| A characteristic of muscle is the ability to produce and conduct action potential. Muscle contraction can be induced through excitation-contraction coupling. Ion channels are a complex group of membrane spanning proteins that orchestrate the production and propagation of the electrical activity required for proper muscular function. Ion channels, as pore-forming proteins, play an important role in setting membrane potential, shaping action potentials, controlling the flow of ions across secretory and epithelial cells, and regulating cell volume. Ion channels are present in the membranes of all cells. Channelopathies are the diseases caused by disturbed function of ion channel subunits or the proteins that regulate them. These diseases may primarily be resulting from a mutation or mutations in the encoding genes, spanning a spectrum from muscle hyperexcitability or hypoexcitability.Hypokalemic periodic paralysis (HypoKPP) is a group of muscle disorders with hypoexcitability, characterized by recurrent muscle weakness, which is accompanied by hypokalemic. HypoKPP can be classified into familial periodic paralysis (FPP), thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP). FPP is an autosomal dominant familial disorder, most common in Caucasians. The pathogenic gene is CACNA1S (encoding the alpha subunits of the skeletal muscle L-type calcium channel) gene or SCN4A (encoding the skeletal muscle sodium channel) gene. However, TPP and SPP are common in Asians. The pathogenic gene and pathogenesis await further explored.TPP is the common neuromuscular complication of hyperthyroidism. Some patients with hyperthyroidism are admitted into hospital for periodic paralysis. TPP is a rare disease, being most prevalent in young male Asians. It is characterized with recurrent muscle weakness and paralysis, accompanied by diminished excitability and tendon reflex. Most TPP patients are complained of Grave’s syndrome, such as weight loss, tachycardia, goiter, tremor and so on. Hypokalemia can be observed when the patients are suffered from episodic paralysis. Paralysis can be effectively relieved by potassium supplementation.SPP patients deny any family history and their thyroid function present normal. The clinical features are similar to FPP. This disorder is also prevalent in young male Asians. Respiratory muscle and heart are spared. Symptoms can be relieved by itself in most patients. However, some may develop into complete tetraparesis, only a few complaining of arythmia. Potassium supplementation is effective for the recovery. It has multiple inducing factors, such as carbohydrate-rich meals, exhaustion and prolonged immobility.In this research, we summarized clinical characterization and genetic findings for TPP and SPP patients. These findings may prompt us to gain more insight into the diagnosis and pathogenesis.Part I Clinical Features of Thyrotoxic Periodic Paralysis and Sporadic Periodic Paralysis and KCNJ18 Gene Mutation ScreeningIn 2010, Ryan et al discovered the new KCNJ18 gene mutations were associated with TPP. This gene encodes the inwardly rectifying potassium channel Kir2.6 of skeletal muscle. Kir2.6 is proved to play an important role in the potassium equilibrium potential and the regulation of the cell excitabilities. Ryan reported that KCNJ18 gene mutations were responsible for 33% Caucasian TPP patients. In 2011, Cheng et al reported that the mutations of this gene were associated with 1.67% TPP in Taiwan. Besides, they also discovered that KCNJ18 mutations were responsible for 3.3% SPP patients. In this research, we conducted KCNJ18 gene screening in all 116 patients. All coding regions and intron-exon boundaries were carried out under standard conditions. The PCR products were subjected to direct bidirectional sequencing.85 SPP and 31 TPP were recruited from 2010 to 2013. Detailed clinical feature and laboratory examinations were collected. One-year follow-up was conducted. DNA were prepared from the peripheral blood.All TPP in our cohort are male. The mean age at onset was 29.5±10.5 years (range, 20-40 years). The symptom of Tetraparesis can be reported by all patients. The blood potassium was observed at 1.5-2.5 mmol/L in 93.6% TPP patients. The muscle strength can be returned to normal in 24h. Respiratory muscle and heart were spared. Potassium supplementation was effective for the recovery. However,25 TPP has suffered more than 3 times attacks, and 6 TPP had twice attack, all of which were induced by the hyperthyroidism.27 patients were diagnosed as Grave’s syndrome,4 as subacutethyroiditis. For SPP patients,2 were female. The mean age at onset was 26.5 ±10.5 years, (range,16-37 years). The blood potassium was 1.6-2.5 mmol/L in in 68.2% SPP patients The induced factors were carbohydrate-rich meals, exhaustion and prolonged immobility.The mutation G169R of KCNJ18 gene was found in a SPP. A heterozygous guanine to cytosine single base substitution at nucleotide 505 (G505C, GGC to CGC) in exon 3, led to a missense mutation from glycine to arginine (G169R) in the second transmembrane domain. None out of 100 healthy control subjects harbored this amino acid change in this gene. This 34-year-old man presented with periodic episodes of paralysis for 17 years. He experienced his first attack of muscle weakness at the age of 17. During this episode, he awoke with quadriplegia. The blood potassium was 1.8 mmol/L. Creatine kinase was 1964 IU/L. No arrhythmia was reported. His symptoms returned to normal after around 24 h without any treatment. Thereafter, similar symptoms occurred once or twice monthly from the age of 17 years, more frequently in September, October and November. Each attack lasted 24-72 h. The patient reported that attack can be induced by exhaustion and prolonged immobility, especially carbohydrate-rich meals. He denied any family history of thyroid or neurological problems. Between attacks, his muscle strength was well maintained, and no fasciculation or percussion myotonia were noted. Thyroid stimulating levels were normal. ET results showed compared to that at rest, CMAPs amplitude increased by 21% immediately after exercise, then the amplitude decreased by 64% 1 h after exercise, which indicated primary periodic paralysis.Part Ⅱ The Functional Analysis and Pathogenesis of Kir2.6 Mutant ChannelsTo date, only four Kir2.6 mutations (Q126X, A200P, K360T, E388K) were identified in the Chinese mainland TPP patients, but their functional data were not available, although A200P mutant has already been verified as a non-functional channel. To provide more insights into the mechanism underlying, electrophysiological analysis of these mutants was further conducted.Based on the recordings of patch clamp, the current-voltage relationship showed that G169R and E388K mutant channels were still characterized with strong inwardly rectifying properties. As for Q126X and K360T, current densities observed showed no significant difference from those in untransfected cells, which indicated that these two mutations of KCNJ18 led to a complete loss of Kir2.6 function. Compared with that from wild type channel, G169R and E388K displayed reduced currents. At the holding potential of -60 mV, the inward current densities of these two mutants were reduced by 65.6% and 29.9%, respectively. In addition, the outward current densities were also decreased by 84.7% and 62.7% respectively, when the holding potential was around 0 mV.To more closely resemble physiological conditions in vitro, we co-transfected wild type and/or mutant KCNJ18 cDNA into HEK293 cells. From the recordings of the whole cell currents, compared to wild type channel, we observed significant changes among the mutants. The current densities of inward and outward were analyzed at -60 mV and 0 mV. Thus, as for cells expressing Q126X with wild type KCNJ18 (1:1), current densities of inward and outward were reduced by 65.7% and 91.4%. For cells co-transfected with K360T and wild type KCNJ18 (1:1), current densities of inward and outward were reduced by 55.1% and 54.3%. Similarly, for G169R with wild type, current densities of inward and outward were reduced by 48.2% and 47.4%, and those for E388K, were reduced by 24.1% and 41.1%.Part Ⅲ Analysis of MicroRNAs Associated With Hypokalemic Periodic ParalysisMicroRNAs (miRNAs, miRs) represent a new class of small, noncoding endogenous RNAs that range in size from 19 to 25 nucleotides (nt) and can negatively regulate target gene expression at the transcriptional and post-transcriptional level. They play an important role in diverse functions, such as proliferation, apoptosis, invasion, cell differentiation, cell cycle progression, and hematopoiesis. Myogenic microRNAs (myomiRs) are defined as the specific microRNAs expressed in skeletal muscle. MyomiRs has been proved to take part in the myogenesis, muscle fiber differentiation, muscle development and so on. In multiple muscle disorders, such as neurodegeneration, muscular dystrophy and metabolic muscle disorders, changes of relative myomiRs expression level can be detected.In this research, muscle sample were obtained from 3 clinically diagnosed SPP and 3 healthy control. Affymetrix miRNA Array was used for detecting miRNA expression profile between the two groups. Microarray results showed that, in SPP,16 miRNAs were down regulated, and 5 miRNAs were up regulated. Furthermore, miRNA-122、 miRNA-181d、miRNA-34a、miRNA-27b analysis were conducted based on real-time PCR. Real-time PCR results were consistent with miRNA array results, which indicated that miRNA may take part in the pathogenesis of SPP.In summary, we conducted KCNJ18 gene screening in 116 SPP and TPP patients, and identified a novel KCNJ18 gene mutation responsible for a SPP. The ET results of this patient also supported his diagnosis. Using patch clamp, electrophysiology of the mutant Kir2.6 associated with SPP and TPP in the Chinese mainland were further analysed, which presented the reduced or even loss function of Kir2.6. Microarray results indicated miRNA may take part in the pathogenesis of SPP. These results may advance our understanding of etiology of SPP and TPP, and provide a new way to further explore the underlying pathogenic machanism. |
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