14例Fabry病肾损害的临床、病理及基因突变特点的分析

目的 探讨Fabry病肾损害的临床病理及α-半乳糖苷酶A(α-Gal A)基因(GLA 基因)突变的特点.方法 回顾性分析14例Fabry病患者的临床、肾脏病理及GLA基因突变等特点.结果 Fabry病肾损害在肾活检患者中检出率为0.074％,平均确诊年龄(30.57±9.32)岁,男∶女=2.5∶1.尿蛋白量中位数为1.71 g/24 h[(0.32～ 4.71)g/24 h].5例有血尿,4例有肾功能受损,肾外受累的表现以血管角质瘤最多见(10/14),其次为心脏病变(6/14).经典型患者9例,迟发型5例,其中6例有肾脏病家族史.肾脏病理光镜下可见明显的肾小球细胞空泡变性,部分患者可见硬化的肾小球.电镜下2例女性患者为部分足细胞内有髓磷脂样小体形成,其余病例所有足细胞内均可见髓磷脂样小体.4例测定α-Gal A活性的先证者均低于正常值.12例先证者进行了GLA基因突变分析,11例发现有GLA基因突变.3个新突变为碱基插入或缺失突变,临床表型均为经典型Fabry病.大多数迟发型患者携带的基因突变位于酶结构的包埋区或部分包埋区(3/11).在已证实的GLA基因突变中,携带I91T、R112H、Q312H的先证者主要表现为“迟发型”;携带W162X、F169S、S201F、N272K及L310R的先证者均表现为“经典型”.结论 本组Fabry病肾损害患者占肾活检的0.074％,常伴有血管角质瘤及心脏受累,且不同的GLA基因突变可能与患者的表型密切相关.     

11个Fabry病家系的α-半乳糖苷酶A活性及GLA基因检测

目的 建立Fabry病α-半乳糖苷酶A（α-gal A）酶活性及基因检测体系,并对基因型临床表型进行分析。方法 检测11个Fabry病家系先证者及家系成员外周血粒细胞α-gal A活性及GLA基因。酶活性检测采用底物荧光法,基因检测采用PCR直接测序法,并进行临床评估。结果 在11个Fabry病家系中检出9种GAL基因突变,包括5个错义突变（R301Q、I91T、G132R、F273L、D165Y）,2个无义突变（W236X、R342X）,1个单碱基缺失（1082delG）和1个大片段缺失（44 bp nt．1077）,其中4种为新突变（D165Y、F273L、1082delG、44 bp nt．1077）。11个家系中通过基因及酶活性检测,确诊男性半合子13例,女性杂合子12例。男性半合子α-gal A酶活性显著下降,女性杂合子α-gal A酶活性部分下降,1/4女性杂合子的α-gal A酶活性处于正常范围内。结论 确诊了11个Fabry病家系的GLA基因突变类型,并筛出所有家系中先证者以外的患者14例。外周血粒细胞α-gal A酶活性和GAL基因检测是筛查和诊断Fabry病的有效手段。     

薄基底膜肾病临床和分子遗传学特征的新认识

薄基底膜肾病（thin basement membrane nephropathy,TBMN）是指以肾小球基底膜（GBM）显著性变薄为特征,临床上以持续性镜下血尿为主要表现的一种遗传性肾脏疾病。该病始于1973年Rogers对一个良性家族性血尿（benign familiar hematuria,BFH）家系的报道,该家系包括4代,共34例患者,这些患者的肾活检光镜及免疫荧光检查均无异常,而电镜显示GBM弥漫变薄^[1]。此后,陆续有学者开始从病理学角度使用“薄基底膜病”或“薄基底膜肾病”对该病进行命名,并得到认可^[1]。近年来,有关TBMN的分子遗传学研究取得了显著进展,进一步提高了对该病的认识。本文就TBMN临床和分子遗传学方面的一些新认识作简要介绍。     

薄基底膜肾病基因点突变后蛋白空间二级结构的改变及与表型的关系

目的 分析薄基底膜肾病（TBMN）患者COL4A4基因点突变后导致编码蛋白中甘氨酸被其它类型氨基酸替代后的结构;探讨基因突变对编码蛋白二级结构的影响及与表型的关系。 方法 以临床确诊的1例常染色体显性连锁遗传型TBMN并发局灶节段性肾小球硬化症（FSGS）患者为研究对象,该患者症状较重,临床表现为血尿、大量蛋白尿,基因检测确定为COL4A4中的g. 1214G>A导致p．G405E。对照选取1例健康人和1例文献报道的单纯TBMN患者（基因检测确定为COL4A4中的g. 1550G>A导致p．G448S）。应用E. coli分别表达患者α4（Ⅳ）链的含有突变位点的结构域及对照α4（Ⅳ）链的相同结构域,圆二色谱检测并比较它们二级结构的差异。 结果 TBMN并发FSGS患者重组蛋白的圆二色谱最低峰所在的波长由正常对照的208 nm变为约220 nm处,而且峰度降低。单纯TBMN对照重组蛋白的检测结果与健康对照相比改变较轻微,最低峰所在的波长不变,峰度仅轻度降低。二级结构分析显示,来自健康对照的重组蛋白中α螺旋、β折叠、转角和无规卷曲均存在,其中前二者各占约1/4。与健康对照蛋白相比,来自TBMN并发FSGS患者的重组蛋白中α螺旋结构增多,约占1/3,无β折叠结构;单纯TBMN对照的重组蛋白与健康对照相似,α螺旋的比例下降而β折叠增多。 结论 位于α4(Ⅳ)链相邻结构域的两个不同位置的甘氨酸被不同的氨基酸替代,它们的临床表型不同,α4(Ⅳ)链的二级结构也存在显著差异。而且,二级结构的改变程度与临床表型的严重性一致。     

薄基底膜肾病并发局灶节段性肾小球硬化症一家系的COL4A3和COL4A4基因突变分析

目的 探讨薄基底膜肾病(TBMN)合并局灶节段性肾小球硬化症(FSGS)的遗传学机制.方法 对一病理学诊断为TBMN合并FSGS患者及其家系的COL4A3和COL4A4基因突变,应用与COL4A3和COL4A4基因连锁的微卫星标记连锁分析方法进行分析.PCR扩增COIAA3和COL4A4全部98个外显子后,直接测序筛查突变.同时测序排除已为公认的FSGS相关基因NPHS1、NPHS2、WT1、TRPC6、ACTN4、CD2AP突变导致FSGS的可能.结果 微卫星标记连锁分析显示此家系与COL4A3和COL4A4基因连锁.直接测序在此家系中发现疾病患者COL4A4基因1214位的鸟嘌呤突变为腺嘌呤,导致Ⅳ型胶原α4链第405位甘氨酸突变为谷氨酸,并且发现COL4A3基因一多态性IVS1-4C＞T.此多态性随疾病分布,可能与致病相关.未发现FSGS相关基因的突变.结论 此家系是在TBMN的基础上发生FSGS.Ⅳ型胶原α4链突变及随疾病分布的基因多态性是否导致TBMN合并FSGS或使其易感性增加尚待更多家系进一步研究.     

Fabry病家系的α-半乳糖苷酶A基因突变研究

目的 通过检测3个Fabry病家系基因突变类型明确基因诊断,并进行家系成员的基因型检测.方法 通过PCR和直接测序的方法,对3个Fabry家系的先证者及部分家系成员外周血DNA进行α-半乳糖苷酶A编码GLA基因7个外显子及其相邻内含子的DNA序列检测.结果 （1）先证者1的GLA基因7号外显子内1142位点发生碱基缺失（1142delG）,1142位碱基G的缺失导致蛋白质翻译在390位氨基酸提前终止,该突变国内外均未见报道;（2）先证者2的GLA基因6号外显子内902位点存在1个错义突变,碱基G被A取代,导致其编码的第301位氨基酸由精氨酸变为谷氨酰胺（902G＞A,R301Q）;（3）先证者3的GLA基因3号外显子内484位点存在1个错义突变,碱基T被C取代,导致其编码的第142位氨基酸由半胱氨酸变为精氨酸（484T＞C,C142R）.在3个家系的部分成员中进行基因检测,检出GLA突变基因携带者共6例,其中男性半合子1例,女性杂合子5例,突变类型均与相应先证者符合.100条正常X染色体对照中均未发现上述位点异常.结论 本研究在3个Fabry病家系中检出3种GLA基因突变,其中1142delG为新发现的突变,并在3个家系的部分家系成员中检出男性半合子1例,女性杂合子5例.     

成人薄基底膜肾小球病的临床病理特点及预后分析

目的:探讨成人薄基底膜肾小球病（thin basement membrane nephropathy,TBMN）的临床病理特征及预后。方法:选取2008年1月1日至2017年12月31日在东部战区总医院肾脏科经肾活检确诊为TBMN的成年患者,回顾性分析临床、病理特点和预后,以及蛋白尿和肾脏慢性病理改变的影响因素。结果:...     

膜性肾病合并薄基底膜肾病患者临床病理分析

目的探讨膜性肾病（MN）合并薄基底膜肾病（TBMN）的临床病理特点。方法选择我院经肾脏活体组织检查（简称：肾活检）确诊为I期MN合并TBMN（I期MN-TBMN）患者7例,分析其临床病理特点,并与同期的I期MN及单纯的TBMN进行比较。结果7例I期MN—TBMN患者占同期所有I期MN的1．49％（7／471例）,均表现为持续性变形性镜下血尿、轻中度蛋白尿[（2．09±0．78）g／24h]和正常肾功能[SCr（65．30±14．09）／2mol／L];电镜超微结构显示肾小球基底膜（GBM）弥漫性变薄[GBM厚度（205．96±45．94）nm]伴上皮下块状电子致密物沉积,免疫荧光显示Ⅳ型胶原a3、a5链呈线条样沿GBM分布与正常肾组织相同。与同期原发性I期MN组相比,I期MN-TBMN组血尿发生率显著升高（P=0．015）,蛋白尿程度显著降低（P=0．019）。随访时间（22．3±16．4）个月,I期MN—TBMN组较I期MN组血白蛋白水平显著增高（P=0．045）,尿蛋白阴性患者比例显著增高（P＝0．017）。结论少数原发性I期MN可与TBMN同时存在,I期MN-TBMN与原发性I期MN相比较血尿发生率升高,蛋白尿程度降低,临床相对较轻,预后相对较好。     

儿童与成人薄基底膜肾病临床病理对比分析

薄基底膜肾病（thin basement membrane nephropathy,TBMN）在儿童与成人并非少见,临床上易误诊为无症状血尿、隐匿性肾炎、急性肾炎,肾活检光镜检查：易误诊为轻度系膜增生性肾炎,肾小球轻微病变。现将我们收治的33例儿童TBMN和37例成人TBMN的临床病理进行对比分析,旨在了解其差异,现报告如下。     

X连锁显性遗传Alport综合征病例报道

正Alport综合征(alport syndrome,AS)又称眼、耳、肾综合征,是以血尿、肾功能渐进性减退、神经性耳聋和晶状体异常为特征的遗传性肾小球基底膜(glomerular basement membrane,GBM)疾病。X连锁显性遗传(x-linked dominant,XL)是其最常见的遗传方式,约占85%。位于X染色体的Ⅳ型胶原蛋白α5链(collagenⅣα5,COL4A5)基因突变破坏了Ⅳ型胶原分子结构,从而影响了基底膜的稳定性。患者既可表现为终末期肾病(end stage renal disease,ESRD),又可表现为隐匿性肾炎,所以基因测序诊断尤为重要。本文报道1例通过COL4A5基因测序确诊的AS患者,对其家系成员进行了调查随访。1先证者病史资料     

Thin basement membrane nephropathy

Thin basement membrane nephropathy. Thin basement membrane nephropathy (TBMN) is the most common cause of persistent glomerular bleeding in children and adults, and occurs in at least 1% of the population. Most affected individuals have, in addition to the hematuria, minimal proteinuria, normal renal function, a uniformly thinned glomerular basement membrane (GBM) and a family history of hematuria. Their clinical course is usually benign. However, some adults with TBMN have proteinuria >500 mg/day or renal impairment. This is more likely in hospital-based series of biopsied patients than in the uninvestigated, but affected, family members. The cause of renal impairment in TBMN is usually not known, but may be due to secondary focal segmental glomerulosclerosis (FSGS) or immunoglobulin A (IgA) glomerulonephritis, to misdiagnosed IgA disease or X-linked Alport syndrome, or because of coincidental disease. About 40% families with TBMN have hematuria that segregates with the COL4A3/COL4A4 locus, and many COL4A3 and COL4A4 mutations have now been described. These genes are also affected in autosomal-recessive Alport syndrome, and at least some cases of TBMN represent the carrier state for this condition. Families with TBMN in whom hematuria does not segregate with the COL4A3/COL4A4 locus can be explained by de novo mutations, incomplete penetrance of hematuria, coincidental hematuria in family members without COL4A3 or COL4A4 mutations, and by a novel gene locus for TBMN. A renal biopsy is warranted in TBMN only if there are atypical features, or if IgA disease or X-linked Alport syndrome cannot be excluded clinically. In IgA disease, there is usually no family history of hematuria. X-linked Alport syndrome is much less common than TBMN and can often be identified in family members by its typical clinical features (including retinopathy), a lamellated GBM without the collagen alpha3(IV), alpha4(IV), and alpha5(IV) chains, and by gene linkage studies or the demonstration of a COL4A5 mutation. Technical difficulties in the demonstration and interpretation of COL4A3 and COL4A4 mutations mean that mutation detection is not used routinely in the diagnosis of TBMN.     

Persistent familial hematuria in children and the locus for thin basement membrane nephropathy

This study examined how often children with persistent familial hematuria were from families where hematuria segregated with the known genetic locus for the condition known as benign familial hematuria or thin basement membrane nephropathy (TBMN) at COL4A3/COL4A4. Twenty-one unrelated children with persistent familial hematuria as well as their families were studied for segregation of hematuria with haplotypes at the COL4A3/COL4A4 locus for benign familial hematuria and at the COL4A5 locus for X-linked Alport syndrome. Eight families (38%) had hematuria that segregated with COL4A3/COL4A4, and four (19%) had hematuria that segregated with COL4A5. At most, eight of the other nine families could be explained by disease at the COL4A3/COL4A4 locus if de novo mutations, non-penetrant hematuria or coincidental hematuria in unaffected family members was present individually or in combination. This study confirms that persistent familial hematuria is not always linked to COL4A3/COL4A4 (or COL4A5) and suggests the possibility of a further genetic locus for benign familial hematuria. This study also highlights the risk of excluding X-linked Alport syndrome on the basis of the absence of a family history or of kidney failure.     

Novel heterozygous COL4A3 mutation in a family with late-onset ESRD

Thin basement membrane nephropathy (TBMN) and Alport syndrome (ATS) are genetically heterogeneous conditions characterized by structural abnormalities in the glomerular basement membrane (GBM). TBMN presents with hematuria, minimal proteinuria, and normal renal function. Although TBMN is an autosomal dominant disease (COL4A3 and COL4A4), ATS can be inherited X-linked (COL4A5), autosomal recessive, or autosomal dominant (both COL4A3 and COL4A4). The clinical course of TBMN is usually benign, whereas ATS typically results in end-stage renal disease (ESRD). Nevertheless, there is a broad spectrum of clinical phenotypes caused by mutations in COL4A3 or COL4A4. We report an Italian family who presented with hematuria and mild proteinuria. Mutational analysis showed a novel heterozygous mutation p.G291E in exon 15 of the COL4A3 gene. Many different mutations in COL4A3 and COL4A4 that cause TBMN have already been identified, but most genetic variability in these genes has been found to cause autosomal ATS. A valid genotype–phenotype correlation for TBMN or ATS is not yet known. Therefore, it is important to identify new mutations by direct sequencing to clarify their clinical importance, to assess the prognosis of the disease, and to avoid renal biopsy.     

Do mutations in <Emphasis Type="Italic">COL4A1</Emphasis> or <Emphasis Type="Italic">COL4A2</Emphasis> cause thin basement membrane nephropathy (TBMN)?

Thin basement membrane nephropathy (TBMN) is the commonest cause of persistent glomerular haematuria and often presents in childhood. Only 40% of affected individuals have mutations identified in the COL4A3 and COL4A4 genes, but mutations in the genes for other COL4A isoforms also result in thinned membranes in humans (COL4A5) and mice (COL4A1). This study examined whether COL4A1/COL4A2 represented a further genetic locus for TBMN. Nine families with TBMN in whom haematuria did not segregate with COL4A3/COL4A4, were examined for linkage to COL4A1/COL4A2 using five micro-satellite markers. In addition, index cases from these families plus a further 14 unrelated individuals with TBMN that was not due to COL4A3 or COL4A4 mutations (n=23) were screened for mutations in each of the 52 exons of COL4A1 and the 47 exons of COL4A2 using single stranded conformational analysis (SSCA). DNA samples that demonstrated bandshifts were sequenced. Haplotype analysis demonstrated that haematuria segregated with the COL4A1/COL4A2 locus in only two small families (2/9, 22%). No definite COL4A1 or COL4A2 mutations were identified in the 23 unrelated individuals with TBMN although novel polymorphisms were demonstrated. This study indicates that COL4A1/COL4A2 does not represent a further major genetic locus for TBMN.     

COL4A5基因多态性与汉族人薄基底膜肾病的关系

目的探讨薄基底膜肾病COL4A5基因突变情况,为其诊断及遗传咨询提供理论基 础。方法使用聚合酶链反应-单链构象多态性分析(PCR-SSCP)的方法,对39例薄基底膜肾病(TBMN)患者COL4A5基因的51个外显子进行分析,对SSCP发现异常者测序。结果8例患者同时发现3个同义突变和1个错义突变,即1297G-C(365Gly-Gly)、1533T-G(444Ile-Ser)、3715A-G(1171Gln-Gln)及4477C-T(1425Asp-Asp)。该4个突变构成一个单体型,在本组TBMN患者中的基因频率为11％(8/70),而正常对照中该单体型的发现率为9％(9/100)。其中1例患者还合并2417C-G(739Pro-Ala)。结论我们研究发现的单体型为正常汉族人COL4A5基因的多态性;该多态性合并其他的基因突变可能与薄基底膜肾病发病有关。     

The clinical features of thin basement membrane nephropathy

Thin basement membrane nephropathy (TBMN) is a common, lifelong condition affecting the kidneys that is characterized by microscopic glomerular hematuria, minimal or no proteinuria, and normal renal function. It often is discovered incidentally, and usually has an excellent prognosis. Many cases are familial and show autosomal-dominant inheritance. The defining characteristic is a glomerular basement membrane (GBM) that is thinned to about half its normal thickness on ultrastructural examination of the renal biopsy specimen. However, occasionally patients with TBMN develop marked proteinuria or renal impairment. It is unclear whether individuals with TBMN and impaired renal function represent part of the spectrum of TBMN associated with heterozygous COL4A3 or COL4A4 mutations, or if their disease is caused by mutations of other genes, or whether it is caused by a second coexistent renal lesion or is misdiagnosed Alport syndrome.     

The role of molecular genetics in diagnosing familial hematuria(s)

Familial microscopic hematuria (MH) of glomerular origin represents a heterogeneous group of monogenic conditions involving several genes, some of which remain unknown. Recent advances have increased our understanding and our ability to use molecular genetics for diagnosing such patients, enabling us to study their clinical characteristics over time. Three collagen IV genes, COL4A3, COL4A4, and COL4A5 explain the autosomal and X-linked forms of Alport syndrome (AS), and a subset of thin basement membrane nephropathy (TBMN). A number of X-linked AS patients follow a milder course reminiscent of that of patients with heterozygous COL4A3/COL4A4 mutations and TBMN, while at the same time a significant subset of patients with TBMN and familial MH progress to chronic kidney disease (CKD) or end-stage kidney disease (ESKD). A mutation in CFHR5, a member of the complement factor H family of genes that regulate complement activation, was recently shown to cause isolated C3 glomerulopathy, presenting with MH in childhood and demonstrating a significant risk for CKD/ESKD after 40?years old. Through these results molecular genetics emerges as a powerful tool for a definite diagnosis when all the above conditions enter the differential diagnosis, while in many at-risk related family members, a molecular diagnosis may obviate the need for another renal biopsy.     

The genetics of thin basement membrane nephropathy

The diagnosis of thin basement membrane nephropathy (TBMN) usually is made on the basis of the clinical features or the glomerular membrane ultrastructural appearance. Only now are we beginning to understand the genetics of TBMN and the role of diagnostic genetic testing. The similarity of clinical and glomerular membrane features first suggested TBMN might represent the carrier state for autosomal-recessive Alport syndrome. This was confirmed subsequently by the demonstration that 40% of families with TBMN have hematuria that segregates with the corresponding locus ( COL4A3/COL4A4 ), and identical mutations occur in both conditions. To date, about 20 COL4A3 and COL4A4 mutations have been shown in TBMN, and these mainly are single nucleotide substitutions that are different in each family. The families in whom hematuria does not appear to segregate with the COL4A3/COL4A4 locus cannot all be explained by de novo mutations, and nonpenetrant or coincidental hematuria. This suggests a further TBMN locus. In patients with persistent hematuria, testing for COL4A3 and COL4A4 mutations to diagnose TBMN is problematic because of the huge size of these genes, their frequent polymorphisms, and the likelihood of a further gene locus. It is far more practicable to perform genetic testing to exclude or confirm X-linked Alport syndrome because this condition is the major differential diagnosis of TBMN and has a very different prognosis.