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.     

Substitution of arginine for glycine 325 in the collagen alpha 5 (IV) chain associated with X-linked Alport syndrome: characterization of the mutation by direct sequencing of PCR-amplified lymphoblast cDNA fragments.

A large kindred with adult-type X-linked Alport syndrome was studied with regard to a defect in the recently described COL4A5 collagen gene. Southern blot analysis with COL4A5 cDNA probes showed loss of a MspI restriction site. Direct sequencing of cDNA amplified from lymphoblast mRNA demonstrated a single-base substitution converting a glycine codon to arginine at position 325 in the alpha 5 chain of type IV collagen. The triple-helical collagenous domain of alpha 5(IV), characterized by a Gly-X-Y repeat sequence, is interrupted 22 times by noncollagenous sequences. The mutation creates an additional interruption in the Gly-X-Y repeat motif, between interruptions 4 and 5. It is interesting that such glycine substitutions inside the COL1A1 or COL1A2 genes have been associated with many cases of osteogenesis imperfecta. This gly325-to-arg substitution presumably alters the triple-helix formation, and, in turn, modifies the ultrastructural and functional characteristics of the type IV collagen network inside the glomerular basement membrane.     

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.     

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.     

Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome

Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and/or proteinuria with structural defects of the glomerular basement membrane. It can be associated with extrarenal manifestations (high-tone sensorineural hearing loss and ocular abnormalities). Somatic mutations in COL4A5 (X-linked), COL4A3 and COL4A4 genes (both autosomal recessive and autosomal dominant) cause Alport syndrome. Somatic mosaicism in Alport patients is very rare. The reason for this may be due to the difficulty of detection.     

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.     

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     

Topoisomerase I and II consensus sequences in a 17-kb deletion junction of the COL4A5 and COL4A6 genes and immunohistochemical analysis of esophageal leiomyomatosis associated with Alport syndrome.

Diffuse esophageal leiomyomatosis (DL), a benign smooth-muscle-cell tumor, is characterized by abnormal cell proliferation. DL is sometimes associated with X-linked Alport syndrome (AS), an inherited nephropathy caused by COL4A5 gene mutations. COL4A5 is tightly linked, in a head-to-head fashion, to the functionally related and coordinately regulated COL4A6 gene. No X-linked AS cases are due to COL4A6 mutations, but all DL/AS cases are always associated with deletions spanning the 5' regions of the COL4A5/COL4A6 cluster. Unlike the COL4A5 breakpoints, those of COL4A6 are clustered within intron 2 of the gene. We identified a DL/AS deletion and the first characterization of the breakpoint sequences. We show that a deletion eliminates the first coding exon of COL4A5 and the first two coding exons of COL4A6. The breakpoints share the same sequence, which, in turn, is closely homologous to the consensus sequences of topoisomerases I and II. Additional DNA evidence suggested that the male patient is a somatic mosaic for the mutation. Immunohistochemical analysis using alpha-chain-specific monoclonal antibodies supported this conclusion, since it revealed the absence of the alpha5(IV) and alpha6(IV) collagen chains in most but not all of the basement membranes of the smooth-muscle-cell tumor. We also documented a similar segmental staining pattern in the glomerular basement membranes of the patient's kidney. This study is particularly relevant to the understanding of DL pathogenesis and its etiology.     

Sequence and localization of a partial cDNA encoding the human alpha 3 chain of type IV collagen.

A novel type IV collagen, alpha 3(IV), has recently been identified in human and bovine basement membranes. Here we describe the cloning and sequencing of a cDNA encoding 218 residues of the NC1 domain of the human alpha 3(IV) chain. Of interest is the possible role of abnormalities of the alpha 3(IV) chain in Alport syndrome, as suggested by the failure to detect the NC1 domain of alpha 3(IV) in the basement membranes of some Alport syndrome patients. To determine whether the alpha 3(IV) gene (COL4A3) may be mutated in Alport syndrome, we localized it, by somatic cell hybrid analysis and in situ hybridization of metaphase chromosomes, to chromosome 2q35-2q37. Mutations in alpha 3(IV) cannot therefore be responsible for the vast majority of cases of Alport syndrome, which have been shown to be X linked. One explanation for the immunochemical data implicating alpha 3(IV) in Alport syndrome pathogenesis is that mutations of the alpha 5(IV) chain, which has been localized to Xq22 and found to be mutated in at least three kindreds with Alport syndrome, lead to failure to incorporate the alpha 3(IV) chains into the multimeric structure of glomerular basement membrane in a stable fashion.     

Glycine substitutions in the triple-helical region of type VII collagen result in a spectrum of dystrophic epidermolysis bullosa phenotypes and patterns of inheritance.

The dystrophic forms of epidermolysis bullosa (DEB) are characterized by fragility of the skin and mucous membranes. DEB can be inherited in either an autosomal dominant or autosomal recessive pattern, and the spectrum of clinical severity is highly variable. The unifying diagnostic hallmark of DEB is abnormalities in the anchoring fibrils, which consist of type VII collagen, and, recently, mutations in the corresponding gene, COL7A1, have been disclosed in a number of families. In this study, we report six families with glycine substitution mutations in the triple-helical region of type VII collagen. Among the six families, two demonstrated a mild phenotype, and the inheritance of the mutation was consistent with the dominantly inherited form of DEB. In the four other families, the mutation was silent in the heterozygous state but, when present in the homozygous state, or combined with a second mutation, resulted in a recessively inherited DEB phenotype. Type VII collagen is, therefore, unique among the collagen genes, in that different glycine substitutions can be either silent in heterozygous individuals or result in a dominantly inherited DEB. Inspection of the locations of the glycine substitutions along the COL7A1 polypeptide suggests that the consequences of these mutations, in terms of phenotype and pattern of inheritance, are position independent.     

Autosomal recessive disorder otospondylomegaepiphyseal dysplasia is associated with loss-of-function mutations in the COL11A2 gene

Otospondylomegaepiphyseal dysplasia (OSMED) is an autosomal recessive skeletal dysplasia accompanied by severe hearing loss. The phenotype overlaps that of the autosomal dominant disorders-Stickler and Marshall syndromes-but can be distinguished by disproportionately short limbs, severe hearing loss, and lack of ocular involvement. In one family with OSMED, a homozygous Gly-->Arg substitution has been described in COL11A2, which codes for the alpha2 chain of type XI collagen. We report seven further families with OSMED. All affected individuals had a remarkably similar phenotype: profound sensorineural hearing loss, skeletal dysplasia with limb shortening and large epiphyses, cleft palate, an extremely flat face, hypoplasia of the mandible, a short nose with anteverted nares, and a flat nasal bridge. We screened affected individuals for mutations in COL11A2 and found different mutations in each family. Individuals from four families, including three with consanguineous parents, were homozygous for mutations. Individuals from three other families, in whom parents were nonconsanguineous, were compound heterozygous. Of the 10 identified mutations, 9 are predicted to cause premature termination of translation, and 1 is predicted to cause an in-frame deletion. We conclude that the OSMED phenotype is highly homogenous and results from homozygosity or compound heterozygosity for COL11A2 mutations, most of which are predicted to cause complete absence of alpha2(XI) chains.     

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.     

Identification of a novel COL4A5 mutation in the proband initially diagnosed as IgAN from a Chinese family with X-linked Alport syndrome

Alport syndrome(AS) is a hereditary progressive nephropathy characterized by hematuria, ultrastructural lesions of the glomerular basement membrane, ocular lesions and sensorineural hearing loss. Germline mutations of COL4 A5 are associated with X-linked AS with an extreme phenotypic heterogeneity. Here, we investigated a Chinese family with Alport syndrome. The proband was a 9-year-old boy with hematuria and proteinuria. Based on the test results of renal biopsy and immunofluorescence,the proband was initially diagnosed as Ig A nephropathy and the treatment was recommended accordingly. Meanwhile, we found that the treatment outcome was poor. Therefore, for proper clinical diagnosis and appropriate treatment, targeted exome-based next-generation sequencing has been undertaken. We identified a novel hemizygous single nucleotide deletion c.1902 del A in COL4 A5 gene. Segregation analysis identified that this novel mutation is co-segregated among the affected family members but absent in unaffected family members. The clinical diagnosis of the proband was revised as AS accompanied by Ig A nephropathy,which has been rarely reported. Our findings demonstrated the significance of the application of Genetic screening, expanded the mutation spectrum of COL4 A5 associated AS patients with atypical renal phenotypes and provided a good lesson to be learned from our detour during the diagnosis.     

Mutation in type II procollagen (COL2A1) that substitutes aspartate for glycine alpha 1-67 and that causes cataracts and retinal detachment: evidence for molecular heterogeneity in the Wagner syndrome and the Stickler syndrome (arthro-ophthalmopathy)

A search for mutations in the gene for type II procollagen (COL2A1) was carried out in affected members of a family with early-onset cataracts, lattice degeneration of the retina, and retinal detachment. They had no symptoms suggestive of involvement of nonocular tissues, as is typically found in the Stickler syndrome. The COL2A1 gene was amplified with PCR, and the products were analyzed by denaturing gradient gel electrophoresis. The results suggested a mutation in one allele for exon 10. Sequencing of the fragment demonstrated a single-base mutation that converted the codon for glycine at position alpha 1-67 to aspartate. The mutation was found in three affected members of the family available for study but not in unaffected members or 100 unrelated individuals. Comparison with previously reported mutations suggested that mutations introducing premature termination codons in the COL2A1 gene are a frequent cause of the Stickler syndrome, but mutations in the COL2A1 gene that replace glycine codons with codons for bulkier amino acid can produce a broad spectrum of disorders that range from lethal chondrodysplasias to a syndrome involving only ocular tissues, similar to the syndrome in the family originally described by Wagner in 1938.     

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.     

DNA rearrangements in the alpha 5(IV) collagen gene (COL4A5) of individuals with Alport syndrome: further refinement using pulsed-field gel electrophoresis.

Alport syndrome (AS), an X-linked kidney disorder, has been shown to be caused by mutations in the gene for the alpha 5-chain of type IV collagen (COL4A5), which maps to Xq22. On the basis of the results of conventional Southern blot analysis of AS patient DNAs, we employed pulsed-field gel electrophoresis to characterize further three gene rearrangements at the 3'-end of alpha 5(IV). We were able to construct long-range restriction maps for all three of these patients and deduce the extent and nature of each rearrangement. One of these mutations is a 450-kb simple deletion that includes 12 kb of the alpha 5(IV) gene. A second mutation has been shown to be a direct duplication of 35 kb of alpha 5(IV) genomic DNA, and a third mutation involves a complex insertion/deletion event resulting in an overall loss of 25 kb.     

Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.     

Canine COL4A3 and COL4A4: sequencing, mapping and genomic organization.

Canine alpha3 and alpha4 chains of collagen type IV genes (COL4A3 and COL4A4) are expressed in the renal glomerular basement membrane, where they provide a critical structural and functional matrix for other basement membrane components. These genes are candidates for hereditary nephritis (Alport syndrome) in several dog breeds (e.g. English Cocker Spaniel and Bull Terrier). Using RACE and PCR, the cDNA of both genes was cloned and sequenced. Both COL4A3 and COL4A4, as well as canine NPPC (Natriuretic Peptide Precursor C), were mapped to CFA25 using an RH panel. Conservation of the tight linkage of COL4A3 and COL4A4 as seen in human and mouse was verified in the dog. Intron-exon boundaries in both genes were determined by BLAST analysis of the Canis Familiaris Trace Archive. The elucidation of the cDNA sequences, genomic organization and the open reading frames of canine COL4A3 and COL4A4 provide the groundwork for screening these genes for mutations in hereditary nephritis in dogs.