The GDAP1 p.Glu222Lys Variant-Weak Pathogenic Effect, Cumulative Effect of Weak Sequence Variants, or Synergy of Both Factors?

Charcot−Marie−Tooth disorders (CMT) represent a highly heterogeneous group of diseases of the peripheral nervous system in which more than 100 genes are involved. In some CMT patients, a few weak sequence variants toward other CMT genes are detected instead of one leading CMT mutation. Thus, the presence of a few variants in different CMT-associated genes raises the question concerning the pathogenic status of one of them. In this study, we aimed to analyze the pathogenic effect of c.664G>A, p.Glu222Lys variant in the GDAP1 gene, whose mutations are known to be causative for CMT type 4A (CMT4A). Due to low penetrance and a rare occurrence limited to five patients from two Polish families affected by the CMT phenotype, there is doubt as to whether we are dealing with real pathogenic mutation. Thus, we aimed to study the pathogenic effect of the c.664G>A, p.Glu222Lys variant in its natural environment, i.e., the neuronal SH-SY5Y cell line. Additionally, we have checked the pathogenic status of p.Glu222Lys in the broader context of the whole exome. We also have analyzed the impact of GDAP1 gene mutations on the morphology of the transfected cells. Despite the use of several tests to determine the pathogenicity of the p.Glu222Lys variant, we cannot point to one that would definitively solve the problem of pathogenicity.

Validation of the Pathogenic Effect of IGHMBP2 Gene Mutations Based on Yeast S. cerevisiae Model.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a heritable neurodegenerative disease characterized by rapid respiratory failure within the first months of life and progressive muscle weakness and wasting. Although the causative gene, IGHMBP2, is well defined, information on IGHMBP2 mutations is not always sufficient to diagnose particular patients, as the gene is highly polymorphic and the pathogenicity of many gene variants is unknown. In this study, we generated a simple yeast model to establish the significance of IGHMBP2 variants for disease development, especially those that are missense mutations. We have shown that cDNA of the human gene encodes protein which is functional in yeast cells and different pathogenic mutations affect this functionality. Furthermore, there is a correlation between the phenotype estimated in in vitro studies and our results, indicating that our model may be used to quickly and simply distinguish between pathogenic and non-pathogenic mutations identified in IGHMBP2 in patients.

Analysis of the Innovative Channel Strut Concept Manufactured by Roll-Forming.

Due to the wide use of channel strut components, manufacturing is implemented in many industrial plants. Standard technology of profiles is based on welding of two parts of the profile and requires the regalvanizing of the joint zone causes. Thus, the production is challenging to automate on a single line. The main idea of the article is to present a concept of a channel strut, a cold-formed continuous metal component with an open or closed profile section. It would serve as a cantilever support instead of a standard solution. In the article, a unique lock system combination is proposed and analyzed both numerically and experimentally to provide steadiness of the strut without welding or other joining techniques. Two main lock shapes-semicircular and triangle-were proposed with some variations in the cutting plane. Analyses were carried out for three main profile cross-sections with different dimensions, based on the current industrial applications. The semicircular type of the lock was found to be the most stable, giving optimal strength to the strut under assumed loading, comparable to traditional solutions. The commercial FEM software MSC Marc was used for the numerical analysis.

Models for IGHMBP2-associated diseases: an overview and a roadmap for the future.

Models are practical tools with which to establish the basic aspects of a diseases. They allow systematic research into the significance of mutations, of cellular and molecular pathomechanisms, of therapeutic options and of functions of diseases associated proteins. Thus, disease models are an integral part of the study of enigmatic proteins such as immunoglobulin mu-binding protein 2 (IGHMBP2). IGHMBP2 has been well defined as a helicase, however there is little known about its role in cellular processes. Notably, it is unclear why changes in such an abundant protein lead to specific neuronal disorders including spinal muscular atrophy with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S). SMARD1 is caused by a loss of motor neurons in the spinal cord that results in muscle atrophy and is accompanied by rapid respiratory failure. In contrast, CMT2S manifests as a severe neuropathy, but typically without critical breathing problems. Here, we present the clinical manifestation of IGHMBP2 mutations, function of protein and models that may be used for the study of IGHMBP2-associated disorders. We highlight the strengths and weaknesses of specific models and discuss the orthologs of IGHMBP2 that are found in different systems with regard to their similarity to human IGHMBP2.

Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation.

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.

Mutations in GDAP1 Influence Structure and Function of the Trans-Golgi Network.

Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative disease that displays great genetic heterogeneity. The genes and mutations that underlie this heterogeneity have been extensively characterized by molecular genetics. However, the molecular pathogenesis of the vast majority of CMT subtypes remains terra incognita. Any attempts to perform experimental therapy for CMT disease are limited by a lack of understanding of the pathogenesis at a molecular level. In this study, we aim to identify the molecular pathways that are disturbed by mutations in the gene encoding GDAP1 using both yeast and human cell, based models of CMT-GDAP1 disease. We found that some mutations in GDAP1 led to a reduced expression of the GDAP1 protein and resulted in a selective disruption of the Golgi apparatus. These structural alterations are accompanied by functional disturbances within the Golgi. We screened over 1500 drugs that are available on the market using our yeast-based CMT-GDAP1 model. Drugs were identified that had both positive and negative effects on cell phenotypes. To the best of our knowledge, this study is the first report of the Golgi apparatus playing a role in the pathology of CMT disorders. The drugs we identified, using our yeast-based CMT-GDAP1 model, may be further used in translational research.

Application of the Numerical Model to Design the Geometry of a Unit Tool in the Innovative RTH Hydroforming Technology.

The article presents a newly patented rapid tube hydroforming (RTH) manufacturing method, perfectly suited to single-piece production. The RTH technology significantly complements the scope of hydroforming processes. Due to the unusual granular material of the die tool, in particular moulding sand or mass, the process design requires the use of numerical modelling calculations. This is related to the complexity and the synergistic effect of process parameters on the final shape of the product. The work presents the results of numerical modelling studies of the process, including the behaviour of the die material and the material of the hydroformed profile. The numerical calculations were performed for a wide range of parameters, and can be used in various applications. The significant properties of moulding material used for the RTH tests were determined and one was chosen to build the die in RTH experiments. The results of the numerical modelling were compared with the results of the experiments, which proved their high compatibility. The final conclusions of the analyses indicate that the RTH technology has many possibilities that are worth further development and research.

Recommendations on the diagnosis of male infertility - genetic testing [Rekomendacje dotyczace diagnostyki genetycznej w nieplodnosci meskiej].

Male infertility is the cause of couples' infertility in about 50% of cases. Current recommendations on the diagnosis and treatment of male infertility advance thorough medical history taking and physical examination, to provide the basis for further genetic evaluation. The extent of genetic testing itself depends on the semen analysis results, which allow the risk of inheritance of chromosomal aberrations to be determined and the root causes of habitual miscarriages to be explained. In azoospermia, once the type of microdeletion has been identified, a decision can be made as to whether a testicular biopsy is required to obtain sperm for the artificial reproductive technology (ART) procedure. The physical examination, genetic interview, and hormonal results are helpful in deciding which genetic tests to perform. Our research facilitates genetic testing in the diagnosis of male infertility.

A Yeast-Based Model for Hereditary Motor and Sensory Neuropathies: A Simple System for Complex, Heterogeneous Diseases.

Charcot-Marie-Tooth (CMT) disease encompasses a group of rare disorders that are characterized by similar clinical manifestations and a high genetic heterogeneity. Such excessive diversity presents many problems. Firstly, it makes a proper genetic diagnosis much more difficult and, even when using the most advanced tools, does not guarantee that the cause of the disease will be revealed. Secondly, the molecular mechanisms underlying the observed symptoms are extremely diverse and are probably different for most of the disease subtypes. Finally, there is no possibility of finding one efficient cure for all, or even the majority of CMT diseases. Every subtype of CMT needs an individual approach backed up by its own research field. Thus, it is little surprise that our knowledge of CMT disease as a whole is selective and therapeutic approaches are limited. There is an urgent need to develop new CMT models to fill the gaps. In this review, we discuss the advantages and disadvantages of yeast as a model system in which to study CMT diseases. We show how this single-cell organism may be used to discriminate between pathogenic variants, to uncover the mechanism of pathogenesis, and to discover new therapies for CMT disease.

Pathogenic Effect of GDAP1 Gene Mutations in a Yeast Model.

The question of whether a newly identified sequence variant is truly a causative mutation is a central problem of modern clinical genetics. In the current era of massive sequencing, there is an urgent need to develop new tools for assessing the pathogenic effect of new sequence variants. In Charcot-Marie-Tooth disorders (CMT) with their extreme genetic heterogeneity and relatively homogenous clinical presentation, addressing the pathogenic effect of rare sequence variants within 80 CMT genes is extremely challenging. The presence of multiple rare sequence variants within a single CMT-affected patient makes selection for the strongest one, the truly causative mutation, a challenging issue. In the present study we propose a new yeast-based model to evaluate the pathogenic effect of rare sequence variants found within the one of the CMT-associated genes, GDAP1. In our approach, the wild-type and pathogenic variants of human GDAP1 gene were expressed in yeast. Then, a growth rate and mitochondrial morphology and function of GDAP1-expressing strains were studied. Also, the mutant GDAP1 proteins localization and functionality were assessed in yeast. We have shown, that GDAP1 was not only stably expressed but also functional in yeast cell, as it influenced morphology and function of mitochondria and altered the growth of a mutant yeast strain. What is more, the various GDAP1 pathogenic sequence variants caused the specific for them effect in the tests we performed. Thus, the proposed model is suitable for validating the pathogenic effect of known GDAP1 mutations and may be used for testing of unknown sequence variants found in CMT patients.

Molecular modelling of mitofusin 2 for a prediction for Charcot-Marie-Tooth 2A clinical severity.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant neuropathy caused by mutations in the mitofusin 2 gene (MFN2). More than 100 MFN2 gene mutations have been reported so far, with majority located within the GTPase domain encoding region. These domain-specific mutations present wide range of symptoms with differences associated with distinct amino acid substitutions in the same position. Due to the lack of conclusive phenotype-genotype correlation the predictive value of genetic results remains still limited. We have explored whether changes in the protein structure caused by MFN2 mutations can help to explain diseases phenotypes. Using a stable protein model, we evaluated the effect of 26 substitutions on the MFN2 structure and predicted the molecular consequences of such alterations. The observed changes were correlated with clinical features associated with a given mutation. Of all tested mutations positive correlation of molecular modelling with the clinical features reached 73%. Our analysis revealed that molecular modelling of mitofusin 2 mutations is a powerful tool, which predicts associated pathogenic impacts and that these correlate with clinical outcomes. This approach may aid an early diagnosis and prediction of symptoms severity in CMT2A patients.

Charcot­Marie­Tooth type 1A drug therapies: role of adenylyl cyclase activity and G­protein coupled receptors in disease pathomechanism.

Charcot­Marie­Tooth type 1A (CMT1A) is a dysmyelinating disease of the peripheral nervous system that results in a slow progressive weakening and wasting of the distal muscles of the upper and lower limbs. Despite extensive research and clinical trials there is still no treatment for CMT1A that results in complete neurological improvement. Recent studies investigating various pharmacological modulators of adenylyl cyclase activity, including ascorbic acid and ligands of G protein­coupled receptors (GPCRs), provide hope for future treatments of this type of hereditary motor and sensory neuropathy. A review of mechanisms of action of several compounds tested for CMT1A in pre­clinical and clinical studies ascorbic acid, onapristone, PXT3003 (baclofen, naltrexone, and sorbitol), and ADX71441, very clearly indicates an important role for adenylyl cyclase activity and GPCRs in the pathomechanism of the disease. Metabotropic γ­aminobutyric acid receptors (GABABR), subtype mu (µ) opioid receptors (MOR), and muscarinic acetylcholine receptors (mACh) appear to be particularly significant in both pathogenesis and treatment, and their activation may exert a similar and synergistic effect on the physiology of Schwann cells as well as neurons. These receptors participate in proliferation and differentiation of Schwann cells and influence excitatory transmission in neurons. We also hypothesize that onapristone might act through a non­classical mechanism via membrane progesterone receptor (mPR) and cAMP signaling. This review endeavors to outline a pathway leading inversely from therapy to an indispensable role for adenylyl cyclase activity and GPCRs in the modulation of dosage sensitive peripheral myelin protein (PMP22) gene expression.

A role for the GDAP1 gene in the molecular pathogenesis of Charcot­Marie­Tooth disease.

In 2002 a series of mutations in the GDAP1 gene were reported in patients suffering from Charcot­Marie­Tooth disease manifesting as early-onset, progressive distal­muscle wasting and weakness. The molecular etiology of Charcot­Marie­Tooth ­GDAP1 disease has been elucidated but its pathogenesis remains unclear, especially given the seemingly contradictory function of the GDAP1 protein. Expression of GDAP1 is observed almost exclusively in neuronal cells, however, the GDAP1 protein is present in mitochondria, where it plays a role in fission, a ubiquitous process occurring in all cells. While GDAP1 contains two glutathione S­transferase (GST) domains, its GST activity is in fact very limited. Additionally, despite GDAP1 affecting mitochondrial functionality, and hence being of great importance to cellular function, the GDAP1­associated Charcot-Marie-Tooth disease is mainly characterized by axonal degeneration. Finally, mutations in the GDAP1 gene may be inherited in a recessive or dominant manner. Given the way such varied observations are hard to reconcile with one another, the investigation of GDAP1 is at one and the same time a difficult but also challenging endeavour. The purpose of this review is to summarize the current knowledge on the GDAP1 protein and its function in the cell. A further part is the characterization of GDAP1­associated Charcot-Marie-Tooth disease, its symptoms and course, as well as an outlining of the possible mechanisms underpinning the disorder.

[Therapeutic perspective in hereditary polyneuropathies]. / Perspektywy terapii w polineuropatiach genetycznie uwarunkowanych.

Hereditary motor and sensory neuropathies (HMSN) also called as Charcot-Marie-Tooth disorders (CMT) are extremely heterogeneous group of disorders of peripheral nervous system. Over 80 genes have been reported in different types of CMT. In all CMT affected patients the main symptoms are slowly progressive wasting of the distal muscles of the lower and upper limbs. To date no efficient therapeutic approach basing upon molecular pathology of CMT has been proposed. This review presents the current state of knowledge concerning clinical, molecular pathogenesis and experimental therapy aspects in CMT disorders. Additionally the possibilities resulting from the use of the yeast model to the identification of new therapeutic substances as well as of neurotoxins are also discussed.

A novel de novo COL6A1 mutation emphasizes the role of intron 14 donor splice site defects as a cause of moderate-progressive form of ColVI myopathy - a case report and review of the genotype-phenotype correlation.

Collagen VI-related myopathy is a group of disorders affecting skeletal muscles and connective tissue. The most common symptoms are muscle weakness and joint deformities which limit the movement and progress over time. Several forms of collagen VI-related myopathies have been described: Bethlem myopathy, an intermediate form and Ullrich congenital muscular dystrophy, which is the most severe. Here we report a novel de novo c.1056+3A>C substitution in intron 14 of the COL6A1 gene encoding alpha-chains of collagen VI in a 13-year-old girl suffering from collagen VI (ColVI) myopathy. Analysis performed on cDNA generated from the RNA obtained from the patient's blood cells showed that the reported variant leads to the entire exon 14 skipping and probably results in an in-frame deletion of 18 amino acids of the COL6A1 protein. Clinical presentation, abnormal secretion of the collagen demonstrated in muscle biopsy and the COL6A1 c.1056+3A>C mutation justify classification of the presented case as ColVI myopathy with moderate-progressive course. Analysis of the literature indicates that the donor splice site of COL6A1 intron 14, associated with the phenotype of Bethlem myopathy or intermediate form, is a hot spot for ColVI myopathies.

The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a de novo Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions.

A novel TPM2 gene splice-site mutation causes severe congenital myopathy with arthrogryposis and dysmorphic features.

To date, only two splice-site mutations within the TPM2 gene have been shown to be causative for congenital myopathies. While the majority of TPM2 gene mutations are causative for nemaline myopathy, cap disease or distal arthrogryposis, some mutations in this gene have been found to be associated with non-specific congenital myopathy. We report on a patient with such an unspecified congenital myopathy associated with distinctive facial dysmorphic features and distal arthrogryposis. Using the whole exome sequencing (WES) approach we were able to identify a novel heterozygous splice-site mutation within the TPM2 gene, showing the utility of WES in molecular diagnostics of congenital myopathies without recognizable morphological hallmarks.

Pathogenic mutations and sequence variants within mitofusin 2 gene in Polish patients with different hereditary motor-sensory neuropathies.

At the time of its first description in 2004, MFN2 was considered the most frequently mutated gene in hereditary motor and sensory neuropathy type 2 (HMSN 2). However recent studies have shown that the frequency of MFN2 gene mutations in HMSN II patients is surprisingly low. To date, no systematic studies devoted to HMSN IIa in Poland have been carried out. In this study, we searched for MFN2 gene mutations in Polish patients representing the population of nearly 40 million. We decided to include a wide spectrum of clinical phenotypes in the study, proving able to detect, in a group of 67 affected patients: 1) 3 pathogenic mutations; 2) 3 sequence variants of unknown pathogenic status; 3) 9 rare MFN2 gene sequence variants; 4) 6 common polymorphisms. The frequency of MFN2 gene mutations in the whole group of patients is 4.5%. Due to the high frequency of MFN2 gene sequence variants within single patients we could not definitely exclude the cumulative effect of these contributing to the HMSN II phenotype. The MFN2 gene should therefore be considered in Polish HMSN II patients, though it is still not possible to determine its position in HMSN II molecular diagnostics.

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.

Molecular pathogenesis, experimental therapy and genetic counseling in hereditary sensory neuropathies.

Hereditary sensory and autonomic neuropathies (HSANs) represent a group of heritable peripheral nerve disorders usually taking a severe clinical course. HSAN-affected patients manifest with deep, poorly-healing ulcerations of the feet and hands. To date no definitive cure for HSANs has been developed and the molecular pathology of these disorders is complex. The aim of this review is therefore to present recent findings in terms of HSAN molecular pathogenesis. So far, mutations in 12 genes coding for different proteins have been reported in association with HSAN and the molecular pathogenesis has been elucidated in HSAN1a, HSAN4 and HSAN5. The genes involved in molecular pathogenesis of HSAN code for a wide spectrum of proteins from enzymes to specific nerve growth factors. As far as HSAN1a is concerned, the enhanced understanding has given rise to achievements in experimental therapy particularly in respect to disease models. Despite a rapid progress in studies on the molecular background of HSAN, numerous loci and genes remain still to be discovered.