Clinical And Basic Studies In A Chinese Family With Paralysis Periodica Paramyotonia

ObjectParalysis periodica paramyotonica are skeletal muscle channelopathies caused by mutations in the skeletal muscle voltage gated sodium channel gene(SCN4A) on chromosome 17q23.1.This disease seriously affects the ability of day to day life of the patients.So the making of a correct early diagnosis,and appropriate clinical intervence will be helpful in decreasing morbidity and fatality rate of patients.This work is aimed at providing a feasible diagnostic method and basis from analysing and observing (using a biomolecular method) a family suffered from the Hyperkalaemic periodic paralysis of the supernumerary muscle with permanent myopathy,and furthermore, probing into possible treatmenting methods at clinic and molecular level.Method1.SubjectThe all subjects were from a chinese family in which 4 generations there were 15 complete dominant inheritance patients with serious paralysis periodica paramyotonica.1.2 Method1.2.1 The clinic method1.2.1.1 Lab test The blood,urine,and stool routine of some subjects from this family were tested; their functions of the liver,kidney,and thyroid gland were examined;and the activities of the hypothalamus-adenohypophyses-adrenaline axis were also observed. Meanwhile,for some patients from this family the serous K⁺ and muscle enzyme level were detected respectively during episode,one week after attack,and the intervals between episodes;For those patients who were treated with diamox,the creatine level in urine were also measured at 24h before and after oral administrations of diamox.1.2.1.2 HE stainness and electron microcopy observationPreparations of the patients' gastrocnemius during episode were made,dyes with HE method,and the ultra microstructure of the preparations were observed under electro microscope.1.2.1.3 Electrophysiological examinationsThe routine electromyography examination,exercise and cold water exposure tests were also done on some patients.1.2.1.4 StatisticsMetagroup T test and the correlation analysis on clinic data of patients were made, compared differences on age and frequency of attack,and correlation the two factors on prognosis.The index of the above comparison:α=0.05.1.2.2 Genetic diagnosis1.2.2.1 Design and analysis of the primerUsing primer3 programmed the special primer directed at 13^th,19^th,23^th,and 24^th SCN4A were designed.1.2.2.2 DNA extraction of genetic groupUsing routine(?)turated phenol-phenochloroform technique DNA of WBC was extracted,and the purity of the DNA extracted was examinated via ultraviolet photometer.1.2.2.3 PCRTaking DNA extracted as the model to expand the exon of 13^th,19^th,23^th,and 24^th of SCN4A gene in the above-mentioned genetic group.1.2.2.4 Test of the PCR productsThe expanded products was tested using 1.5%agar gel,1.2.2.5 Using SSCP system the PCR products were detected1.2.2.6 Sequence testPurifying the questionable preparations of genetic mutation,and then these preparations were examinated using ABI,PRISMIN 3700 DNA sequence meter,and the results were tested at http://www.Ncbi.nlm.nih.gov/blast to determine if there were any changes in amino acid sequence,and if there were/was,positioning is done.1.2.3 Treatment1.2.3.1 The primary culture of the skeletal muscle cellThe gastrocnemius preparations from patient and normal subjects were cultured using primary culture method,and then were observed dynamically under inversed phase difference microscope to achieve the growth of cells.1.2.3.2 Dying the skeletal muscular cellPutting the skeletal muscle cell cultured for 10 days to the growth slides,and using the routine SP staining method to perform Desmin test.1.2.3.3 Intervention of bFGF on the skeletal muscular cells from patientsIncubating the muscular cells of patients during logarithmic phase into the culture-board with 96 pores,and were divided into 3groups:the control group(cultured fluid plus the cultured cells),concentration group:(the culture solution plus 8 different concentrations of bFGF),zero control group(only culture solution).All preparations were cultured for 2 days,and then changed culture solution of 10%FBS to culture 48hs as to synchronized all the cultured cells to G0 phase.After doing this,different concentrations of bFGF without serum were used to continuingly culture the cells for 72hs.1.2.3.3 1 Using MTT method to test the proliferation of the cells1.2.3.3.2 Determinations activities of the lactic dehydrogenase(LDH) and creatine phosphokinase1.2.3.3.3 Determinations of protein content of the cells using Coomassie brilliant blue1.2.3.4 StatisticsAll comparative figure data were expressed as mean value±SD,then were analyzed the SPSS10.0 program.Meanwhile,the comparisons among the multigroups data were performed via S.N.K method.Result1.Among the all subjects only one patient showed a higher level of serous K⁺ concentration during episode,while the other subjects' serious K⁺ concentration ranged in normal limits but higher than that before the episode.The CK level in all the patients without PM increased after each attack,and reached its highest level at 72h.Meanwhile, the CK level of PM patients showed a sustaining rise as high as 2 folds of the normal. At 24h of oral administrations of diamox,the level of the urine reatine begins to drop.2.The results of the muscle biopsy demonstrated retrograde degenerations in skeletal muscle cells.3.The myographic examination showed that titanic discharges were found in the all subjects tested during episodes,and as the skin temperature of the subjects decreased the frequency of this titanic discharge increased significantly.In PM patients, the positive sharp wave and shorter duration of action potential were observed.The conduction velocity of the nerve in the all patients was at normal ranges.The longer duration course of the insertion potential during episode,titanic discharges,and the decrease in number,duration,and the altitude of the action potential,all these were accorded with the levels of myasthenia.4.The altitude of patients' CMAP decrease more than 40%after the exercise test. In the cold water test,as the skin temperature decreased,the altitude of CMAP decreased also,and was greater than that in the normal temperature water test. 5.At the age of onset of the disease,frequency of attack,and age developing to PM,all these showed no significantly difference between male and female patients(P＞0.05).Between the age of disease onset and the prognosis there was no correlation ship(r=0.2058,t=0.7285,p=0.48),while the frequency of attack showed a positive correlationship with prognosis(P＜0.05).6.The 24^th exon 4774bp of SCN4A in all the patients showed a mantle spike, i.e.A/G.The changes in sequences of amino acids were caused by AACATGTA→AACGTGTA,which lead to replacing the methionine with valine (Met1592Val).7.The skeletal muscle cell can be cultured in vitro,including 3 growthing phases: lag period,division-proliferate period,and myotubule formation period,which accorded with those in vivo.8.The initial development of the preparations from the patients was much slower than that from the normal subjects,and the size of the muscular cells from the patients were different.Two weeks latter,the muscle cells from patients began to growth sharply, and some tubules were formed,but was significantly lagged than that from the normal subjects.9.There were significant concentration-dependent relationship between bFGF and development of the muscle cells,the optimal dose was 120mg/ml.10.The LDH and CK activities and protein synthesis of the muscle cells from patients showed higher bFGF concentration dependent than that from the normal subjects(P＜0.01).Conclusion1.Decreasing the frequency of attack,alleviating gravity of each episode,and avoiding overworking may be helpful and effective to prevented PM.2.The exercise test and cold water test may be simple and easy to be used,and reliable for diagnosing this disease.According to the classifications of the disease the screening field of the focus genes can be narrowed,and thus directing Genetic diagnosis.3.The age of this disease onset,the frequency of attacks of disease,and the age of the disease developing to the PM phase were all no different between male and fen[male.The age of the disease onset showed no correlation with prognosis.And the higher of attack frequency was,and the earlier patients developing to the myopathy,the more serious the prognosis would be.4.SCN4A Met1592Val was the focus gene of Chinese patients with normal serum K⁺ concentration periodic paralysis.While for patients with PM,Thr704Met, Met1592Val should be considered as hot sites.5.Between the patients with normal serous K⁺ concentration and those with higher serous K⁺ concentration,there was no different in nature.6.The environmental factors may be involved in onset of this disease,gene may not be the only factor.A propriate medical intervention may be very helpful for alleviating disease development.7.The damaged muscles of patients with PM showed some regeneration ability, but not in the formation of the myotubules in vitro.This suggested that in patients the damaged muscles may not be regenerated normally,so the progressive muscle exhaustion progress definitely exists.8.The exogenous bFGF can promote the division and proliferation of muscle cells in vitro,this may suggest that bFGF may be used to prevent and treat PM patients in clinic.9.Our results suggested that using automyocell transplantation method to treat PM patient may be reasonable.