Absence of the alpha6(IV) chain of collagen type IV in Alport syndrome is related to a failure at the protein assembly level and does not result in diffuse leiomyomatosis.
ABSTRACT: X-linked Alport syndrome is a progressive nephropathy associated with mutations in the COL4A5 gene. The kidney usually lacks the alpha3-alpha6 chains of collagen type IV, although each is coded by a separate gene. The molecular basis for this loss remains unclear. In canine X-linked hereditary nephritis, a model for X-linked Alport syndrome, a COL4A5 mutation results in reduced mRNA levels for the alpha3, alpha4, and alpha5 chains in the kidney, implying a mechanism coordinating the production of these 3 chains. To examine whether production of alpha6 chain is under the same control, we studied smooth muscle cells from this animal model. We determined the canine COL4A5 and COL4A6 genes are separated by 435 bp, with two first exons for COL4A6 separated by 978 bp. These two regions are >/= 78% identical to the human sequences that have promoter activity. Despite this potential basis for coordinated transcription of the COL4A5 and COL4A6 genes, the alpha6 mRNA level remained normal in affected male dog smooth muscle while the alpha5 mRNA level was markedly reduced. However, both alpha5 and alpha6 chains were absent at the protein level. Our results suggest that production of the alpha6 chain is under a control mechanism separate from that coordinating the alpha3-alpha5 chains and that the lack of the alpha6 chain in Alport syndrome is related to a failure at the protein assembly level, raising the possibility that the alpha5 and alpha6 chains are present in the same network. The lack of the alpha6 chain does not obviously result in disease, in particular leiomyomatosis, as is seen in Alport patients with deletions involving the COL4A5 and COL4A6 genes.
Project description: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.
Project description:Collagen IV is a major structural component of basement membranes. In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes. To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus. Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner. In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly. The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease. This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.
Project description:Hereditary hearing loss is the most common human sensorineural disorder. Genetic causes are highly heterogeneous, with mutations detected in >40 genes associated with nonsyndromic hearing loss, to date. Whereas autosomal recessive and autosomal dominant inheritance is prevalent, X-linked forms of nonsyndromic hearing impairment are extremely rare. Here, we present a Hungarian three-generation family with X-linked nonsyndromic congenital hearing loss and the underlying genetic defect. Next-generation sequencing and subsequent segregation analysis detected a missense mutation (c.1771G>A, p.Gly591Ser) in the type IV collagen gene COL4A6 in all affected family members. Bioinformatic analysis and expression studies support this substitution as being causative. COL4A6 encodes the alpha-6 chain of type IV collagen of basal membranes, which forms a heterotrimer with two alpha-5 chains encoded by COL4A5. Whereas mutations in COL4A5 and contiguous X-chromosomal deletions involving COL4A5 and COL4A6 are associated with X-linked Alport syndrome, a nephropathy associated with deafness and cataract, mutations in COL4A6 alone have not been related to any hereditary disease so far. Moreover, our index patient and other affected family members show normal renal and ocular function, which is not consistent with Alport syndrome, but with a nonsyndromic type of hearing loss. In situ hybridization and immunostaining demonstrated expression of the COL4A6 homologs in the otic vesicle of the zebrafish and in the murine inner ear, supporting its role in normal ear development and function. In conclusion, our results suggest COL4A6 as being the fourth gene associated with X-linked nonsyndromic hearing loss.
Project description:Type IV collagen is present ubiquitously in basement membranes. A bifunctional promoter regulates the expression of the alpha1/alpha2 genes, and the alpha3/alpha4 and the alpha5/alpha6 genes are also considered to be regulated by putative bifunctional promoters. Unlike the other type IV collagen chains, the alpha5(IV) and alpha6(IV) chains do not always co-localize and are present in distinct basement membranes. To address such dichotomy in the alpha5(IV) and alpha6(IV) gene regulation, we cloned a mouse genomic DNA fragment containing the promoter region between the two transcription start sites of these genes and we then placed this putative promoter sequence between the chloramphenicol acetyltransferase and Luciferase reporter genes, so that these genes would be transcribed in opposite directions in this unique construct. Glomerular endothelial cells and mesangial cells generate the kidney glomerular basement membrane, which always contains the alpha5(IV) chain but not the alpha6(IV) chain. In contrast, the basement membranes of Bowman's capsule and distal tubuli (produced by the tubular epithelial cells) contain the alpha6(IV) chain. We demonstrate that, in response to TGF-beta (transforming growth factor beta), epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor, expression from the alpha5(IV) gene is significantly enhanced in the glomerular endothelial cells and mesangial cells, but not expression from the alpha6(IV) gene. In contrast, the expression from the alpha6(IV) gene, and not that from the alpha5(IV) gene, was significantly enhanced in response to growth factors in the tubular epithelial cells. Our results demonstrate that the proximal bifunctional promoter regulates the expression of the alpha5(IV) and alpha6(IV) genes in a cell-specific manner and offers the first demonstration of the promoter plasticity in growth factor regulation of type IV collagen genes in different tissues of the body.
Project description:Type IV collagen is a predominant component of basement membranes, and glomeruli of a kidney filter approximately 70-90 liters of plasma every day through a specialized glomerular basement membrane (GBM). In Alport syndrome, a progressive disease primarily affecting kidneys, mutations in GBM-associated type IV collagen genes (COL4A3, COL4A4, or COL4A5) lead to basement membrane structural defects, proteinuria, renal failure, and an absence of all three GBM collagen triple helical chains because of obligatory posttranslational assembly requirements. Here, we demonstrate that transplantation of wild-type bone marrow (BM) into irradiated COL4A3(-/-) mice results in a possible recruitment of BM-derived progenitor cells as epithelial cells (podocytes) and mesangial cells within the damaged glomerulus, leading to a partial restoration of expression of the type IV collagen alpha3 chain with concomitant emergence of alpha4 and alpha5 chain expression, improved glomerular architecture associated with a significant reduction in proteinuria, and improvement in overall kidney histology compared with untreated COL4A3(-/-) mice or irradiated COL4A3(-/-) mice with BM from adult COL4A3(-/-) mice. The alpha3(IV) collagen produced by BM-derived podocytes integrates into the GBM and associates with other alpha-chains to form type IV collagen triple helical networks. This study demonstrates that BM-derived stem cells can offer a viable strategy for repairing basement membrane defects and conferring therapeutic benefit for patients with Alport syndrome.
Project description:The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues.
Project description:Deletions encompassing the 5' termini of the paired type IV collagen genes COL4A5 and COL4A6 on chromosome Xq22 give rise to Alport syndrome (AS) and associated diffuse leiomyomatosis (DL), a syndrome of disseminated smooth-muscle tumors involving the esophagus, large airways, and female reproductive tract. In this study, we report isolation and characterization of two deletion junctions. The first, in a patient described elsewhere, arose by a nonhomologous recombination event fusing a LINE-1 (L1) repetitive element in intron 1 of COL4A5 to intron 2 of COL4A6, resulting in a 13.4-kb deletion. The second, in a previously undescribed family, arose by unequal homologous recombination between the same L1 and a colinear L1 element in intron 2 of COL4A6, resulting in a>40-kb deletion. L1 elements have contributed to the emergence of this locus as a site of frequent recombinations by diverse mechanisms. These give rise to AS-DL by disruption of type IV collagen and perhaps other as yet unidentified genes, evidenced by deletions as small as 13.4 kb.
Project description:X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the alpha 5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant alpha 5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine alpha 5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the alpha1(IV) and alpha2(IV), but not the alpha 3(IV) to alpha 6(IV) collagen chains. By Northern blotting, an alpha 5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the alpha1(IV) and alpha2(IV) chains but the recombinant alpha 5(IV) chain was not incorporated. These findings are consistent with the concept that the alpha 5(IV) chain requires one or more of the alpha 3(IV), alpha 4(IV), or alpha 6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the alpha 5(IV) transgene was injected into bladder smooth muscle that lacks both the alpha 5(IV) and alpha 6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the alpha 5(IV) and alpha 6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant alpha 5(IV) chain is capable of restoring expression of a second alpha(IV) chain that requires the presence of the alpha 5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome.
Project description:Introduction:X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. Methods:Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV ?5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL2) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. Results:Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 (P = 0.01 and P = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels (P < 0.01), and reduced ER size (P < 0.01 by EM and P < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P < 0.01). Extracellular collagen IV ?5 chain was increased in the M1 line only (P = 0.06). Discussion:Sodium 4-phenylbutyrate increases collagen IV ?5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV ?5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.
Project description:Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.