Missense mutations of the proline-rich transmembrane protein 2 gene cosegregate with mild paroxysmal kinesigenic dyskinesia and infantile convulsions in a Chinese pedigree

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

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Clinical features of patients with paroxysmal kinesigenic dyskinesia, mutation screening of PRRT2 and the effects of morning draughts of oxcarbazepine

BMC Pediatrics ◽

10.1186/s12887-019-1798-7 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Gang Pan ◽

Linmei Zhang ◽

Shuizhen Zhou

Keyword(s):

Clinical Features ◽

Transmembrane Protein ◽

Mutation Screening ◽

Venous Blood ◽

Gene Mutations ◽

Paroxysmal Kinesigenic Dyskinesia ◽

Starting Dose ◽

Dyskinetic Movement ◽

Effective Doses ◽

Polymerase Chain

Abstract Background The objective of this study was to summarize clinical features and PRRT2 mutations of paediatric paroxysmal kinesigenic dyskinesia (PKD) patients and observe the tolerability and effects of morning draughts of oxcarbazepine. Methods Twenty patients diagnosed with PKD at Children’s Hospital of Fudan University between January 2011 and December 2015 were enrolled. These patients’ medical records were reviewed. Peripheral venous blood was obtained from all enrolled patients, and polymerase chain reaction (PCR) and Sanger sequencing were used to sequence proline-rich transmembrane protein 2 (PRRT2) gene mutations. Clinical features of PKD patients with and without PRRT2 mutations were compared. All enrolled patients were treated with morning draughts of oxcarbazepine (OXC). The starting dose was 5 mg/kg·d, and the dose was increased by 5 mg/kg·d each week until attacks stopped. Effective doses and adverse effects were recorded. Results For all enrolled patients, dyskinesia was triggered by sudden movement. Dyskinetic movement usually involved the limbs and was bilateral; the majority of enrolled patients exhibited both dystonia and choreoathetosis. We identified PRRT2 mutations in 5 patients, including 4 familial patients and 1 sporadic patient. All 20 patients took low doses of OXC (5–20 mg/kg·d) as draughts in the morning, and dyskinesia attacks stopped in 19 patients. Conclusions Paediatric PKD patients have various phenotypes. PRRT2 mutations are common in familial cases. OXC taken as morning draughts can be a treatment option for paediatric PKD patients.

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The Spectrum of PRRT2-Associated Disorders: Update on Clinical Features and Pathophysiology

Frontiers in Neurology ◽

10.3389/fneur.2021.629747 ◽

2021 ◽

Vol 12 ◽

Author(s):

Annamaria Landolfi ◽

Paolo Barone ◽

Roberto Erro

Keyword(s):

Movement Disorders ◽

Neurodevelopmental Disorders ◽

Transmembrane Protein ◽

Brain Disorders ◽

Hemiplegic Migraine ◽

Causative Gene ◽

Paroxysmal Kinesigenic Dyskinesia ◽

Infantile Epilepsy ◽

Biallelic Mutations

Mutations in the PRRT2 (proline-rich transmembrane protein 2) gene have been identified as the main cause of an expanding spectrum of disorders, including paroxysmal kinesigenic dyskinesia and benign familial infantile epilepsy, which places this gene at the border between epilepsy and movement disorders. The clinical spectrum has largely expanded to include episodic ataxia, hemiplegic migraine, and complex neurodevelopmental disorders in cases with biallelic mutations. Prior to the discovery of PRRT2 as the causative gene for this spectrum of disorders, the sensitivity of paroxysmal kinesigenic dyskinesia to anticonvulsant drugs regulating ion channel function as well as the co-occurrence of epilepsy in some patients or families fostered the hypothesis this could represent a channelopathy. However, recent evidence implicates PRRT2 in synapse functioning, which disproves the “channel hypothesis” (although PRRT2 modulates ion channels at the presynaptic level), and justifies the classification of these conditions as synaptopathies, an emerging rubric of brain disorders. This review aims to provide an update of the clinical and pathophysiologic features of PRRT2-associated disorders.

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Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

10.21203/rs.2.19199/v1 ◽

2019 ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background: Dysferlin is a type-II transmembrane protein and the causative gene of dysferlinopathies, which are characterized by absence or marked reduction in dysferlin protein and muscle weakness. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. The muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Release of thrombospondin-1 (TSP-1) by dysferlin deficient muscle has been reported as a possible factor of the inflammation observed in the muscle of both human and mouse models of dysferlinopathy. It has also been reported that treatment with vitamin D3 enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic implication in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patient carrying two missense mutationsMethods: Dysferlin deficient myotubes were treated with EB1089, a vitamin D3 analog, oprozomib and ixazomib to assess proteasome inhibition. Western blot was performed to analyze the effect of the different treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using Enzyme Linked Immunosorbent Assay to analyze the effect of these drugs on its release.A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury. Data were analyzed using a one-way ANOVA test followed by by Tukey post hoc test and analysis of variance. Ap≤0.05 was considered statistically significant. Results : Treatment with proteasome inhibitors and EB1089 resulted in a slight increase of dysferlin expression which was accompanied by a low increase of myogenin expression. Also, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes from a dysferlinopathy patient. However, the increase of dysferlin had no effect on the repair of muscle membrane after injury. Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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Proteasome inhibitors reduce thrombospondin-1 release in human dysferlin-deficient myotubes

BMC Musculoskeletal Disorders ◽

10.1186/s12891-020-03756-7 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44. Methods To assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p ≤ 0.05 was considered statistically significant. Results Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury. Conclusions Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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PRRT2 Gene Analysis of Paroxysmal Kinesigenic Dyskinesia (PKD) in Thai Children

Journal of Health Science and Medical Research ◽

10.31584/jhsmr.2021853 ◽

2021 ◽

Author(s):

Pantaree Laosuebsakulthai ◽

Surachai Likasitwattanakul ◽

Theerapong Pho-iam ◽

Wanna Thongnoppakhun ◽

Mongkol Chanvanichtrakool

Keyword(s):

Hot Spot ◽

Transmembrane Protein ◽

Complete Response ◽

Gene Analysis ◽

Detection Rates ◽

Mutation Status ◽

Familial Cases ◽

Paroxysmal Kinesigenic Dyskinesia ◽

Siriraj Hospital ◽

Sporadic Cases

Objective: To examine the frequency of the proline-rich transmembrane protein-2 (PRRT2) gene mutation in Thai patients with paroxysmal kinesigenic dyskinesia (PKD). Material and Methods: A retrospective study of children aged 0-18 years with a diagnosis of PKD at Siriraj Hospital. The genetic analyses of the PRRT2 gene were done by bidirectional Sanger sequencing.Results: Twelve patients with PKD were included. The known PRRT2 mutation, c.649dupC (p.Arg217Profs*8), was identified in three of the patients (25.0%), one of the nine sporadic cases (11.1%) and two of the three familial cases (66.6%), all from different families. PKD had a complete response to carbamazepine treatment regardless of PRRT2 mutation status. Conclusion: Our study provided the new details of the clinical phenotypes and PRRT2 gene analysis findings for Thai PKD. PRRT2 mutations were identified in our Thai PKD patients with increased detection rates in the familial PKD cases. The c.649dupC (p.Arg217Profs*8) was also found to be a hot-spot mutation in our Thai PKD patients. Furthermore, this study demonstrates the importance of PRRT2 gene analysis in order to properly diagnose and treat these patients.

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Proline-rich transmembrane protein 2 gene mutation in a sporadic paroxysmal kinesigenic dyskinesia

Journal of Pediatric Neurosciences ◽

10.4103/jpn.jpn_148_16 ◽

2017 ◽

Vol 12 (1) ◽

pp. 112

Author(s):

Puneet Jain ◽

Suvasini Sharma ◽

Guido Breedveld ◽

Vincenzo Bonifati ◽

Satinder Aneja

Keyword(s):

Gene Mutation ◽

Transmembrane Protein ◽

Paroxysmal Kinesigenic Dyskinesia

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Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

10.21203/rs.2.19199/v2 ◽

2020 ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background: Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy from patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of human and mouse models of dysferlinopathy suggest dysferlin-deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patient with two missense mutations in exon 44. Methods: To assess proteasome inhibition we treated dysferlin deficient myotubes with oprozomib, ixazomib, and EB1089, a vitamin D3 analogue. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury. We also calculated the fusion index in response to the different treatments. Data were analysed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p≤0.05 was considered statistically significant. Results: Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on muscle membrane repair after injury. Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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PRRT2 Related Epilepsies: A Gene Review

Journal of Pediatric Neurology ◽

10.1055/s-0041-1728683 ◽

2021 ◽

Author(s):

Carmela Rita Massimino ◽

Laura Portale ◽

Annamaria Sapuppo ◽

Francesco Pizzo ◽

Laura Sciuto ◽

...

Keyword(s):

Basal Ganglia ◽

De Novo ◽

Transmembrane Protein ◽

Common Disease ◽

Hemiplegic Migraine ◽

Pathogenic Variants ◽

Disease Spectrum ◽

Paroxysmal Kinesigenic Dyskinesia ◽

Paroxysmal Disorders ◽

Infantile Epilepsy

Abstract PRRT2 encodes for proline-rich transmembrane protein 2 involved in synaptic vesicle fusion and presynaptic neurotransmitter release. Mutations in human PRRT2 have been related to paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions with choreoathetosis, benign familial infantile epilepsies, and hemiplegic migraine. PRRT2 mutations cause neuronal hyperexcitability, which could be related to basal ganglia or cortical circuits dysfunction, leading to paroxysmal disorders. PRRT2 is expressed in the cerebral cortex, basal ganglia, and cerebellum. Approximately, 90% of pathogenic variants are inherited and 10% are de novo. Paroxysmal attacks in PKD are characterized by dystonia, choreoathetosis, and ballismus. In the benign familial infantile epilepsy (BFIE), seizures are usually focal with or without generalization, usually begin between 3 and 12 months of age and remit by 2 years of age. In 30% of cases of PRRT2-associated PKD, there is an association with BFIE, and this entity is referred to as PKD with infantile convulsions (PKD/IC). PRRT2 mutations are the cause of benign family childhood epilepsy and PKD/IC. On the other hand, PRRT2 mutations do not seem to correlate with other types of epilepsy. The increasing incidence of hemiplegic migraine in families with PRRT2-associated PKD or PKD/IC suggests a common disease pathway, and it is possible to assert that BFIE, paroxysmal kinesigenic dyskinesia, and PKD with IC belong to a continuous disease spectrum of PRRT2-associated diseases.

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