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Deletion of the Paired α5(IV) and α6(IV) Collagen Genes in Inherited Smooth Muscle Tumors
Abstract
The gene encoding alpha 6(IV) collagen, COL4A6, was identified on the human X chromosome in a head-to-head arrangement and within 452 base pairs of the alpha 5(IV) collagen gene, COL4A5. In earlier studies, intragenic deletions of COL4A5 were detected in a subset of patients with Alport syndrome (AS), a hereditary defect of basement membranes. In some families, AS cosegregates with diffuse leiomyomatosis (DL), a benign smooth muscle tumor diathesis. Here it is shown that patients with AS-DL harbor deletions that disrupt both COL4A5 and COL4A6. Thus, type IV collagen may regulate smooth muscle differentiation and morphogenesis.
... 2,5,6,[8][9][10][11][12][13][14][15][16][17][18][19][20] Eventually all 6 type IV collagen isoforms, α1(IV) to α6(IV) chains, were reported, cloned, and described to share similar structures including: a C-terminal non-collagenous (NC1) domain of w230 residues, a w1,400 residue collagenous Gly-X-Y repeat that forms a triple-helix with 2 other chains, and an N-terminal '7S' domain. 10,[21][22][23][24][25][26][27] The glycine residue is frequently mutated in Alport syndrome. 28 Unlike the ubiquitous basement membrane α1(IV) and α2(IV) chains, the other isoforms were observed to have more limited tissue distribution. ...
... The age at which kidney failures occurs, if it is to happen, has broad variability. 68,69,73 In one retrospective study, the median ages at kidney failure for X-linked Alport syndrome males with large deletion/proteintruncating, splice site, and missense variants were 25 (95% CI, 21-31), 28 (95% CI, [26][27][28][29][30][31][32], and 37 (95% CI, 34-40) years, respectively (P < 0.001 for the former 2 versus the latter), indicating allelic heterogeneity. 69 In another study, 5 heterozygous X-linked Alport syndrome females had a progressive course that varied from 12 to 83 years at the time of kidney failure. ...
... P < 0.001). 96 The geometric mean UACR ratio was 19% lower in the treated compared with placebo group (95% CI, [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. There was no increase in serious adverse events in the empagliflozin group. ...
Alport syndrome is a hereditary disorder characterized by kidney disease, ocular abnormalities, and sensorineural hearing loss. Work in understanding the cause of Alport syndrome and the molecular composition of the glomerular basement membrane ultimately led to the identification of COL4A3, COL4A4 (both on chromosome 2q36), and COL4A5 (chromosome Xq22), encoding the α3, α4, and α5 chains of type IV collagen, as the responsible genes. Subsequent studies suggested that autosomal recessive Alport syndrome and males with X-linked Alport syndrome have more severe disease, whereas autosomal dominant Alport syndrome and females with X-linked Alport syndrome have more variability. Variant type is also influential-protein-truncating variants in autosomal recessive Alport syndrome or males with X-linked Alport syndrome often present with severe symptoms, characterized by kidney failure, extrarenal manifestations, and lack of the α3-α4-α5(IV) network. By contrast, mild-moderate forms from missense variants display α3-α4-α5(IV) in the glomerular basement membrane and are associated with protracted kidney involvement without extrarenal manifestations. Regardless of type, therapeutic intervention for kidney involvement is focused on early initiation of angiotensin-converting enzyme inhibitors. There are several therapies under investigation including sodium/glucose cotransporter 2 inhibitors, aminoglycoside analogs, endothelin type A antagonists, lipid-modifying drugs, and hydroxychloroquine, although targeting the underlying defect through gene therapy remains in preclinical stages.
... It is important to know that these two genes are found together head to head on chromosome X and that they share the same promoter ( Figure 2a). However, a couple of cases have also been published in which deletions in COL4A6 or in the promoter region are not essential for the development of DL [2,12,13]. Figure 2. (a) Type IV collagen genes (COL4A1-COL4A6) are located in three different chromosomes pairwise, that encode the corresponding α-chains (α1-α6). (b) α-chains can be combined among each other in three different ways, forming triple helices (trimers). ...
Alport syndrome is a genetic and hereditary disease, caused by mutations in the type IV collagen genes COL4A3, COL4A4 and COL4A5, that affects the glomerular basement membrane of the kidney. It is a rare disease with an underestimated prevalence. Genetic analysis of population cohorts has revealed that it is the second most common inherited kidney disease after polycystic kidney disease. Renal involvement is the main manifestation, although it may have associated extrarenal manifestations such as hearing loss or ocular problems. The degree of expression of the disease changes according to the gene affected and other factors, known or yet to be known. The pathophysiology is not yet fully understood, although some receptors, pathways or molecules are known to be linked to the disease. There is also no specific treatment for Alport syndrome; the most commonly used are renin–angiotensin–aldosterone system inhibitors. In recent years, diagnosis has come a long way, thanks to advances in DNA sequencing technologies such as next-generation sequencing (NGS). Further research at the genetic and molecular levels in the future will complete the partial vision of the pathophysiological mechanism that we have, and will allow us to better understand what is happening and how to solve it.
... Lastly, deletions in the X chromosome affecting the collagen IV genes COL4A5 and COL4A6 associated with Alport syndrome have also been associated with development of smooth muscle tumors (diffuse leiomyomatosis), including uterine leiomyomas [106][107][108]. ...
Uterine leiomyomas represent the most common benign gynecologic tumor. These hormone-dependent smooth-muscle formations occur with an estimated prevalence of ~70% among women of reproductive age and cause symptoms including pain, abnormal uterine bleeding, infertility, and recurrent abortion. Despite the prevalence and public health impact of uterine leiomyomas, available treatments remain limited. Among the potential causes of leiomyomas, early hormonal exposure during periods of development may result in developmental reprogramming via epigenetic changes that persist in adulthood, leading to disease onset or progression. Recent developments in unbiased high-throughput sequencing technology enable powerful approaches to detect driver mutations, yielding new insights into the genomic instability of leiomyomas. Current data also suggest that each leiomyoma originates from the clonal expansion of a single transformed somatic stem cell of the myometrium. In this review, we propose an integrated cellular and molecular view of the origins of leiomyomas, as well as paradigm-shifting studies that will lead to better understanding and the future development of non-surgical treatments for these highly frequent tumors.
... and COL4A6 leading to the original hypothesis that absence of 345(IV) and 556(IV) causes the disease (Zhou et al., 1993). However, this was challenged by deletions extending beyond exon 3 of COL4A6 that do not cause diffuse leiomyomatosis (Heidet et al., 1995), and by the identification of a deletion contained with COL4A5 that caused AS-diffuse leiomyomatosis (Sá et al., 2013). ...
While traditionally the function of the extracellular matrix and the basement membrane was considered to be providing structural support, it is now clear that this only covers one aspect of its multiple functions. This is also illustrated by our growing knowledge of the role of collagen IV, a major component of basement membranes, in development, health, and disease. With the extracellular matrix and collagen IV increasingly being recognised as key players in a growing number of diseases from stroke and vascular defects to kidney disease, deafness, and eye abnormalities, it is paramount that we increase our fundamental understanding of these complex molecules ranging from their biosynthesis to their role in human disease. Recently, exciting progress has been made in delineating the mechanisms by which mutations in collagen IV cause disease, and these are being exploited to develop mechanism-based treatments. Yet many important questions remain that need addressing to develop treatments for diseases associated with collagen IV.
Familial glomerular hematuria syndromes result from variants that affect the genes that encode type IV collagen, the major collagenous constituent of glomerular basement membranes (GBM): Alport syndrome (AS) and hereditary angiopathy with nephropathy, aneurysms and cramps (HANAC) syndrome. Persistent hematuria is a cardinal feature of each of these disorders. Pathogenic variants in any of three type IV collagen genes, COL4A3, COL4A4 or COL4A5, can cause AS, which is characterized by progressive deterioration of kidney function, with associated hearing and ocular involvement in many affected individuals. Heterozygous variants in these genes are also significant and link to a wider spectrum of kidney disease. Variants in COL4A3, COL4A4 or COL4A5 account for about 30–50% of children with isolated glomerular hematuria seen in pediatric nephrology clinics. HANAC syndrome arises from variants in COL4A1.KeywordsAlport syndromeType IV collagenFamilial nephritisSensorineural hearing lossFocal segmental glomerulosclerosis
Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha-1(IV), alpha-2(IV), alpha-3(IV), and alpha-5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha-4(IVC been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha-4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha-4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha-2(IV), alpha-1(IV), alpha-5(IV), and alpha-3(IV), respectively. Alignment of the peptides also shows that alpha-4(IV) is most closely related to alpha-2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha-4(IV). Cloning of the region of alpha-4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.
We have generated and characterized cDNA clones providing the complete amino acid sequence of the human type IV collagen chain whose gene has been shown to be mutated in X chromosome-linked Alport syndrome. The entire translation product has 1,685 amino acid residues. There is a 26-residue signal peptide, a 1,430-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a Gly-Xaa-Yaa-repeat sequence interrupted at 22 locations, and a 229-residue carboxyl-terminal noncollagenous domain. The calculated molecular weight of the mature alpha 5(IV) chain is 158,303. Analysis of genomic DNA from members of a kindred with Alport syndrome revealed a new HindIII cleavage site within the coding sequence of one of the cDNA clones characterized. The proband had a new 1.25-kilobase HindIII fragment and a lack of a 1.35-kilobase fragment, and his mildly affected female cousin had both alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin. The results were confirmed by allele-specific hybridization analyses.
We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.
Mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen have been found in linkage with X-chromosomal Alport syndrome (AS). To identify COL4A5 mutations in patients from Germany with clinically defined AS, DNA from 20 unrelated patients was analyzed by conventional Southern blotting. By using full length alpha 5(IV) cDNA probes, large COL4A5 deletions could be detected in two patients. In one case, a 34 kb deletion affecting the 14 most 3' exons of the gene was observed. The second patient harbored a complete COL4A5 deletion. In both cases, functional alpha 5(IV) mRNA was unlikely to be present. Clinically, both patients developed end-stage renal failure before age 30. Furthermore, they had characteristic retinal flecks, and sensorineural hearing loss with typical changes on the audiogram. The patient with the complete deletion of COL4A5 lost the renal allograft due to an anti-GBM mediated glomerulonephritis.
The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.
We have determined the primary structure of the α1(IV)-chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the α1(IV)-chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy-terminal globular domain. The human α1(IV)-chain contains a total of 21 interruptions in the collagenous Gly-X-Y repeat sequence. These interruptions vary in length between two and eleven residues. The α1(IV)-chain contains four cysteine residues in the triple-helical domain, four cysteines in the 15-residue long noncollagenous sequence at the amino-terminus and 12 cysteines in the carboxy-terminal NC-domain
We have isolated two overlapping cDNA clones that provide the complete nucleotide sequence coding for the NC-1 domain and 3′-untranslated region of the α2 chain of human type IV collagen as well as a sequence encoding 232 residues of the collagenous domain. An extensive homology was observed between the sequences of the NC-1 domain of the α1(IV) and α2(IV) chains, but considerably less between the sequences encoding collagenous and 3′-untranslated regions. There were four interruptions in the collagenous sequence studied whereas the comparable region of the α1(IV) chain had only two. A potential oligosaccharide attachment site was found in a 6-residue long interruption of the collagenous domain but none in the NC-1 domain.
Alport syndrome and diffuse leiomyomatosis: Deletions in the 5' end of the COL4A5 collagen gene. Alport syndrome (AS) is an hereditary glomerulonephritis that is mainly inherited as a dominant X-linked trait. Structural abnormalities in the type IV collagen 5 chain gene (COL4A5), which maps to Xq22, have recently been detected in several patients with AS. The association of AS with diffuse esophageal leiomyomatosis (DL) has been reported in 24 patients, most of them also suffering from congenital cataract. The mode of transmission and the location of the gene(s) involved in this association have not been elucidated. Southern blotting using cDNA probes spanning the whole COL4A5 and a 5' end COL4A5 genomic probe showed that three out of three patients with the DL-AS association had a deletion in the 5' part of the COL4A5 gene extending beyond its 5' end. This indicates that the same gene, COL4A5, is involved in classical AS and in DL-AS and that the transmission of DL-AS is X-linked dominant. These results also suggest that leiomyomatosis might be due to the alteration of a second gene involved in smooth muscle cell proliferation, which is located upstream of the COL4A5 gene, and that there might be a contiguous gene deletion syndrome, involving at least the genes coding for congenital cataract, DL and AS.
