OBJECTIVE: To explore the clinical features of a Chinese pedigree affected with skeletal muscle sodium channelopathies due to variation of SCN4A gene. METHODS: Potential variation of the 24 exons of the SCN4A gene was screened using PCR and Sanger sequencing. RESULTS: Four family members were affected with the disease in an autosomal dominant inheritance pattern. Three patients had normekalemic periodic paralysis, while 1 showed paramyotonia congenita. Genetic analysis detected a missense variation c.2078T>C (p.Ile693Thr) in exon 13 of the SCN4A gene in the proband and other 3 affected relatives. CONCLUSION Normokalemic periodic paralysis and paramyotonia congenita can occur in different family members with skeletal muscle sodium channelopathies due to c.2078T>C(p.Ile693Thr) variation of SCN4A gene.
Channelopathies ; genetics ; Humans ; Muscle, Skeletal ; physiopathology ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Pedigree

Humans ; Myotonia Congenita/genetics* ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel/genetics*

Adult ; Female ; Humans ; Hypokalemic Periodic Paralysis ; genetics ; Infant, Newborn ; Male ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Pedigree ; Potassium Channels, Voltage-Gated ; genetics ; Young Adult

OBJECTIVE: To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T. METHODS: The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp. RESULTS: Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the of voltage-dependence of fast inactivation of I1363T mutant[(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type[(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type. CONCLUSIONS I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na+ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.
Gene Expression Profiling ; Humans ; Ion Channel Gating ; genetics ; Muscle, Skeletal ; physiopathology ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Sequence Analysis, Protein

BACKGROUNDNormokalaemic periodic paralysis (normoKPP) is characterized by transient and recurrent myoasthenia, and some patients also show muscle stiffness induced by cold exposure (paramyotonia congenita, PMC). It is caused by a mutation in the muscle voltage gated sodium channel alpha subunit (SCN4A) gene. Due to the diversity of the clinical manifestations of patients, it is difficult for clinicians to differentiate some of patients with atypical normoKPP from those who suffer from other periodic paralysis and nondystrophic myotonia. So far, for normoKPP there are almost no ways to assist definite diagnosis besides genetic screening. This research was designed to evaluate an exercise test (ET) in confirming the diagnosis of normoKPP and in assessing the therapeutic effectiveness of some drugs on this disease.

METHODSET, described by McMains, was performed on six subjects from a Chinese family, including four patients with overlapping disease of normoKPP and PMC caused by a mutation of SCN4A Met1592Val that is identified by genetic analysis and two normal control members. The change of compound muscle action potential (CMAP) was recorded. Besides the family, two patients were also tested during treatments with acetazolamide.

RESULTSAll patients showed a slight increase in CMAP immediately after exercise, followed by an abnormal gradual decline, which reached its nadir 25-30 minutes after exercise. CMAP amplitude dropped by more than 40% in patients but less than 23% in controls. In the patients who received treatment with acetazolamide, the change of CMAP amplitude was less than 28% and, at any fixed times, less than pretreatment values.

CONCLUSIONSThe ET may be used as a predictive, easy and reliable method of diagnosing normoKPP under conditions without genetic screening help, and is an objective way to evaluate the therapeutic effectiveness. According to different response patterns, the ET may also be helpful in reducing the scope of genetic screening.

Action Potentials ; Adult ; Electromyography ; Exercise Test ; Female ; Humans ; Male ; Middle Aged ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; Paralyses, Familial Periodic ; genetics ; physiopathology ; Sodium Channels ; genetics

OBJECTIVEPeriodic paralysis (PP) is one type of skeletal muscle channelopathies characterized by episodic attacks of weakness. It is usually classified into hyperkalemic periodic paralysis (HyperPP), hypokalemic periodic paralysis (HypoPP) and normokalemic periodic paralysis (NormoPP) based on the blood potassium levels. HypoPP is the most common type of these three and NormoPP is the rarest one. The aim of this study was to explore the clinical and genetic features of a Chinese family with normokalemic periodic paralysis (NormoKPP).

METHODClinical features of all patients in the family with NormoKPP were analyzed. Genomic DNA was extracted from peripheral blood leukocytes and amplified with PCR. We screened all 24 exons of SCN4A gene and then sequence analysis was performed in those who showed heteroduplex as compared with unaffected controls.

RESULT(1) Fifteen members of the family were clinically diagnosed NormoKPP, and their common features are: onset within infacy, episodic attacks of weakness, the blood potassium levels were within normal ranges, high sodium diet or large dosage of normal saline could attenuate the symptom. One muscle biopsy was performed and examination of light and electronic microscopy showed occasionally degenerating myofibers. (2) Gene of 12 patients were screened and confirmed mutations of SCN4A genes--c. 2111 T > C/p. Thr704Met.

CONCLUSIONThe study further defined the clinical features of patients with NormoKPP, and molecular genetic analysis found SCN4A gene c. 2111 T > C/p. Thr704Met point mutation contributed to the disease. In line with the autosomal dominant inheritance laws, this family can be diagnosed with periodic paralysis, and be provided with genetic counseling. And the study may also help the clinical diagnosis, guide treatment and genetic counseling of this rare disease in China.

Amino Acid Sequence ; Channelopathies ; diagnosis ; genetics ; pathology ; Child ; DNA Mutational Analysis ; Female ; Humans ; Male ; Muscle, Skeletal ; pathology ; physiopathology ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Paralyses, Familial Periodic ; diagnosis ; genetics ; pathology ; Pedigree ; Polymerase Chain Reaction ; Potassium ; blood

OBJECTIVETo detect SCN4A gene mutation in a pedigree with paramyotonia congenita in order to facilitate genetic counseling and assisted reproduction.

METHODSClinical data of the family was collected. DNA was extracted from peripheral blood samples. Potential mutation of the SCN4A gene was screened using PCR-Sanger sequencing. Potential mutation was detected in 3 affected relatives, 4 unaffected relatives and 100 unrelated healthy controls. Bioinformatics software was used to predict the effect of mutation on the protein function and conservation of the sequence at the mutation site across various species.

RESULTSA novel missense mutation c.4427T>C (p.Met1476Thr) was detected in the exon 24 of the SCN4A gene in the proband and other 3 affected relatives, but not in 4 unaffected relatives and 100 unrelated controls. Bioinformatic analysis indicated that the codon is highly conserved across various species, and that the mutation has caused damage to the structure and function of SCN4A protein.

CONCLUSIONThe c.4427 T>C (p.Met1476Thr) mutation of the SCN4A gene may contribute to the paramyotonia congenita. Detection of SCN4A gene mutation is an effective method for the diagnosis of paramyotonic congenita.

Adult ; Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Exons ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation, Missense ; Myotonic Disorders ; genetics ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Pedigree ; Point Mutation ; Sequence Alignment

OBJECTIVEIn this report are reviewed two unrelated patients with typical normokalemic periodic paralysis (normoKPP) features and the results of screening the SCN4A gene for the disease-related mutation.

METHODSTwo sporadic cases with normoKPP were screened for previously known mutations in SCN4A gene (T704M, A1156T, M1360V, I1495F, M1592V) that lead to hyperKPP; denaturing high performance liquid chromatography (DHPLC) was used. Then the rest exons of SCN4A gene were screened by DHPLC, and sequence analysis was performed on those with DHPLC chromatogram variation when compared with unaffected control.

RESULTSTwo cases and one patient's father were detected with V781I, which was proved to be a singular missense mutation in SCN4A gene.

CONCLUSIONThe mutation V781I exists in Chinese patients with normoKPP and may be responsible for normoKPP.

Adult ; Amino Acid Sequence ; Base Sequence ; Child ; Chromatography, High Pressure Liquid ; methods ; DNA ; analysis ; genetics ; Exons ; Female ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; NAV1.4 Voltage-Gated Sodium Channel ; Paralyses, Familial Periodic ; genetics ; Point Mutation ; Sequence Analysis, DNA ; Sodium Channels ; genetics

OBJECTIVEThe present study is to observe in vitro the proliferation ability of the muscle cells from permanent myopathy (PM) patients of nomokalaemic periodic paralysis (normKPP), which is caused by mutations of Met1592Val in the skeletal muscle voltage gated sodium channel (SCN4A) gene on chromosome 17q23.1. We also evaluate the possible effect of the foreign basic fibroblast growth factor (bFGF) in preventing and curing PM.

METHODSThe gastrocnemius muscle cells were taken from two male patients with PM of the same Chinese family with Met1592Val mutation of SCN4A, determined by gene screening. Four male patients suffering from the skeletal injury without PM were taken as control. All preparations were protogenerationally cultured in vitro. Proliferation of the cultured preparations was measured by MTT. Activities of the lactic dehydrogenase (LDH), creatine kinase (CK), and protein content in these cells were also detected. The effects of bFGF with different doses (10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 120 ng/mL and 160 ng/mL) on the above mentioned parameters were also evaluated.

RESULTSCells from both PM and control subjects were successfully cultured in vitro. The cultivation of the muscle cells from PM patients in vitro was not yet seen. Results indicated the obvious stimulation of bFGF on cell proliferation, activities of LDH and CK, protein synthesis, in a dose dependent manner. The optimal dose of bFGF was 120 ng/mL (P<0.05), beyond which greater dose caused a less effect. The effect of bFGF on 160 ng /mL was stronger than that on 80 ng/mL, but there was no significant difference (P>0.05).

CONCLUSIONMyoblastic cells from patients with PM had a weaker ability of developing into the myotubules, thus they were unable to perform effective regeneration, which resulted in a progressive necrosis. The exogenous bFGF could promote the division and proliferation of the muscle cells in vitro. These results shield a light on bFGFos potential role in preventing and treating PM.

Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Creatine Kinase ; metabolism ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Male ; Methionine ; genetics ; Middle Aged ; Muscle Development ; genetics ; physiology ; Muscular Diseases ; genetics ; pathology ; Mutation ; genetics ; Myoblasts ; drug effects ; NAV1.4 Voltage-Gated Sodium Channel ; Sodium Channels ; genetics ; Valine ; genetics

OBJECTIVETo construct and investigate the cell model of a novel mutation R675Q in the skeletal muscle Na channel type 4 alpha subunit gene (SCN4A) identified from a Chinese family with normokalemic periodic paralysis.

METHODScDNA encoding the adult isoform of SCN4A was used as a template for in vitro site-directed mutagenesis by PCR method. The mutated plasmid was transiently transfected into HEK-293 cells by calcium phosphate precipitation. Twenty four and 48 hours after transfection, the expression level of SCN4A was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Whole cell voltage-clamp recording was used to study the current of sodium channels.

RESULTSThe site-mutagenesis of the plasmid was confirmed by sequencing. The expression of SCN4A gene was significantly elevated 24 h and 48 h after transfection. The relative current of R675Q is smaller than that of wide type before reaching peak current under the same test voltage, but larger than that of wild type current after reaching peak current. They both had the largest peak current under 0 mV test pulse.

CONCLUSIONA cell model of normokalemic periodic paralysis was successfully constructed. The R675Q mutation of the SCN4A gene enhances the activation and inactivation of the sodium channel, and the S4 transmembrane segment may have intimate relationship with the attack of weakness in normoKPP patients.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Cell Line ; Electric Conductivity ; Family ; Humans ; Models, Biological ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; Paralyses, Familial Periodic ; genetics ; metabolism ; pathology ; Patch-Clamp Techniques ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Channels ; genetics ; metabolism ; Transfection