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Juliane S Muller - One of the best experts on this subject based on the ideXlab platform.
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global n linked glycosylation is not significantly impaired in myoblasts in congenital myasthenic syndromes caused by defective glutamine fructose 6 phosphate transaminase 1 GFPT1
Biomolecules, 2015Co-Authors: Qiushi Chen, Juliane S Muller, Hanns Lochmuller, Pohchoo Pang, S Laval, Stuart M Haslam, Anne DellAbstract:Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.
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a 3 utr mutation creates a microrna target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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A 3′-UTR mutation creates a microRNA target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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mutations in GFPT1 that underlie limb girdle congenital myasthenic syndrome result in reduced cell surface expression of muscle achr
Human Molecular Genetics, 2013Co-Authors: Katarzyna Marta Zoltowska, Richard Webster, Sarah Finlayson, Susan Maxwell, Judith Cossins, Juliane S Muller, Hanns Lochmuller, David BeesonAbstract:: Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, e, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder.
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g p 25 a 3 utr mutation creates a potential microrna target site in the GFPT1 gene of lg cms patients
Neuromuscular Disorders, 2012Co-Authors: Juliane S Muller, Hanns Lochmuller, Marina Dusl, Anja Pertl, Angela AbichtAbstract:Abstract Recently, we described mutations in the GFPT1 (glucosamine-fructose-6-phosphate aminotransferase 1) gene causing congenital myasthenic syndrome with tubular aggregates. One of the mutations detected by us was a change in the 3′-untranslated region (UTR) of GFPT1:c.∗22C>A. We identified GFPT1 c.∗22C>A in six patients from three independent families of Spanish and German origin as compound heterozygous mutation accompanying a different second heterozygous pathogenic mutation in each case. However, the pathogenic mechanism of the mutation c.∗22C>A remained unclear. GFPT1 protein amounts were reduced in muscle biopsy samples of two of those patients as shown by Western blot. On mRNA level, transcripts from both alleles were present in equal amounts, indicating that c.∗22C>A does not increase mRNA degradation or lower transcription rates for the allele carrying c.∗22C>A. microRNAs are well known to alter gene expression on post-transcriptional level. The mutation GFPT1 c.∗22C>A creates new binding sites for two miRNAs. Co-expression of reporter luciferase constructs containing the mutated or the wild type GFPT1 3′UTR together with the miRNAs resulted in a reduction of luciferase expression, indicating that c.∗22C>A indeed influences miRNA binding and thereby protein translation. Our results suggest that alteration of miRNA binding sites by mutations might be a relevant pathogenic factor and that variants in the 5′- and 3′-UTRs should not be disregarded during routine genetic diagnostic procedures.
Angela Abicht - One of the best experts on this subject based on the ideXlab platform.
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a 3 utr mutation creates a microrna target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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A 3′-UTR mutation creates a microRNA target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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g p 25 a 3 utr mutation creates a potential microrna target site in the GFPT1 gene of lg cms patients
Neuromuscular Disorders, 2012Co-Authors: Juliane S Muller, Hanns Lochmuller, Marina Dusl, Anja Pertl, Angela AbichtAbstract:Abstract Recently, we described mutations in the GFPT1 (glucosamine-fructose-6-phosphate aminotransferase 1) gene causing congenital myasthenic syndrome with tubular aggregates. One of the mutations detected by us was a change in the 3′-untranslated region (UTR) of GFPT1:c.∗22C>A. We identified GFPT1 c.∗22C>A in six patients from three independent families of Spanish and German origin as compound heterozygous mutation accompanying a different second heterozygous pathogenic mutation in each case. However, the pathogenic mechanism of the mutation c.∗22C>A remained unclear. GFPT1 protein amounts were reduced in muscle biopsy samples of two of those patients as shown by Western blot. On mRNA level, transcripts from both alleles were present in equal amounts, indicating that c.∗22C>A does not increase mRNA degradation or lower transcription rates for the allele carrying c.∗22C>A. microRNAs are well known to alter gene expression on post-transcriptional level. The mutation GFPT1 c.∗22C>A creates new binding sites for two miRNAs. Co-expression of reporter luciferase constructs containing the mutated or the wild type GFPT1 3′UTR together with the miRNAs resulted in a reduction of luciferase expression, indicating that c.∗22C>A indeed influences miRNA binding and thereby protein translation. Our results suggest that alteration of miRNA binding sites by mutations might be a relevant pathogenic factor and that variants in the 5′- and 3′-UTRs should not be disregarded during routine genetic diagnostic procedures.
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the clinical phenotypic spectrum of GFPT1 associated congenital myasthenic syndrome
Journal of Neurology Neurosurgery and Psychiatry, 2012Co-Authors: Amina Chaouch, David Beeson, Velina Guergueltcheva, Angela Abicht, Volker Straub, Kate Bushby, J Palace, J S Mueller, Francesco Muntoni, Hanns LochmullerAbstract:We report on the clinical features of a distinct group of DOK 7 negative limb girdle congenital myasthenic syndrome (LG-CMS), the cause of which has only recently been unravelled. Congenital myasthenic syndromes are a rare group of inherited neuromuscular disorders associated with distinct clinical and genetic abnormalities, in which neuromuscular transmission is impaired. An interesting and often difficult group to recognise is the LG-CMS. This typically manifests with shoulder and pelvic girdle muscle weakness with or without additional features including ocular and bulbar involvement. Until recently, DOK 7 gene mutations have been the only recognised genetic cause of this phenotype and were found in half of all LG-CMS patients. Mutations in a novel CMS gene (GFPT1) were recently implicated in an undiagnosed, DOK 7 negative, LG-CMS cohort of 24 patients. All patients had proximal limb weakness with no ocular or bulbar features and showed a positive response to pyridostigmine. Although age of onset of disease was usually in early childhood, a great number of patients presented to medical attention well into adulthood. Furthermore, tubular aggregates arising from the sarcoplasmic reticulum were seen in the majority of patients.
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congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations velina guergueltchevajuliane s mullermarina dusljan senderekanders oldfors christopher lindberghsusan maxwelljaume colomercecilia jimenez mallebreraandres nascimento jua
2012Co-Authors: Velina Guergueltcheva, Jan Senderek, Marina Dusl, Angela Abicht, Beate Schlotter, Benedikt Schoser, Amina Chaouch, Volker Straub, Kate Bushby, Anders OldforsAbstract:Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1, in a series of DOK7-negative cases. How- ever, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic
Hanns Lochmuller - One of the best experts on this subject based on the ideXlab platform.
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global n linked glycosylation is not significantly impaired in myoblasts in congenital myasthenic syndromes caused by defective glutamine fructose 6 phosphate transaminase 1 GFPT1
Biomolecules, 2015Co-Authors: Qiushi Chen, Juliane S Muller, Hanns Lochmuller, Pohchoo Pang, S Laval, Stuart M Haslam, Anne DellAbstract:Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.
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Congenital Myasthenic Syndromes with Predominant Limb Girdle Weakness.
Journal of neuromuscular diseases, 2015Co-Authors: Teresinha Evangelista, Michael G. Hanna, Hanns LochmullerAbstract:Congenital myasthenic syndromes are a heterogeneous group of genetically determined disorders characterized by impaired neuromuscular transmission. They usually present from birth to childhood and are characterised by exercise induced weakness and fatigability. Genotype-phenotype correlations are difficult. However, in some patients particular phenotypic aspects may point towards a specific genetic defect. The absence of ptosis and ophthalmoparesis in patients with limb-girdle weakness makes the diagnosis of a neuromuscular transmission defect particularly challenging (LG-CMS). This is illustrated by a well-documented case published by Walton in 1956. The diagnosis of LG-CMS is secured by demonstrating a neuromuscular transmission defect with single fibre EMG or repetitive nerve stimulation, in the absence of auto-antibodies. Ultimately, a genetic test is required to identify the underlying cause and assure counselling and optimization of treatment. LG-CMS are inherited in autosomal recessive traits, and are often associated with mutations in DOK7 and GFPT1, and less frequently with mutations in COLQ, ALG2, ALG14 and DPAGT. Genetic characterization of CMS is of the upmost importance when choosing the adequate treatment. Some of the currently used drugs can either ameliorate or aggravate the symptoms depending on the underlying genetic defect. The drug most frequently used for the treatment of CMS is pyridostigmine an acetylcholinesterase inhibitor. However, pyridostigmine is not effective or is even detrimental in DOK7- and COLQ-related LG-CMS, while beta-adrenergic agonists (ephedrine, salbutamol) show some sustained benefit. Standard clinical trials may be difficult, but standardized follow-up of patients and international collaboration may help to improve the standards of care of these conditions.
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a 3 utr mutation creates a microrna target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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A 3′-UTR mutation creates a microRNA target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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mutations in GFPT1 that underlie limb girdle congenital myasthenic syndrome result in reduced cell surface expression of muscle achr
Human Molecular Genetics, 2013Co-Authors: Katarzyna Marta Zoltowska, Richard Webster, Sarah Finlayson, Susan Maxwell, Judith Cossins, Juliane S Muller, Hanns Lochmuller, David BeesonAbstract:: Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, e, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder.
Marina Dusl - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of congenital myasthenic syndromes in Spain
Neuromuscular Disorders, 2017Co-Authors: D. Natera-de Benito, Ana Töpf, Juan J. Vílchez, L. González-quereda, J. Domínguez-carral, J. Diaz-manera, Carlos Ortez, M. Bestué, P. Gallano, Marina DuslAbstract:Abstract Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management.
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a 3 utr mutation creates a microrna target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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A 3′-UTR mutation creates a microRNA target site in the GFPT1 gene of patients with congenital myasthenic syndrome
Human Molecular Genetics, 2015Co-Authors: Marina Dusl, Juliane S Muller, Hanns Lochmuller, Jan Senderek, Johannes G Vogel, Anja Pertl, Rolf Stucka, Robert David, Angela AbichtAbstract:Abstract Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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g p 25 a 3 utr mutation creates a potential microrna target site in the GFPT1 gene of lg cms patients
Neuromuscular Disorders, 2012Co-Authors: Juliane S Muller, Hanns Lochmuller, Marina Dusl, Anja Pertl, Angela AbichtAbstract:Abstract Recently, we described mutations in the GFPT1 (glucosamine-fructose-6-phosphate aminotransferase 1) gene causing congenital myasthenic syndrome with tubular aggregates. One of the mutations detected by us was a change in the 3′-untranslated region (UTR) of GFPT1:c.∗22C>A. We identified GFPT1 c.∗22C>A in six patients from three independent families of Spanish and German origin as compound heterozygous mutation accompanying a different second heterozygous pathogenic mutation in each case. However, the pathogenic mechanism of the mutation c.∗22C>A remained unclear. GFPT1 protein amounts were reduced in muscle biopsy samples of two of those patients as shown by Western blot. On mRNA level, transcripts from both alleles were present in equal amounts, indicating that c.∗22C>A does not increase mRNA degradation or lower transcription rates for the allele carrying c.∗22C>A. microRNAs are well known to alter gene expression on post-transcriptional level. The mutation GFPT1 c.∗22C>A creates new binding sites for two miRNAs. Co-expression of reporter luciferase constructs containing the mutated or the wild type GFPT1 3′UTR together with the miRNAs resulted in a reduction of luciferase expression, indicating that c.∗22C>A indeed influences miRNA binding and thereby protein translation. Our results suggest that alteration of miRNA binding sites by mutations might be a relevant pathogenic factor and that variants in the 5′- and 3′-UTRs should not be disregarded during routine genetic diagnostic procedures.
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Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations
Journal of Neurology, 2012Co-Authors: Velina Guergueltcheva, Susan Maxwell, Juliane S Muller, Jan Senderek, Marina Dusl, Anders Oldfors, Christopher Lindbergh, Jaume Colomer, Cecilia Jimenez Mallebrera, Andres NascimentoAbstract:Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1 , in a series of DOK7 -negative cases. However, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic limb-girdle weakness and fatigue. A common feature was beneficial and sustained response to acetylcholinesterase inhibitor treatment. Most of the patients who had a muscle biopsy showed tubular aggregates in myofibers. Analysis of endplate morphology in one of the patients revealed unspecific abnormalities. Our study delineates the phenotype of CMS associated with GFPT1 mutations and expands the understanding of neuromuscular junction disorders. As tubular aggregates in context of a neuromuscular transmission defect appear to be highly indicative, we suggest calling this condition congenital myasthenic syndrome with tubular aggregates (CMS-TA).
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Chapter 99 – Congenital Myasthenic Syndromes
Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease, 2020Co-Authors: Andrew G EngelAbstract:Congenital myasthenic syndromes (CMS) are heterogeneous disorders. Clinical, electrophysiologic, and morphologic studies have paved the way for detecting CMS-related mutations in proteins residing in the nerve terminal, the synaptic basal lamina, and in the postsynaptic region of the motor endplate. The disease proteins identified to date include choline acetyltransferase (ChAT), the endplate species of acetylcholinesterase (AChE), β2-laminin, the acetylcholine receptor (AChR), rapsyn, plectin, and Nav1.4. Other mutations occur in proteins subserving endplate development and maintenance, namely agrin, MuSK, and Dok-7; and in proteins that subserve glycosylation, such as GFPT1, DPAGT1, ALG2 and ALG14. Myasthenic syndromes can also occur in combination with centronuclear myopathy defects in plectin. Analysis of the properties of the expressed mutants contributed to finding improved therapy for most CMS. Despite these advances, the molecular basis of some phenotypically characterized CMS remains elusive. Moreover, other types of CMS likely exist and await discovery.
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Chapter 26 – Congenital Myasthenic Syndromes
Neuromuscular Disorders of Infancy Childhood and Adolescence, 2020Co-Authors: Andrew G EngelAbstract:Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Clinical, electrophysiologic, morphologic, and molecular genetic studies have paved the way for defining phenotypic features of the different syndromes, identifying disease genes and proteins, and recommending appropriate therapy. Disease proteins reside in the nerve terminal, the synaptic basal lamina, or the postsynaptic region of the neuromuscular junction, or are distributed in many tissues including the junction. Those identified to date include choline acetyltransferase, the endplate species of acetylcholinesterase β2-laminin, each subunit of the acetylcholine receptor, rapsyn, plectin, Nav1.4, MuSK, agrin, Dok-7, GFPT1, DPAGT1, ALG2, and ALG14. Analyses of properties of expressed mutant proteins contribute to finding improved therapy for most CMS. Despite these advances, the molecular basis of some phenotypically characterized CMS remains elusive. Moreover, other types of CMS and disease genes likely exist and await discovery.
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Genetic basis and phenotypic features of congenital myasthenic syndromes.
Handbook of Clinical Neurology, 2020Co-Authors: Andrew G EngelAbstract:Abstract The congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. The disease proteins reside in the nerve terminal, the synaptic basal lamina, or in the postsynaptic region, or at multiple sites at the neuromuscular junction as well as in other tissues. Targeted mutation analysis by Sanger or exome sequencing has been facilitated by characteristic phenotypic features of some CMS. No fewer than 20 disease genes have been recognized to date. In one-half of the currently identified probands, the disease stems from mutations in genes encoding subunits of the muscle form of the acetylcholine receptor (CHRNA1, CHRNB, CHRNAD1, and CHRNE). In 10–14% of the probands the disease is caused by mutations in RAPSN, DOK 7, or COLQ, and in 5% by mutations in CHAT. Other less frequently identified disease genes include LAMB2, AGRN, LRP4, MUSK, GFPT1, DPAGT1, ALG2, and ALG 14 as well as SCN4A, PREPL, PLEC1, DNM2, and MTM1. Identification of the genetic basis of each CMS is important not only for genetic counseling and disease prevention but also for therapy, because therapeutic agents that benefit one type of CMS can be harmful in another.
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Congenital myasthenic syndromes in adult neurology clinic: A long road to diagnosis and therapy
Neurology, 2018Co-Authors: Margherita Milone, Andrew G Engel, Duygu Selcen, Xin Ming Shen, Teerin LiewluckAbstract:Objective To investigate the diagnostic challenges of congenital myasthenic syndromes (CMS) in adult neuromuscular practice. Methods We searched the Mayo Clinic database for patients with CMS diagnosed in adulthood in the neuromuscular clinic between 2000 and 2016. Clinical, laboratory, and electrodiagnostic data were reviewed. Results We identified 34 patients with CMS, 30 of whom had a molecular diagnosis (14 DOK7 , 6 RAPSN , 2 LRP4 , 2 COLQ , 2 slow-channel syndrome, 1 primary acetylcholine receptor deficiency, 1 AGRN , 1 GFPT1 , and 1 SCN4A ). Ophthalmoparesis was often mild and present in 13 patients. Predominant limb-girdle weakness occurred in 19 patients. Two patients had only ptosis. Age at onset ranged from birth to 39 years (median 5 years). The median time from onset to diagnosis was 26 years (range 4–56 years). Thirteen patients had affected family members. Fatigable weakness was present when examined. Creatine kinase was elevated in 4 of 23 patients (range 1.2–4.2 times the upper limit of normal). Repetitive nerve stimulation revealed a decrement in 30 patients. Thirty-two patients were previously misdiagnosed with seronegative myasthenia gravis (n = 16), muscle diseases (n = 15), weakness of undetermined cause (n = 8), and others (n = 4). Fifteen patients received immunotherapy or thymectomy without benefits. Fourteen of the 25 patients receiving pyridostigmine did not improve or worsen. Conclusion Misdiagnosis occurred in 94% of the adult patients with CMS and causes a median diagnostic delay of nearly 3 decades from symptom onset. Seronegative myasthenia gravis and muscle diseases were the 2 most common misdiagnoses, which led to treatment delay and unnecessary exposure to immunotherapy, thymectomy, or muscle biopsy.
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Congenital Myasthenic Syndromes in 2018
Current Neurology and Neuroscience Reports, 2018Co-Authors: Andrew G EngelAbstract:Purpose of Review Summarize features of the currently recognized congenital myasthenic syndromes (CMS) with emphasis on novel findings identified in the past 6 years. Recent Findings Since the last review of the CMS in this journal in 2012, several novel CMS were identified. The identified disease proteins are SNAP25B, synaptotagmin 2, Munc13-1, synaptobrevin-1, GFPT1, DPAGT1, ALG2, ALG14, Agrin, GMPPB, LRP4, myosin 9A, collagen 13A1, the mitochondrial citrate carrier, PREPL, LAMA5, the vesicular ACh transporter, and the high-affinity presynaptic choline transporter. Summary Exome sequencing has provided a powerful tool for identifying novel CMS. Identifying the disease genes is essential for determining optimal therapy. The landscape of the CMS is still unfolding.