Clinical,Myopathological And Molecular Biological Studies Of Skeletal Muscle Ion Channelopathies

Skeletal muscle channelopathies(SMCs)are a group of diseases with clinical and genetic heterogeneity,which are caused by mutations in genes encoding skeletal muscle cell membrane voltage-gated chloride,calcium,sodium,and potassium ion channels.According to the excitability of the muscle cell membrane and the characteristics of clinical manifestations,SMCs are divided into non-dystrophic myotonias(NDMs)and primary periodic paralyses(PPPs).NDMs are a group of skeletal muscle disorders that have myotonia as their common feature,in reference to a delayed muscle relaxation after voluntary or evoked muscle contraction.NDMs are presenting clinically as muscle stiffness,muscle hypertrophy(a body-builder appearance),which may be accompanied by muscle weakness(continuous/periodical attacks)and myalgia.The onset of recurrent attacks is usually within the first or second decade of age.The phenotype of NDMs involves almost exclusively the skeletal muscle and,no relevant extra-muscular involvement has been described so far.NDMs are differently from dystrophic myotonias,because of the absence of muscle atrophy and systemic features.NDMs comprise myotonia congenital(MC),paramyotonia congenital(PMC),and sodium channel myotonia(SCM),which are caused by mutations of skeletal muscle chloride channel(CLCN1)and sodium channel(SCN4A)genes.PPPs are a group of skeletal muscle ion channelopathies characterized by episodic flaccid muscle weakness.The attacks may last hours to days or weeks,and often are associated with altered serum potassium levels.Patients usually complete recovery between the attacks,although some develop permanent fixed weakness later in life.A subset of PPP patients accompany with cardiac arrhythmia or sudden cardiac death.PPPs are classified as hyperkalemic periodic paralysis(Hyper PP),normokalemic periodic paralysis(normo PP),or hypokalemic periodic paralysis(Hypo PP)based on serum potassium levels during paralytic attacks.Andersen-Tawil syndrome(ATS)is a rare PPP characterized by a triad of periodic paralysis,cardiac arrhythmia,and skeletal and craniofacial anomalies,with high,low,or normal serum potassium levels.PPPs are caused by mutations in CACNA1S,SCN4A,KCNJ2 and KCNJ5genes.Voltage-gated sodium(Na_V)channels are the structural basis of governing membrane excitability in excitable cells.In humans,there are nine subtypes of Na_V channels(Na_V1.1-Na_V1.9).Na_V1.4 channels are transmembrane glycoproteins,which are mainly expressed in skeletal muscles.Na_V1.4 channel is composed of the?-pore-forming subunit encoded by SCN4A and a single auxiliary?-subunit encoded by SCN1B through noncovalent binding.The a-subunit forms the core of the channel that carries the majority of the inward Na~+current.SCN4A mutations can cause skeletal muscle Na_V1.4 channelopathies,which have rich clinical heterogeneity.Two main clinical phenotypes are paroxysmal weakness with reduced excitability,including Hyper PP,normo PP,Hypo PP2 and congenital myasthenic syndrome(CMS),and myotonia with improved excitability,including PMC and SCM.In recent years,it has been reported that SCN4A mutations can cause Hyper PP or normo PP overlapped with PMC.SMCs have remarkable clinical and genetic heterogeneity.Here,we retrospectively reviewed clinical and genetic features of patients with SMCs in China,including 20 cases of NDMs,8 cases of PPPs and 9 cases of skeletal muscle Na_V1.4 channelopathies,whose main clinical manifestations were paroxysmal weakness or myotonia,and confirmed by genetic testing at the Third Hospital of Hebei Medical University between June 2010 and December2020.Part one Clinical,myopathological and genetic analysis of non-dystr-ophic myotoniaObjective:To explore the clinical,myopathological and genetic characteristics of NDM.Methods:We reviewed clinical manifestations,laboratory results,electrocardiogram,electromyography(EMG),muscle biopsy,genetic analysis,treatment and follow-up of 20 patients with NDM.Results:There were 20 patients with NDM(from 18 families),including17 patients with MC and three patients with PMC.Significant clinical overlap exists between MC and PMC,presenting clinically muscle stiffness and hypertrophy,grip and percussion myotonia,myotonia relieved after repeated activity(warm up phenomenon),and myotonia aggravated by exposure to cold.Facial stiffness,eye closure myotonia and cold sensitivity are more common in PMC patients,and may be accompanied by permanent weakness.Nine patients with MC and two patients with PMC had cardiac involvement,mainly manifested as cardiac arrhythmia or conduction block defects on the electrocardiogram or 24 hr holter.Among them,six patients complained of palpitations,chest tightness,chest pain,etc,and the father of one patient died of sudden cardiac death.Creatine kinase was normal or slightly elevated.Myotonic potentials were found in all patients on the EMG,and seven patients with MC had mild myopathic potentials,while PMC didn't have.There is no difference in muscle pathology between MC and PMC,most of which are abnormal muscle fiber type distribution or selective muscle fiber atrophy.A total of 19 CLCN1 mutation sites were found in 17 patients with MC,in which c.1277C>A(p.T426N)and c.795T>G(p.D265E)were novel mutations,and two SCN4A mutation sites were found in three patients with PMC.The patients were treated with mexiletine and/or carbamazepine orally,and the symptoms of myotonia were partially improved.Conclusion:NDMs are a group of hereditary skeletal muscle ion channel diseases characterized by myotonia and caused by CLCN1 or SCN4A mutations,mainly including MC and PMC.The clinical symptoms and signs of MC and PMC are similar.PMC is more common in facial rigidity and eye closure myotonia,which may be accompanied by permanent weakness.The muscle pathology of NDM lacks specific changes.This study found that both MC and PMC can be accompanied by cardiac arrhythmias or conduction block.Sudden cardiac death occurs in severe case.The clinical manifestations of NDM with cardiac involvement are worthy of attention,and cardiac evaluation should be performed regularly.Sodium channel blockers(mexiletine,carbamazepine)partially relieve the symptoms of myotonia.Part two Clinical,myopathological and genetic analysis of primary per-iodic paralysisObjective:To explore the clinical,myopathological and genetic characteristics of PPP.Methods:We reviewed clinical manifestations,laboratory results,electrocardiogram,EMG,muscle biopsy,genetic analysis,treatment and follow-up of eight patients with PPP.Results:Eight patients with PPP included:one patient with Hypo PP1(CACNA1S,1/8),two patients with Hypo PP2(SCN4A,2/8,from one family),three patients with normo PP(SCN4A,3/8,two cases of normo PP overlapped with PMC),one patient with Hyper PP(SCN4A,1/8,overlapped with PMC)and one patient with ATS(KCNJ2,1/8).The basic clinical manifestations of eight patients were consistent with PPP,presenting with paroxysmal muscle weakness,with or without abnormal serum potassium.Hyper PP and two patients with normo PP accompanied with difficulty in relaxing his grip and diffuse myotonia discharge at rest in EMG,who were diagnosed with Hyper PP/normo PP overlapped with PMC.ATS was accompanied by ventricular arrhythmias,and skeletal and craniofacial anomalies,developing with a permanent fixed myopathy later.The EMG showed diffuse myopathic discharge.Permanent fixed myopathy develops later in two patients with AST and normo PP overlapped with PMC,and muscle biopsy showed tubular aggregates.Genetic testing revealed eight patients with PPP carried CACNA1S(R1242S),SCN4A(R675Q/W?T704M?I693T),and KCNJ2(R218Q)respectively,in which CACNA1S(R1242S)was novel mutation.Acute attack management and chronic prevention therapy can reduce attack time and frequency.Conclusion:PPPs are a group of autosomal-dominant genetic ion channelopathies caused by mutations in skeletal muscle calcium,sodium,and potassium channel genes(CACNA1S,SCN4A,KCNJ2,KCNJ5),which have significant clinical and genetic heterogeneity.PPPs are classified as Hyper PP,normo PP or Hypo PP based on the concomitant serum K~+level.ATS is different from the other phenotypes of PPPs,characterized by periodic paralysis,ventricular arrhythmias,and skeletal and craniofacial anomalies,and high,low or normal serum potassium levels.Tubular aggregates are more common in patients with persistent muscle weakness.Diagnosis is based on the characteristic clinical presentation then confirmed by genetic testing.Recovery of blood potassium levels and avoidance of triggers are crucial in reducing paralytic attacks.Part three The clinical and genetic heterogeneity analysis of skeletal muscle Na_V1.4 channelopathiesObjective:To report the clinical features and SCN4A mutations of nine patients with skeletal muscle Na_V1.4 channelopathies.Methods:We reviewed clinical,laboratory,EMG,muscle biopsy and genetic data from nine patients with skeletal muscle Na_V1.4 channelopathies.Results:Nine patients with skeletal muscle Na_V1.4 channelopathies included two patients with Hypo PP2(2/9,from one family),one patient with normo PP(1/9),two patients with normo PP overlapped with PMC(2/9),one patients with Hyper PP overlapped with PMC(1/9)and three patients with PMC(3/9).There are three clinical phenotypes of Hyper PP,PMC and Hyper PP overlapped with PMC in the family of Hyper PP overlapped with PMC,which has significant clinical heterogeneity within the family.Nine patients with skeletal muscle Na_V1.4 channelopathies carried six different SCN4A mutations(R675Q,R675W,T704M,I693T,L689F,V1293I).T704M and L689F mutations respectively appeared in two families.Conclusions:The main clinical manifestations of skeletal muscle Na_V1.4channelopathies caused by SCN4A mutations include episodic muscle weakness and/or myotonia,which have significant clinical heterogeneity and the clinical phenotypes within the same family can also be different.SCN4A mutations cause Na_V1.4 gain-of-function or loss-of-function and abnormal excitability of skeletal muscle,which lead to significant clinical heterogeneity of skeletal muscle Na_V1.4 channelopathies.