补体旁路途径异常活化相关肾脏疾

发布时间：2018-07-09 16:51

本文选题：C3肾病 + 致密物沉积病　；参考：《南京大学》2014年博士论文

【摘要】：补体系统是固有免疫的重要组成部分,有三条激活途径,其中补体旁路系统活化受10余种调节蛋白调控,主要参与抗感染的早期防御。生理情况下,补体旁路处于低度活化平衡状态。调控蛋白基因变异和/或自身抗体的获得等因素,可导致此途径异常活化,并参与多种肾脏疾病的发生发展。现有的研究表明,补体旁路的异常活化直接引发非典型溶血尿毒综合征(atypical hemolytic uremic syndrome, aHUS)和C3肾病(C3 glomerulopathy, C3G);该系统的活化也参与并加重了IgA肾病、狼疮性肾炎和缺血再灌注肾脏损伤等疾病的进程。认识这一病理生理过程和相关疾病的特征,对于指导临床诊疗具有重要的意义。目前,此类疾病的研究主要见于队列报道,集中于欧美高加索人群。在亚洲地区,只有小样本的aHUS、致密物沉积病((lense deposit disease, DDD)和C3肾炎(C3 glomerulonephritis, C3GN)病例报道,更缺乏补体旁路调节蛋白的基因研究报道。本研究首先基于目前亚洲最大样本量的C3G患者,总结分析了该疾病汉族人群的临床病理特征及预后特点,并将亚型DDD和C3GN进行了比较。其次,对aHUS相关的基因突变进行了荟萃分析,预测评估其可能的致病性,以了解aHUS相关基因突变位点分布和致病性间关系；同时对本中心确诊的24例aHUS患者进行了基因筛查分析,探讨基因突变和临床表型间的关系。研究一：C3肾病的临床病理特征及预后分析目的：C3肾病(C3G, C3 glomerulopathy)是一类新认识的疾病,主要包括致密物沉积病(dense deposit disease,DDD)和C3肾小球肾炎(C3GN, C3 glomerulo-nephritis)。本研究旨在分析该疾病的临床、病理及预后特征,以加深临床医生对该疾病的认识。方法：入选了1985年到2014年于解放军肾脏病研究所确诊的C3肾病患者54例,其中DDD15例、C3GN39例。记录、评估了患者的临床表型、肾脏受累、病理特征及预后。结果：C3肾病患者男性占多数(32例,59%),中位发病年龄32(8～71)岁。起病时,44%表现为肾病综合征,尿蛋白定量4.1(0.2～14.5)g/24h；78%肾功能正常；并有37%起病即伴有高血压,35%伴有贫血。另82%出现低补体血症,血清C3水平均值0.52(正常值0.8～1.8)g/L,61%抗“O”阳性。C3G病理上肾小球以膜增殖性(61%)和系膜增生性病变(28%)为主,补体C3主要沉积于血管袢(98%)和系膜区(63%),另59%伴有免疫球蛋白沉积。与C3GN相比,DDD患者发病年龄轻(P0.05),蛋白尿水平相似,但低蛋白血症更显著(P0.05),伴发高血压的比例显著低(P0.05),循环补体水平接近。病理上,DDD患者的膜增殖性病变及新月体形成较G3GN更为常见,但无统计学差异；除血管袢和系膜区的C3沉积外,DDD亦常伴有肾小管基底膜及间质血管区的沉积(P0.001)。电镜下,DDD患者电子致密物主要沉积于肾小球基膜内、肾小管基底膜和包囊壁,C3GN患者则分布于系膜区、内皮下和上皮侧(P0.001)。两组随访5年的累积肾生存率无差异。结论：本研究是目前亚洲人群最大样本C3肾病的报道,可以帮助临床医生更好地认识这类疾病及其亚型,促进临床诊疗。研究二：aHUS遗传机制初探目的：补体调节蛋白的遗传缺陷在aHUS发病中起着重要作用,目前报道的相关基因突变很多,但相关基因功能明确的不多。本研究通过对既往报道的基因突变的荟萃分析和生物信息学预测来构建评估体系,明确基因型和表型间的关系,加深了解aHUS的遗传机制。方法：荟萃分析了截止至2014年5月所有报道的aHUS相关的补体旁路调节蛋白的基因突变。据已报道功能的基因突变评估了5种基因突变功能预测软件及其联合预测方法,运用预测准确率最高的软件对所有基因突变进行了致病性预测,观察了不同基因突变位点的二级结构分布特点与相关蛋白功能间关系。并对本中心确诊的24例aHUS患者进行了补体旁路调节蛋白基因外显子测序,观察基因突变和患者临床表型、预后间的关系。结果：荟萃分析了aHUS相关的基因突变375个,其中错义突变262个,涉及12个基因,约6513个患者,其中高加索人种占99.2%,亚洲人65例(0.5%)。各基因涉及的研究患者数差异大,基因突变的发生率变异也大。研究最多的基因为CFH,有112个错义突变报道,突变发生率高的基因为CFH(66%)、C3(54%)和MCP(31%)。71(27.1%)个错义突变进行了实验功能研究,据此发现SNAP预测软件的敏感性、阴性和阳性预测价值、准确率及MCC值均显著高于PolyPhen2、SIFT、 SNAP、 Align GVGD、 Pmut及联合预测。进一步以SNAP软件分析发现：CFH突变主要在C端(42.8%),CCPs19-20占预测致病性突变总数的48.3%。CFI突变主要在丝氨酸蛋白酶区(47.7%),占总的致病性突变比例为48%。膜蛋白MCP和THBD的突变主要位于跨膜区。对24个aHUS患者进行了补体旁路调节蛋白及补体成分的外显子捕获定制测序分析,共检测了11个基因,发现了15个未报道的和2个已报道的错义突变。12个患者携带有基因突变,aHUS患者总的基因突变率为50%,其中CD46、 C3、 CFB及CFHR5各发现3个新的基因变异,CFH、 CFHR3和THBD各为1个,据SNAP预测,8个基因突变有致病性。据基因突变及致病性分组比较,基因突变组补体C3减低更明显(P=0.003),余两组间临床表现及预后无明显统计学差异。结论：本研究通过对既往报道的补体旁路调节蛋白基因突变的荟萃分析,明确了aHUS相关基因突变谱和突变特征,并评估了生物信息学基因突变功能预测方法,了解了基因突变位点二级结构分布和功能的关系,并应用于aHUS患者的基因筛查分析,帮助分析基因型和表型间的关系,加深了对汉族人群aHUS遗传机制的
[Abstract]:The complement system is an important part of the innate immunity, and there are three activation pathways, in which the activation of complement bypass system is regulated by more than 10 kinds of regulatory proteins, mainly involved in the early defense of anti infection. In physiological conditions, the complement bypass is in a low activation equilibrium state. This approach is abnormally activated and participates in the development of a variety of renal diseases. Existing studies have shown that the abnormal activation of complement pathway directly leads to atypical hemolytic uremic syndrome (aHUS) and C3 nephropathy (C3 glomerulopathy, C3G). The activation of this system also participates in and aggravates IgA nephropathy and lupus nephritis. The understanding of this pathophysiological process and the characteristics of related diseases is of great significance for guiding clinical diagnosis and treatment. At present, the study of such diseases is mainly in the cohort report, concentrated in the European and American Caucasus. In Asia, only small samples of aHUS, compact matter deposition disease (lense Deposit disease, DDD) and C3 nephritis (C3 glomerulonephritis, C3GN) cases reported more lack of gene research on complement bypass regulation protein. First, based on the largest sample of C3G in Asia, this study summarized the clinicopathological features and prognostic characteristics of the Han population of the disease, and compared the subtype DDD and C3GN to the disease. Secondly, a meta-analysis of aHUS related gene mutations was conducted to predict the possible pathogenicity of the aHUS related gene mutations and the relationship between the mutation sites and the pathogenicity of the gene. At the same time, the genetic screening analysis of 24 patients with aHUS in the center was carried out to explore the relationship between gene mutation and clinical phenotype. 1: C3 nephropathy Clinical pathological features and prognostic analysis objective: C3 nephropathy (C3G, C3 glomerulopathy) is a new class of diseases, which mainly include dense deposit disease, DDD, and C3 glomerulonephritis (C3GN, C3 glomerulo-nephritis). The purpose of this study is to analyze the clinical, pathological and prognostic features of the disease in order to deepen the clinicians. Methods: 54 patients with C3 nephropathy confirmed by the PLA kidney disease institute from 1985 to 2014 were selected, including DDD15 and C3GN39 cases. The clinical phenotypes, renal involvement, pathological features and prognosis were recorded. Results: the majority of the patients with C3 nephropathy (32 cases, 59%), the median age of 32 (8~71) years. At the time, 44% were nephrotic syndrome, urine protein was 4.1 (0.2 ~ 14.5) g/24h; 78% renal function was normal; 37% onset was accompanied by hypertension, 35% accompanied by anemia. 82% had hypocomplements, the mean value of serum C3 was 0.52 (0.8 ~ 1.8) g/L, 61% anti "O" positive.C3G and mesangial proliferative venereal disease (61%) and mesangial proliferative disease (61%) and mesangial hyperplasia Change (28%) mainly, complement C3 mainly deposited in vascular loop (98%) and mesangial region (63%), and 59% with immunoglobulin deposition. Compared with C3GN, DDD patients have light onset age (P0.05), the level of proteinuria is similar, but hypoproteinemia is more significant (P0.05), the proportion of hypertension is significantly lower (P0.05), and the level of circulating complement is close. Pathological, membrane of DDD patients The proliferative lesions and crescent formation were more common than G3GN, but there was no statistical difference. In addition to the C3 deposition in the vascular loops and mesangial regions, DDD was often accompanied by the deposition of the basement membrane of the renal tubules and the interstitial vascular area (P0.001). Under the electron microscope, the electron dense substance in the DDD patients was mainly deposited in the glomerular basement membrane, the basilar membrane of the renal tubules and the cyst wall, and the C3GN patients. Distribution in mesangial area, subendothelium and epithelial side (P0.001). Two groups of 5 years of cumulative renal survival rate of no difference. Conclusion: This study is the largest sample of C3 nephropathy in Asian population, can help clinicians better understand the disease and its subtype, promote clinical treatment. Study two: aHUS genetic mechanism objective: complement The genetic defects of the regulatory proteins play an important role in the pathogenesis of aHUS. There are a lot of mutations in the related genes, but the function of the related genes is not very clear. This study constructs the evaluation system by meta-analysis and bioinformatics prediction of previously reported gene mutations, clearly the relationship between genotypes and phenotypes, and a deeper understanding of the aHUS Genetic mechanism. Methods: a meta analysis was conducted to analyze the mutation of aHUS related complement bypass regulation proteins that were reported in May 2014. According to the reported genetic mutation, 5 kinds of gene mutation function prediction software and its combined prediction method were reported, and all the gene mutations were caused by the most accurate prediction software. The relationship between the two stage structure distribution characteristics of different gene mutation sites and the function of related proteins was observed. The relationship between the complement bypass regulation protein gene exon sequencing was carried out in 24 cases of aHUS patients diagnosed by the center, and the relationship between the gene mutation and the clinical phenotype and prognosis of the patients was observed. Results: a meta analysis of aHUS related bases was made. There were 375 mutations, of which 262 were missense mutations, involving 12 genes, about 6513 patients, 99.2% of the Caucasus and 65 in Asia (0.5%). The number of patients involved in the study was large, the mutation rate was also large. The most studied gene was CFH, there were 112 missense mutations, and the high mutation rate was CFH (66). The experimental functions of C3 (54%) and MCP (31%).71 (27.1%) missense mutations were studied. Accordingly, the sensitivity, negative and positive predictive value of the SNAP prediction software, the accuracy rate and the MCC value were significantly higher than that of PolyPhen2, SIFT, SNAP, Align GVGD, Pmut and joint prediction. The 48.3%.CFI mutation of the total number of predicted pathogenicity mutations was mainly in the serine protease region (47.7%). The total proportion of the total pathogenicity mutation was 48%. membrane protein MCP and THBD in the transmembrane region. A total of 24 aHUS patients were sequenced by the exons of complement by-pass protein and complement components, and 11 were detected. 11 15 unreported and 2 reported missense mutations were found in.12 patients with genetic mutations. The total gene mutation rate in aHUS patients was 50%, of which 3 new gene mutations were found in CD46, C3, CFB and CFHR5, and 1 were CFH, CFHR3 and THBD. According to SNAP, 8 gene mutations were pathogenicity. According to gene mutation and pathogenicity In the group comparison, the decrease of complement C3 in the gene mutation group was more obvious (P=0.003). There was no significant difference in clinical manifestation and prognosis between the remaining two groups. Conclusion: This study identified the mutation spectrum and mutation characteristics of the aHUS related gene by a meta-analysis of the previously reported mutation of the complement bypass regulation protein gene, and evaluated the mutation of the bioinformatics gene. The function prediction method is used to understand the relationship between the two level structure distribution and function of the gene mutation site. It is applied to the gene screening analysis of aHUS patients to help analyze the relationship between genotype and phenotype and enhance the genetic mechanism of aHUS in the Han population.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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