本文選題：C3腎病 + 致密物沉積病　； 參考：《南京大學(xué)》2014年博士論文

【摘要】：補體系統(tǒng)是固有免疫的重要組成部分,有三條激活途徑,其中補體旁路系統(tǒng)活化受10余種調(diào)節(jié)蛋白調(diào)控,主要參與抗感染的早期防御。生理情況下,補體旁路處于低度活化平衡狀態(tài)。調(diào)控蛋白基因變異和/或自身抗體的獲得等因素,可導(dǎo)致此途徑異�；罨�,并參與多種腎臟疾病的發(fā)生發(fā)展�，F(xiàn)有的研究表明,補體旁路的異�；罨苯右l(fā)非典型溶血尿毒綜合征(atypical hemolytic uremic syndrome, aHUS)和C3腎病(C3 glomerulopathy, C3G);該系統(tǒng)的活化也參與并加重了IgA腎病、狼瘡性腎炎和缺血再灌注腎臟損傷等疾病的進程。認(rèn)識這一病理生理過程和相關(guān)疾病的特征,對于指導(dǎo)臨床診療具有重要的意義。目前,此類疾病的研究主要見于隊列報道,集中于歐美高加索人群。在亞洲地區(qū),只有小樣本的aHUS、致密物沉積病((lense deposit disease, DDD)和C3腎炎(C3 glomerulonephritis, C3GN)病例報道,更缺乏補體旁路調(diào)節(jié)蛋白的基因研究報道。本研究首先基于目前亞洲最大樣本量的C3G患者,總結(jié)分析了該疾病漢族人群的臨床病理特征及預(yù)后特點,并將亞型DDD和C3GN進行了比較。其次,對aHUS相關(guān)的基因突變進行了薈萃分析,預(yù)測評估其可能的致病性,以了解aHUS相關(guān)基因突變位點分布和致病性間關(guān)系；同時對本中心確診的24例aHUS患者進行了基因篩查分析,探討基因突變和臨床表型間的關(guān)系。研究一：C3腎病的臨床病理特征及預(yù)后分析目的：C3腎病(C3G, C3 glomerulopathy)是一類新認(rèn)識的疾病,主要包括致密物沉積病(dense deposit disease,DDD)和C3腎小球腎炎(C3GN, C3 glomerulo-nephritis)。本研究旨在分析該疾病的臨床、病理及預(yù)后特征,以加深臨床醫(yī)生對該疾病的認(rèn)識。方法：入選了1985年到2014年于解放軍腎臟病研究所確診的C3腎病患者54例,其中DDD15例、C3GN39例。記錄、評估了患者的臨床表型、腎臟受累、病理特征及預(yù)后。結(jié)果：C3腎病患者男性占多數(shù)(32例,59%),中位發(fā)病年齡32(8～71)歲。起病時,44%表現(xiàn)為腎病綜合征,尿蛋白定量4.1(0.2～14.5)g/24h；78%腎功能正常；并有37%起病即伴有高血壓,35%伴有貧血。另82%出現(xiàn)低補體血癥,血清C3水平均值0.52(正常值0.8～1.8)g/L,61%抗“O”陽性。C3G病理上腎小球以膜增殖性(61%)和系膜增生性病變(28%)為主,補體C3主要沉積于血管袢(98%)和系膜區(qū)(63%),另59%伴有免疫球蛋白沉積。與C3GN相比,DDD患者發(fā)病年齡輕(P0.05),蛋白尿水平相似,但低蛋白血癥更顯著(P0.05),伴發(fā)高血壓的比例顯著低(P0.05),循環(huán)補體水平接近。病理上,DDD患者的膜增殖性病變及新月體形成較G3GN更為常見,但無統(tǒng)計學(xué)差異；除血管袢和系膜區(qū)的C3沉積外,DDD亦常伴有腎小管基底膜及間質(zhì)血管區(qū)的沉積(P0.001)。電鏡下,DDD患者電子致密物主要沉積于腎小球基膜內(nèi)、腎小管基底膜和包囊壁,C3GN患者則分布于系膜區(qū)、內(nèi)皮下和上皮側(cè)(P0.001)。兩組隨訪5年的累積腎生存率無差異。結(jié)論：本研究是目前亞洲人群最大樣本C3腎病的報道,可以幫助臨床醫(yī)生更好地認(rèn)識這類疾病及其亞型,促進臨床診療。研究二：aHUS遺傳機制初探目的：補體調(diào)節(jié)蛋白的遺傳缺陷在aHUS發(fā)病中起著重要作用,目前報道的相關(guān)基因突變很多,但相關(guān)基因功能明確的不多。本研究通過對既往報道的基因突變的薈萃分析和生物信息學(xué)預(yù)測來構(gòu)建評估體系,明確基因型和表型間的關(guān)系,加深了解aHUS的遺傳機制。方法：薈萃分析了截止至2014年5月所有報道的aHUS相關(guān)的補體旁路調(diào)節(jié)蛋白的基因突變。據(jù)已報道功能的基因突變評估了5種基因突變功能預(yù)測軟件及其聯(lián)合預(yù)測方法,運用預(yù)測準(zhǔn)確率最高的軟件對所有基因突變進行了致病性預(yù)測,觀察了不同基因突變位點的二級結(jié)構(gòu)分布特點與相關(guān)蛋白功能間關(guān)系。并對本中心確診的24例aHUS患者進行了補體旁路調(diào)節(jié)蛋白基因外顯子測序,觀察基因突變和患者臨床表型、預(yù)后間的關(guān)系。結(jié)果：薈萃分析了aHUS相關(guān)的基因突變375個,其中錯義突變262個,涉及12個基因,約6513個患者,其中高加索人種占99.2%,亞洲人65例(0.5%)。各基因涉及的研究患者數(shù)差異大,基因突變的發(fā)生率變異也大。研究最多的基因為CFH,有112個錯義突變報道,突變發(fā)生率高的基因為CFH(66%)、C3(54%)和MCP(31%)。71(27.1%)個錯義突變進行了實驗功能研究,據(jù)此發(fā)現(xiàn)SNAP預(yù)測軟件的敏感性、陰性和陽性預(yù)測價值、準(zhǔn)確率及MCC值均顯著高于PolyPhen2、SIFT、 SNAP、 Align GVGD、 Pmut及聯(lián)合預(yù)測。進一步以SNAP軟件分析發(fā)現(xiàn)：CFH突變主要在C端(42.8%),CCPs19-20占預(yù)測致病性突變總數(shù)的48.3%。CFI突變主要在絲氨酸蛋白酶區(qū)(47.7%),占總的致病性突變比例為48%。膜蛋白MCP和THBD的突變主要位于跨膜區(qū)。對24個aHUS患者進行了補體旁路調(diào)節(jié)蛋白及補體成分的外顯子捕獲定制測序分析,共檢測了11個基因,發(fā)現(xiàn)了15個未報道的和2個已報道的錯義突變。12個患者攜帶有基因突變,aHUS患者總的基因突變率為50%,其中CD46、 C3、 CFB及CFHR5各發(fā)現(xiàn)3個新的基因變異,CFH、 CFHR3和THBD各為1個,據(jù)SNAP預(yù)測,8個基因突變有致病性。據(jù)基因突變及致病性分組比較,基因突變組補體C3減低更明顯(P=0.003),余兩組間臨床表現(xiàn)及預(yù)后無明顯統(tǒng)計學(xué)差異。結(jié)論：本研究通過對既往報道的補體旁路調(diào)節(jié)蛋白基因突變的薈萃分析,明確了aHUS相關(guān)基因突變譜和突變特征,并評估了生物信息學(xué)基因突變功能預(yù)測方法,了解了基因突變位點二級結(jié)構(gòu)分布和功能的關(guān)系,并應(yīng)用于aHUS患者的基因篩查分析,幫助分析基因型和表型間的關(guān)系,加深了對漢族人群aHUS遺傳機制的
[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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R692

【參考文獻】

相關(guān)期刊論文 前1條

1 Maurizio Salvadori;Elisabetta Bertoni;;Update on hemolytic uremic syndrome:Diagnostic and therapeutic recommendations[J];World Journal of Nephrology;2013年03期

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本文編號：2110007