ADPKD伴发IA患者PKD1基因突变的研究与分析

发布时间：2018-04-21 00:15

本文选题：常染色体显性遗传多囊肾疾病 + 颅内动脉瘤　；参考：《郑州大学》2017年硕士论文

【摘要】：背景和目的常染色体显性遗传多囊肾病(Autosomal Dominant Polycystic Kidney Disease,ADPKD)是一种医学上较为容易遇到的、具有致命风险的常染色体单基因遗传疾病。ADPKD患者通常是在中青年时确诊,全世界大概有1/1000—1/400的人患有此病,1200多万人达到放射诊断标准而诊断为此病。相关研究表明,ADPKD通常由PKD1和PKD2两种基因突变导致,PKD1位于16号常染色体短臂一区3带3亚带,PKD2位于4q21区。PKD1通过转录翻译得到的产物为多囊蛋白1(polycystin-1,PC1),PKD2通过转录翻译得到的产物多囊蛋白2(polycystin-2,PC2)。多囊蛋白1是一种血浆膜样受体蛋白,主要分布在细胞膜上,维持细胞之间或是细胞与细胞外液之间的相互作用;多囊蛋白2是一种非选择性的阳离子通道,PC2与PC1结合后,形成复合物,该复合物对钙离子有较强的通透性,对钙离子起到门控通道的作用。位于6号染色体短臂一区2带的PKHD1基因发生突变则会导致隐性遗传多囊肾病的形成,PKHD1通过转录翻译得到的产物FPC蛋白。对已发现的多囊肾病基因的致病位点的研究发现,大片段缺失、重复或重排突变发现极少(约为3%—4%),ADPKD主要以点突变为主。因此,对患者的致病基因序列进行直接测序仍然是对多囊肾病的基因诊断的主要技术。ADPKD是一种明确的基因座异质性疾病,它的基因诊断相当复杂,PKD1或PKD2两个基因有一个出现致病突变即可引发ADPKD,其中多囊肾疾病多是由PKD1基因突变引起的,约为85%,临床表型也最为严重,导致病人发展至终末期肾病的时间比PKD2要早20年。其次,从目前已有的研究来看,ADPKD基因突变并没有像其它疾病一样有固定的位点,它的突变可能会发生在PKD1和PKD2基因的任何一个位点,较难检测。ADPKD的特点是双肾多发囊肿和早发性慢性肾功能衰竭,最终将导致终末期肾病(ESRD),只能进行肾替代治疗或肾移植维持生命。除肾脏囊肿外,ADPKD通常还伴有肝囊肿、脾囊肿、胰囊肿、卵巢囊肿和精囊囊肿等肾外表现,它也是颅内动脉瘤发生发展的重要危险因素;多囊肾患者患动脉瘤的概率为4%至41%。由于ADPKD有向家族聚集的倾向,而且该病一般发病较晚,多于成年引发,十分不利于患者自身的治疗,大多患者在结婚生子以后才被确诊为多囊肾病,生出携带有致病突变的孩子概率很大,这对患者及其家属和保健系统也造成了沉重的负担。颅内动脉瘤(intracrartial aneurysm,IA)系颅内动脉血管壁发生病理性局限性扩张形成的血管瘤样病变,一般多会引发自发性蛛网膜下腔出血。颅内动脉瘤一旦发生破裂,具有极高的致死性和致残性,而且目前并没有具体的药物来稳定或预防其破裂,所以它是给患者生命和健康带来很大威胁的一种疾病,也给家庭和社会也带来了繁重的负担。IA的发病机制尚未清楚,通常认为它的发病是由多种因素共同引起的。目前研究已经证实年龄、性别、抽烟、酗酒、高血压、吸毒、季节、家族史是颅内动脉瘤的高危因素,也很可能是血流动力改变化及后天退行性变等综合作用的结果。然则相关研究报道,遗传因素对于引发IA也起到了十分重要的作用。心血管并发症是ADPKD的首要死因,而IA引起的蛛网膜下腔出血是其中最重要的并发症。ADPKD中IA的诊断和治疗策略还没有被全面建立起来。IA是所有ADPKD患者心血管并发症中最严重的一种,这是因为血管瘤的破裂后随之而来的是50%的高死亡率。尽管多囊蛋白在动脉平滑肌的表达,这一PKD基因突变诱发动脉瘤的形成机制仍然不完全清楚,PKD1和PKD2的突变对于IAs的发病风险相等。靠近PKD1 5’端一半距离的位点的突变更容易发展成为IAs。数学建模表明,多流体填充肾间质囊肿的形成起始于儿童期,从而导致早期颅内动脉瘤较难检测。因此我们希望建立一个高效特异的检测突变的体系,研究ADPKD伴发IA的患者PKD1突变与一般ADPKD患者突变的区别,找出基因层面上辅助诊断的方法,使ADPKD和IA在形成和潜伏期就可以被检测出,以便及早治疗,从而减免患者的痛苦。方法在上海交通大学附属第六人民医院收集并整理从2013年7月到2014年8月在该院接受治疗的ADPKD患者,所有患者均经B超、CT、磁共振血管成像法筛选伴有颅内动脉瘤的患者,共收集ADPKD伴发ICA患者23例。用乙二胺四乙酸(EDTA)抗凝管采取23例患者术前早晨空腹外周血4mL,置于冰箱中,试剂盒提取23例患者的基因组DNA。设计巢式PCR反应,分析所需的特异性引物,选择六对长片段引物,分别扩增PKD1的1号外显子、2到12号外显子、13到15D号外显子、外显子15E到22、外显子22到32、36到46号外显子,选择特异性好的PCR反应条件进行扩增。把扩增所得小片段产物进行一代测序,对结果进行分析。结果1所得PCR产物经电泳验证,条带高亮且产物唯一,特异性较好。2 23例样本进行测序后共检出突变85个。这些突变均为点突变,其中47个错义突变,35个同义突变,1个无义突变,1个移码突变。而且所有样品均在7号外显子检测到同一突变。3共发现未知突变占19种,其中错义突变14种,同义突变3种,无义突变1种,移码突变1种。结论1 PKD1 7号外显子检测到的同一突变,与普通PKD患者和正常人均不同,为多囊肾伴发动脉瘤的研究提供了指引。2 PKD1靠近5'端一半距离共发现72个突变,占发现突变总数的84.70%,证明靠近PKD1 5'端一半距离的位点的突变更容易发展成为颅内动脉瘤。
[Abstract]:Background and objective autosomal dominant hereditary polycystic kidney disease (Autosomal Dominant Polycystic Kidney Disease, ADPKD) is a medicine that is relatively easy to meet. The fatal risk of autosomal monogenic genetic disease of.ADPKD patients is usually diagnosed in young and middle-aged people, and the world probably has 1/1000 1/400 people with this disease, more than 1200 The study showed that ADPKD was usually caused by two mutations of PKD1 and PKD2, PKD1 was located in the 3 band and 3 subband of the 16 autosomal short arm, and the product of PKD2 in 4q21 region.PKD1 through transcriptional translation was polycystic protein 1 (polycystin-1, PC1), and PKD2 was more than the product of transcriptional translation. The cysts 2 (polycystin-2, PC2). Polycystic protein 1 is a plasma membrane like receptor protein, mainly distributed on the cell membrane, maintaining the interaction between cells or between cells and extracellular fluid; polycystic protein 2 is a non selective cation channel, and the combination of PC2 and PC1 forms a complex, and the complex has a strong passage to calcium ions. PKHD1 gene mutation at the 2 band of the short arm of chromosome 6 will lead to the formation of recessive polycystic kidney disease and the product of PKHD1 through the transcriptional translation of FPC protein. Mutations found very little (about 3% to 4%) and ADPKD was mainly point mutation. Therefore, direct sequencing of the patient's gene sequence is still the main technique for genetic diagnosis of polycystic kidney disease (.ADPKD), a specific loci heterogeneity disease. Its genetic diagnosis is complex, and the two genes of PKD1 or PKD2 have one disease. Mutation can cause ADPKD, in which polycystic kidney disease is caused by a mutation of PKD1 gene, about 85%, and the most severe clinical phenotype, which causes the patient to develop to end-stage kidney disease 20 years earlier than PKD2. Secondly, from the present study, the mutation of the ADPKD gene does not have a fixed site like other diseases, and its mutation It may occur at any site of the PKD1 and PKD2 genes. It is difficult to detect.ADPKD, which is characterized by double renal multiple cysts and premature chronic renal failure, which will eventually lead to end-stage renal disease (ESRD), only renal replacement therapy or renal transplantation to maintain life. Except for renal cysts, ADPKD is usually accompanied by liver cysts, splenic cysts, and pancreatic cysts. Ovarian cysts and seminal vesicle cysts are also an important risk factor for the development of intracranial aneurysms. The incidence of aneurysms in patients with polycystic kidney disease is 4% to 41%. because ADPKD has a tendency to gather in the family, and the disease usually occurs later, more than the adult, and is very detrimental to the patient's own treatment, most of the patients are married. The child has been diagnosed with polycystic kidney disease after being diagnosed as polycystic kidney disease. It has a great probability to produce a child with a pathogenic mutation. It also causes a heavy burden on the patients and their families and health care systems. Intracranial aneurysms (intracrartial aneurysm, IA) are the angiomatoid lesions formed by the pathological localized dilatation of the vascular walls of the intracranial arteries. Onset of subarachnoid hemorrhage. Once intracranial aneurysm is ruptured, it is extremely lethal and disabled, and there is no specific drug to stabilize or prevent its rupture, so it is a disease that poses a great threat to the life and health of the patient, and also brings a heavy burden to the family and society with the pathogenesis of.IA. It is not clear that it is usually considered to be caused by a variety of factors. The current study has confirmed that age, sex, smoking, alcohol, hypertension, drug use, season, family history are the high risk factors for intracranial aneurysms, and may also be the results of the combined effects of changes in hemodynamic changes and acquired degeneration. Genetic factors also play an important role in the initiation of IA. Cardiovascular complications are the leading causes of ADPKD, and IA induced subarachnoid hemorrhage is the most important complication in.ADPKD. The diagnosis and treatment strategy of IA has not been fully established, and.IA is the most serious of all cardiovascular complications in ADPKD patients. It is because of the rupture of a hemangioma that follows a high death rate of 50%. Although polycystic protein is expressed in the arterial smooth muscle, the mechanism of the PKD gene mutation is still not completely clear, the mutation of PKD1 and PKD2 is equal to the risk of IAs. Mutations near the half distance of the PKD1 5 'end are more likely to occur. IAs. mathematical modeling shows that the formation of multifluid filling of renal interstitial cysts begins in childhood, which leads to early intracranial aneurysms which are difficult to detect. Therefore, we hope to establish an efficient and specific mutation detection system to identify the difference between the PKD1 mutation in the patients with IA and the mutation of a ADPKD patient, and to find out the gene level. The method of auxiliary diagnosis can be detected in the formation and incubation period of ADPKD and IA so as to treat early and reduce the sufferings of the patients. Methods at the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, the patients were collected and collected from July 2013 to August 2014. All patients were treated by B-ultrasound, CT, and magnetic resonance angiography. A total of 23 patients with intracranial aneurysms with intracranial aneurysms were selected, and 23 patients with ICA were collected. 23 patients with EDTA (EDTA) anticoagulant were placed in the fridge in the fridge. The genomic DNA. of 23 patients was extracted by the kit to design nested PCR reaction. The specific primers needed were analyzed, and six long segments were selected. We amplified PKD1 exon 1, exon 2 to 12, 13 to 15D exon, exon 15E to 22, exon 22 to 32,36 to exon 46, and selected specific PCR reaction conditions to amplify. The small fragment of the amplified fragment was sequenced and analyzed. The result 1 obtained the PCR product to be verified by electrophoresis. 23 samples were sequenced and 85 of the 23 samples were sequenced. All of these mutations were point mutations, including 47 missense mutations, 35 synonymous mutations, 1 unsense mutations and 1 code mutation. And all the samples were detected by the same sudden change.3 in 19 of the same sudden change, of which the missense mutation was 14. 3 kinds of synonymous mutagenesis, 1 non sense mutations and 1 kinds of shift mutation. Conclusion 1 PKD1 7 exons were detected by the same mutation, which were different from normal PKD patients and normal persons. The study provided a guide to the study of the polycystic kidney associated aneurysm by guiding the.2 PKD1 near the 5'end to find 72 sudden changes, accounting for 84.70% of the total number of mutations, which proved to be close to PKD1 5'. The mutation at the half distance is more likely to develop into intracranial aneurysms.

【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】：R692;R743

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