Mutation c.359_363delGTATTinsATAC in the COL4A5 Causes alport syndrome in a Chinese family

The X-linked form of Alport syndrome is associated with mutations in the COL4A5 gene, which is located at Xq22.3 and encodes the α5 chain of type IV collagen. Here we clinically characterized a Chinese family with Alport Syndrome, but no ocular or hearing abnormalities have been observed in any patient in the family. Through Linkage analysis and direct DNA sequencing, a novel complex deletion/insertion mutation c.359_363delGTATTinsATAC in the COL4A5 gene was identified in the family. The mutation was found in all affected family members, but was not present in the unaffected family individuals or the 200 controls. The predicted mutant protein in the family is a truncated protein consisting of only 153 residues. Our report for the first time revealed that the frameshift mutation in the type IV collagen chain α5 causes only renal disease, without extrarenal lesion. Our study broadens genotypic and phenotypic spectrum of COL4A5 mutations associated with Alport syndrome.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

Structure of the Human Type IV CollagenCOL4A6Gene, Which Is Mutated in Alport Syndrome-Associated Leiomyomatosis

Basement membrane (type IV) collagen, a subfamily of the collagen protein family, is encoded by six distinct genes in mammals. Three of those,COL4A3, COL4A4,andCOL4A5,are linked with Alport syndrome (hereditary nephritis). Patients with leimoyomatosis associated with Alport syndrome have been shown to have deletions in the 5′ end of theCOL4A6gene, in addition to having deletions inCOL4A5(Zhouet al., Science261: 1167–1169, 1993). The humanCOL4A6gene is reported to be 425 kb as determined by mapping of overlapping YAC clones by probes for its 5′ and 3′ ends. In the present study we describe the complete exon/intron size pattern of the humanCOL4A6gene. The 12 λ phage clones characterized in the study spanned a total of 110 kb, including 85 kb of the actual gene and 25 kb of flanking sequences. The overlapping clones contained all 46 exons of the gene and all introns, except for intron 2. Since the total size of the exons and all introns except for intron 2 is about 85 kb, intron 2 must be about 340 kb. All exons of the gene were assigned toEcoRI restriction fragments to facilitate analysis of the gene in patients with leiomyomatosis associated with Alport syndrome. The exon size pattern ofCOL4A6is highly homologous with that of the human and mouseCOL4A2genes, with 27 of the 46 exons ofCOL4A6being identical in size between the genes.     

Clonal overgrowth of esophageal smooth muscle cells in diffuse leiomyomatosis-Alport syndrome caused by partial deletion in COL4A5 and COL4A6 genes

This is a study of a patient who manifests all of the features of a diffuse leiomyomatosis-Alport syndrome (DL-ATS), and her two-year-old son who has already been diagnosed with Alport syndrome. Fourteen years ago, the patient underwent a partial esophageal resection followed by a replacement with jejunum. Recently, she underwent a surgical resection of the esophagus due to esophageal dysfunction. Genetic analyses of COL4A5 and COL4A6 on the X-chromosome were efficiently performed using the genomic DNA of her son. We have identified a novel deletion of 194-kb in length, encompassing COL4A5-COL4A6 promoters as well as nearly the entire large intron 1 of COL4A5 and intron 2 of COL4A6. To uncover the relationship of the esophagus-specific occurrence of the tumor and the expression of those genes, immunohistochemical analyses of type IV collagen α chains were conducted in the non-affected individuals. The esophageal smooth muscle-specific expression of α5(IV) and α6(IV) chains in the gastrointestinal tract was observed. Moreover, CAG repeat analysis of the androgen receptor gene and an immunohistochemical analysis in the leiomyoma revealed clonal overgrowth of the cells which received X-inactivation on the non-affected allele. These results may suggest that the dominant effect was caused by the partial deletion of the esophageal smooth muscle-specific genes, COL4A5 and COL4A6.     

Efficient Targeted Next Generation Sequencing-Based Workflow for Differential Diagnosis of Alport-Related Disorders

Alport syndrome (AS) is an inherited type IV collagen nephropathies characterized by microscopic hematuria during early childhood, the development of proteinuria and progression to end-stage renal disease. Since choosing the right therapy, even before the onset of proteinuria, can delay the onset of end-stage renal failure and improve life expectancy, the earliest possible differential diagnosis is desired. Practically, this means the identification of mutation(s) in COL4A3-A4-A5 genes. We used an efficient, next generation sequencing based workflow for simultaneous analysis of all three COL4A genes in three individuals and fourteen families involved by AS or showing different level of Alport-related symptoms. We successfully identified mutations in all investigated cases, including 14 unpublished mutations in our Hungarian cohort. We present an easy to use unified clinical/diagnostic terminology and workflow not only for X-linked but for autosomal AS, but also for Alport-related diseases. In families where a diagnosis has been established by molecular genetic analysis, the renal biopsy may be rendered unnecessary.     

Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.     

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.     

Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.

Autosomal recessive Alport syndrome is a progressive hematuric glomerulonephritis characterized by glomerular basement membrane abnormalities and associated with mutations in either the COL4A3 or the COL4A4 gene, which encode the alpha3 and alpha4 type IV collagen chains, respectively. To date, mutation screening in the two genes has been hampered by the lack of genomic structure information. We report here the complete characterization of the 48 exons of the COL4A4 gene, a comprehensive gene screen, and the subsequent detection of 10 novel mutations in eight patients diagnosed with autosomal recessive Alport syndrome. Furthermore, we identified a glycine to alanine substitution in the collagenous domain that is apparently silent in the heterozygous carriers, in 11.5% of all control individuals, and in one control individual homozygous for this glycine substitution. There has been no previous finding of a glycine substitution that is not associated with any obvious phenotype in homozygous individuals.     

Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment

Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.     

Evaluation of canine COL4A3 and COL4A4 as candidates for familial renal disease in the Norwegian elkhound

The collagen type IV alpha3 and alpha4 chains (COL4A3 and COL4A4) are part of the specialized glomerular basement membrane in the kidney. In human these genes are responsible for Alport syndrome (a type of hereditary nephritis). Histopathological similarities between kidneys of Norwegian elkhound dogs affected with familial renal disease and human Alport syndrome were the basis for a candidate gene approach in Norwegian elkhounds. Three microsatellites-tightly linked to canine COL4A3 and COL4A4--were developed. The microsatellites were used to analyze linkage between COL4A3 and COL4A4 and familial renal disease in a Norwegian elkhound pedigree segregating this disease. Presence of one recombinant between familial renal disease and COL4A3/COL4A4 suggests that these genes are not likely candidates for familial renal disease in this breed.     

Loss of collagen-receptor DDR1 delays renal fibrosis in hereditary type IV collagen disease

Alport syndrome is a hereditary type IV collagen disease leading to progressive renal fibrosis, hearing loss and ocular changes. End stage renal failure usually develops during adolescence. COL4A3?/? mice serve as an animal model for progressive renal scarring in Alport syndrome. The present study evaluates the role of Discoidin Domain Receptor 1 (DDR1) in cell–matrix interaction involved in pathogenesis of Alport syndrome including renal inflammation and fibrosis.DDR1/COL4A3 Double-knockouts were compared to COL4A3?/? mice with 50% or 100% expression of DDR1, wildtype controls and to DDR1?/? COL4A3+/+ controls for over 6 years. Double-knockouts lived 47% longer, mice with 50% DDR1 lived 29% longer and showed improved renal function (reduction in proteinuria and blood urea nitrogen) compared to animals with 100% DDR1 expression. Loss of DDR1 reduced proinflammtory, profibrotic cells via signaling of TGFβ, CTGF, NFκB and IL-6 and decreased deposition of extracellular matrix. Immunogold-staining and in-situ hybridisation identified podocytes as major players in DDR1-mediated fibrosis and inflammation within the kidney.In summary, glomerular epithelial cells (podocytes) express DDR1. Loss of DDR1-expression in the kidney delayed renal fibrosis and inflammation in hereditary type IV collagen disease. This supports our hypothesis that podocyte–matrix interaction via collagen receptors plays an important part in progression of renal fibrosis in Alport disease. The blockade of collagen-receptor DDR1 might serve as an important new therapeutic concept in progressive fibrotic and inflammatory diseases in the future.     

Hearing impairment in Stickler syndrome: a systematic review

Background

Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely COL2A1, COL11A1 and COL11A2 (autosomal dominant inheritance), and COL9A1 and COL9A2 (autosomal recessive inheritance). The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype.

Methods

English-language literature was reviewed through searches of PubMed and Web of Science, in order to find relevant articles describing auditory features in Stickler patients, along with genotype. Prevalences of hearing loss are calculated and correlated with the different affected genes and type of mutation.

Results

313 patients (102 families) individually described in 46 articles were included. Hearing loss was found in 62.9%, mostly mild to moderate when reported. Hearing impairment was predominantly sensorineural (67.8%). Conductive (14.1%) and mixed (18.1%) hearing loss was primarily found in young patients or patients with a palatal defect. Overall, mutations in COL11A1 (82.5%) and COL11A2 (94.1%) seem to be more frequently associated with hearing impairment than mutations in COL2A1 (52.2%).

Conclusions

Hearing impairment in patients with Stickler syndrome is common. Sensorineural hearing loss predominates, but also conductive hearing loss, especially in children and patients with a palatal defect, may occur. The distinct disease-causing collagen genes are associated with a different prevalence of hearing impairment, but still large phenotypic variation exists. Regular auditory follow-up is strongly advised, particularly because many Stickler patients are visually impaired.

     

Targeted Exome Sequencing Integrated with Clinicopathological Information Reveals Novel and Rare Mutations in Atypical,Suspected and Unknown Cases of Alport Syndrome or Proteinuria

We applied customized targeted next-generation exome sequencing (NGS) to determine if mutations in genes associated with renal malformations, Alport syndrome (AS) or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR), recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS), a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and provide insights into etiology of conditions with equivocal clinical and pathologic presentations.     

Epistatic Role of the MYH9/APOL1 Region on Familial Hematuria Genes

Familial hematuria (FH) is explained by at least four different genes (see below). About 50% of patients develop late proteinuria and chronic kidney disease (CKD). We hypothesized that MYH9/APOL1, two closely linked genes associated with CKD, may be associated with adverse progression in FH. Our study included 102 thin basement membrane nephropathy (TBMN) patients with three known COL4A3/COL4A4 mutations (cohort A), 83 CFHR5/C3 glomerulopathy patients (cohort B) with a single CFHR5 mutation and 15 Alport syndrome patients (cohort C) with two known COL4A5 mild mutations, who were categorized as “Mild” (controls) or “Severe” (cases), based on renal manifestations. E1 and S1 MYH9 haplotypes and variant rs11089788 were analyzed for association with disease phenotype. Evidence for association with “Severe” progression in CFHR5 nephropathy was found with MYH9 variant rs11089788 and was confirmed in an independent FH cohort, D (cumulative p value = 0.001, odds ratio = 3.06, recessive model). No association was found with APOL1 gene. Quantitative Real time PCR did not reveal any functional significance for the rs11089788 risk allele. Our results derive additional evidence supporting previous reports according to which MYH9 is an important gene per se, predisposing to CKD, suggesting its usefulness as a prognostic marker for young hematuric patients.     

Alport syndrome: a genetic study of 31 families

Thirty one families with Alport syndrome including 3 families with associated syndromes were studied. The location of the COL4A5 gene, responsible for the Alport syndrome, was determined by linkage analysis with eight probes of the Xq arm and by a radiation hybrid panel. Concordant data indicated the localization of the Alport gene between DXS17 and DXS11. Four deletions and one single base mutation of the COL4A5 gene were detected. Homogeneity tests failed to show any evidence of genetic heterogeneity superimposed on clinical heterogeneity for ophthalmic signs and end-stage renal disease age.     

一性连锁Alport综合征中国家系COL4A5基因新突变位点的检测

通过PCR和直接测序的方法,对一性连锁Alport综合征家系17个受检个体的COL4A5基因所有51个外显子及其相邻内含子的DNA序列进行检测。结果发现,在第26外显子2240位点,男患者存在C碱基缺失(2240delc),女患者存在杂合缺失,同时对女患者相应的PCR产物进行克隆和测序以验证PCR测序结果的可靠性,而在正常家系成员和80例对照中均未发现此位点异常,说明2240delc为引起该家系临床病变的突变位点,不是多态性位点。在性连锁Alport综合征中,COL4A5基因的这个单碱基缺失突变位点为首次报道。     

Common ancestry of three Ashkenazi-American families with Alport syndrome and COL4A5 R1677Q

Mutations in the basement membrane collagen gene COL4A5 cause the progressive renal glomerular nephropathy and typical hearing loss that occur in X-linked Alport syndrome. Nearly all cases involve distinct mutations, as expected for an X-linked disease that significantly reduces the fitness of affected males. A few exceptional COL4A5 mutations appear to be associated with a reduced disease severity and may account for a significant proportion of late-onset Alport syndrome in populations where a founder effect has occurred. The novel mutation reported here, COL4A5 arg1677gln, has been detected in three independently ascertained Ashkenazi-American families, causes a relatively mild form of nephritis with typical onset in the fourth or fifth decade, and may be involved in the etiology of a large proportion of adult-onset hereditary nephritis in Ashkenazi Jews. Received: 14 October 1996 / Revised: 11 December 1996     

Genetic cause of X-linked Alport syndrome in a family of domestic dogs

Alport syndrome is a hereditary disease of type IV (basement membrane) collagens that occurs spontaneously in humans and dogs. In the human, X-linked Alport syndrome (XLAS) is caused by mutations in COL4A5, resulting in absence of type IV collagen alpha5 chains from the glomerular basement membrane (GBM) of affected individuals. The consequence of this defect is progressive renal failure, for which the only available treatments are dialysis and transplantation. Recent studies support the prospect of gene transfer therapy for Alport syndrome, but further development of required technologies and demonstration of safety and efficacy must be accomplished in a suitable animal model. We previously identified and have propagated a family of mixed-breed dogs with an inherited nephropathy that exhibits the clinical, immunohistochemical, pathological, and ultrastructural features of human XLAS. To identify the causative mutation, COL4A5 cDNAs from normal and affected dogs were sequenced in their entirety. Sequence analyses revealed a 10-bp deletion in exon 9 of affected dogs. This deletion causes a frame-shift that results in a premature stop codon in exon 10. Characterization of the causative mutation was followed by development of an allele-specific test for identification of dogs in this kindred that are destined to develop XLAS.     

Identification of a novel COL4A5 mutation in a Chinese family with X-linked Alport syndrome using exome sequencing

Alport syndrome (AS) is an inherited disorder and clinically characterized by glomerulonephritis and end-stage kidney disease (ESRD). The aim of this study was to identify the gene responsible for glomerulopathy in a 4-generation Chinese pedigree. Exome sequencing was conducted in four patients of the family, and then direct sequencing was performed in other members of the pedigree. A novel missense mutation c.368G>A (p.Gly123Glu) in the collagen type IV alpha-5 gene (COL4A5) was found to be the genetic cause. The p.Gly123Glu mutation occurs prior to Gly-X-Y repeats in the alpha-5 chain of type IV collagen. Neither sensorineural hearing loss nor ocular abnormalities were present in patients of this family. Other clinical features, such as age of onset, age of ESRD, disease severity and complications, varied among patients of this family. Our finding may provide new insights into the cause and diagnosis of AS, and also have implications for genetic counseling.     

Spectrum of mutations in the COL4A5 collagen gene in X-linked Alport syndrome.

Alport syndrome is a mainly X-linked hereditary disease of basement membranes that is characterized by progressive renal failure, deafness, and ocular lesions. It is associated with mutations of the COL4A5 gene located at Xq22 and encoding the alpha5 chain of type IV collagen. We have screened 48 of the 51 exons of the COL4A5 gene by SSCP analysis and have identified 64 mutations and 10 sequence variants among 131 unrelated Alport syndrome patients. This represents a mutation-detection rate of 50%. There were no hot-spot mutations and no recurrent mutations in our population. The identified mutations were 6 nonsense mutations, 12 frameshift mutations, 17 splice-site mutations, and 29 missense mutations, 27 of the latter being glycine substitutions in the collagenous domain. Two of these occurred on the same allele in one patient and segregated with the disease in the family. We showed that some of the glycine substitutions could be associated with the lack of immunological expression of the alpha3(IV)-alpha5(IV) collagen chains in the glomerular basement membrane.