【作者】 陈冲；

【导师】 沈璐；

【作者基本信息】 中南大学 ， 神经病学， 2010， 硕士

【摘要】 背景：遗传性痉挛性截瘫(hereditary spastic paraplegia,HSP或SPG)是一组具有高度临床和遗传异质性的神经退行性疾病。SPG4为AD-HSP最常见的亚型,约占单纯型HSP的40%-45%。国外研究显示,在家系内和家系间,SPG4患者的发病年龄、伴随症状、疾病进展速度、病情严重程度等都存在明显的差异。另外,SPG4临床表型还存在外显不全、性别差异、遗传早现等现象,给临床诊断带来困难,某些家系内的轻症病人更是常常被漏诊。目前尚无中国汉族人群大样本SPG4患者临床特点研究。线粒体功能障碍直接或间接地参与了许多神经退行性疾病如遗传性痉挛性截瘫(HSP)、脊髓小脑性共济失调(SCA)、亨廷顿舞蹈病(HD)、阿尔茨海默病(AD)的致病过程。在脊髓小脑性共济失调(SCA)、亨廷顿舞蹈病(HD)和阿尔茨海默病(AD)患者中,线粒体DNA均存在较高的突变率,且与疾病的临床表现有一定相关性。但至今尚无遗传性痉挛性截瘫患者线粒体DNA突变研究报道。目的：明确中国汉族人群遗传性痉挛性截瘫SPG4基因型临床特点；探讨SPG4基因型患者线粒体DNA突变特点,及其与临床表型之间的关系,为揭示线粒体DNA在HSP发病机制中的可能作用提供线索。方法：通过回访获得本课题组前期经基因检测确诊的15个中国汉族人群SPG4家系的所有家系成员的临床资料,应用PCR-直接测序结合MLPA技术对所有家系成员进行SPAST基因突变检测；对上述15个SPG4家系所有家系成员及70例正常对照,应用PCR-直接测序技术进行线粒体DNA中MT-LT1、MT-ND1、MT-C02、MT-TK、MT-ATP8、MT-ATP6基因点突变及线粒体DNA重排突变检测。结果：15个中国汉族人群SPG4家系中的51名家系成员回访后临床诊断为HSP患者,平均发病年龄21.65±14.67岁,平均病程22.90±12.93年；大部分为单纯型,多以双下肢僵硬为首发症状,常伴弓形足(52.9%)、大便障碍(17.6%),而智能障碍(2.0%)少见；男性患者双下肢肌张力障碍较女性常见(p<0.05),部分家系男女临床表型存在明显差异；存在遗传早现现象,均为母系遗传家系。SPAST基因检测诊断SPG4基因型患者共64例,除51例临床诊断患者外,尚有13例外显不全者；1-19岁年龄段外显不全率为40%,20～40岁年龄段外显不全率为21.1%,40岁之后外显不全率为14.3%。线粒体DNA点突变检测中共发现12种新的序列变异,其中C8513T和T8825C同时在SPAST基因突变检测阳性者和家系内正常人中发现,所有序列变异均完全依照母系遗传方式在代间传递,所有子代中均未发现新发mtDNA序列变异。68.8%的SPG4基因型患者、73.3%的家系正常成员、58.6%的正常对照存在至少一种类型的线粒体DNA重排,在SPG4基因型患者、SPG4家系正常人及正常对照各组之间,一种类型和多种类型重排突变的线粒体DNA重排突变阳性率无显著性差异(P>0.05)；SPG4基因型患者和外显不全者各种类型的线粒体DNA重排突变阳性率无显著性差异(P>0.05)；无线粒体DNA重排突变组的SPG4患者平均发病年龄低于线粒体DNA重排突变组,有显著性差异(p<0.05)。结论：1、本组中国汉族人群SPG4患者以单纯型为主,多在青年起病,常以双下肢僵硬或无力为首发症状,与其他人群相比,弓形足、大便障碍常见,智能障碍少见；2、本组中国汉族人群SPG4患者具有明显的遗传和临床异质性,也存在外显不全、性别差异、遗传早现等现象；3、本组中国汉族人群SPG4基因型患者线粒体DNA点突变率低,且与SPG4基因突变和疾病表型无关；4、本组中国汉族人群SPG4基因型患者线粒体DNA重排突变与SPG4基因突变及外显不全无关,但无线粒体DNA重排的SPG4患者发病年龄较早。更多还原

【Abstract】 Background:Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group neurodegenerative disorders. SPAST-associated HSP is the most common type of pure autosomal dominant HSP, accounting for 40% to 45% of such cases.Within families and between families, the age of onset, the accompanying symptoms, the disease progression and the severity index of SPG4 patients are all significantly different. In addition, SPG4 clinical phenotype is still incomplete penetrance, gender differences, genetic anticipation phenomena, which brings difficulties in clinical diagnosis, some patients with mild symptoms within the family are often missed. But there is still no big sample study on the clinical characteristics of Chinese patients with SPG4.Mitochondrial dysfunction, directly or indirectly involved in many neurodegenerative diseases such as hereditary spastic paraplegia (HSP), spinocerebellar ataxia (SCA), Huntington disease (HD), and Alzheimer’s disease (AD) pathogenesis.It has been reported that a higher mitochondrial DNA (mtDNA) mutation rate was found in SCA, HD and AD patients, in addition, these mutations were also correlated with the clinical characteristics of certain degree.So far, there is no report about mtDNA mutation analysis in patients with SPG4.Objective:To identify the clinical features of Chinese Han patients with SPG4.To discuss the mtDNA mutations of SPG4 genotype and its correlation with the phenotype, this might provide clues to reveal the role of mtDNA in HSP pathogenesis.Methods:Detail follow-up and clinical investigation one by one was carried out to obtain clinical data of 15 Chinese Han population SPG4 family members, in which the probands have been diagnosed as SPG4 in our previous study. Sequencing and MLPA technologies were carried out to detect mutation of SPAST gene in all family members.PCR and direct sequencing technologies were carried out to screen for micro-mutations of MT-LT1,MT-ND1,MT-CO2,MT-TK, MT-ATP8,and MT-ATP6 genes and rearrangement mutations of mtDNA in all SPG4 family members and 70 healthy controls.Results:Fifty-one family members from the 15 Chinese Han population SPG4 pedigrees were diagnosed as HSP patients in clinic after follow-up study. Their mean age at onset was 21.65±14.67, and the mean duration was 22.90±12.93 years. Most patients manifested as "pure" forms, and the stiffness in the legs was often the earliest symptom at the beginning of the disease.Compared with other populations,these SPG4 patients often accompanied with arch foot (52.9%) and bowel disorders(17.6%), while mental retardation (2.0%) was rare.Male patients with lower limb dystonia was more frequent than female (P<0.05),and there was obviously difference between male and female phenotype in certain families. The phenomenon of genetic anticipation was also existed in some maternally inherited families. SPAST gene mutations were found in 64 family members from the 15 SPG4 pedigrees by genetic testing.In addition to 51 cases diagnosed by clinical investigation, there were 13 family members without any symptoms and were considered as "incomplete penetrance".The nonpenetrance rate of these SPG4 patients was from 40% in the group of younger age(1-19years) to 21.1% in the age of 20 to 40, and 14.3% after 40.Twelve novel sequence variations were found by micro-mutation detection of mtDNA genes, in which variations C8513T and T8825C were found in both SPG4 patients and normal family members.All of the sequence variations were in full accordance with maternal inheritance in generational transmission, and no novel mtDNA sequence variation was found in filial generation.At least one type of mtDNA rearrangement was found in 68.8% of SPG4 patients,73.3% of normal family members, and 58.6% of normal controls. There was no significant difference of either one type or multiple type rearrangement rates existed among the three groups (P>0.05).In addition, there is also no significant difference of various rearrangement mutation rates existed between SPG4 patients and incomplete penetrance (P>0.05). But patients with no mtDNA rearrangement mutation had a significantly earlier age at onset (P<0.05).Conclusions:Firstly, most of the Chinese Han patients with SPG4 in this group manifest as "pure" forms, with a young onset and stiffness of both lower extremities as the first symptom, and arched feet and bowel disorder symptoms is common while mental retardation is rare compared with other populations.Secondly, Chinese Han patients with SPG4 in this group have significant genetic and clinical heterogeneity, and obviously incomplete penetrance, gender differences, and genetic anticipation. Thirdly, the rate of mtDNA micro-mutation is low in these Chinese SPG4 patients, furthermore, there is no relationship between the SPG4 gene mutation and disease phenotype.Fourthly, mtDNA rearrangement of these Chinese SPG4 patients has no relationship with either SPG4 gene mutation or incomplete penetrance, while patients with no mtDNA rearrangement have a significantly earlier age at onset.更多还原