【作者】 李楠；

【导师】 唐北沙；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 家族性皮质性震颤伴癫痫(familial cortical tremor with epilepsy, FCTE)是一种较为罕见的特发性癫痫综合征,呈常染色体显性遗传模式,于成人期起病。由日本Okuma等于1997年首先命名提出。临床表现为与特发性震颤相像的以肢体远端为主的皮质性震颤；频率稀发的癫痫；神经电生理检查显示为皮质反射性肌阵挛的特点；震颤和癫痫对抗癫痫药物反应良好；非进展性病程、预后良好。同时表现有震颤、癫痫和肌阵挛等极其类似症状的,还见于另外四种分类命名有争议的特发性癫痫综合征：家族性特发性肌阵挛与癫痫(FEME)、良性成人家族性肌阵挛性癫痫(BAFME)、家族性成人肌阵挛癫痫(FAME)、常染色体显性遗传的皮质性肌阵挛与癫痫(ADCME),其中BAFME、FAME和ADCME的致病基因分别被定位于8q23.3-q24.1、2p11.1-q12.2。而FCTE的致病基因尚未定位。我们在中国湖南省收集到一个中国人FCTE大家系,所有患者均具有FCTE典型临床表现。为定位和克隆该FCTE家系的致病基因,我们应用微卫星遗传标记连锁分析的方法首先对其进行位点8q23.3-q24.1、2p11.1-q12.2排除分析,结果排除该FCTE家系与2p11.1-q12.2的连锁关系,而提示与8号染色体连锁。基于此我们进一步精细定位,在常染色体显性遗传模式下,重组率θ为0时于D8S 199处获得最大两点LOD值7.21,经单体型分析,将此FCTE家系致病基因定位于D8S555与D8S1753之间约30.5 cM的区间8q23.3-24.23。结合目前已知特发性癫痫致病基因以离子通道基因为主的特点,我们选择此区间内和紧毗邻区的钾离子通道基因KCNQ3、KCNV1、KCNS2,运用聚合酶链反应(PCR)扩增、直接测序法进行突变检测,结果未发现任何的序列变异,排除三个候选基因为该FCTE家系致病基因可能,为进一步候选克隆奠定了重要基础。这是在中国首次发现报道FCTE家系,并首次在世界范围内定位了FCTE致病基因位点。良性家族性婴儿惊厥(benign familial infantile seizures, BFIS)是一种较为罕见的呈常染色体显性遗传的特发性癫痫综合征。临床表现为3月-10月龄尤其4月-7月龄出现、1岁-3岁内可自然消退的无热性惊厥,精神运动发育无异常,体查、发作间期脑电图及神经影像学检查正常。目前已定位3个致病基因位点19q12-13.1、16p12-q12和2q24。随后发现部分家系与上述位点并无连锁,提示BFIS还存在其它致病基因位点。我们在中国湖南省收集到两个BFIS大家系,前期工作中,在排除已报道的BFIS致病位点19q12-q13.1、16p12-q12、2q24和排除KCNQ2、KCNQ3、SCN2A基因突变后,其中一个家系(家系1)的致病基因提示位于其它新的位点；另一个家系(家系2)运用微卫星遗传标记的全基因组扫描和精细定位将其致病基因定位于新位点1p36.12-p35.1,并对区间内的33个候选基因进行了相关突变检测,但未发现致病性改变。本研究中,我们采用最新一代SNP遗传标记连锁分析微珠芯片进一步对BFIS家系1进行定位,结果得到三个候选区域5q22.1-5q32、9p21.3-9p21.2和22q11.21-22q12.1。经微卫星遗传标记分析确证与5号染色体连锁,单体型分析将此BFIS家系致病基因定位至极可能位点D5S2057与D5S500之间约5.47 cM、相当于5q31.1-31.2的区域。对紧邻的GABRG2基因,运用聚合酶链反应(PCR)扩增、直接测序法进行突变检测,未发现序列变异。而对定位于1p36.12-p35.1的另一BFIS家系,继续筛选区间内和紧毗邻区的另外14个新候选基因(SYTL1、FUCA1、EPHA8、SH3BGRL3、AK2、SDC3、MAN1C1、ID3、LAPTM5、PAQR7、RCC1、RUNX3、KCNQ4、GABRD)进行突变检测,结果共发现13个SNP改变,其中2个为新发现的SNP。排除这14个基因为BFIS新致病基因的可能,为进一步候选克隆和最终揭示BFIS的发病机制奠定了重要基础。全面性(遗传性)癫痫伴热性惊厥附加症(generalized/genetic epilepsy with febrile seizures plus, GEFS+)是一种家族遗传性的特发性癫痫综合征,具有高度的表型和遗传异质性。目前已克隆6个致病基因：SCN1A、SCN1B、SCN2A、SCN9A、GABRD、GABRG2.我们对三个中国GEFS+家系临床特点进行详细分析,发现其临床表型谱包括有热性惊厥(FS)、热性惊厥附加症(FS+)、失神癫痫(AS)和无热性癫痫(AFS),提示存在表型异质性。对突变比例相对较大的基因SCNIA、SCNIB、GABRG2,运用聚合酶链反应(PCR)扩增、直接测序法进行突变检测,结果发现其中一个GEFS+家系在SCN1A基因产生突变c.5383G>A(氨基酸E1795K),为一新的突变点。其余两个GEFS+家系无SCNIA改变及SCNIB、GABRG2基因的改变,提示致病性改变位于其它基因,或可能不是外显子区的常规点突变,尚需进一步探索。对新发现的突变c.5383G>A,我们后续工作中将进行相关功能研究,以探明其发病的病理生理机制。更多还原

【Abstract】 Familial cortical tremor with epilepsy (FCTE) is a rare autosomal dominant idiopathic epilepsy syndrome, characterized by adult-onset fine finger cortical tremor resembling essential tremor, infrequent seizures, features of cortical reflex myoclonus in neuroelectrophysiological study, good response to anticonvulsants, non-progressive course and benign prognosis. Similar symptoms were described within other controversial idiopathic epilepsy syndromes including FEME, BAFME, FAME, ADCME. Causative genes of BAFME, FAME and ADCME were mapped to 8q23.3-q24.1 and 2p11.1-q12.2. However, Causative gene of FCTE was not mapped yet. A large pedigree of Chinese origin affected with FCTE was found in Hunan Province of China. All of affected individuals manifested typical symptoms of FCTE. By linkage analysis, we excluded this family from linkage to 2p11.1-q12.2, while indicated evidence of linkage to chromosome 8. Using fine microsatellite markers, a maximum two-point LOD score of 7.21 for microsatellite D8S199 at recombination fractionθ0.0 was obtained. Recombination results suggest the locus on chromosome 8q23.3-24.23 over 30.5 cM interval between D8S555 and D8S1753. Considering ion channel genes play an important role in the epileptogenesis of idiopathic epilepsies, three potassium channel genes including KCNQ3, KCNV1 and KCNS2 located in and near 8q23.3-24.23 were subjected to mutation detection through polymerase chain reaction (PCR) and direction sequencing. No sequence variants were found in three candidate genes, therefore excluded from the FCTE pedigree. This is the first family case report of FCTE found in China, and for the first time in the world the causative gene locus of FCTE has been identified.Benign familial infantile seizures (BFIS) is an autosomal dominant idiopathic epilepsy syndrome characterized by afebrile seizures, which typically onset from three to twelve months and remit spontaneously within one to three years old, normal psychomotor development outcome and normal finding in physical examination, interictal EEG and neuroimaging. Genetic studies have revealed three causative loci on 19q12-13.1,16p12-q12 and 2q24, and further studies indicate more susceptibility loci may exist. We identified two large BFIS pedigrees from Hunan Province of China. In the previous studies, after exclusion of known loci 19q12-13.1,16p12-q12 and 2q24 as well as mutation in genes KCNQ2, KCNQ3 and SCN2A, causative gene of one BFIS family (Pedigree 1) was indicated to be on novel locus, while that of another BFIS family (Pedigree 2) was mapped to novel locus on 1p36.12-p35.1 through genome-wide scan and fine mapping using microsatellite markers. Then thirty-three candidate genes located on 1p36.12-p35.1 were subjected to mutation detection for Pedigree 2, but no causative variants were found. In this study, we applied SNP linkage beadchip to map causative gene for Pedigree 1. Three candidate loci on 5q22.1-5q32, 9p21.3-9p21.2 and 22q11.21-22q12.1 were obtained. Through linkage analysis using fine microsatellite markers, the most likely locus was linked to 5q31.1-31.2, with a maximum two-point LOD score of 2.84 for microsatellite marker D5S2117 at recombination fractionθ0.0 between D5S2057 and D5S500 over 5.47 cM interval. Subsequently, one candidate gene (GABRG2) closed to 5q31.1-31.2 of Pedigree 1 was selected and subjected to mutation detection by PCR and direct sequencing, but no sequence variant was found. Meanwhile, fourteen candidate genes (SYTL1, FUCA1, EPHA8, SH3BGRL3, AK2, SDC3, MAN1C1, ID3, LAPTM5, PAQR7, RCC1, RUNX3, KCNQ4, GABRD) located/closed to 1p36.12-p35.1 of Pedigree 2, were selected and subjected to mutation detection. Thirteen SNPs were detected, two of which are not reported yet. It is unlikely that the above-mentioned genes are involved in the pathogenesis of the two Chinese BFIS families respectively. The results have laid indispensable basis for further analysis of other candidate genes for to elucidate the molecular pathogenesis of BFIS.Generalized/Genetic epilepsy with febrile seizures plus (GEFS+) is a familial idiopathic syndrome with high phenotypic heterogeneity and genetic heterogeneity. To date, six causative genes have been identified including SCN1A, SCN1B, SCN2A, SCN9A, GABRD and GABRG2. We studied three GEFS+ pedigrees of Chinese origin and found their clinical spectrum include FS, FS+, AS and AFS. Three genes, SCN1A, SCN1B and GABRG2 which are more frequently involved in the etiology of GEFS+, were subjected to mutation detection. By PCR and direct sequencing, a novel mutation c.5383G>A in SCN1A was identified in one of pedigrees. But no sequence variants were found in SCN1A, SCN1B or GABRG2 for two other GEFS+ families, indicating causative variants lie in other candidate genes or are probably not routine point mutations. Further investigations are needed for two other GEFS+ families as to finally disclose the corresponding causative gene(s). Functional study will be carried out to investigate the pathogenesis of the novel mutation c.5383G>A.更多还原