【作者】 邹欣；

【导师】 刘晓蓉；

【作者基本信息】 广州医学院 ， 神经病学， 2011， 硕士

【摘要】 目的在部分性癫痫伴热性惊厥附加症(PEFS+)患者中筛查SCN1A基因突变,研究SCN1A突变与PEFS+的关系;研究不同内含子突变对SCN1A基因剪切的影响;总结PEFS+的SCN1A突变特点,从突变的角度探讨PEFS+作为Dravet综合征(DS)和全面性癫痫伴热性惊厥附加症(GEFS+)过渡表型的可能。研究对象和方法收集符合以下标准的病例的临床资料及基因组DNA:(1)起病前精神运动发育正常,起病后无明显精神发育迟滞或仅有轻度异常;(2)于3个月至6岁间开始出现热性惊厥,随后可出现非热性癫痫发作或6岁以后仍有热性痫性发作;(3)除GTCS外,非热性发作仅表现为部分性发作或部分继发全面性发作,无其它痫性发作形式;(4)排除肿瘤、外伤、感染、局灶性皮质发育异常等症状性癫痫;(5)排除Dravet综合征等特发性癫痫。采用高效液相色谱仪(DHPLC)与直接测序结合的方法,对患者SCN1A基因外显子和剪切相关内含子区进行突变筛查。对新发现的突变,通过检索多态性位点数据库及设置正常对照组(100例)进行验证;通过构建迷你基因模型转染真核细胞的方法研究内含子突变c.473+5 G>A、c.473+110 A>G对剪切的影响;通过检索SCN1A突变数据库及复习文献,结合本研究结果总结PEFS+的SCN1A突变特点,对比GEFS+和DS,分析PEFS+作为GEFS+和DS过渡表型的可能性。结果共收集PEFS+病例37例,发现4个SCN1A基因突变:c.473+5 G>A、c.473+110 A>G、c.1028+35 T>C、c.2378 C>T,其中c.473+5 G>A可引起前体mRNA异常剪切导致蛋白质截短;c.473+110 A>G对剪切无影响,可能为非致病性突变。截止目前,共报道PEFS+表型SCN1A突变20个,包括:错义突变17个、剪切位点突变2个、移码突变1个。PEFS+主要由错义突变引起,52%错义突变位于电压感受区和中央孔区(S4～S6),比例明显高于GEFS+(27.3%)接近于DS(60.2%))。剪切位点突变和移码突变虽然可以导致PEFS+,但不是其主要突变形式。结论(1)SCN1A很可能是PEFS+致病基因。(2)PEFS+主要由SCN1A错义突变引起,突变位点主要位于重要功能区域(S4-S6);剪切位点突变和移码突变亦可导致PEFS+,但不是其主要突变形式。(3)PEFS+在SCN1A突变形式和突变位点方面的特点提示它可能是DS和GEFS+间的一种过渡表型。(4)SCN1A剪切位点突变可能通过引起蛋白质截短使钠通道功能丧失,最终导致PEFS+患者发病。更多还原

【Abstract】 PurposeTo screen SCN1A mutations in patients with partial epilepsy with febrile seizures plus(PEFS+)and investigate the mechanism of intronic mutations effecting on precursor mRNA(pre-mRNA) splicing. To characterize SCN1A mutations of PEFS+ and discuss the possibility that PEFS+ is a distinct phenotype of SCN1A mutation and different from both Dravet syndrome (DS) and generalized epilepsy with febrile seizures plus (GEFS+).Patients and methods37 patients with PEFS+ were collected. All the 26 exons and splicing regions of SCN1A were screened with denaturing high performance liquid chromatography (DHPLC) and direct sequencing. A splicing assay by mini-gene construction was conducted to determine the effect of the c.473+5 G>A and c.473+110A>G mutation on pre-mRNA splicing. All the update SCN1A mutations which were identified in PEFS+ patients had been reviewed to detect a common feature and to find the relationship between PEFS+、GEFS+ and DS. Results4 heterozygous mutations were detected: c.473+5 G>A、c.473+110 A>G、c.1028+35 T>C、c.2378 C>T. The splice site mutation c.473+5 G>A could disrupt normal splcing and finally result in a truncation of SCN1A. The intron mutation c.473+110 A>G did not effect on splicing. 20 SCN1A mutations in PEFS+ were reviewed and PEFS+ was mainly induced by missense mutations. 52% of these mutations located in the S4-S6 segments. The proportion of mutations in the S4-S6 segments in PEFS+ is much higher than GEFS+(27.3%) and close to DS(60.2%). Splicing-site mutation and frame-shift mutation could also lead to PEFS+,but they were not the main mutation types for PEFS+.ConclusionSCN1A is a candidate gene for PEFS+. Missense mutation of SCN1A is the most common mutation type for PEFS+. Most of missense mutations in PEFS+ were located in the S4-S6 segments. Splicing-site mutations and frame-shift mutations were also related to PEFS+. The pathogenesis of intron mutations for PESF+ was to result in SCN1A truncation by altering splicing sites of SCN1A pre-mRNA. According to the mutation characteristics, PEFS+ seems to be a transition phenotype between DS and GEFS+.更多还原