原发性肌张力障碍的遗传学研究

发布时间：2018-05-04 14:08

本文选题：原发性 + 肌张力障碍　；参考：《北京协和医学院》2014年博士论文

【摘要】：背景肌张力障碍(dystonia)(包括扭转痉挛、眼睑痉挛、口下颌肌张力障碍、痉挛性斜颈、痉挛性构音障碍、书写痉挛等)是一类病理生理复杂、机制未明的运动障碍病。临床症状以肌肉不自主收缩为特征,常导致扭转、重复性运动或异常姿势,部分患者仅表现为震颤。迄今为止,全世界共发现20余种基因与原发性肌张力障碍有关,其中DYT25/GNAL基因于2012年新近发现,国内外对其研究处于初步阶段,国内尚缺乏大规模人群研究。目的本研究旨在一个大样本的中国原发性肌张力障碍患者中进行GNAL基因突变筛查,明确我国肌张力障碍人群GNAL基因突变频率及常见突变位点。方法纳入就诊于北京协和医院神经内科运动障碍病门诊的原发性肌张力障碍患者214例(阳性家族史患者12例),同时募集100例健康者作为对照组,留取外周血提取基因组DNA。对GNAL基因全部12个外显子和5'UTR区进行PCR扩增,产物直接测序。测序发现的变异在对照组中用同样方法进行筛查,并运用生物信息学软件分析预测变异对蛋白的影响。同时检索国内外GNAL基因突变筛查研究,计算各研究中GNAL基因的突变频率,并与本研究结果进行对比。结果本研究对214例原发性肌张力障碍患者进行GNAL基因突变筛查,结果发现11个变异位点,分别是：c.-37AG, c.-41TC, c.-111CT, c.-116AG, c.-142GC, c.-485TC, c.15CT (p.G5=), c.30GT (p.T10=), c.44GA (p.G15D), c.1059CT (p.A353=), c.360CT (p.S120=)。正常对照组未发现以上变异。其中c.360CT(p.S120=), c-111CT, c.-41TC, c.-37AG, c.30GT (p.T10=)和c.44GA(p.G15D)为已知SNP位点,编号分别为rs76888098, rs61495657, rs9303742, rs200432196, rs78167172和-199761315,认为以上6个变异位点为良性变异,非致病突变。c.15CT(p.G5=),c.30GT(1).T10=),c.1059CT(p.A353=)和c.360CT(p.S120=)属同义突变,编码氨基酸未发生变化,认为是非致病突变。应用Mutation Taster对c.-485TC, c.-142GC, c.-116AG进行预测,结果显示为多态性改变。总体而言,本研究未发现明确致病突变。对国内外文献进行回顾,不同种族人群中原发性肌张力障碍患者GNAL基因突变频率差异很大,在0%-15.38%不等。结论本研究对中国214例原发性肌张力障碍患者进行GNAL基因突变筛查,结果未发现明确致病突变,认为GNAL基因突变非中国人群原发性肌张力障碍的常见致病基因。背景痉挛性斜颈(cervical dystonia,CD)是临床上最常见的局灶型肌张力障碍,以颈部肌肉不自主收缩导致头颈部运动和姿势异常为特征。病因尚不明确,遗传因素可能参与其中。最近研究发现,DYT23/CIZ1、DYT24/AN03和DYT25/GNAL基因突变可能与原发性肌张力障碍,特别是颅颈段肌张力障碍相关。目前国内外关于以上3个基因的研究较少,国内尚无原发性痉挛性斜颈家系的遗传学研究。目的本研究旨在对中国汉族人群原发性痉挛性斜颈家系进行CIZ1、AN03和GNAL基因突变筛查,明确我国家族性原发性痉挛性斜颈患者的常见突变基因及其突变频率。方法纳入就诊于北京协和医院神经内科运动障碍病门诊的中国汉族原发性痉挛性斜颈家系20个,同时募集100名健康受试者作为对照组,留取外周血提取基因组DNA。对先证者的CIZ1、AN03.GNAL基因全部外显子和GNAL基因5'UTR区进行PCR扩增,产物直接测序。测序发现的变异在对照组中用同样方法进行筛查,并运用生物信息学软件分析预测变异对蛋白的影响。计算我国原发性痉挛性斜颈家系各基因的突变频率,并与既往研究结果进行对比。结果本研究对中国汉族20个原发性痉挛性斜颈家系进行CIZl、AN03和GNAL基因突变筛查,结果：在CIZ1基因发现两个同义突变,分别为c.696GA(p.P232＝)和c.1227CA(p.P409=),编码氨基酸未发生变化,SNP编号为rs45579839和rs45559035,考虑非致病突变；在AN03基因发现7个变异位点,分别为c.-11GT,c.1158AG(p.L386=),c.1692AC(p.A564=),c.2682CT(p.P894=),c.2478CG,(p.T826=),c.2520TG(p.R840=)和c.2540AG(p.Y847C),健康对照组检测到c.1158AG(p.L386=),c.1692AC(p.A564=)和c.2682CT(p.P894=)。其中c.-11GT,c.1158AG(p.L386=),c.1692AC(p.A564=)和c.2682CT(p.P894=)为已知SNP位点,编号分别为rS143269109,rs2663168,rs11604768和rs10835051.c.2478CG(p.T826=)和c.2520TG(p.R840=)为同义突变,编码氨基酸未发生变化,认为以上6个变异为非致病突变。在家系S中发现错义突变c.2540AG(p.Y847C),编码氨基酸发生变化,由酪氨酸变为半胱氨酸,对照组未见此变异,既往研究未见报道,应用软件Sift、Polyphen-2和Mutation Taster对其进行预测,结果均显示有害,应用Clustal Omega软件分析发现Y847氨基酸在物种进化中相对保守,认为c.2540AG(p.Y847C)是致病突变。家系S中患者的主要临床特征是痉挛性斜颈和肌张力障碍性震颤。先证者中未检测到GNAL基因突变。结论本研究对中国汉族20个原发性痉挛性斜颈家系进行CIZl、ANO3、GNAL基因突变筛查,结果在家系S中发现AN03基因新的致病突变：c.2540AG(p.Y847C),突变频率为5%(1/20)。未见CIZl和GNAL基因致病突变,认为CIZ1和GNAL基因突变非中国汉族痉挛性斜颈家系的常见原因。背景肌张力障碍(dystonia)(包括扭转痉挛、眼睑痉挛、口下颌肌张力障碍、痉挛性斜颈、痉挛性构音障碍、书写痉挛等)是一类病理生理复杂、机制未明的运动障碍病。临床以肌肉不自主收缩为特征,常导致扭转、重复性运动或异常姿势,部分患者仅表现为震颤。研究显示感觉运动皮层的可塑性异常可能参与肌张力障碍的发生。脑源性神经生长因子(brain-derived neurotrophic factor, BDNF)是突触和神经可塑性的重要调节因子,BDNF基因中的单核苷酸多态性Val66Met (G196A)可能与肌张力障碍相关,但各个研究结果不一致。目的探讨BDNF基因Val66Met (rs6265)多态性与我国原发性肌张力障碍间的相关性。方法纳入就诊于北京协和医院神经内科运动障碍病门诊的原发性肌张力障碍患者252例,同时招募214例既往健康、无神经系统疾病、性别年龄与患者相匹配的健康体检者作为对照组,留取外周血提取基因组DNA。采用SNaPshot技术对BDNF基因中Val66Met (rs6265)多态性进行检测。用SPSS13.0统计软件进行数据处理,运用方差分析验证组间基因型及等位基因分布差异。结果本研究对中国252例原发性肌张力障碍患者和214例健康对照进行BDNF基因Val66Met (rs6265)多态性检测,结果发现原发性肌张力障碍组与对照组、痉挛性斜颈组和对照组之间基因型和等位基因分布无统计学差异(P值分别为0.309和0.803),各基因型组别之间起病年龄也未见明显差异(P0.05)。结论本研究在中国原发性肌张力障碍和健康人群中进行BDNF基因Val66Met(rs6265)多态性检测,结果未发现两者基因型和等位基因分布存在统计学差异(P0.05),提示Val66Met (rs6265)多态性与中国原发性肌张力障碍无相关性,非中国原发性肌张力障碍人群的遗传易感因素。
[Abstract]:background
Dystonia (dystonia) (including torsional spasm, blepharospasm, dyspomia, spasmodic torticollis, spasmodic dysarthria, writing spasm, etc.) is a kind of pathophysiological complex, dysfunctional dyskinesia. Clinical symptoms are characterized by involuntary contraction of muscles, often causing torsion, repetitive movement or abnormal posture, and partial suffering. So far, more than 20 genes in the world have been found to be related to the primary dystonia, in which the DYT25/GNAL gene was found in 2012, and the research at home and abroad is in the initial stage, and there is still a lack of large population research at home.
objective
This study aims to screen the GNAL gene mutation in a large sample of Chinese patients with primary dystonia and to identify the frequency of GNAL gene mutation and the common mutation loci in the people with dystonia in China.
214 patients with primary dystonia (12 cases of positive family history) were enrolled in the Department of Neurology, Peking Union Medical College Hospital, and 100 healthy people were recruited as the control group. The genomic DNA. was extracted from the peripheral blood, and all the 12 exons and 5'UTR regions of the GNAL gene were amplified by PCR, and the products were sequenced and sequenced. The current mutation was screened in the control group by the same method, and the effect of mutation on the protein was predicted by bioinformatics software. At the same time, the GNAL gene mutation screening study at home and abroad was searched to calculate the mutation frequency of the GNAL gene in each study, and the results were compared with the results of the study.
Result
In this study, 214 patients with primary dystonia were screened for GNAL gene mutation. The results showed that 11 variation sites were: c.-37AG, c.-41TC, c.-111CT, c.-116AG, c.-142GC, c.-485TC, c.15CT (p.G5=), c.30GT (p.T10=), c.44GA. C.360CT (p.S120=), c-111CT, c.-41TC, c.-37AG, c.30GT (p.T10=) and c.44GA (p.G15D) are known SNP loci. ) and c.360CT (p.S120=) is synonymous mutation, the encoding amino acid is not changed, considered as a non pathogenic mutation. Mutation Taster was used to predict c.-485TC, c.-142GC, c.-116AG, and the results showed polymorphic change. The frequency of GNAL gene mutation in dystonia patients is quite different from that in 0%-15.38%.
conclusion
In this study, the GNAL gene mutation was screened in 214 patients with primary dystonia in China. The results were not found to be clear, and the GNAL gene mutation was not a common cause of primary dystonia in Chinese people.
background
Cervical dystonia (CD) is the most common focal muscular dystonia in the clinic, which is characterized by involuntary contraction of the neck muscles. The etiology is not clear and genetic factors may be involved. Recent studies have found that the mutations in the DYT23/CIZ1, DYT24/AN03 and DYT25/GNAL genes may be associated with the primary muscle. Dystonia, especially the dystonia of cranioceptic segment, is related to dystonia. There are few studies on the above 3 genes at home and abroad, and there is no genetic study of primary spasmodic torticollis at home.
objective
The purpose of this study was to screen the CIZ1, AN03 and GNAL mutations of primary spasmodic torticollis in Chinese Han population, and to identify the common mutation genes and the frequency of mutation of the familial primary spasmodic torticollis.
Method
20 Chinese Han Primary spasmodic torticollis families were enrolled in the neurology department of Peking Union Medical College Hospital, Peking Union Medical College Hospital, and 100 healthy subjects were recruited as the control group. The genomic DNA. of the peripheral blood was extracted from the peripheral blood, and the whole exon of the AN03.GNAL gene and the 5'UTR region of the GNAL gene were amplified by PCR, and the product was directly produced. The mutations were screened in the control group by the same method, and the effects of the variation on the protein were predicted by the bioinformatics software. The mutation frequencies of the genes in the primary spasmodic torticollis of our country were calculated and compared with the previous research results.
Result
In this study, CIZl, AN03 and GNAL mutations were screened in 20 Chinese primary spasmodic torticollis families. The results showed that two synonymous mutations were found in the CIZ1 gene, c.696GA (p.P232 =) and c.1227CA (p.P409=), the encoded amino acids were not changed, the SNP number was rs45579839 and rs45559035, and the non pathogenic mutation was considered, and the AN03 gene was in the AN03 gene. C.-11GT, c.1158AG (p.L386=), c.1692AC (p.A564=), c.2682CT (p.P894=), c.2478CG, c.2520TG (p.T826=), c.2520TG (p.R840=) and c.2540AG. ) for the known SNP loci, rS143269109, rs2663168, rs11604768 and rs10835051.c.2478CG (p.T826=) and c.2520TG (p.R840=) are synonymous mutations, and the encoded amino acids are not changed. The above 6 variations are considered as non pathogenic mutations. The missense mutagenesis c.2540AG (p.Y847C) is found in the family S, and the coded amino acids change, and the tyrosine changes are changed. For cysteine, no previous study was found in the control group. The application software Sift, Polyphen-2 and Mutation Taster were used to predict it. The results were all harmful. The Clustal Omega software analysis found that the Y847 amino acids were relatively conservative in the evolution of species, and c.2540AG (p.Y847C) was a pathogenic mutation. The main factors in the family S were the patients. The clinical features were spasmodic torticollis and dystonia tremor. No mutations in the GNAL gene were detected in the proband.
conclusion
In this study, the CIZl, ANO3, GNAL gene mutations were screened in 20 Chinese primary spasmodic torticollis families. The results showed a new AN03 gene mutation in family S: c.2540AG (p.Y847C), and the mutation frequency was 5% (1/20). No CIZl and GNAL gene mutations were found, and CIZ1 and GNAL genes were identified as non Chinese Han spasmodic torticollis families. Common reasons.
background
Dystonia (dystonia) (including torsional spasm, blepharospasm, dyspomia, spasmodic torticollis, spasmodic dysarthria, writing spasms, etc.) is a kind of pathophysiological complex, dysfunctional dyskinesia characterized by involuntary contraction of muscles, often causing torsion, repetitive movement or abnormal posture, and partial patients only Brain-derived neurotrophic factor (BDNF) is an important regulator of synapse and nerve plasticity, and the single nucleotide polymorphisms Val66Met (G196A) in the BDNF gene may be associated with dystonia. Guan, but the results of the various studies are inconsistent.
objective
Objective to investigate the correlation between BDNF gene Val66Met (rs6265) polymorphism and primary dystonia in China.
Method
252 patients with primary dystonia in the clinic of Neurology Department of Neurology of Peking Union Medical College Hospital were enrolled, and 214 cases of health, no nervous system disease, sex age and patients matched with healthy persons were recruited as control group, and SNaPshot technique was used for Val66M in the BDNF gene in the BDNF gene of the peripheral blood extraction group. ET (rs6265) polymorphism was detected. Data were processed by SPSS13.0 statistical software. Variance analysis was used to verify the difference of genotype and allele distribution among groups.
Result
In this study, the BDNF gene Val66Met (rs6265) polymorphism was detected in 252 patients with primary dystonia and 214 healthy controls. The results showed that the distribution of genotypes and alleles between the primary dystonia group and the control group had no statistical difference between the spastic torticollis group and the control group (P value was 0.309 and 0.803, respectively). There was no significant difference in age between groups (P0.05).
In this study, the BDNF gene Val66Met (rs6265) polymorphism was detected in Chinese primary dystonia and healthy people. The results were not found to be statistically different between the genotype and allele distribution (P0.05), suggesting that Val66Met (rs6265) polymorphism was not associated with primary dystonia in China and non Chinese primary dystonia. Genetic susceptibility factors of the population.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R746

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