Influence of SCN1A intronic mutations in mRNA splicing and relation of mRNA splicing changes with phenotype in febrile seizures related epilepsy / 中华神经医学杂志

Objective To study the influence of SCNIA intronic mutations in mRNA splicing in febrile seizures related epilepsy,and investigate the association between splicing changes and genotype-phenotype-inheritance pattern.Methods Molecular cloning of 5 SCN1A intronic mutations was performed in patients with partial epilepsy with antecedent febrile seizures plus (PEFS+) and Dravet syndrome (DS) through constructing mutant and wild-type plasmids of pTragetE2-3-4-5 and E24-25-26 by using Minigene splicing assay,and the in vitro expressions in HENK293 cells were detected.The mRNA splicing changes were analyzed qualitatively and quantitatively by reverse transcription (RT)-PCR and real time quantitative (q)-PCR.Results (1) Using RT-PCR,DS mutants presented a whole exon skipping without significant remain of normal mRNA transcripts,while PEFS+ mutants showed partial exon skipping or intronic insertion with coexistence of normal and aberrant mRNA transcripts.(2) Statistical differences were found between relative quantity (RQ) of aberrant and normal mRNA in PEFS+ mutant (c.473+5G＞A:4.92％±1.05％ and 6.10％±0.21％;c.473+5G＞C:7.97％±1.12％ and 3.94％ ±1.25％) and that in DS mutant (c.602+1G＞A:60.51％±1.81％ and 0.060％±0.022％,P＜0.05);similarly,there were statistical differences between relative RQ of normal and aberrant mRNA in PEFS+ mutant c.4853-25T＞A (71.22％±11.92％ and 7.38％±1.61％) and that in DS mutant c.4853-1G＞C (0.08％±0.01％ and 22.11％±2.83％,P＜0.05).Conclusion The position and difference of splicing patterns of SCNIA intronic mutations are potential molecular pathogenesis for phenotypic difference of febrile seizures related epilepsy.

Changes of neuregulin 1 receptor ErbB4 and their significance in FMR1 gene knockout mouse / 中华神经医学杂志

Objective To study the expression changes ofneuregulin 1 (NRGI) receptor ErbB4 in the cortex and hippocampus of fragile X mental retardation 1 (FMR1) gene knockout (KO) mice,and explore the role of ErbB4 in epilepsy susceptibility in fragile X syndrome (FXS).Methods Two weeks old male FMR1 KO mice (n=3),4 weeks old FMR1 KO mice (n=3) and 2 weeks old male wild type (WT) mice (n=3),4 weeks old WT mice (n=3) were chosen in our study.Immunohistochemical staining and Western blotting were performed to detect the ErbB4 positive neurons and protein expression in the cerebral cortex and hippocampus of each group.Double-labeling imnmunofluorescence and laser confocal microscope were used to observe the ErbB4 protein expression in the parvalbumin (PV) inter-neurons of cortex and hippocampus.Results As compared with those in the 2 and 4 weeks old WT mice,the number of ErbB4 positive neurons was significantly smaller and ErbB4 protein expressions in the cerebral cortex and hippocampus of 2 and 4 weeks old KO mice were significantly decreased (P＜0.05).The number of PV and ErbB4 co-expression neurons in cortex and CA1 and CA3 hippocampus of 2 and 4 weeks old KO mice was significantly smaller than that in the 2 and 4 weeks old WT mice (P＜0.05).Conclusion The decreased ErbB4 expression is related to the decreased number of PV positive inter-neurons in the FMR1 KO mice,which is involved in the pathogenesis of FXS epilepsy susceptibility.

Analysis of fragilex mental retardation 1 and fragilex mental retardation 2 gene mutations in intellectually disabled population of unknown causes in Southern China / 中华神经医学杂志

Objective To screen the fragilex mental retardation 1 (FMR1) gene mutations and explore the frequency of FMR1 gene mutation in the population with mental retardation in South China.Methods Seventy-two patients (65 males and 7 females) with suspected fragile X syndrome (FXS) in South China were enrolled in our hospitals from October 2009 to April 2014.The CGG trinucleotide repeats in 5'UTR of FMR1 gene and CCG trinucleotide repeats in FMR2 gene were screened respectively by PCR.Southern blotting and capillary electrophoresis sequencing were performed in male patients without normal target bands and suspected female patients;patients with normal CGG alleles were,then,performed exons and 3'-UTR ofFMR1 gene amplification and sequencing.The frequency of FMR1 gene mutation in patients with mental retardation in different countries and regions was compared with statistical analysis.Results Six pedigrees with full mutation (one female and five males being the probands),one pedigree (mother and son) with FMR1 gene deletion and one pedigree (mother and son) with mutation in the transition region were identified in 72 patients with mental retardation.The prevalence of total mutation was 9.7％ (7/72) and that in male patients was 9.2％ (6/65).These results showed significant differences in prevalence as compared with the results from different countries and areas (P＜0.05);there were no variations in 3'UTR ofFMR1 gene and FMR2 gene mutation in the patients with FXS-like phenotype.Conclusions FMR1 mutation frequency may be higher in mental retardation population in southem China as compared with that in developed countries or areas.Targeted screening on the unexplained mental retardation pedigrees (family history) can improve the diagnosis of FXS.Importantly,deletion mutations screening should also be performed in suspected FXS subjects with normal CGG repeats.

Expression of Nav1.1 and its significance in brain tissues of FMR1 knockout mice / 中华神经医学杂志

Objective To explore the possible role of Nav 1.1 in the pathogenesis of increased susceptibility to epileptic seizures in FMR1 knockout (FMR1 KO) mice.Methods FVB strain FMR1 KO mice and wild type (WT) controls at ages of 2 and 4 weeks old were chosen;immunohistochemistry was used to determine the expression of Nav 1.1 in different brain regions (striate cortex,temporal cortex,piriform cortex,hippocampus CA1,CA3 and dentate gyrus),and Western blotting was used to determine the Nav1.1 level in the cerebral acustici cortex and hippocampus.Results The mean optical density of Nav1.1 was significantly increased in the striate cortex,temporal acustici cortex,piriform cortex,regions of CA1 and dentate gyrus in FMR1 KO mice at ages of 2 and 4 weeks (2 weeks:0.058±0.006,0.054± 0.006,0.130±0.015,0.090±0.009 and 0.142±0.010;4 weeks:0.066±0.007,0.060±0.007,0.159±0.018,0.102±0.015 and 0.192±0.025) as compared with the age-matched WT mice (2 weeks:0.049±0.007,0.046±0.007,0.118±0.012,0.080±0.009 and 0.133±0.010;4 weeks:0.051±0.007,0.048±0.005,0.127± 0.012,0.089±0.012 and 0.175±0.024,P＜0.05).The levels of Nav1.1 in the cerebra1 cortex and hippocampus in FMR1 KO mice at ages of 2 and 4 weeks (2 weeks:0.635±0.082 and 0.954±0.111;4 weeks:0.819 ±0.064 and 1.145 ±0.159) were also significantly increased as compared with the age-matched WT mice (2 weeks:0.382±0.025 and 0.555±0.056;4 weeks:0.550±0.040 and 0.847±0.127,P＜0.05).Conclusion Increased expression of Nav1.1 might play an important role in the increased susceptibility to epileptic seizures in FMR1 KO mice.

Effect of Kv channel-interacting protein 1 over-expression on excitability of primary cultured hippocampal neurons / 中华神经医学杂志

Objective To investigate the effect of Kv channel-interacting protein 1 (KCNIP1) over-expression on K+ currents and neuronal excitability in primary cultured hippocampal neurons.Methods Enhanced green fluorescent protein plasmids carried KCNIP1 (pEGFP-KCNIP1) were established; empty pEGFP vectors were used as controls; primary cultured hippocampal neurons were transfected with pEGFP-KCNIP1 and control vectors.Whole-cell patch clamp technique was used for electrophysiological recording.Results The cultured neurons transfected with pEGFP-KCNIP1 led to KCNIP1 over-expression.The amplitudes of A-type K+ currents in the KCNIP1-overexpress neurons were significantly higher than that in the control group ([0.96±0.17] nA vs.[0.72±0.09] nA,P＜0.05),while no significant difference was found between the component of steady-state outwards K+ currents and controls.Current clamp analysis revealed significantly decreased frequency of evoked discharges and subthreshold membrane potential oscillations,and statistically increased membrane resistance of the hippocampal neurons in the group of KCNIP1 over-expression as compared with those in the controls (P＜0.05).Conclusion Over-expression of KCNIP1 could inhibit neuronal discharges possibly via its potentiation on A-type K+ currents.

Correlation analysis of clinic and SCN1A mutation in febrile seizures plus with autism / 实用医学杂志

Objective To study the clinical characteristics of autism in febrile seizures plus (FS+) and the relationship between autism and SCN1A mutation. Methods Clinical data of 103 patients with FS+ treated in epilepsy centre of the Second Affiliated Hospital of Guangzhou Medical University were collected and analyzed. According to the international criteria, generalized epilepsy with febrile seizures plus (GEFS+), partial seizures with febrile seizures plus (PEFS+), Dravet syndrome (DS) and autism were diagnosed. Genomic DNA was obtained from blood samples. SCN1A were PCR amplified and mutations were detected by DHPLC and sequencing. Result Mental retardation was found in 53.8%of patients with GEFS+, 69.2%of patients with PEFS+, and all patients with DS, respectively. One in GEFS+, one in PEFS+and nine in DS patients were accompanied with autism (P<0.01). Among FS+patients with autism, one SCN1A mutation was found in PEFS+patients, while six SCN1A mutations were found in DS patients. Conclusions Majority of GEFS+and PEFS+patients showed mental retardation, while all the DS patients were accompanied with retardation. The occurrence of autism with DS is higher than GEFS+and PEFS+. No definite relationship between autism and SCN1A mutation was indicated.

De novo sodium channel αl-subuult mutation of monozygotic twins with borderland severe myoclonic epilepsy in infancy / 中华神经科杂志

Objective To study the sodium channel α1-subunit (SCN1A) gene in a pair of monozygotic twins with borderland severe myoclonic epilepsy in infancy (SMEB) and its characteristic of clinical manifestations. Methods The clinical features of 2 monozygotic twins were summarized. All 26 exons of SCNIA genes were screened with denaturing high performance liquid chromatography (DHPLC), and direct sequence analysis was performed on those with abnormal elution peak. Results The proband and her sister showed typical clinical features of SMEB. The same heterozygous mutations on exon 26 which caused the related amino acid change were found among them (c. 5348C > T, A1783E). Conclusion Monozygotic twins with similar clinical phenotype of SMEB have same SCN1A gene mutation.

Mosaic SCN1A mutation in a family with partial epilepsy with febrile seizures plus / 中华神经科杂志

Objective To study the SCN1A gene in a family with partial epilepsy with febrile seizures plus ( PEFS+ ) and its characteristics of inheritance. Methods The clinical features of the 2 patients and their father were summarized. All 26 exons of SCN1A gene were screened with denaturing high performance liquid chromatography (DHPLC), and direct sequence analysis was pedormed on those with abnormal elution peak. Pyrosequencing was subsequently performed in those without abnormality in direct sequence analysis. Results The proband and his sister had the phenotype of PEFS+ . The same heterozygous mutations (AS768G) on exon 26 which caused the related amino acid change (Q1923R) were found among them. Their father had frequent febrile seizures (FS) in childhood, and seizures stopped spontaneously. No abnormality was found in direct sequence but mosaic mutation in the same site was discovered with pyrosequencing (mutation quantity was 25% ). Conclusions The mutatin of SCN1A could cause partial epilepsy. PEFS+ could be inherited, the relatives carrying the affected gene may have mild clinical symptoms, possibly resulting from the low concentration of the mutated gene due to mosaic mutation.

Teaching epilepsy according to the international classification of epilepsy and epilepsy syndrome in different level students / 中华医学教育探索杂志

In order to improve the quality of epilepsy teaching,the appropriate version of the international classification of epilepsy and epileptic syndrome were selected to teach in the different level students by the way of PBL and clinical case analysis.The clinical thoughts and enthusiasm were improved.The classification of epilepsy could be grasped and easily used in their clinical work.

Comparison of Behavioral and Histological Changes between Pilocarpine-Induced Temporal Epilepsy Model and Pentylenetetrazole Kindling Absence Model in Rats / 中国康复理论与实践

@#ObjectiveTo investigate and compare the behavioral changes, neuron loss of hippocampus and mossy fiber sprouting between pilocarpine-induced status epilepticus (SE) model and pentylenetetrazole (PTZ) kindling model in rats.MethodsAfter two different epilepsy models were made, Vedio was adopted to observe the behavioral changes. Nissl staining and Neo-timms' staining were separately used to observe and compare the neuron loss of hippocampus and mossy fiber sprouting in the dentate gyrus (DG) at different time points during epileptogenisis.ResultsNo recurrent spontaneous seizure, no neuron loss and no mossy fiber sprouting were found in PTZ kindling model; whereas obvious neuron loss was found in CA1, CA3 of hippocampus and hilus of DG, and mossy fiber sprouting were found in pilocarpine model in parallel with recurrent spontaneous seizures. ConclusionPTZ kindling model resembles absence epilepsy in human, while pilocarpine-induced status epilepticus model resembles chronic temporal epilepsy in human. Neuron loss and mossy fiber sprouting may play an important role in epileptogenisis. Pilocarpine-induced epilepsy model can be regarded as an ideal chronic temporal epilepsy model.

Functional evaluation of hypertensive cerebrovascular injury by critical closing pressure / 中国组织工程研究

BACKGROUND: Critical closing pressure (CCP) is recently thought to play a key role in cerebral blood flow autoregulation as an effective downstream pressure of cerebral circulation and can objectively reflect the cerebrovascular tone, namely the vascular smooth muscle contraction and diastole, which is subjected to dynamic modulation.OBJECTIVE: To dynamically assess the hypertension-induced damage of the contraction function of cerebral microvascular smooth muscles and its correlation with morphological changes based on CCP evaluation.DESIGN: Randomized controlled experiment.SETTING: Institute of Neural Science of Second Hospital Affiliated to Guangzhou Medical College and Department of Neurology, First Hospital Affiliated to Sun Yet-san University.MATERIALS: The experiment was carried out at the Laboratory of Physiological Science of Sun Yet-san University between July 2002 and August 2003. Totally 160 health male SD rats were randomized into control group and hypertension group with 80 rats in each group. METHODS: Stroke-prone renovas cular hyp ortonsive rats were established in rats of the hypertension group by bilateral renal artery occlusion with two clips. The rats in the control group were not subjected to the occlusion with other treatments identical to those of the hypertension group. At the time points of 2, 4, 6, 8, 10, 12, 14 and 16 weeks after operation, respectively, 10 rats were randomly selected from each of the two groups for determination of arterial pressure and CCP. After the measurements the frontal-parietal lobe was obtained from the anaesthetized rats and cut into slices for quantitative analysis of the morphological changes in cerebral microvessels.different postoperative time points.mean arterial pressure in hypertension group obviously increased from the 6th postoperative week with significant difference from that of the control after operation to a level significantly higher than that of the control group at postoperative 14 and 16 weeks [(63.75±7.43) vs (37.28±3.68) mm Hg and (67.37±15.57) vs (38.39t7.41) mm Hg, respectively, P ＜ 0.05].significance from that of the control group at the 8th postoperative week (Paverage arterial pressure and cerebral arteriole tunica media (r=0.906 93,0.811 36, respectively, P ＜ 0.05). The changes in CCP was more obvious in the early and advanced stages of blood pressure elevation, but not so manifest during obvious blood pressure increment, displaying an inverted S-shaped curve of changes (R2=0.996 2, P ＜ 0.05).CONCLUSION: Contraction of the cerebrovascular smooth muscles is enhanced with the dynamic increment of arterial pressure after the development of hypertension. Vascular tone increase is more manifest during the early and advanced stages of hypertension.