Correlating motor unit morphology with bioelectrical activity - A simulation study.

OBJECTIVES: The aim was to determine motor unit morphology underpinning the various MUP waveforms using MUP analysis. METHOD: The simulation method is based on the decomposition of MUP into single fiber potentials. Number of fibers, fiber diameters and fiber to electrode distances were determined. The impact of each muscle fiber on the MUP waveform was determined and quantified by its percentage contribution. RESULTS: The origin of the four examined MUPs of distinct waveforms have been explained by showing the histograms of fiber diameters and their distance to the electrode. In the case of a low amplitude MUP it was found that it originated from fibers of smaller than normal diameters with no dominant fiber. In another case of a MUP of short duration its shape was due to a single fiber close to the electrode which contributed to about 80% of the MUP. In case of polyphasic MUP, muscle fiber diameters variability was responsible for MUP characteristic. MUP from normal muscle originated from few fibers of similar diameters. Correlation between MUP's characteristic and morphological features has been indicated. Our findings are consistent with the neurophysiological knowledge about the origins of MUP. The approximation method enables MUP analysis that provides quantitative description of motor unit morphology. CONCLUSION: MUP analysis using an approximation method enables to get an insight into motor unit morphology and therefore increases understanding of the way the motor unit structure correlates with MUP waveform. SIGNIFICANCE: Extending the amount of information available from EMG examinations.

Dysfunctional lamins as mediators of oxidative stress in Emery-Dreifuss muscular dystrophy.

Deficit of lamin A/C or emerin causes genetically transmitted Emery-Dreifuss muscular dystrophy (EDMD). As lamins are considered to be mediators of oxidative stress, the antioxidant/oxidant status was examined. The total oxidant/antioxidant status in serum was examined in 29 cases of Emery-Dreifuss muscular dystrophy. The study included 12 autosomal-dominant laminopathies (AD-EDMD), 17 X-linked emerinopathies (X-EDMD) and 20 age-matched normal subjects. Total oxidant status (TOS) was reduced in all cases, and the total antioxidant capacity (TAC) was found to be decreased in the majority of the patients (in 82.8%). A relationship between TOS level and disease progression was noted. No correlation between TOS/TAC level and cardiological or neurological parameters was detected. The results of the study indicate disturbances of redox balance in EDMD patients. Determination of TOS/TAC might help to assess the progress of the disease and the potential effectiveness of antioxidant therapy.

Novel Col12A1 variant expands the clinical picture of congenital myopathies with extracellular matrix defects.

INTRODUCTION: Mutations in the COL12A1 (collagen, type XII, alpha 1) gene have been described in a milder Bethlem-like myopathy in 6 patients from 3 families (dominant missense), and in a severe congenital form with failure to attain ambulation in 2 patients in a single pedigree (recessive loss-of-function). METHODS: We describe an 8-year-old girl of Polish origin who presented with profound hypotonia and joint hyperlaxity at birth after a pregnancy complicated by oligohydramnios and intrauterine growth retardation. RESULTS: We identified a novel, potentially pathogenic heterozygous missense COL12A1 c.8329G>C (p.Gly2777Arg) variant using a targeted sequencing panel. Patient fibroblast studies confirmed intracellular retention of the COL12A1 protein, consistent with a dominant-negative mutation. CONCLUSIONS: As our patient showed a more intermediate phenotype, this case expands the phenotypic spectrum for COL12A1 disorders. So far, COL12A1 disorders seem to cover much of the severity range of an Ehlers-Danlos/Bethlem-like myopathy overlap syndrome associated with both connective tissue abnormalities and muscle weakness. Muscle Nerve 55: 277-281, 2017.

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.

Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases.

BACKGROUND: Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. OBJECTIVE: To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. METHODS: We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. RESULTS: We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. CONCLUSIONS: Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

Cardiac pacing in 21 patients with Emery-Dreifuss muscular dystrophy: a single-centre study with a 39-year follow-up.

BACKGROUND: Emery-Dreifuss muscular dystrophy (EDMD) is a genetic condition associated with cardiac arrhythmias. The patients typically develop early, asymptomatic bradyarrhythmia, which may lead to sudden death, preventable with a cardiac implantable electronic device (CIED). EDMD may be characterised by atrial electrical silence. Intra-operative electrophysiological evaluation of the myocardium helps ultimately determine the true nature of the disorder and select an appropriate CIED. AIM: To analyse permanent electrotherapy procedures in EDMD patients: atrial pacing limitations that stem from the electrophysiological properties of the myocardium and long-term follow-up of implanted devices. METHODS: A total of 21 EDMD patients (mean age 29 ± 9 years) with a CIED implanted (1976-2014) due to bradyarrhythmia were included in the study. The implantation procedures and factors determining the CIED type selection were analysed. RESULTS: CIEDs were implanted in five women and in 16 men with EDMD types 1 and 2 (mean follow-up: 11 ± 8 years). Intra-operatively assessed atrial electrophysiology resulted in changing the planned CIED type during the procedure in three men with EDMD type 1. Eventually, we implanted: eight DDD, one VDD, 11 VVI, and one CD-DR device, with four of the patients' devices switched later from DDD to VVI mode in response to electrophysiological changes in the atria. CONCLUSIONS: Intra-operative assessment of atrial electrophysiological properties resulted in changing the planned DDD mode for VVI in 19% of patients with EDMD type 1. Progression of the underlying disease over a 39-year follow-up resulted in a later change of the initially selected pacing mode from DDD to VVI in 40% of cases.

Emery-Dreifuss muscular dystrophy type 2 associated (?) with mild peripheral polyneuropathy.

In recent years numerous mutations in the LMNA gene encoding lamin A/C were shown to segregate with a wide spectrum of phenotypes. A recurrent p.R377H mutation in the LMNA gene was reported in patients with Emery-Dreifuss dystrophy (EDMD2) with various ethnic backgrounds. We present a patient with EDMD2 caused by a p.R377H mutation, associated with mild peripheral polyneuropathy. The analysis of peripheral myelin protein 22 (PMP22), ganglioside induced differentiation-associated protein 1 (GDAP1), gap junction ß-1 protein (GJB1), and myelin protein zero (MPZ) genes did not reveal mutations; however, we identified a new sequence intronic variant in the mitofusin 2 (MFN2) gene of unknown pathogenic significance. A complex phenotype in the presented patient might depend either on single mutation in the LMNA gene or on bigenic defect; therefore, a wide genetic investigation is needed to elucidate the molecular background of EDMD2/polyneuropathy in this case.

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.

Tissue inhibitors of matrix metalloproteinases in serum are cardiac biomarkers in Emery-Dreifuss muscular dystrophy.

BACKGROUND: Tissue inhibitors of matrix metalloproteinases (TIMPs) are known to be involved in cardiovascular diseases. Hitherto, they have not been examined in dilated cardiomyopathy in the course of Emery-Dreifuss muscular dystrophy (EDMD). AIM: To define TIMPs in serum because they might help in defining cardiac dysfunction at the early cardiological stages of this disease and detect preclinical stages of cardiomyopathy. METHODS: Twenty-five EDMD patients connected with lamin A/C (AD-EDMD) or emerin (X-EDMD) deficiency and 20 healthy age-matched controls were examined. The serum levels of the tissue inhibitors TIMP-1, -2, -3 were quantified using the ELISA sandwich immunoassay procedure with appropriate antibodies. RESULTS: Serum levels of TIMP-1 were normal in autosomal AD-EDMD and increased in the majority of X-linked EDMD. The level of TIMP-2 was decreased in 25%/21% of AD-EDMD/X-EDMD cases. TIMP-3 serum level was significantly reduced in all the examined patients. Receiver operating curves indicated that in terms of sensitivity and specificity characteristics the performance of TIMP-3 (less that of TIMP-2) makes them the best markers of cardiac involvement among the examined TIMPs. CONCLUSIONS: Evidence shows that the levels of TIMP-3, and in some cases also TIMP-2, are decreased in EDMD. The decrease might be associated with an adverse effect on matrix metalloproteinases and remodelling of the myocardial matrix. The specific decrease of TIMP-3 indicates that this biomarker might help in early detection of cardiac involvement in EDMD. Up-regulation of TIMP-1 in the majority of patients with X-EDMD indicates increased myocardial extracellular matrix turnover, early onset of tissue remodelling, and may contribute to arrhythmia, frequently occurring in this form of the disease.

The LITAF/SIMPLE I92V sequence variant results in an earlier age of onset of CMT1A/HNPP diseases.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) represent the most common heritable neuromuscular disorders. Molecular diagnostics of CMT1A/HNPP diseases confirm clinical diagnosis, but their value is limited to the clinical course and prognosis. However, no biomarkers of CMT1A/HNPP have been identified. We decided to explore if the LITAF/SIMPLE gene shared a functional link to the PMP22 gene, whose duplication or deletion results in CMT1A and HNPP, respectively. By studying a large cohort of CMT1A/HNPP-affected patients, we found that the LITAF I92V sequence variant predisposes patients to an earlier age of onset of both the CMT1A and HNPP diseases. Using cell transfection experiments, we showed that the LITAF I92V sequence variant partially mislocalizes to the mitochondria in contrast to wild-type LITAF which localizes to the late endosome/lysosomes and is associated with a tendency for PMP22 to accumulate in the cells. Overall, this study shows that the I92V LITAF sequence variant would be a good candidate for a biomarker in the case of the CMT1A/HNPP disorders.

Early-onset facioscapulohumeral muscular dystrophy type 1 with some atypical features.

Facioscapulohumeral muscular dystrophy cases with facial weakness before the age of 5 and signs of shoulder weakness by the age of 10 are defined as early onset. Contraction of the D4Z4 repeat on chromosome 4q35 is causally related to facioscapulohumeral muscular dystrophy type 1, and the residual size of the D4Z4 repeat shows a roughly inverse correlation with the severity of the disease. Contraction of the D4Z4 repeat on chromosome 4q35 is believed to induce a local change in chromatin structure and consequent transcriptional deregulation of 4qter genes. We present early-onset cases in the Polish population that amounted to 21% of our total population with facioscapulohumeral muscular dystrophy. More than 27% of them presented with severe phenotypes (wheelchair dependency). The residual D4Z4 repeat sizes ranged from 1 to 4 units. In addition, even within early-onset facioscapulohumeral muscular dystrophy type 1 phenotypes, some cases had uncommon features (head drop, early disabling contractures, progressive ptosis, and respiratory insufficiency and cardiomyopathy).

Truncating and missense mutations in IGHMBP2 cause Charcot-Marie Tooth disease type 2.

Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-µ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5' region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.

A severe recessive and a mild dominant form of Charcot-Marie-Tooth disease associated with a newly identified Glu222Lys GDAP1 gene mutation.

Charcot-Marie-Tooth (CMT) disease caused by mutations in the GDAP1 gene has been shown to be inherited via traits that may be either autosomal recessive (in the majority of cases) [CMT4A] or autosomal dominant [CMT2K]. CMT4A disease is characterized by an early onset, and a severe clinical course often leading to a loss of ambulation, whereas CMT2K is characterized by a mild clinical course of benign axonal neuropathy beginning even in the 6th decade of life. Clinical data from a GDAP1 mutated patient suggests that the presence of a particular mutation is associated with a certain trait of inheritance. The association of a particular GDAP1 gene mutation and a dominant or recessive trait of inheritance is of special importance for genetic counseling and the prenatal diagnostics as regards severe forms of CMT. In the present study we report on two CMT families in which a newly identified Glu222Lys mutation within the GDAP1 gene segregates both in autosomal dominant and recessive traits. Our study shows that at least some GDAP1 gene mutations may segregate with the CMT phenotype as both dominant and recessive traits. Thus, genetic counseling for CMT4A/CMT2K families requires more extensive data on GDAP1 phenotype-genotype correlations.

Novel point mutations in survival motor neuron 1 gene expand the spectrum of phenotypes observed in spinal muscular atrophy patients.

The aim of our study was to identify point mutations in a group of 606 patients diagnosed for spinal muscular atrophy with excluded biallelic loss of the SMN1 gene. Point missense mutations or small deletions in the SMN1 gene were ultimately identified in 18 patients. Six patients were found to have small deletions, the c.429_435del mutation in 3 cases, the c.431delC mutation in 2 and c.722delC in one. Those mutations, not described previously, were characteristic of patients presenting a severe phenotype. The most frequent missense mutation - p.Thr274Ile, was identified in 9 patients presenting a rather mild phenotype. Three other missense mutations, i.e., p.Ser230Leu, p.Ala111Gly and p.Pro244Leu, were identified in a further 3 SMA3 patients. Mutation p.Pro244Leu, not described so far, was identified in a patient with a mild form of SMA and more distal distribution of muscle weakness. Our results suggest a specific point mutation spectrum in the Polish population. The existence of small deletions not identified thus far could suggest a possible founder effect. In patients with preserved one SMN1 allele without common exon 7 deletion, presenting a mild form of SMA, a special consideration should be given to the p.Thr274Ile mutation.

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.

Advances in basic and clinical research in laminopathies.

Lamins (LMNA) are the main proteins of the nuclear lamina considered to be the ancestors of all intermediate filament proteins. They form complex protein assemblies with integral proteins of the inner nuclear membrane, transcriptional regulators, histones and chromatin modifiers. During recent years, interest in lamins has greatly increased due to the identification of many distinct heritable human disorders associated with lamin mutations. These disorders, collectively termed laminopathies, range from muscular dystrophies to premature aging. They may affect muscle, fat, bone, nerve and skin tissues. The workshop was addressed to understand lamin organization and its roles in nuclear processes, mutations in lamins affecting cell and tissues functions, the biology of the nucleus and laminopathic disease mechanisms, all aspects important for designing future therapies.

Relationships between clinical data and quantitative EMG findings in facioscapulohumeral muscular dystrophy.

BACKGROUND AND PURPOSE: In recently published reports, electrophysiological findings were analysed, in some facioscapulo-humeral muscular dystrophy (FSHD) cases without genetic disease confirmation. In several reports, some electrophysiological findings were described, not specific for myopathy. The aim of study was to analyse electrophysiological findings in a genetically homogeneous FSHD group to find possible relationships between electromyography (EMG) abnormalities and clinical symptoms. MATERIAL AND METHODS: 37 patients with genetically proven FSHD (23 men and 14 women) aged 7-58 years (mean 28.8 years) were studied. Electromyographic examinations were done according to a uniform scheme for FSHD. Quantitative EMG examination was performed in vastus lateralis, tibialis anterior, deltoid and biceps brachii muscles. RESULTS: There was no correlation between clinical features and electrophysiological findings. EMG confirmed myopathic changes in all patients with most advanced changes in tibialis anterior and deltoid muscles. Some of these changes were unspecific for myopathy and the degree of their intensity differed in particular muscles. The most advanced changes were observed in the tibialis anterior and deltoid muscles. The usefulness of the size index for myopathic processes assessment was confirmed. Analysis of so-called outliers for motor unit activity potential parameters did not show any new data for evaluation of the myopathic process. Myopathic changes in our material were not as advanced as those described in classical dystrophies. Histopathological examinations of skeletal muscle were normal in about 1/3 of patients. CONCLUSIONS: We established that myopathic changes are clearly present in FSHD, with different degrees of intensity, most pronounced in tibialis anterior and deltoid muscles. There was no correlation between electrophysiological findings and clinical features. The size index provided the highest motor unit potential diagnostic sensitivity in FSHD.