兒童癲癇KCNQ2基因突變之功能性分析與基因型-表現型相關性探討

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Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/18925

Title:	兒童癲癇KCNQ2基因突變之功能性分析與基因型-表現型相關性探討 Functional analysis and genotype-phenotype correlation of childhood epilepsy caused by KCNQ2 mutation
Authors:	李英齊 Lee, Inn-Chi
Contributors:	李宣佑
Keywords:	KCNQ2;癲癇;功能性研究;兒童;次世代定序 KCNQ2;epilepsy;functional study;children;next-generation sequencing
Date:	2017
Issue Date:	2018-03-20T08:27:35Z (UTC)
Abstract:	背景:癲癇佔人口約1%，而兒童至少有60%為找不到原因的癲癇(epilepsy without an identified cause)。KCNQ2與KCNQ3為細胞膜鉀離子通道調控基因。對帶有KCNQ2突變的人，可能會導致癲癇、智障與嚴重的腦病變，但目前尚未完全瞭解。本研究目的主要在於探討癲癇病人的下列幾點: (1) 探討KCNQ2突變在兒童非病灶性癲癇 (non-lesional epilepsy)角色。(2) 對特有的突變點，進一步利用HEK293細胞膜式做功能性的分析。 (3)探討不同KCNQ2突變點基因型與表現型的關係，並進一步分析突變基因造成細胞電流的改變，與病人症狀是否相關性。病人與方法: 從2005到2016年我們選取75 位病人均符合下列條件:[1]兒童癲癇無法找到原因; [2]第一次抽搐年齡小於18歲，且最後一次追蹤時間小於18歲;[3]至少有一次腦部核磁共診掃描(MRI)且無發現腦部病灶與癲癇相關。在這些病人中，34 (45%) 病人第一次抽搐年齡小於2個月，其中8個病人有新生兒癲癇腦病變 (epileptic encephalopathy); 41 (55%) 病人第一次抽搐年齡介於2個月與18歲之間。使用次世代定序(next-generation sequencing )方法來偵測KCNQ2與KCNQ3是否有突變。並對55個正常且無抽搐病史的成人做KCNQ2基因的篩檢。對於突變點的功能性影響用(1) sited directed mutagenesis 把突變點植入HEK293 cell;(2)雙電極電位鉗定技術(two-electrode voltage-clamp，TEVC)分別以野生型(wild type)KCNQ2、突變型KCNQ2與野生型(wild type)KCNQ3以不同比率殖入HEK293 cell，探討是否造成細胞功能電位的改變;(3)免疫螢光法與西方點墨(western blot)來決定KCNQ2突變造成膜蛋白的影響。結果: 我們完成55個正常人和75個非病灶性癲癇 (non-lesional epilepsy)病人的KCNQ2與KCNQ3基因檢查，經過初步病人組與對照組比較後，發現病人帶有KCNQ2基因變異(variants)，但不知這些變異是否與疾病有相關性。再經過FATHMM、 PolyPhen2與SIFT電腦軟體分析後，8(11%)個病人帶的突變基因極可能與疾病有相關性。會導致的疾病的可能突變點包括: c.1545 G>C (p.E515D); c. 2264 G>A (p.Y755C); c.1627G>A (p.V543M)與 c.1294 C > T (p.R432C)。這些突變分布在8個家族中。在功能性分析顯示c.1545 G>C (p.E515D)與 c.1627G>A (p.V543M)造成HEK293 cell電位的改變，且與疾病的嚴重度有相關性。 E515D突變可造成良性新生兒癲癇症後群(benign familial neonatal convulsions)與晚發性兒童癲癇腦病變(continuous spikes and waves during slow-wave sleep); V543M可造成良性新生兒癲癇症後群。進一步探討三個不同基因型(c.1545 G>C (p.E515D)、 c.1627G>A (p.V543M) 與c.638G>A (p.R213Q))的症狀與HEK293 cell細胞電流的改變有顯著的關係。但在HEK293 cell KCNQ2膜蛋白的表現量上，三個不同基因雖與野生型(wild type)KCNQ2有顯著差別，但比對膜蛋白的表現量與症狀並無顯著相關性。結論: KCNQ2突變c.1545 G>C (p.E515D)、 c.1627G>A (p.V543M)與c.1294 C > T (p.R432C)與癲癇有相關性。KCNQ2基因突變造成細胞電流的改變量與症狀嚴重度有明顯關係。基因型 (genotype)對於病人的症狀佔重要的因素。本研究對於釐清:(1)在嬰幼兒癲癇中，KCNQ2基因突變的與疾病的相關性與(2)不同的KCNQ2突變點造成腦部病變及癲癇症狀的差異，其突變點的基因型與表現型的關係有很大的助益。Background: Epilepsy without an identified cause in newborns and children is usually genetic heterogeneity. Pediatric epilepsy caused by a KCNQ2 gene mutation usually manifests as benign familial neonatal convulsions (BFNC) during the first week of life. However, the exact mechanism, phenotype, and genotype of the KCNQ2 mutation are unclear. We analyzed the KCNQ2 wild-type gene and mutations to highlight the important association between the KCNQ2 phenotype and genotype. Patients and methods: We studied the KCNQ2 and KCNQ3 genotype from 75 nonconsanguineous patients with childhood epilepsy without an identified cause (age range: from 2 days to 18 years) and from 55 healthy adult controls without epilepsy. The clinical phenotypes of mutations were compared. KCNQ2, wild-type, and mutant KCNQ2 alleles were transfected into HEK293 cells before whole-cell patch-clamp analysis to HEK293 cells to investigate what functional changes they induced. We also did a functional analysis of the allele proved a benign polymorphism of c.2339A>C (p.N780T) (rs1801475) as negative controls and of the c.638G>A (p.R213Q) mutation that has been confirmed to cause early onset epileptic encephalopathy as a positive control. Results: KCNQ2 variants were detected in 75 patients after comparing them with the control group. After additional FATHMM, SIFT and PolyPhen2 computer-based functional analyses, probable mutation variants indicating disease-associated KCNQ2 mutations were detected in 8 (11%) of the 75 patients. The mutations were c.1545 G>G/C (p.E515D) in four; c.1627G>A (p.V543M) in one; c.1294 C>T (p.R432C) in one; and c.2264 G>A (p.Y755C) in two. All mutation variants were predicted to be deleterious by computer-based functional analyses. Four (5%) of the patients had the E515D KCNQ2 mutation. Their seizure outcomes were favorable, but three had an intellectual disability. Two patients with E515D presented with continuous spikes and waves during slow-wave sleep (CSWS), and the other 2 presented with BFNC. We also analyzed 10 affected family members with the same KCNQ2 mutation: all had epilepsy (8 had BFNC and 2 had CSWS). A functional analysis showed that the recordings of the E515D currents were significantly different (p < 0.05), which suggested that channels with KCNQ2 E515D variants are less sensitive to voltage and require stronger depolarizations to reach opening probabilities than do those with the wild-type or N780T (a benign polymorphism). Neurodevelopmental outcomes were worst in patients with the p.R213Q mutation, better in patients with the p.E515D mutation, and best in patients with the novel p.V543M mutation. The currents in p.E515D and in p.V543M were significantly lower than in the wild-type in homomeric and heteromeric transfected HEK293 cells (p < 0.05). The opening threshold shifted to values that were more positive, and the maximal current induced by strong depolarization was higher in cells with the p.E515D and p.R213Q mutations. Conclusion: KCNQ2 mutations can cause different epileptic phenotypes in children: they lead to BFNC and CSWS. We hypothesize that patients with the KCNQ2 E515D, V543M and R432C mutation are susceptible to seizures. We provide evidence that genotype is involved in determining clinical phenotype, including the seizure frequency and outcome. KCNQ2 should be a candidate gene when diagnosing idiopathic childhood epilepsy.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/18925
Appears in Collections:	[醫學研究所] 博碩士論文

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