良性成人家族性肌阵挛性癫痫的遗传学特征及致病基因的定位

发布时间：2018-08-03 19:01

【摘要】：良性成人家族性肌阵挛癫痫(benign adult familial myoclonic epilepsy, BAFME)是一种常染色体显性遗传的，主要以四肢远端震颤、肌阵挛伴或不伴全面强直-阵挛发作的癫痫综合征。至今，全世界约有100个家系在日本、意大利、荷兰、法国、土耳其及中国等地被相继发现并报道。但本病尚未被纳入到2001年国际抗癫痫联盟的癫痫综合征的分类当中。本病早期报道源于日本，日本学者Wakeno在1975年报道了一个以家族性震颤和癫痫发作为表现的家系，而后陆续报道了多个类似家系，，并经电生理研究确定其震颤均来源于大脑皮质，且均为常染色体显性遗传。1991年Yasuda在报道两个家系的基础上，总结了本病具有良性病程和常染色体显性遗传的特点，首次提出了“良性成人家族性肌阵挛癫痫”的概念。在1999年，Mikami使用连锁分析的方法将患有该病的一个3代27人的家系致病基因定位于8q23.3-24.1，从而发现了本病的第一个致病基因位点。2003年，Strianon对一个意大利的BAFME家系进行基因连锁分析后发现，其致病基因与2p11-q12.2具有连锁关系，又发现了一个新的致病基因位点，从而说明了该病的遗传异质性。2006年，我国邓飞燕等人通过对一BAFME大家系的遗传学分析，将致病基因定位于10p15；2010年Depienne等人将一法国FCMTE家系致病基因定位于5p15.31-p15；2012年Yeetong将一泰国BAFME家系致病基因定位在3q26.32-3q28。然而，目前为止尚未克隆出该病的致病基因。仅有日本学者Atsushi等人在2003年提出了CSMD3可能为BFAME的致病候选基因。CSMD3是一种编码跨膜蛋白的较大的基因，位于人类染色体8q23.3-q24.1区段，即BAFME被定位的区段。该基因包含73个外显子，长度跨越1.2Mb。主要在成人及胎儿的脑组织中表达。自1991年Yasuda首次提出了BAFME的概念以来，BAFME综合征的研究不超过20余年历史，其中大多为病例报道，仅有屈指可数的几个遗传学研究报道，虽取得了一些研究成果，但是尚缺乏系统的对BAFME致病基因研究报道。尤其我国对BAFME家系很少有研究报道，对多代多人发病的大家系报道更是少见。我国作为人口大国，家系患病人群数量也相对较多，而BAFME的基因突变特点却不明确，因此对于中国BAFME家系的调查研究有很大意义。而且积极开展本病的临床及分子遗传学研究有助于提高临床医生对本病的认识、了解本病及其他特发性癫痫的分子发病机制，并为本病的诊断、治疗及遗传咨询提供基础。课题组调查的为辽宁省沈阳市的一个家系，对家系调查分析后，确诊为良性成人家族性肌阵挛性癫痫。课题组与该家系成员进行沟通并讲述实验研究的目的以及意义，在其签署知情同意书后，对其进行遗传学调查、病史采集、临床资料搜集、辅助检查及采集外周血。该家系共4代43人，患者9人，其中男患2人，女患7人。现存4代40人，男女患病机会均等，除第4代尚未到发病年龄外，其余每代均有发病患者，符合常染色体显性遗传特点。患者发病年龄多在30～40岁左右，呈良性病程。所有发病患者的临床表现都比较相似，大多以四肢远端震颤为首发症状，随后或几年后出现全身性强直-阵挛发作，服用抗癫痫药物对缓解症状有效，β受体阻滞剂或饮酒无效，可除外原发性震颤。该家系是一个遗传关系很明确，发病人数较多的呈常染色体显性遗传的BAFME大家系。采集该家系成员及部分成员配偶的外周血，提取白细胞DNA进行致病基因定位研究。实验思路为首先采用聚合酶链式反应（PCR）及PCR产物测序法对家系中先证者进行BAFME可疑致病基因CSMD3的突变检测；如果结果为阴性，则对已知的BAFME基因进行STR荧光标记物连锁分析，明确是否与目前已知的几个染色体区段连锁，尽可能将该家系的致病基因定位到某个染色体区段；如果结果仍为阴性，与现已知的基因位点均不连锁，致病基因不在几个候选染色体区段，则可以选择全基因组扫描定位法。将致病基因定位在某染色体的特定区域后，进行候选基因筛选，选择该区域内与癫痫有关的基因进行突变检测，从而明确该家系的致病基因。首先，应用PCR产物测序分析法对先证者CSMD3基因的73个外显子进行PCR扩增产物测序，测序结果与GenBank人类CSMD3gDNA序列进行比较，未发现任何DNA序列变异，既没有发现多态也没有发现与疾病相关的突变，说明本家系不存在CSMD3基因突变。而后，对目前已报道的5个染色体区段进行连锁分析，结果显示染色体8q23.3-q24.1、2p11.1-q12.2、3q26.32-3q28、10p15区段的多个STR标记连锁分析结果显示不支持连锁，因此考虑本家系致病基因并不在上述4个染色体区段。而针对5p15.31-p15首先选择了4个STR标记，连锁分析结果表明现不能完全肯定与否定致病基因是否位于该区段，之后，为进一步明确结果，我们在该区段增加了8个STR位点，结果显示，D5S486在θ=0.0时，LOD值为2.8，实验结果支持该家系致病基因与该位点的连锁,考虑致病基因在5p15.31-p15染色体区段。本实验研究了一良性成人家族性肌阵挛性癫痫的临床症状、遗传学特点并定位了致病基因位点。为良性成人家族性肌阵挛性癫痫的研究提供了经验，也为该病的遗传学研究提供了思路,并首次将国内的良性成人家族性肌阵挛性癫痫家系致病基因定位在染色体5p15.31-p15区段。
[Abstract]:Benign adult familial myoclonus epilepsy (benign adult familial myoclonic epilepsy, BAFME) is a kind of autosomal dominant hereditary, mainly with distal tremor, myoclonus, or no total tonic clonic seizure syndrome. Up to now, about 100 families in the world are in Japan, Italy, Holland, France, Turkey and China. The disease has not been included in the classification of epilepsy syndrome of the International Antiepileptic Union in 2001.
The early report of this disease originated in Japan. In 1975, Japanese scholar Wakeno reported a family with familial tremor and epileptic hair. After that, several similar families were reported, and their tremors were all derived from the cerebral cortex by electrophysiological study, and all two families were reported to be the dominant hereditary.1991 year Yasuda. On the basis of this, the concept of "benign adult familial myoclonus epilepsy" was first proposed. In 1999, Mikami used the method of linkage analysis to locate the pathogenic gene of a family of 3 generation and 27 people with the disease in 8q23.3-24.1, and found the first one of the disease. The genetic linkage analysis of a Italy BAFME family in.2003 was found by Strianon. The pathogenic gene was linked to 2p11-q12.2, and a new gene locus was found. The genetic heterogeneity of the disease was explained in.2006 years. Deng Feiyan and others in our country passed the genetics of a BAFME family. Analysis, the pathogenic gene was located in 10p15; in 2010, Depienne et al. Located a French FCMTE family gene in 5p15.31-p15; in 2012 Yeetong, a BAFME family in Thailand was located in 3q26.32-3q28., but so far, the pathogeny gene of the disease has not been cloned. Only Japanese scholar Atsushi et al. In 2003 3 the possible pathogenetic candidate gene for BFAME,.CSMD3, is a large gene encoding the transmembrane protein, located in the 8q23.3-q24.1 section of the human chromosome, the section of the BAFME that is located. The gene contains 73 exons, and the length of the 1.2Mb. is mainly expressed in the brain tissue of the adult and the fetus.
Since Yasuda first proposed the concept of BAFME in 1991, the study of BAFME syndrome is not more than 20 years old, most of which are cases reported, only a few of the few genetic studies have been reported. Although some research results have been obtained, there is no systematic report on the study of BAFME pathogenic genes. Especially in China, there are few BAFME families in China. It is more rare to report on the incidence of the onset of multiple generation of people. As a country with a large population, the number of families with families is relatively large, but the genetic mutation characteristics of BAFME are not clear. Therefore, it is of great significance for the investigation and study of the Chinese BAFME family. To improve the understanding of the disease by clinicians and to understand the molecular pathogenesis of the disease and other idiopathic epilepsy, and to provide the basis for the diagnosis, treatment and genetic counseling of this disease.
The subject group investigated a family in Shenyang city of Liaoning province. After the investigation and analysis of the family, the group was diagnosed as familial myoclonic epilepsy of benign adults. The group communicated with the members of the family and explained the purpose and significance of the experimental study. After signing the informed consent book, the group had a genetic investigation, a history collection, and a clinical data search. There were 4 generations and 43 patients and 9 patients, including 2 men and 7 women, 4 generation 40, equal opportunities for both men and women, except for fourth generations that had not reached the age of onset, and the rest of the generation had the characteristics of autosomal dominant inheritance. The age of the patients was about 30~40 years old and had a benign course. The clinical manifestations of all the patients were similar, mostly with distal tremor of the extremities as the first symptom, followed by a generalized tonic clonic seizure later or a few years later, taking antiepileptic drugs to relieve the symptoms, the beta blocker or alcohol ineffective, except for the primary tremor. The family is a clear genetic relationship and the number of patients. A large number of autosomal dominant BAFME families.
The peripheral blood of the spouses of the family members and some members of the family was collected to extract the leukocyte DNA for the location of the pathogenic gene. The experiment was to detect the mutation of the suspected BAFME gene CSMD3 by polymerase chain reaction (PCR) and the sequencing of PCR products. If the result was negative, the known BAFME gene was found. A linkage analysis of STR fluorescent markers is carried out to determine whether it is possible to locate the family's pathogenic gene to a certain chromosome segment as far as possible, and if the result is still negative, it is not linked to the known gene loci, and the pathogenic gene is not in several candidate chromosome segments, then the whole gene can be selected. After locating the pathogenic gene in a specific region of a chromosome, the candidate gene was screened and the genes related to epilepsy in the region were selected for mutation detection so as to identify the pathogenic genes of the family.
First, the PCR product sequencing analysis was used to sequence the PCR amplification products of the 73 exons of the CSMD3 gene of the precursor. The sequencing results were compared with the CSMD3gDNA sequence of the GenBank human, and no mutation of the DNA sequence was found. Neither the polymorphism nor the disease related mutations were found, which showed that the family did not have the CSMD3 gene mutation. After the linkage analysis of the 5 reported chromosomal segments, the results showed that the results of multiple STR marker linkage analysis in the chromosome 8q23.3-q24.1,2p11.1-q12.2,3q26.32-3q28,10p15 section showed that the linkage was not supported. Therefore, the pathogenic genes in the family line were not in the 4 chromophore segments, and 4 were selected for 5p15.31-p15. STR markers, the result of linkage analysis showed that it was not fully affirmed and denying that the pathogenic gene was located in the area, and then, to further clarify the results, we added 8 STR loci to the section. The results showed that the LOD value was 2.8 when D5S486 was at theta =0.0. The experimental results supported the linkage of the pathogenic gene of the family with the site, considering the pathogenic gene in 5. P15.31-p15 chromosome section.
This study studied the clinical symptoms, genetic characteristics and loci of a benign adult familial myoclonic epilepsy, providing experience for the study of familial myoclonic epilepsy in benign adults, and for the genetic study of the disease, and for the first time the family myoclonic epilepsy home in China. The pathogenic gene was located in chromosome 5p15.31-p15 region.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1

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