APOL1基因变异对中国FSGS患者影响的研究

发布时间：2018-08-14 11:33

【摘要】：局灶节段肾小球硬化(FSGS)是难治性肾病综合征和终末期肾病最常见的原因之一,遗传因素在其发病和进展中起重要作用。前期研究显示APOL1的G1和G2变异与FSGS的发病与进展密切相关。但该结果并没有在中国人群中得到重复。提示我国FSGS可能存在其他APOL1变异。本研究的目的是:(1)对APOL1基因单核苷酸多态性和单体型进行分析,探索APOL1基因单体型对不同人群中疾病的易感性。(2)检测FSGS患者和正常对照组中APOL1基因拷贝数的情况,通过比较拷贝数变异在两组的分布情况,探索APOL1基因拷贝数变异对中国FSGS患者临床治疗与预后之间的可能意义。(3)利用本课题组前期发现的APOL1新突变,构建突变质粒和野生型质粒转染到永生化的足细胞,研究该APOL1突变对细胞标志蛋白、细胞骨架和离子通道的影响,并探索其参与FSGS足细胞损伤的可能机制。我们的研究方法包括(1)从千人基因组数据中筛选APOL1基因SNP,统计不同区域APOL1单体型,并比较每个单体型在不同人群中的分布情况。(2)筛选符合条件的FSGS患者DNA样本;通过设计APOL1引物和探针进行Taqman拷贝数变异实验;使用Copy Caller软件分析结果,SPSS分析FSGS患者与正常对照组中APOL1基因拷贝数分布差异及不同拷贝数的FSGS患者临床预后的差异。(3)将突变质粒转染至细胞后动态追踪细胞形态的改变,western blot检测足细胞标志蛋白,自噬标志物,信号转导蛋白分子的表达水平;激光共聚焦检测足细胞骨架的改变及线粒体损伤情况,并用膜片钳技术检测足细胞膜离子通道的变化。研究结果显示(1)APOL1基因各个区域的单体型在不同人群的分布都具有明显差异。大部分单体型在混合人群和欧洲人群的频率相对较高,有2个单体型在亚洲人群中的分布频率高于其他种群。(2)FSGS患者和正常组之间APOL1基因拷贝数分布情况没有统计学意义。多拷贝数和低拷贝数的FSGS患者之间治疗后的缓解率无显著差异。(3)转染突变质粒的足细胞细胞骨架蛋白排列紊乱,细胞发生溶解,细胞间隙增大,间接反映了足细胞足突的收缩;足细胞标志蛋白表达下降,P38MAPK、SAPKs和JNK等信号通路被激活;此外,APOL1新突变可能破坏了细胞膜氯离子通道蛋白。在本研究中,我们从三个层面分析了APOL1基因变异与FSGS的关系。结果表明,APOL1基因的单核苷酸多态性在不同人群中分布有明显差异。其拷贝数变异与我国FSGS易感性无密切相关性。我们前期发现的APOL1位点突变可能从多方面影响足细胞的形态和功能,表明APOL1基因突变可能参与我国FSGS的发病,其机制还需要深入研究。
[Abstract]:Focal segmental glomerulosclerosis (FSGS) is one of the most common causes of refractory nephrotic syndrome and end-stage nephropathy. Genetic factors play an important role in its pathogenesis and progression. Previous studies have shown that G 1 and G 2 mutations of APOL1 are closely related to the pathogenesis and progression of FSGS. But the results were not repeated in Chinese. These results suggest that there may be other APOL1 variations in FSGS in China. The purpose of this study was: (1) to analyze the mononucleotide polymorphism and haplotype of APOL1 gene, and to explore the susceptibility of APOL1 haplotype to disease in different population. (2) to detect the copy number of APOL1 gene in FSGS patients and normal controls. By comparing the distribution of copy number variation in the two groups, the possible significance of copy number variation of APOL1 gene to the clinical treatment and prognosis of Chinese patients with FSGS was explored. (3) A new mutation of APOL1 was found in the early stage of our research group. The mutant plasmids and wild-type plasmids were constructed and transfected into immortalized podocytes. The effects of the APOL1 mutation on cell marker proteins, cytoskeleton and ion channels were studied, and the possible mechanisms involved in the damage of FSGS podocytes were explored. Our research methods include: (1) screening APOL1 gene from thousands of human genome data, counting APOL1 haplotypes in different regions, and comparing the distribution of APOL1 haplotypes in different populations. (2) screening DNA samples from eligible FSGS patients; Taqman copy number variation experiments were carried out by designing APOL1 primers and probes. The results of Copy Caller software were used to analyze the difference of copy number distribution of APOL1 gene between FSGS patients and normal controls and the difference of clinical prognosis between FSGS patients with different copy numbers. (3) the mutant plasmid was transfected into the cells and the morphology of the cells was dynamically tracked. Western blot was used to detect podocyte marker protein. The changes of cytoskeleton and mitochondrial damage of podocytes were detected by laser confocal focusing and ion channels of podocyte membrane were detected by patch clamp technique. The results showed that (1) the distribution of haplotypes in different regions of APOL1 gene was significantly different in different populations. The frequencies of most haplotypes were relatively high in mixed population and European population, and the frequencies of two haplotypes in Asian population were higher than those in other populations. (2) there was no significant difference in the distribution of APOL1 gene copy number between FSGS patients and normal population. There was no significant difference in remission rate between FSGS patients with multiple copy number and low copy number. (3) the cytoskeleton protein of podocyte transfected with mutated plasmids was disordered, the cells dissolved and the cell gap increased, which indirectly reflected the contraction of podocyte foot process. The expression of P38 MAPK- SAPKs and JNK were activated, and the new mutation of APOL1 may destroy the membrane chloride channel protein. In this study, we analyzed the relationship between APOL1 gene variation and FSGS on three levels. The results showed that the single nucleotide polymorphisms of APOL1 gene were significantly different in different populations. There was no close correlation between copy number variation and susceptibility to FSGS in China. The mutation of APOL1 locus discovered in our earlier period may affect the morphology and function of podocyte in many ways, which indicates that the mutation of APOL1 gene may be involved in the pathogenesis of FSGS in China, and its mechanism needs further study.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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