患与未患IGE的同卵双胞胎致病基因定位与功能的初步研究

Idiopathic generalized epilepsy (IGE) is a group of epileptic disorders that are believed to have a strong underlying genetic basis. Patients with an IGE subtype are typically otherwise normal and have no structural brain abnormalities. Patients also often have a family history of epilepsy and seem to have a genetically predisposed risk of seizures. IGE tends to manifest itself between early childhood and adolescence although it can be eventually diagnosed later. The genetic cause of some IGE types is known, though inheritance does not always follow a simple monogenicmechanism. Monozygotic (MZ) or “identical” twins have been widely studied to dissect the relative contributions of genetics and environment in human diseases.We plan to use second-generation methods for targeted sequencing of all protein-coding regions (‘exomes’), to find a gene of monozygotic twins discordant for IGE. We demonstrate that targeted capture and massively parallel sequencing represents a cost-effective, reproducible and robust strategy for the sensitive and specific identification of variants causing proteincoding changes in individual human genomes. Also, to detect genomic alteration, the copy number analysis of the IGE case was performed using Affymetrix Genome-Wide HumanSNP Array 6．0 and Affymetrix Genotyping Console 3.0 software was used for quality control. It is generally believed that most affected individuals were suffering from an unknown genetic defect resulting in manifesting the clinical features. So the discovery of biological basis of IGE will contribute to the scientific understanding of this syndrome, as well as idiopathic epilepsy. It is expectable that the convincing genetic findings of IGE were extraordinarily rare in all available publications. Fortunately, the collection of academic valued pedigrees, and application of exome sequencing and the emergence of modern high-resolution microarray-based technologies combined with genomewide linkage analysis skills provided the researchers a great opportunity to localize the genetic loci of IGE and to investigate its pathogenic mutations in detail.

特发性全面性癫痫（IGE）特发性癫痫是一类具有高度临床表现异质性和遗传异质性的神经系统疾病,其致病机理尚不清楚。本课题组已收集一对IGE同卵双胞胎，一为IGE患者，另一为正常对照，临床表现典型，IGE诊断明确。我们拟采用全基因组外显子测序技术与芯片全基因组扫描相结合寻找IGE 的致病基因。通过全基因组外显子测序与芯片全基因组扫描，定位IGE疾病基因位点；筛选候选基因，对IGE双胞胎进行突变分析，确定IGE致病基因。并根据致病基因的结构和功能特点，进一步进行功能分析，初步探讨基因突变致IGE的机理，为揭示特发性癫痫的发病机制及开发新的抗癫痫药物提供理论依据。

癫痫是一种常见的中枢神经系统疾病，依据病因可分为特发性、症状性和隐源性，按部位又可分为全身性和局灶性。群体遗传学、双生子和脑电图研究都表明，多种类型.的癫痫都具有一定的遗传性。本课题组已收集收集了同卵双胞胎一对，女性，24岁，一为癫痫患者，另一为正常对照。我们通过全基因组外显子测序结合连锁分析查找A.DCTE的致病基因。明确家系诊断后，采集家系成员（患者5例，正常人11例）外周血，提取DNA。在2p11.1-q12.2区域选取5个，5p15.31-p15区域4个以及8q23.3-q24.1区域10个微卫星标记，进行部分性基因组扫描。采用LINKAGE 软件包v5.1中Mlink软件进行两点间连锁分析，计算LOD值。并利用Cyrillic 2.1软件，对含有基因扫描数据的患者文件进行单体型分析。根据单体型在患者个体中共享的情况，并根据交换的发生可得到最终由所有患者共享的一段最小区域。将1个病例、1个正常对照进行全基因组外显子测序。通过PCR测序方法，将筛选得到的致病基因及其突变位点在家系内的另外2例正常对照进行家系内验证，之后，在更大范围的正常人群中验证在前两步中发现的存在于候选致病基因中的所有功能突变。结果发现，该名患者存在VWF基因exon23:c.2602A>T杂合突变，对照为纯合子，存在共分离现象。该突变位点在正常人群中不存在。从而在遗传学上初步证实 VWFc.2602A>T 为该家系的致病突变。本研究采用最先进的全基因组外显子测序方法，成功发现了IGE的致病基因VWF，并进一步证实了全基因组外显子测序为一种发现稀有单基因疾病致病基因有力、有效的方法。

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