常染色体隐性遗传早发性帕金森病2例家系分子遗传学研究

发布时间：2018-03-29 04:16

本文选题：早发性帕金森病　切入点：常染色体隐性遗传　出处：《郑州大学》2017年硕士论文

【摘要】：背景帕金森病(Parkinson’s disease,PD)是一种常见的具有复杂的临床表现的神经变性疾病,其特征性病理改变在于黑质中多巴胺能神经元的进行性丧失以及残存神经元中路α-突触核蛋白的聚集。临床表现主要为出现锥体外系的功能紊乱的典型症状,如运动迟缓、静止性震颤、肌强直和姿势反射异常等,严重亦可有记忆障碍和痴呆。全球65岁以上人口中,该病的发病率为1-2%,80岁以上人口的发病率为4%。目前已明确散发性PD与各种环境因素相关,包括暴露于神经毒素(MPTP)、杀虫剂和除草剂,如鱼藤酮、百草枯等。然而随着研究的日益深入,有越来越多的证据表明遗传因素在疾病的发病过程中起着重要的作用,由基因突变引起的家族性PD约占PD患者的10-15%。早发型帕金森病(Early-onset Parkinson’s disease,EPOD)是帕金森病的一种形式,首发症状主要出现在40岁之前,且多为常染色体隐性遗传,称为常染色体隐性遗传性早发型帕金森病(Autosomal recessive early-onset parkinsonism,AREP),有较为特征性的临床表现,如起病年龄小(≤40岁),病程长,进展缓慢,病程早期出现肌张力障碍,睡眠可使症状缓解,认知功能影响小,对左旋多巴制剂反应好等。现已证实一些基因的变异与该病的发病有关,包括Parkin,PINK1,DJ1和LRRK2等,其中以Parkin基因最为常见,进一步研究各基因突变引起选择性黑质多巴胺能神经元变性的机制,明确各临床表型与不同基因之间对应的关系,对于我们最终搞清帕金森病的发病机制、研制开发相应的治疗药物具有极其重要的意义。目的对河南省两个四代多人发病的AREP家系的临床资料进行分析,对该疾病有更深层次的认识,并进行基因突变位点检测,为AREP的病因及发病机制研究提供遗传学线索,寻找基因突变与临床表型之间的关联。方法1.对河南省某县两个四代多个家系成员中多人发病的PD大家系进行实地调查,与家系成员签署知情同意书后搜集临床资料,进行PD量表测定及相关辅助检查,绘制家系遗传学图谱,总结该家系PD发病特点,临床诊断为常染色体隐性遗传性早发型帕金森病。2.两个家系共24人的血液由随访获得,酚/氯仿法提取其基因组DNA,采用聚合酶链式反应(polymerase chain reaction,PCR)方法特异性扩增,琼脂糖凝胶电泳检测扩增产物,经分离纯化后通过DNA测序方法获得其序列,然后与正常序列进行对比分析。结果1.2个AREP家系共有6名患者,发病年龄17~29岁,平均(22.5±5.5)岁,男5例,女1例。2.临床表型:家系Ⅰ:先证者(Ⅲ6)开始出现双下肢无力为主,肌力Ⅳ级,后逐渐出现四肢僵硬伴静止性震颤,运动迟缓,行走困难,并呈“晨轻暮重”,午睡后减轻,口服左旋多巴治疗,开始0.125g/次,每12小时1次,症状基本消失;该家系先证者的弟弟有类似临床表现。家系Ⅱ:先证者(Ⅲ12)开始出现发作性肌张力障碍,表现为姿势异常、易跌倒,后出现四肢静止性震颤伴肌张力增高,呈“晨轻暮重”,午睡后减轻,发病1年后口服美多芭治疗,开始服0.0625g/次,每天1次,症状可完全缓解;该家系中先证着大哥、二哥有类似的更严重的临床表现。3.基因检测结果:家系Ⅰ:先证者送检标本发现Parkin基因exon3杂合和exon4纯合缺失突变,先证者母亲(Ⅱ4)、伯伯(Ⅱ7)弟弟(Ⅲ11)发现Parkin基因exon3和exon4杂合缺失突变;先证者父亲(Ⅱ3)、叔叔(Ⅱ5)发现Parkin基因exon4杂合缺失突变;家系Ⅱ:先证者Parkin基因exon7上第850号核苷酸由鸟嘌呤变异为胞嘧啶(c.850GC)导致第284号氨基酸由甘氨酸变异为精氨酸(p.G284 R),先证者之子(Ⅳ10)、先证者大哥(Ⅲ5)、先证者二哥(Ⅲ7)该位点杂合变异,先证者其余家系成员该位点无变异。结论1.结果可得Parkin基因突变是该研究收录的两个AREP家系的致病基因。2.研究可得Parkin基因突变方式有点突变、片段缺失,常见为exon3、exon4、exon7突变。3.Parkin基因突变导致的AREP的家族成员患者存在临床表型异质性或外显不全的可能性,也存在基因突变的异质性。4.对于临床症状提示可疑AREP的患者,在排除了多巴胺反应性肌张力障碍以及继发性PD可能性后,可行Parkin基因及其他常见突变基因筛查。5.本研究得出结果所示致病基因为已知类型,未发现新突变,但以此工作流程形式继续搜集患病家系,可能发现新的基因突变,以利于临床诊断或针对位点的药物研发。
[Abstract]:The background of Parkinson's disease (Parkinson 's disease, PD) is a common clinical manifestation with complex neurodegenerative disease, characterized by pathological changes in the substantia nigra dopaminergic neurons were lost and the remaining neurons, aggregation of alpha synuclein. Clinical manifestations were typical symptoms of disorders extrapyramidal, such as bradykinesia, tremor, rigidity and postural reflex abnormalities, may have serious memory disorders and dementia. The global population over the age of 65, the incidence of the disease is 1-2%, the incidence of the population over the age of 80 at a rate of 4%. has been clearly sporadic PD associated with various environmental factors, including exposure to the neurotoxin (MPTP), pesticides and herbicides, such as rotenone, paraquat. However, with the deepening research, there is growing evidence that genetic factors in the pathogenesis of the disease in An important role by gene mutation in familial PD caused by about PD 10-15%. in patients with early onset Parkinson disease (Early-onset Parkinson s disease, EPOD) is a form of Parkinson's disease. The first symptom mainly occurred before the age of 40, and is known as autosomal recessive, autosomal recessive early onset Parkinson disease (Autosomal recessive early-onset parkinsonism, AREP), clinical manifestations are relatively characteristic, such as age of onset is small (less than 40 years old), long course of disease, slow progress in the early course of emergence of dystonia, sleep can ease the symptoms, cognitive function, little influence on good response to levodopa. It has been confirmed that a few mutations associated with the onset of the disease, including Parkin, PINK1, DJ1 and LRRK2, in which Parkin gene is the most common, further study the mutations induced by selective dopaminergic neurons The degeneration mechanism, the corresponding relationship between the clinical phenotype and different genes, we finally find out the pathogenesis of Parkinson's disease, has very important significance to develop the corresponding drugs. AREP family on the incidence of Henan province two four generations of clinical data analysis, have a deeper understanding for the disease, and gene mutation detection, provide the genetic clues for studies on the etiology and pathogenesis of AREP, to find the gene mutation associated with clinical phenotypes between 1.. Methods on the incidence of a county in Henan province two four generation multiple family members more than PD pedigree investigation, signed informed consent the book collected the clinical data and the family members of PD, measurement and related auxiliary examination, drawing the pedigree genetic map, summarized the characteristics of the PD pathogenesis, clinical diagnosis of autosomal recessive. Transfer of early onset Parkinson's disease.2. two pedigrees and 24 human blood by follow-up, phenol / chloroform extraction of the genomic DNA by polymerase chain reaction (polymerase chain reaction, PCR) specific amplification method, agarose gel electrophoresis of PCR products, purified by DNA sequencing method to obtain the sequence. Then compared with the normal sequence. The results of 1.2 AREP families a total of 6 patients, age 17~29 years old, the average (22.5 + 5.5) years old, male 5 cases, female 1 cases clinical phenotype of.2.: family of the proband (III 6) began to appear in lower limb weakness, muscle strength IV, after the gradual emergence of stiff limbs with resting tremor, bradykinesia, difficulty walking, and a "morning light sunset", after a nap reduced, oral levodopa therapy, 0.125g/ time, 1 times every 12 hours, the symptoms disappeared; the proband's brother had similar clinical manifestations. Pedigree II: the proband (III 12) began to appear in episodes of dystonia, manifested as abnormal posture, easy to fall, after limb tremor associated with increased muscle tension, a morning evening light weight, reduce the incidence of 1 years after a nap, after oral Madopar treatment started for 0.0625g/ times, 1 once a day, the symptoms can be relieved completely; the family of a brother, brother had more severe clinical manifestations of.3. gene detection results of similar pedigree of the proband samples were found Parkin gene exon3 heterozygous and Exon4 homozygous deletion mutations in the proband's mother (4, uncle (II) II 7) brother (III 11) found that the Parkin gene exon3 and Exon4 heterozygous deletion mutation; the proband's father (II 3), uncle (II 5) found that Parkin Exon4 gene heterozygous deletion mutation; pedigree of the proband Parkin gene exon7 850th nucleotide variation from guanine to cytosine (c.850GC) 284th amino acids caused by Gansu The amino acid mutation to arginine (p.G284 R), the proband's son (IV 10), the proband (III 5), brother of the proband brother (III 7) the heterozygous mutation, the proband of the members of the site more than no variation. Conclusion the results obtained in 1. Parkin gene mutation.2. is the two AREP families were included in the study of Parkin gene mutations are point mutations, deletions, common exon3, Exon4, exon7,.3.Parkin gene mutation mutation in the family of AREP patients have clinical phenotype heterogeneity or explicit possibility is not full, there is gene mutation the heterogeneity of.4. for clinical symptoms suggestive of suspected AREP patients in the exclusion of dopamine responsive dystonia and secondary PD possibility, feasible Parkin gene and other common gene mutation screening of.5. the results of the study are shown in the genes because of known types, not found new mutations, However, the continuing collection of diseased families in the form of this workflow may find new mutations in the gene for clinical diagnosis or the development of needle counterpart.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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