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Cardiac involvement in a patient with congenital muscular dystrophy related to POMT2 gene mutation
Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249
CONGENITAL MUSCULAR DYSTROPHIES: DYSTROGLYCANOPATHIES AND OTHERS P.35 Translational biochemistry for dystroglycanopathies H. Wang, R. Sprute, H. Daimagüler, S. Cirak University Childrens Hospital Cologne, Cologne, Germany ISPD (isoprenoid synthase domain containing) mutations cause a wide clinical spectrum of muscular dystrophies (dystroglycanopathies). Human ISPD is a cytidyltransferase, catalyses the reaction from CTP and Ribitol-5phosphate to CDP-Ribitol, a novel nucleotide sugar for functional alphadystroglycan. hISPD has one conserved cytidyltransferase domain and a second functional unknown non-conserved domain. We aim to correlate clinical phenotypes to structural impacts of ISPD mutations to decipher the underlying molecular mechanisms. First we established a high throughput in vitro protein thermostablity screening assay (Thermofluor assay, TF), by which we screened for the ligands of recombinant hISPD (purified from E.coli) and found out that Ribitol-5-Phosphate can only alter the protein thermostability in the presence of CTP. The thermostablities of missense mutants are also assessed by TF assay, together with protein half-life cycloheximide assay in HEK cells transfected with mutants revealing that some mutants have significant lower stabilities than wild type (p.Thr238Ile). Surprisingly, few mutants have a higher stability than wild type. Interestingly, we found that C-terminal truncating mutations of the second domain showed very low protein levels in HEK cells (p.Arg395, p.Arg396*), consistent with the fact that secondary domain is essential for protein stability and dimerization and with the associated severe form of CMD. Enzyme activates were measured by Pyrophosphate Fluormetric assay, revealed that most missense mutants have 30%-50% reduced activity. However, few mutations have a high enzymatic activity, those are currently being investigated for their ligand binding affinity (Km). In summary, we assessed the effects of the single amino acid changes on ISPD protein stability, protein structure, enzyme activities, and correlated this with the patient phenotype. Ultimately, using the high throughput Thermofluor assay, we screened a small chemical chaperones library as a proof-of-principle for personalized treatment of dystroglycanopathy patients. http://dx.doi.org/10.1016/j.nmd.2017.06.065
P.36 Limb girdle muscular dystrophy type 2I: Lack of correlation between clinical severity, histopathological alterations and levels of glycosylated α-dystroglycan in patients homozygous for the common FKRP mutation S. Lindal 1, M. Alhamidi 2, V. Brox 2, E. Stensland 2, M. Liset 1, O. Nilssen 3 1 University hospital of Northern Norway, Tromsø, Norway; 2 University Hospital of North-Norway, Tromsø, Norway; 3 Department of Medical Genetics Division of Child and Adolescent Health, University Hospital of North-Norway, Tromsø, Norway Limb girdle musclular dystrophy type 2I (LGMD2I) is a progressive disorder caused by mutations in the FuKutin-Related Protein gene (FKRP). LGMD2I displays clinical heterogeneity with onset of severe symptoms in early childhood to mild calf and thigh hypertrophy in the second or third decade. Patients homozygous for the common FKRP mutation c.826C > A (p.Leu276Ile) show phenotypes within the milder end of the clinical spectrum. However, this group also manifests substantial clinical variability. FKRP deficiency causes hypoglycosylation of α-dystroglycan; a component of the dystrophin associated glycoprotein complex. α-Dystroglycan hypoglycosylation is in turn associated with loss of interaction with -, and hence, depletion of laminin α2. Here, we have attempted to clarify if the clinical variability seen in patients homozygous for c.826C > A, is related to alterations in muscle fiber pathology, α-DG glycosylation levels, levels of laminin α2 as well as the capacity of α-DG to bind to laminin. We have assessed vastus lateralis muscle biopsies from 25 LGMD2I patients harboring the c.826C > A/c.826C > A genotype by histological
S109
examination, immunohistochemistry and immunoblotting. No clear correlation was found between clinical severity, as determined by self-reported walking function, and the above features, suggesting that more complex molecular processes are contributing to the progression of disease. http://dx.doi.org/10.1016/j.nmd.2017.06.066
P.37 Cardiac involvement in a patient with congenital muscular dystrophy related to POMT2 gene mutation M. Sframeli 1, M. La Rosa 1, M. Distefano 1, C. Barcellona 1, G. Vita 2, G. Nicocia 1, G. Astrea 3, A. D’Amico 4, E. Bertini 4, F. Santorelli 3, G. Vita 1, S. Messina 1 1 Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy; 2 NEMO SUD Clinical Centre for Neuromuscular Disorders, Messina, Italy; 3 Molecular Medicine and Neurogenetics, IRCCS Fondazione Stella Maris, Calambrone, Pisa, Italy; 4 Unit of Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children’s Hospital, Roma, Italy Dystroglycanopathies are a genetically heterogeneous group of congenital muscular dystrophies (CMD) with autosomal recessive inheritance, presenting with a broad spectrum of phenotypes. Cardiac involvement appears to frequently occur in patients with FKRP and FKTN gene mutations. To date, only two unrelated patients with CMD due to POMT2 mutations and cardiac involvement have been reported. We describe a boy with clinical history of delayed motor milestones, proximal weakness, intellectual disability and cardiac involvement. His maximum motor ability was walking with support at the age of 4 years. At the age of 9 years, a muscle biopsy was performed and he received the diagnosis of CMD with depletion of merosin and alphadystroglycan glycosylated complex. He was referred to our attention at the age of 14 years. He was wheelchair-bound and his neurological examination showed: generalized hypotonia, muscle weakness more proximal than distal, absent tendon reflexes. The creatine kinase value was elevated, 2000 U/L. Brain MRI showed a mild brain atrophy. The ophthalmologic evaluation was normal. The cardiological evaluation showed: mild left ventricular dysfunction with ejection fraction (EF)of 49% (mildly decreased), mitral regurgitation and electrocardiographic features of incomplete right bundle branch block. Given the depressed EF systolic function, the patient was started with Enalapril 2.5 mg/day and is on regular cardiological follow-up. Genetic analysis showed the homozygous missense mutation R72H in POMT2 gene. Our data show that cardiac evaluation and follow-up are warranted in this form. Moreover, further cohort studies might help to define possible genotype-phenotype correlations of the cardiac involvement. http://dx.doi.org/10.1016/j.nmd.2017.06.067
P.38 The phenotype of POGLUT1 mutations: Broad clinical expression and distinctive muscle imaging pattern E. Servián-Morilla 1, M. Cabrera-Serrano 1, H. Takeuchi 2, N. Muelas 3, E. Rivas-Infante 1, G. Cantero 1, F. Mavillard 1, J. Vilchez 3, C. Paradas 1 1 Hospital Universitario Virgen del Rocío/IBiS, Sevilla, Spain; 2 Nagoya University School of Medicine, Nagoya, Japan; 3 Hospital Universitario La Fe, Valencia, Spain Our group has recently described the human POGLUT1 p.D233E mutation that impairs Notch posttranslational modification and maintenance of muscle stem cells and leads to muscular degeneration and α-dystroglycan hypoglycosylation. Our patients display an adult-onset pure muscular phenotype, in contrast to the lethal, multisystemic phenotypes observed in animals with complete loss of POGLUT1. In order to better define the phenotype associated to mutations in POGLUT1 we screened a candidate group of 54 patients from five Spanish national reference centers and one Italian
CONGENITAL MUSCULAR DYSTROPHIES: DYSTROGLYCANOPATHIES AND OTHERS P.35 Translational biochemistry for dystroglycanopathies H. Wang, R. Sprute, H. Daimagüler, S. Cirak University Childrens Hospital Cologne, Cologne, Germany ISPD (isoprenoid synthase domain containing) mutations cause a wide clinical spectrum of muscular dystrophies (dystroglycanopathies). Human ISPD is a cytidyltransferase, catalyses the reaction from CTP and Ribitol-5phosphate to CDP-Ribitol, a novel nucleotide sugar for functional alphadystroglycan. hISPD has one conserved cytidyltransferase domain and a second functional unknown non-conserved domain. We aim to correlate clinical phenotypes to structural impacts of ISPD mutations to decipher the underlying molecular mechanisms. First we established a high throughput in vitro protein thermostablity screening assay (Thermofluor assay, TF), by which we screened for the ligands of recombinant hISPD (purified from E.coli) and found out that Ribitol-5-Phosphate can only alter the protein thermostability in the presence of CTP. The thermostablities of missense mutants are also assessed by TF assay, together with protein half-life cycloheximide assay in HEK cells transfected with mutants revealing that some mutants have significant lower stabilities than wild type (p.Thr238Ile). Surprisingly, few mutants have a higher stability than wild type. Interestingly, we found that C-terminal truncating mutations of the second domain showed very low protein levels in HEK cells (p.Arg395, p.Arg396*), consistent with the fact that secondary domain is essential for protein stability and dimerization and with the associated severe form of CMD. Enzyme activates were measured by Pyrophosphate Fluormetric assay, revealed that most missense mutants have 30%-50% reduced activity. However, few mutations have a high enzymatic activity, those are currently being investigated for their ligand binding affinity (Km). In summary, we assessed the effects of the single amino acid changes on ISPD protein stability, protein structure, enzyme activities, and correlated this with the patient phenotype. Ultimately, using the high throughput Thermofluor assay, we screened a small chemical chaperones library as a proof-of-principle for personalized treatment of dystroglycanopathy patients. http://dx.doi.org/10.1016/j.nmd.2017.06.065
P.36 Limb girdle muscular dystrophy type 2I: Lack of correlation between clinical severity, histopathological alterations and levels of glycosylated α-dystroglycan in patients homozygous for the common FKRP mutation S. Lindal 1, M. Alhamidi 2, V. Brox 2, E. Stensland 2, M. Liset 1, O. Nilssen 3 1 University hospital of Northern Norway, Tromsø, Norway; 2 University Hospital of North-Norway, Tromsø, Norway; 3 Department of Medical Genetics Division of Child and Adolescent Health, University Hospital of North-Norway, Tromsø, Norway Limb girdle musclular dystrophy type 2I (LGMD2I) is a progressive disorder caused by mutations in the FuKutin-Related Protein gene (FKRP). LGMD2I displays clinical heterogeneity with onset of severe symptoms in early childhood to mild calf and thigh hypertrophy in the second or third decade. Patients homozygous for the common FKRP mutation c.826C > A (p.Leu276Ile) show phenotypes within the milder end of the clinical spectrum. However, this group also manifests substantial clinical variability. FKRP deficiency causes hypoglycosylation of α-dystroglycan; a component of the dystrophin associated glycoprotein complex. α-Dystroglycan hypoglycosylation is in turn associated with loss of interaction with -, and hence, depletion of laminin α2. Here, we have attempted to clarify if the clinical variability seen in patients homozygous for c.826C > A, is related to alterations in muscle fiber pathology, α-DG glycosylation levels, levels of laminin α2 as well as the capacity of α-DG to bind to laminin. We have assessed vastus lateralis muscle biopsies from 25 LGMD2I patients harboring the c.826C > A/c.826C > A genotype by histological
S109
examination, immunohistochemistry and immunoblotting. No clear correlation was found between clinical severity, as determined by self-reported walking function, and the above features, suggesting that more complex molecular processes are contributing to the progression of disease. http://dx.doi.org/10.1016/j.nmd.2017.06.066
P.37 Cardiac involvement in a patient with congenital muscular dystrophy related to POMT2 gene mutation M. Sframeli 1, M. La Rosa 1, M. Distefano 1, C. Barcellona 1, G. Vita 2, G. Nicocia 1, G. Astrea 3, A. D’Amico 4, E. Bertini 4, F. Santorelli 3, G. Vita 1, S. Messina 1 1 Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy; 2 NEMO SUD Clinical Centre for Neuromuscular Disorders, Messina, Italy; 3 Molecular Medicine and Neurogenetics, IRCCS Fondazione Stella Maris, Calambrone, Pisa, Italy; 4 Unit of Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children’s Hospital, Roma, Italy Dystroglycanopathies are a genetically heterogeneous group of congenital muscular dystrophies (CMD) with autosomal recessive inheritance, presenting with a broad spectrum of phenotypes. Cardiac involvement appears to frequently occur in patients with FKRP and FKTN gene mutations. To date, only two unrelated patients with CMD due to POMT2 mutations and cardiac involvement have been reported. We describe a boy with clinical history of delayed motor milestones, proximal weakness, intellectual disability and cardiac involvement. His maximum motor ability was walking with support at the age of 4 years. At the age of 9 years, a muscle biopsy was performed and he received the diagnosis of CMD with depletion of merosin and alphadystroglycan glycosylated complex. He was referred to our attention at the age of 14 years. He was wheelchair-bound and his neurological examination showed: generalized hypotonia, muscle weakness more proximal than distal, absent tendon reflexes. The creatine kinase value was elevated, 2000 U/L. Brain MRI showed a mild brain atrophy. The ophthalmologic evaluation was normal. The cardiological evaluation showed: mild left ventricular dysfunction with ejection fraction (EF)of 49% (mildly decreased), mitral regurgitation and electrocardiographic features of incomplete right bundle branch block. Given the depressed EF systolic function, the patient was started with Enalapril 2.5 mg/day and is on regular cardiological follow-up. Genetic analysis showed the homozygous missense mutation R72H in POMT2 gene. Our data show that cardiac evaluation and follow-up are warranted in this form. Moreover, further cohort studies might help to define possible genotype-phenotype correlations of the cardiac involvement. http://dx.doi.org/10.1016/j.nmd.2017.06.067
P.38 The phenotype of POGLUT1 mutations: Broad clinical expression and distinctive muscle imaging pattern E. Servián-Morilla 1, M. Cabrera-Serrano 1, H. Takeuchi 2, N. Muelas 3, E. Rivas-Infante 1, G. Cantero 1, F. Mavillard 1, J. Vilchez 3, C. Paradas 1 1 Hospital Universitario Virgen del Rocío/IBiS, Sevilla, Spain; 2 Nagoya University School of Medicine, Nagoya, Japan; 3 Hospital Universitario La Fe, Valencia, Spain Our group has recently described the human POGLUT1 p.D233E mutation that impairs Notch posttranslational modification and maintenance of muscle stem cells and leads to muscular degeneration and α-dystroglycan hypoglycosylation. Our patients display an adult-onset pure muscular phenotype, in contrast to the lethal, multisystemic phenotypes observed in animals with complete loss of POGLUT1. In order to better define the phenotype associated to mutations in POGLUT1 we screened a candidate group of 54 patients from five Spanish national reference centers and one Italian