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.     

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。     

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。     

常染色体隐性遗传Alport 综合征一家系COL4A3及COL4A4基因突变分析

目的 通过对有近亲婚配史的Alport综合征一家系Ⅳ型胶原α3和α4链的 COL4A3/COL4A4 基因分析，明确常染色体隐性遗传Alport综合征的基因突变,为该病的基因诊断和家系遗传咨询提供更为全面的理论基础｡ 方法 PCR扩增先证者DNA COL4A3/COL4A4 基因的共98个外显子，经直接测序，寻找突变位点，对有意义的突变经限制性内切酶AvaⅡ酶切在家系中分析验证｡ 结果 在该患者中共发现1个错义突变和10个序列变异｡其中在COL4A3 基因上发现一个位于42号外显子上的错义突变 G3725A,导致蛋白质Gly1242Asp的突变｡错义突变在患者中是纯合子，携带者中是杂合子，其他正常家系成员及筛查100条正常人染色体，未发现该突变｡10个序列变异为单核苷酸多态性改变｡ 结论 报道了一个国内较少见的常染色体隐性遗传Alport 综合征家系，同时经基因突变筛查发现Ⅳ型胶原α3链的一个新的致病性的基因突变｡     

薄基底膜肾病的基因突变研究进展

薄基底膜肾病(TBMN)又称良性家族性血尿(BFH),为编码Ⅳ型胶原α链的基因突变导致的一种较常见的常染色体显性遗传性肾脏疾病.其临床表现主要为单纯性镜下血尿,可合并有轻度蛋白尿,通常预后良好~([1-3]).TBMN的诊断主要依靠电镜下见到弥漫性变薄的肾小球基底膜(GBM)~([4]).国外于20世纪60年代中期由McConville~([5])首先报道此病;国内1990年由章友康~([6])首先报道.     

常染色体显性遗传Alport综合征致病基因 COL4A4杂合剪接突变的致病性分析及基因型-表型关联分析

目的探讨常染色体显性遗传Alport综合征(autosomal dominant Alport syndrome, ADAS)致病基因COL4A4杂合剪接突变的致病机制及基因型与表型的关联, 以加深对COL4A4剪接突变的认识以及对ADAS表型异质性的理解。方法本研究为病例系列分析。从3家医院收集5个ADAS家系先证者及家系成员的临床资料。对从先证者中经全外显子组测序(whole exome sequencing, WES)发现的COL4A4杂合剪接变异, 通过RNA体内剪接或Minigene体外实验分析其对mRNA正常剪接的影响。结果在此5个ADAS家系患者中经WES发现了4个COL4A4杂合剪接变异位点。家系1、家系2、家系3和家系4中多数患者呈孤立性镜下血尿或合并微量蛋白尿, 个别患者合并显性蛋白尿、进入老年期后出现肾功能轻度减退。家系5中4例患者均呈快速肾功能进展, 于28～41岁进展至终末期肾病。家系1、家系2患者携带的c.735+3A>G和家系3患者携带的c.694-1G>C均可引起COL4A4的第12号外显子跳跃导致42 bp核苷酸框内缺失(c.694     

一例女性Fabry病并发薄基底膜肾病及其家系调查

目的 了解具有两种遗传性疾病,即Fabry病并发薄基底膜肾病（TBMN）的临床病理和基因突变特点以及家系患病情况。 方法 总结分析本院收治的1例41岁女性Fabry病并发TBMN患者的临床病理特征和基因突变情况,同时对家系成员进行调查及相关检测。 结果 先证者呈现典型的Fabry病的肾外临床表现,包括皮疹、神经痛、眩晕、耳鸣、肥厚型心肌病等,同时亦有蛋白尿、镜下血尿及高血压等肾脏受累表现;肾活检光镜下病理改变为局灶性节段性肾小球硬化（FSGS）,部分足细胞空泡变性;电镜下肾小球脏层上皮细胞胞质内多数髓磷脂小体形成,肾小球基底膜（GBM）弥漫性变薄,厚度为（216±31） nm。家系调查及基因突变检测显示先证者女儿除有典型Fabry病肾外表现外,亦有以血尿为主的肾脏症状。先证者的1个妹妹仅表现为镜下血尿。先证者及其女儿α-半乳糖苷酶 A（α-Gal A）活性分别为33和75活性单位（正常参考值为100~500活性单位）,且2人均携带新发现的GLA基因突变——1208ins21 bp及COL4A3基因多态性——c:3627 G>A（p:M1209I）。仅表现为镜下血尿的先证者的妹妹仅携带COL4A3基因的c:3627 G>A（p:M1209I）多态性,α-Gal A活性正常,无GLA基因突变。 结论 对于Fabry肾病患者呈现血尿,尤其是表现为家族性血尿时,应考虑并认真排除并发TBMN的可能。     

家族性IgA肾病--777例中国IgA肾病回顾性调查分析

目的报道中国家族性IgA肾病(FIgAN)发生情况以及临床病理特征,提高对家族性IgA肾病的认识。方法利用肾脏疾病数据库IgA肾病子库中我院1988～2001年收集的777例IgA肾病患者,进行家族史调查,以及部分家庭成员中行尿常规肾功能检查确定FIgAN发病情况,比较家族性与家族史阴性的IgA肾病患者临床表型的差异。结果(1)在调查的777例IgA肾病患者中,35例患者同时合并薄基底膜肾病,另外742例患者中,65例IgA肾病患者家族史阳性,占8.7%,其中10例(1.3%)患者为FIgAN,55例(7.4%)为可疑家族性IgA肾病。(2)与家族史阴性的患者相比,FIgAN患者在发病年龄、性别、肾穿时的血压、血尿、蛋白尿、肾功能上差异无显著性意义。结论FIgAN在中国人并非少见,加强家系调查特别是在IgA肾病患者亲属中常规进行尿检查将有助于发现该病。家族性IgA肾病诊断应强调同时电镜检查以除外薄基底膜肾病和早期Alport综合征。初步研究提示,本组患者与家族史阴性的IgAN患者相比,其临床病理表现不具有特征性。     

遗传性肾炎一家系COL4A5基因微卫星遗传标记单倍体图研究

目的 通过分析COL4A5基因附近的12个微卫星遗传标记形成的单倍体图，排除性定位一个Alpon综合征家系的致病相关基因。方法应用聚合酶链反应(PCR)扩增COL4A5基因附近的微卫星遗传标记DXS993、DXS991、DXS986、DXS990、DXS1106、DXS8048、DXSl220、DXS8055、DXS1001、DXS1047、DXS122、DXS8043，绘制单倍体图分析。结果该家族中来自不同家庭的患者COL4A5基因的微卫星标记单倍体型不同，并在遗传过程中在不同个体的COL4A5基因附近发生了交换。结论排除该Alport综合征家系致病基因是通过X染色体连锁的方式遗传，而可能是常染色体上基因突变致病。     

成年人肾小球基底膜厚度及基底膜变薄标准研究

目的 了解成年人肾小球基底膜（GBM）厚度及拟建议薄基底膜肾病（TBMN）的GBM弥漫变薄的标准。 方法 选取肾癌根治性切除患者29例,分析性别、年龄、尿常规、Scr以及既往史、家族史等临床资料。选取远离病灶的肾皮质组织,进行光镜、免疫荧光及透射电镜检查,并进行GBM厚度测量和Ⅳ型胶原α3、α5链免疫荧光检查。 结果 29例中,男15例、女14例,年龄（55.9±14.9）岁（20~80岁）,所有病例均无肾脏病家族史。肾组织GBM厚度为（363.6±46.8） nm。GBM厚度与性别相关,男性为（384.0±41.7） nm,女性为（335.0±39.2） nm,差异有统计学意义（P = 0.008）。建议以均数减去两倍标准差作为GBM变薄的标准,即GBM厚度＜270 nm。 结论 成年人肾组织的GBM厚度为（363.6±46.8） nm。GBM厚度和性别相关,男性GBM厚度大于女性,差异有统计学意义。TBMN的GBM弥漫变薄的诊断标准建议为GBM厚度＜270 nm,也建议今后制定TBMN标准中应考虑男女的差异。     

No linkage to the COL4A3 gene locus in Japanese thin basement membrane disease families

Summary: Patients with thin basement membrane disease (TBMD) exhibit persistent haematuria with a diffuse thinning of the glomerular basement membrane (GBM), especially of the lamina densa. It appears to be an autosomal dominant trait. It has been reported that the Goodpasture epitope, which is located in the non-collagenous domain of type IV collagen α 3 chain, may be reduced in patients with TBMD. We speculated that the candidate gene for TBMD could be the type IV collagen α 3 chain gene ( COL4A3 ), which is present closely to type IV collagen α 4 chain gene ( COL4A4 ) on chromosome 2q35–37. We conducted a linkage analysis to investigate the relationship between familial TBMD and COL4A3 gene, using COL4A3 cDNA polymorphism and a (CA)_n microsatellite marker located in the COL4A3 gene. We examined 32 individuals from four Japanese families with TBMD. There were no associations between the patients with haematuria and certain alleles of the two markers in the pedigrees of three families. It has been reported that type IV collagen α 1 chain gene ( COL4A1 ) and α 2 chain gene ( COL4A2 ) are not involved in TBMD, and that α 5 chain gene ( COL4A5 ) and a 6 chain gene ( COL4A6 ) map to chromosome X. In conclusion, our findings suggested that familial TBMD is not caused by the genetic abnormalities of type IV collagen genes isolated thus far.     

COL4A4 mutation in thin basement membrane disease previously described in Alport syndrome

BACKGROUND: Carriers of autosomal-recessive and X-linked Alport syndrome often have a thinned glomerular basement membrane (GBM) and have mutations in the COL4A3/COL4A4 and COL4A5 genes respectively. Recently, we have shown that many individuals with thin basement membrane disease (TBMD) are also from families where hematuria segregates with the COL4A3/COL4A4 locus. This study describes the first COL4A4 mutation in an individual with biopsy-proven TBMD who did not have a family member with autosomal-recessive or X-linked Alport syndrome, inherited renal failure, or deafness. METHODS: The index case and all available family members were examined for dysmorphic hematuria> 50,000/mL using phase contrast microscopy and for segregation of hematuria with the COL4A3/COL4A4 and COL4A5 loci using DNA satellite markers. COL4A4 exons from the index case were then studied using the enzyme mismatch cleavage method, and exons that demonstrated abnormal cleavage products were sequenced. RESULTS: Hematuria in this family segregated with a haplotype at the COL4A3/COL4A4 locus (P = 0.031) but not with haplotypes at the COL4A5 locus. A mutation in COL4A4 that changed C to T resulting in an arginine residue being replaced by a stop codon (R1377X) was demonstrated in exon 44, which encodes part of the alpha 4(IV) collagen sequence close to the junction with the noncollagenous domain. This mutation was present in all five family members with hematuria, but not in the four unaffected family members, 33 unrelated individuals with TBMD, or 22 nonhematuric normals. CONCLUSIONS: R1377X has been described previously in a compound heterozygous form of autosomal-recessive Alport syndrome. Our observation is evidence that TBMD can represent a carrier state for autosomal-recessive Alport syndrome in at least some individuals.     

Alport-like glomerular basement membrane changes with renal-coloboma syndrome

BACKGROUND: Autosomal dominant mutations in paired box gene 2 (PAX2), on chromosome 10q24, are responsible for renal coloboma syndrome (RCS). The role of PAX2 in glomerular basement membrane (GBM) formation and maintenance remains unknown. CASE-DIAGNOSIS: We report a case of a 13-year-old Japanese girl who had both optic disk coloboma and renal insufficiency. Her father and sister also had both coloboma and renal dysfunction. Renal pathological findings revealed a basket-weave pattern of the GBM, which was compatible with Alport syndrome, but type IV collagen α5 staining was normal. The patient's findings of coloboma and renal dysfunction suggested that she had RCS, and genetic analysis revealed a PAX2 heterozygous mutation in exon 2 (c.76dup, p.Val26Glyfsx27) without any mutations of COL4A3, COL4A4, and COL4A5, which are responsible for autosomal and X-linked Alport syndrome. CONCLUSIONS: PAX2 mutations may result in abnormal GBM structure.     

Identification of COL4A5 defects in Alport's syndrome by immunohistochemistry of skin

BACKGROUND: The COL4A3-COL4A4-COL4A5 network in the glomerular basement membrane is affected in the inherited renal disorder Alport's syndrome (AS). Approximately 85% of the AS patients are expected to carry a mutation in the X-chromosomal COL4A5 gene and 15% in the autosomal COL4A3 and COL4A4 genes. The COL4A5 chain is also present in the epidermal basement membrane (EBM). It is predicted that approximately 70% of the COL4A5 mutations prevent incorporation of this chain in basement membranes. METHODS: We investigated whether or not COL4A5 defects could be detected by immunohistochemical analysis of the EBM. Punch skin biopsies were obtained from 22 patients out of 17 families and two biopsy specimens from healthy males were used as controls. RESULTS: In four cases with the COL4A5 frameshift or missense mutations, the COL4A5 chain was either lacking from the EBM (male) or showed a focally negative pattern (female). In three other patients with a COL4A5 missense mutation, a COL4A3 and a COL4A4 mutation, respectively, the COL4A5 staining was normal. A (focally) negative EBM-COL4A5 staining was found in three patients of six families with a diagnosis of AS and in one family of a group of four families with possible AS. CONCLUSIONS: The (focal) absence of COL4A5 in the EBM of skin biopsy specimens can be used for fast identification of COL4A5 defects. Combined with polymorphic COL4A5 markers, both postnatal and prenatal DNA diagnosis are possible in the family of the patient.     

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.     

A large tandem duplication within the COL4A5 gene is responsible for the high prevalence of Alport syndrome in French Polynesia

A large tandem duplication within the COL4A5 gene is responsible for the high prevalence of Alport syndrome in French Polynesia. Background. The prevalence of X-linked Alport syndrome, a progressive inherited nephropathy associated with mutations in the type IV collagen gene COL4A5, is remarkably high in French Polynesia. Methods. A vast clinical, genealogic, and molecular study was undertaken in Polynesia, based on public records, patients' interviews, linkage analysis, and mutation screening. Results and Conclusions. We show that the high frequency of Alport syndrome in this region is due to a founder mutation that occurred onto a common haplotype shared by affected and unaffected individuals, the presence of which precludes indirect molecular diagnosis. We have characterized the mutation as a tandem duplication of 35 COL4A5 exons, resulting in a approximately 65% increase in the length of the collagenous domain of the alpha 5(IV) chain, which is still able to assemble into type IV collagen network as shown by immunofluorescence analysis. That mutation is associated with severe and highly penetrant ocular symptoms and with uniformly thin glomerular basement membrane (GBM) in male adult patients. However, the rate of progression of the renal disease is very variable from one male patient to another, demonstrating the importance of strong modifier factors. Our results suggest that the 20% to 50% of "missing"COL4A5 mutations in X-linked Alport syndrome may be rearrangements similar to that reported here, which was not detectable by sequencing of either individual COL4A5 exons or overlapping cDNA fragments. Finally, we provide the basis for a polymerase chain reaction (PCR) assay that accurately identifies female carriers and allows adequate genetic counseling in this population.     

COL4A3/COL4A4 mutations producing focal segmental glomerulosclerosis and renal failure in thin basement membrane nephropathy

Mutations in the COL4A3/COL4A4 genes of type IV collagen have been found in approximately 40% of cases of thin basement membrane nephropathy, which is characterized by microscopic hematuria and is classically thought to cause proteinuria and chronic renal failure rarely. Here we report our observations of 116 subjects from 13 Cypriot families clinically affected with thin basement membrane nephropathy. These families first came to our attention because they segregated microscopic hematuria, mild proteinuria, and variable degrees of renal impairment, but a dual diagnosis of focal segmental glomerulosclerosis (FSGS) and thin basement membrane nephropathy was made in 20 biopsied cases. Molecular studies identified founder mutations in both COL4A3 and COL4A4 genes in 10 families. None of 82 heterozygous patients had any extrarenal manifestations, supporting the diagnosis of thin basement membrane nephropathy. During follow-up of up to three decades, 31 of these 82 patients (37.8%) developed chronic renal failure and 16 (19.5%) reached end-stage renal disease. Mutations G1334E and G871C were detected in seven and three families, respectively, and were probably introduced by founders. We conclude that these particular COL4A3/COL4A4 mutations either predispose some patients to FSGS and chronic renal failure, or that thin basement membrane nephropathy sometimes coexists with another genetic modifier that is responsible for FSGS and progressive renal failure. The findings presented here do not justify the labelling of thin basement membrane nephropathy as a benign condition with excellent prognosis.     

Alport syndrome associated with diffuse leiomyomatosis: COL4A5-COL4A6 deletion associated with a mild form of Alport nephropathy.

BACKGROUND: The X-linked Alport syndrome (AS) is an inherited nephropathy due to mutations in the COL4A5 gene, encoding the alpha5 chain of type IV collagen, a major component of the glomerular basement membrane (GBM). Here, we report a new kindred with the rare association of X-linked AS and diffuse leiomyomatosis (DL), which is a tumourous process involving smooth muscle cells of the oesophagus, the tracheobronchial tree and, in females, the genital tract. For this syndrome, an almost constant association of large COL4A5 rearrangements with a severe juvenile form of nephropathy has been described for male patients. METHODS: DNA rearrangement at the COL4A5-COL4A6 locus was studied in several members of this family using polymerase chain reaction and pulsed field gel electrophoresis. Furthermore, immunohistochemical staining of tumour and skin samples was performed. RESULTS: The affected patients in this family carry a 120 kb deletion by which the COL4A5 exon 1 and COL4A6 exons 1, 1', and 2 are removed. Immunohistochemical investigation of a skin biopsy of an affected male patient confirmed the absence of both the alpha5 and the alpha6 chains of type IV collagen in the basement membrane of the skin. Surprisingly, both affected male patients had a rather mild renal phenotype. CONCLUSIONS: This report shows that, contrary to what has been reported to date, patients suffering from AS associated with DL can be associated with a late onset renal failure (adult) form of nephropathy.