【作者】 王梦阳；

【导师】 吴立文；

【作者基本信息】 中国协和医科大学 ， 神经病学， 2009， 博士

【摘要】 全面性癫痫伴热性惊厥附加症(GEFS+)的概念提出已超过10年,被国际抗癫痫联盟(ILAE)2001年癫痫及癫痫综合征诊断草案列为有待进一步明确的癫痫综合征。随着分子遗传学研究的不断深入,GEFS+概念的提出者Sheffer将其更名为遗传性癫痫伴热性惊厥附加症(GeEFS+),以覆盖更多FS相关的遗传性癫痫。概念的扩大化虽然解决了命名上存在的矛盾,但遗传异质性导致的表型和基因型间不确定的对应关系仍困扰着研究者们。【目的】通过对三个北方GEFS+家系的基因定位研究,结合对GeEFS+患者突变筛查阳性率的统计及GEFS+患者表型与基因型关系的分析,探讨GeEFS+的可能分子遗传机制及可行的研究方法,强调继续GeEFS+分子遗传学研究对癫痫病因学、癫痫综合征分类学及临床实践的意义。【方法】第一部分,采用共享等位基因分析及连锁分析对收集到的田氏及柴氏GEFS+家系进行5个候选基因及4个基因座的排查及定位。按照θ=0时,Lod值≥2提示连锁,≤-2排除连锁的标准,对所有STR进行筛选。用Cylliric2.0软件对可能连锁区域进行精确定位,构建单体型,之后对区域内候选基因编码外显子、剪接位点及启动子测序,寻找突变。邸氏家系只通过等位基因共享对5个候选基因进行排查。第二部分,首先,搜索Pubmed及万方数据库,选择与FS、GEFS+、PEFS+、家族性TLE伴FS相关的文献;其次,汇总3个常见候选基因SCN1B、SCN1A、GABRG2突变及表型的详细信息;第三,计算GeEFS+中SCN1B、SCN1A、GABRG2、SCN2A及GABRD基因突变筛查阳性率;第四,比较携带SCN1B、SCN1A、GABRG2突变GEFS+家系之间、携带突变与未发现突变GEFS+家系之间表型的差异。【结果】第一部分显示,田氏家系在5q34区域D5S422处得到最大LOD值2.043,根据单体型重建,提示该家系的致病位点与D5S820-D5S422间6Mb的区域存在连锁关系。对GABRG2、GABRA1编码外显子、剪接位点及启动子测序,结果未见突变,发现6个SNP,分别为rs11135176、rs211037(GABRG2)和rs11575999、rs41275339、rs11576004、rs2279020(GABRA1)。rs11575999、rs41275339、rs11576004未与家系共分离,rs211037与GEFS+无关联。柴氏及邸氏与所有候选基因及基因座无肯定连锁关系。第二部分显示,有SCN1A突变的GEFS+家系表型特点更鲜明,表现为:FS+多于FS,表型分布更丰富,外显率更高,FS起病早且孤立发作罕有,突变的部位、性质及对离子通道功能的影响均与表型特点有关。GABRG2家系FS患者明显多于FS+患者,部分性发作罕有,苯二氮卓作用位点的突变与失神发作密切相关。SCN1B的FS及FS+百分比更接近SCN1A,但没有后者表型丰富。没有找到突变的家系FS多而FS+少,虽然表型较丰富,但缺乏严重表型,如MAE和SMEI。【结论】虽然大多数常染色体显性FS家系、表型单一且缺乏严重表型的GEFS+家系将难以找到致病突变,但加强GeEFS分子遗传学的研究并非没有意义,这很可能对表型严重的癫痫或难治性癫痫分子机制的研究有促进作用。本研究扩大了候选基因排查的范围,优化了微卫星遗传标记,并在常规外显子测序的基础上,增加了候选基因启动子测序、SNP的共分离分析及基于家系的关联分析,这为进一步的基因定位研究打下了坚实的基础。[目的]青少年癫痫患者的生活质量研究开展较晚,目前国内还缺乏相应的调查问卷。由美国耶鲁大学医学院Cramer设计的QOLIE-AD-48是目前唯一专门用于青少年癫痫患者的自陈式生活质量调查问卷,具有良好的信度、效度及敏感度,已被翻译为多种语言版本,并逐步应用于临床研究。本研究的目的在于将英文版QOLIE-AD-48翻译为中文,并检验其信度、效度及敏感度,为临床研究提供一个良好的测量工具。【方法】根据欧洲癌症研究及治疗机构(EORTC)生活质量小组关于量表翻译过程的指南,将QOLIE-AD-48翻译成中文。总样本量的选择及测量指标验证的最低期望值根据欧洲生活质量评估小组指南。用PASS2008软件估计研究所需总样本量及重测信度所需样本量。进入心理测量学特性分析过程患者的入选标准为:1)活动性癫痫(2年内至少1次发作);2)癫痫病程超过2年;3)过去的4个月内AEDs无变化;4)正常上学,具备阅读及书写中文能力。有脑外科手术史、同时应用其他中枢神经系统药物或者有进行性神经或精神疾病的青少年患者被排除。参加重测信度分析的患者要求在两次测验间的健康及社会状态无变化。检验的信度指标包括内部一致信度(Cronbach’sα系数)及重测信度(组内相关系数,intraclass correlationcoefecient,ICC),效度指标包括聚合及判别效度、结构效度。敏感度分析通过比较不同发作严重程度总分的差异是否显著来评价。【结果】在OOLIE-AD-48翻译为中文的过程中碰到的主要问题是对动词时态及其准确含义的理解。另外,跨文化的问题也比较突出。PASS2008软件估计总样本量不低于105例,重测信度样本量不低于43例。从2007年8月至2009年3月,总计145例患者参加了第一次QOLIE-AD-48测验,其中60例在14-30天内进行第二次测验,45例符合重测信度统计的入选标准。75.9%的受试者完成了所有题目,完成时间平均为16分钟(范围10-25分钟)。分量表的平均分数在25.4到91.9分之间,总分为67.5。“对待癫痫的态度”分量表(20.0%)有明显的地板效应,“躯体功能”(20.7%)及“学校表现”分量表(53.8%)有明显的天花板效应。总分无天花板及地板效应。聚合/判别效度结果显示,分量表内的每道题目与所在分量表的相关系数在0.4以上,每一题目与所在分量表的相关性高于其他分量表。此外,QOLIE-AD-48总分与PedsQL总分显著相关。主轴因子分析显示没有二级因子产生,所有的分量表与总分显著相关(r=0.37-0.91)。总分内部一致信度为0.93,分量表在0.60-0.91之间(表5),PedsQL的Cronbach’sα系数为0.92。QOLIE-AD-48的总分重测信度为0.76。总分随发作严重程度的增加而显著下降,显示敏感度良好。【结论】总而言之,QOLIE-AD-48中文版的翻译过程经过反复斟酌,测验结果显示基本满足HROOL量表所需的心理测量学特性标准,具有良好的效度、信度及敏感度。因此,该量表完全可以胜任中国青少年癫痫患者HRQOL研究之需。毫无疑问,未来的临床研究必将会从该量表的应用中受益。更多还原

【Abstract】 The name of generalized epilepsy with febrile seizures plus(GEFS+) has been used for more than ten years and was listed as "syndromes in development" by the 2001 ILAE Proposed Diagnostic Scheme for People with Epileptic Seizures and with Epilepsy. With the development of molecular genetics,Scheffer recently proposed changing the name of GEFS+ to "genetic epilepsy with febrile seizures plus" from "generalized epilepsy with febrile seizures plus".This was because although generalized epilepsies predominate in GEFS+ families,focal or partial epilepsies are well recognized with and without preceding FS.The problematic name was changed,but the uncertain relationship between the phenotype and the genotype still puzzles the researchers.Objective We mapped the genes of three GEFS+ families from the north of China, calculate mutation rate of GeEFS+,and analyze the relationship between the phenotype and the genotype of GEFS+ in order to explore the possible molecular mechanisms of GeEFS+ and the feasible study methods。We emphasize that further study on molecular genetic mechanisms is very important to epilepsy etiology,classification of epilepsy syndromes and clinical practice.Methods In the first part,we used allele sharing and linkage analysis to genotype the family members in Family Tian and Family Chai with microsatellites for reported 5 candidate genes and 4 candidate loci for GEFS+ and FS.We screened all STRs according to the evaluation criterion of LOD score(≥2:hint of linkage atθ=0;≤-2:hint of no linkage atθ=0).The Cylliric2.0 software was used to reconstruct haplotype for fine mapping.Then,a mutation search by means of direct sequencing was performed in the coding exons,flanking splice sites and promotor of two genes(GABRG2 and GABRA1). Only allele sharing was performed to screen 5 candidate genes in Family Di.In the second part,first,the literatures retrieval was performed at Pubmed and Wanfang database.The articles on FS、GEFS+、PEFS+ and familial TLE with febrile seizures were included.Second,the information on characteristics of mutations in three genes(SCN1B, SCN1A and GABRG2) was collected.Third,mutation rate of SCN1B,SCN1A, GABRG2,SCN2A and GABRD in GeEFS+ was calculated.Fourth,the phenotypes among the families with mutations in three genes(SCN1B,SCN1A and GABRG2) and between the families with and without mutations were compared.Results Family Tian had a maximum LOD score(Zmax=2.043) atθ=0 for D5S422.In this family,linkage to 6Mb region between D5S820 and D5S422 could be possible based on haplotype analysis.Direct sequencing in GABRG2 and GABRA1 was performed,and no causal mutations were found.Six SNPs were detected,including rs11135176 and rs211037 in GABRG2 and rs11575999、rs41275339、rs11576004 and rs2279020 in GABRA1.Co-segregation of rs11575999、rs41275339 and rs11576004 with phenotype was excluded and association on rs211037 with phenotype was not found.Linkage to all candidate genes and loci could be excluded in Family Chai and Di.The families with mutation in SCN1A had more specific characters in phenotype: FS+ much more than FS,wide phenotype distribution,higher penetrance,early onset and rare single FS.Location and type of the mutations as well as effect of the mutations on ion Channel were associated with phenotype.In the families with mutation in GABRG2, the patients with FS were more than those with FS+,partial seizures were rare,and the mutations in benzodiazepin binding pocket were associated with absence seizures.In the families with mutations in SCN1B,the patients with FS and FS+ were similar to those with mutations in SCN1A,but the quantity of phenotype was not as much as the latter. FS was more oftern than FS+ in the families in which mutations could not be found. Although the quantity of phenotype was much,severe phenotype was not found,such as MAE and SMEI..Conclusion Although it is very difficult to find the causal mutations in most of the Autosome dominant FS families and GEFS+ with simple phenotype and without severe phenotype,further study on molecular genetics in GeEFS is by no means nonsignificant, which could facilitate study on molecular mechanisms of epilepsies with severe phenotype or refractory epilepsies.We supplemented the candidate genes and loci, optimized the genetic markers,performed direct senquencing in promoters, co-segregation analysis in three SNPs besides sequencing in the coding exons and family-based association tests in one SNP,which established a substantial foundation for further gene localization. Research on health-related quality of life(HRQOL) in adolescents with epilepsy began relatively late in China,even though significant advances had been made in Western societies.One reason for this was the unavailability of valid measures for measuring and evaluating adolescents’ HRQOL in China.The Quality of Life in Epilepsy Inventory for Adolescents(QOLIE-AD-48) is the most frequently recommended questionnaire for measuring HRQOL in youths with epilepsy and has been used in different studies to date.The QOLIE-AD 48 has been validated in several languages,including Spanish,Serbian,and Brazilian Portuguese recently,and these translations have been shown to be as specific and sensitive as the original version.Objective The aim of this study was to translate into Chinese the Quality of Life in Epilepsy Inventory for Adolescents(QOLIE-AD-48) and to evaluate its psychometric properties.Methods The QOLIE-AD-48 was translated according to the procedure of the European Organization for Research and Treatment of Cancer(EORTC) Quality of Life Group. After a multistage translation and cultural adaptation,the final Chinese version was administered to 145 adolescents with epilepsy to evaluate its validity and reliability.The inclusion criteria were:(1) active epilepsy(i.e.,at least one seizure in the last 2 years); (2) epilepsy duration longer than 2 years;(3) no change in antiepileptic drug regimens in the past 4 months;(4) regular school attendance with ability to read and write Chinese.Adolescents who had had brain surgery,used a concomitant medication with central nervous system effects,or had progressive neurological or psychiatric illness were not considered eligible for the study.Test-retest analysis was administered to 45 adolescents without significant changes in health and social status during a two-four weeks’ interval between test and retest.Interitem correlations,correlations with the Pediatric Quality of Life Inventory(PedsQL),internal consistency,test-retest reliability and sensitivity were examined.Results The main translational problems encountered in developing the Chinese QOLIE-AD-48 were due to discrepancies in verbs form and exact meanings,which involved mainly the content of some items.Cross-cultural problems were also difficult to he solved.The acceptability,as well as the feasibility,of the Chinese version appeared satisfactory,considering that 75.9%of the adolescents completed the entire instrument within 10-25 min.Mean total score was 67.5,and all subscales contributed significantly to the summary measure.Considering the validity of the translation,each item correlated Research on health-related quality of life(HRQOL) in adolescents with epilepsy began relatively late in China,even though significant advances had been made in Western societies.One reason for this was the unavailability of valid measures for measuring and evaluating adolescents’ HRQOL in China.The Quality of Life in Epilepsy Inventory for Adolescents(QOLIE-AD-48) is the most frequently recommended questionnaire for measuring HRQOL in youths with epilepsy and has been used in different studies to date.The QOLIE-AD 48 has been validated in several languages,including Spanish,Serbian,and Brazilian Portuguese recently,and these translations have been shown to be as specific and sensitive as the original version.Objective The aim of this study was to translate into Chinese the Quality of Life in Epilepsy Inventory for Adolescents(QOLIE-AD-48) and to evaluate its psychometric properties.Methods The QOLIE-AD-48 was translated according to the procedure of the European Organization for Research and Treatment of Cancer(EORTC) Quality of Life Group. After a multistage translation and cultural adaptation,the final Chinese version was administered to 145 adolescents with epilepsy to evaluate its validity and reliability.The inclusion criteria were:(1) active epilepsy(i.e.,at least one seizure in the last 2 years); (2) epilepsy duration longer than 2 years;(3) no change in antiepileptic drug regimens in the past 4 months;(4) regular school attendance with ability to read and write Chinese.Adolescents who had had brain surgery,used a concomitant medication with central nervous system effects,or had progressive neurological or psychiatric illness were not considered eligible for the study.Test-retest analysis was administered to 45 adolescents without significant changes in health and social status during a two-four weeks’ interval between test and retest.Interitem correlations,correlations with the Pediatric Quality of Life Inventory(PedsQL),internal consistency,test-retest reliability and sensitivity were examined.Results The main translational problems encountered in developing the Chinese QOLIE-AD-48 were due to discrepancies in verbs form and exact meanings,which involved mainly the content of some items.Cross-cultural problems were also difficult to he solved.The acceptability,as well as the feasibility,of the Chinese version appeared satisfactory,considering that 75.9%of the adolescents completed the entire instrument within 10-25 min.Mean total score was 67.5,and all subscales contributed significantly to the summary measure.Considering the validity of the translation,each item correlated更多还原