a mutation c.137G > A in the LAMP-2 gene.

BACKGROUND: Danon disease is caused by a primary deficiency of lysosome-associated membrane protein-2 (LAMP-2). MATERIALS AND METHODS: Our study describes a 19-year-old man with isolated hypertrophic cardiomyopathy, in whom we performed DNA analysis and compared results of microscopic analysis of skeletal and cardiac muscles. RESULTS: Sequencing of the LAMP-2 gene revealed a novel point mutation c.137G > A in exon 2, leading to premature stop codon. Ultrastructural analysis of cardiac and skeletal muscles revealed the presence of unusual autophagic vacuoles in both. Although some vacuoles in skeletal muscle reacted strongly with dystrophin, ß-sarcoglycan, and laminin, those in cardiomyocytes showed no immunoreactivity. CONCLUSION: Our immunohistochemical and ultrastructural findings reinforce the claim that in Danon disease the pathomechanism of chaperone-mediated autophagy in cardiomyocytes differs from that in skeletal muscle.

Remodeling of mitochondrial interior in cardiac lipofuscinosis.

Ultrastructural analysis was performed in cardiac ceroidlipofuscinosis to confirm the presence and the nature of storage material. Granular osmophilic deposits characteristic of GROD structures coincidented with particularly aberrant mitochondria. Remodeling of mitochondrial interior with the appearance of several form of abnormal inclusions was never observed in cardiac ceroidlipofuscinosis. The presence of dense osmophilic bodies, glycogen conglomerates, balloon-like and onion-like structures in mitochondrial interior seem to be early events of this storage process.

Did giant mitochondria delay muscle maturation? An uncommon congenital myopathy.

INTRODUCTION: Mitochondria are semi-autonomous organelles that are able to change their shape, size, location, and number inside the living cell. Mitochondrial division is an extremely important process, because cell survival depends on there being an adequate number of mitochondria in each cell. The dynamics of these organelles are particularly important for normal embryonic cell development. We studied morphological and ultrastructural features of muscle-cell immaturity, with the appearance of abnormal giant mitochondria, in a child with an unusual congenital myopathy. METHODS: The biceps brachii and quadriceps femoris muscles were analyzed at the light and electron microscopy levels. RESULTS: Ultrastructural features of muscle-cell immaturity were manifested in the presence of primary and mature myotubes coexisting with abnormal giant mitochondria. CONCLUSIONS: Failure of mitochondrial fusion/fission machinery at an early stage of development may lead to delayed muscle-cell maturation.

[Myopathy in the course of carnitine palmitoyltransferase II deficiency]. / Miopatia w przebiegu niedoboru palmitylotransferazy karnityny II.

Congenital deficiency of carnitine palmitoyltransferase (CPT) II is a disease with an autosomal recessive inheritance of phenotypic variability which depends on age at the onset of symptoms. Three entities associated with deficiency of CPT II are known: the perinatal, the infantile and the adult form. The perinatal disease is the most severe form and is invariably fatal. On the other hand, the adult CPT II clinical phenotype is benign and requires additional external triggers such as high-intensity exercise to provoke myopathic symptoms. We report a case of adult CPT II deficiency presenting with the subtle symptoms of myopathy. A 32-year-old man was admitted to the hospital complaining of muscle pain after exercise. Athletic appearance drew attention, because the patient denied practicing sport. Neurological examination revealed marked tiredness during the single-leg hop test without other abnormalities. Electromyography (EMG) and serum biochemistry were not typical for myopathy. Routine histopathological examination did not reveal any abnormalities of structure of muscle fibers. Diagnosis was established after ultrastructural and biochemical analysis which revealed changes typical for CPT II deficiency.

Are cardiomyocytes able to generate pre-amyloid peptides?

We performed ultrastructural testing of a cardiac biopsy taken from a heart with amyloidosis in which transthyretin mutation and light chain A amyloidosis were excluded. Cardiomyocytes of the affected heart showed accumulation of endosomal-like structures in which soluble amyloid oligomeric conformation was deposited. Intracellular accumulation of ß -amyloid as well as phosphorylated tau protein seen in the immunohistochemical study suggest that the heart tissue may generate an amyloidogenic peptide leading to cardiomyocyte destruction and heart dysfunction.

Late form of Pompe disease with glycogen storage in peripheral nerves axons.

Pompe disease is caused by the deficiency of acid α-glucosidase (GAA), which degrades glycogen into glucose. Its manifestation is characterized by a broad and continuous spectrum of clinical severity ranging from severe infantile to relatively benign adult form. We describe a 12-year-old girl diagnosed at a presymptomatic stage of late form Pompe disease due to fortuitous detection of an elevated level of serum creatine kinase (CK) at the age of 4. Biopsies were taken from the quadriceps muscle and studied with histological and histochemical techniques, as well as in electron microscope. Sporadic muscle cells showed the accumulation of lysosomal glycogen, suggesting Pompe disease. Interestingly, we found lysosomal bound glycogen, located in the axons of intramuscular nerves. The diagnosis was confirmed by deficient GAA activity in leukocytes. Mutation analysis revealed changes IVS1-13T>G and p.C103G in the GAA gene. The patient was able to obtain enzyme replacement therapy in the early asymptomatic stage of the disease.

Motor endplate remodeling in some cases with congenital myasthenic syndrome.

The architecture of motor endplates in three cases with congenital myasthenic syndrome (CMS) was compared with ultrastructure of the normal control neuromuscular junction (NMJ). The remodeling of postsynaptic region was observed in all three individuals. The most conspicuous abnormalities seen in the slow channel syndrome was the vacuolization and disorganization of secondary synaptic clefts which extended for beyoned the border of NMJ. Degenerated postsynaptic nuclei and junctional sarcoplasm were an additional feature of presented syndromes. The quite different feature of NMJ was observed in the DOK-7 deficient syndrome. The appearance of small, pale terminal axons, poorly developed postsynaptic membrane with the sparse secondary synaptic clefts and degenerated postsynaptic nuclei suggested impairment of postsynaptic region maturation. The conjunction of postsynaptic membrane paucity and its degeneration was a specific structural feature observed in the third syndrome with no established genetic defects.

Ultrastructural evidence of myocardial capillary remodeling in peripartum cardiomyopathy.

BACKGROUND: The etiology of peripartum cardiomyopathy (PPCM) is not well understood. Potential causal mechanisms include infection, autoimmune disease, and abnormal response to the hemodynamic stress of pregnancy. Recently it was shown that the development of PPCM in an experimental model is associated with the generation of a cleaved antiangiogenic and proapoptotic 16-kDa form of the nursing hormone prolactin, which impairs the cardiac capillary network and its function. The purpose of this study was to evaluate the ultrastructure of the myocardium in a patient with PPCM and in a comparative group of patients and to identify structural characteristics that may predispose to myocardium dysfunction. CASE REPORT: A 28-year-old woman with PPCM had fulminant heart failure leading to the implantation of a biventricular assist device, but no myocarditis was found on histological examination of her myocardial tissue. An ultrastructural study of myocardial tissue taken from three patients (mean age: 22 years, 2 females) with fulminant heart failure not related to PPCM (2 myocarditis, 1 dilated cardiomyopathy) served as a comparison. The ultrastructural analysis revealed the presence of small capillary damage. Vascular lumen occlusion, endothelial cell remodeling, and apoptosis with the appearance of mast cells, plasma cells, and preadipocytes were the most characteristic features of the damaged myocardial tissue with secondary interstitial fibrosis. No vascular pathology of cardiac capillaries was observed in the comparative group. CONCLUSIONS: A myocardial tissue study in PPCM revealed ultrastructural remodeling of small capillaries with the presence of endothelial cell apoptosis and impairment of the microcirculation.

Usefulness of the ultrastructural and immunohistochemical analysis of cardiac biopsy in affected heart.

In the last few years endomyocardial biopsy becomes a useful diagnostic tool for the investigation of idiopathic dilated cardiomyopathy. The aim of our current study was to try to identify ultrastructural and immunohistochemical specificity of truncated cardiac proteins in affected heart. The focal loss of plasma membrane continuity together with the lack of dystrophin activity in affected myocytes facilitated to find mutation in dystrophin gene. The accumulation of granulofilamentous desmin-positive material in cytoplasm of myocytes was the main indicator of presented mutation in the desmin gene. Nuclear structure remodeling, concomitantly with loss of lamin A/C activity, contributed to identify mutation in lamin A/C gene. Analysis of hypertrophic heart with disarray of sarcomeres and lack of I-Z-I bands suggested embryonic faiulure in titin activity. All this findings indicate that endomyocardial biopsy reperesent a useful method for a correct diagnosis of heart dysfunction.

Genetic and ultrastructural studies in dilated cardiomyopathy patients: a large deletion in the lamin A/C gene is associated with cardiomyocyte nuclear envelope disruption.

Major nuclear envelope abnormalities, such as disruption and/or presence of intranuclear organelles, have rarely been described in cardiomyocytes from dilated cardiomyopathy (DCM) patients. In this study, we screened a series of 25 unrelated DCM patient samples for (a) cardiomyocyte nuclear abnormalities and (b) mutations in LMNA and TMPO as they are two DCM-causing genes that encode proteins involved in maintaining nuclear envelope architecture. Among the 25 heart samples investigated, we identified major cardiomyocyte nuclear abnormalities in 8 patients. Direct sequencing allowed the detection of three heterozygous LMNA mutations (p.D192G, p.Q353K and p.R541S) in three patients. By multiplex ligation-dependant probe amplification (MLPA)/quantitative real-time PCR, we found a heterozygous deletion encompassing exons 3-12 of the LMNA gene in one patient. Immunostaining demonstrated that this deletion led to a decrease in lamin A/C expression in cardiomyocytes from this patient. This LMNA deletion as well as the p.D192G mutation was found in patients displaying major cardiomyocyte nuclear envelope abnormalities, while the p.Q353K and p.R541S mutations were found in patients without specific nuclear envelope abnormalities. None of the DCM patients included in the study carried a mutation in the TMPO gene. Taken together, we found no evidence of a genotype-phenotype relationship between the onset and the severity of DCM, the presence of nuclear abnormalities and the presence or absence of LMNA mutations. We demonstrated that a large deletion in LMNA associated with reduced levels of the protein in the nuclear envelope suggesting a haploinsufficiency mechanism can lead to cardiomyocyte nuclear envelope disruption and thus underlie the pathogenesis of DCM.