Molecular defects of type III procollagen in Ehlers-Danlos syndrome type IV

Summary Fibroblasts from most patients with Ehlers-Danlos syndrome (EDS) type IV, a disorder characterized by fragility of skin, blood vessels, and internal organs, secrete reduced amounts of type III procollagen. In 7 of 8 cell strains analyzed, we found evidence of structural defects in half of the type III procollagen chains synthesized, such as deletions or bona fide amino acid substitutions, which cause delayed formation and destabilization of the collagen triple helix and, as a consequence, reduced secretion of the molecule. The data suggest that EDS type IV is often caused by heterozygosity for mutations at the COL3A1 locus, which affect the structure of type III procollagen. The triple-helical region of the molecule, like the homologous region of type I procollagen, appears to be particularly vulnerable.Parts of this work have been presented at the 2nd International Conference on Molecular Biology and Pathology of Matrix, Philadelphia, June 15–18, 1988     

A single base mutation that substitutes serine for glycine 790 of the alpha 1 (III) chain of type III procollagen exposes an arginine and causes Ehlers-Danlos syndrome IV

Previous observations (Stolle, C.A., Pyeritz, R.E., Myers, J.C., and Prockop, D.J. (1985) J. Biol. Chem. 260, 1937-1944) indicated that fibroblasts from a proband with dominantly inherited Ehlers-Danlos syndrome type IV synthesized type III procollagen with a structural defect near the collagenase cleavage site at amino acid 781 and near the trypsin-sensitive site at 789. The type III procollagen was unusually sensitive to proteinases and cleaved by trypsin into a three-quarter fragment at 0 degrees C. Here we demonstrate that the mutation in the type III procollagen gene is a single base mutation that converts the codon for glycine at amino acid 790 of the alpha 1(III) chain to a codon for serine. The mutation probably makes the procollagen molecule unusually sensitive to proteases because it causes local unfolding of the triple helix and exposes the adjacent arginine residue. The results provide the first indication that not all glycine substitutions in the triple helices of fibrillar collagens are equivalent in terms of their effects of the biological function of the molecule.     

Multiexon deletion in the procollagen III gene is associated with mild Ehlers-Danlos syndrome type IV

We have characterized a deletion of approximately 9 kilobases which spans from intron 33 to exon 48 of one pro-alpha 1 (III) collagen allele in a patient with Ehlers-Danlos syndrome type IV. The mutation results in the production of an in-frame species of mRNA which lacks the sequences corresponding to residues 595-1,008 of the triple-helical domain. Thus, half of the pro-alpha 1 (III) chains synthesized by the patient's fibroblasts are nearly 30% shorter than normal. The procollagen III molecules composed of either three normal length or three shortened chains are thermally stable and efficiently secreted. In contrast, the procollagen III molecules that contain one or two shortened chains are unstable and are not secreted. Failure to secrete unstable molecules and a residual functional role of the shortened but stable homotrimers may explain the somewhat milder phenotype of this individual compared with that of another Ehlers-Danlos type IV patient bearing a deletion of similar size in the amino-terminal portion of the alpha 1 (III) collagen chain.     

Ehlers-Danlos syndrome type IV: cosegregation of the phenotype to a COL3A1 allele of type III procollagen

Summary Ehlers-Danlos syndrome (EDS) type IV is a rare and catastrophic genetic disorder of the connective tissue. Individuals from two families with this disorder were studied for a restriction fragment length polymorphism (RFLP) associated with the COL3A1 gene. Our results suggested cosegregation of the EDS type IV phenotype with a COL3A1 RFLP allele. Biochemical studies in cultured skin fibroblasts indicated the presence of different mutations affecting the stability and secretion of the pro1(III) chains of type III procollagen in the two families, thus suggesting that EDS type IV is biochemically heterogeneous. Our data demonstrated the feasibility of molecular diagnosis in this condition using COL3A1 gene related RFLPs.     

Deletion of 24 amino acids from the pro-alpha 1(I) chain of type I procollagen in a patient with the Ehlers-Danlos syndrome type VII

A child with the type VII form of the Ehlers-Danlos syndrome was shown to have a structural defect in the amino terminus of the pro-alpha 1(I) chain of type I procollagen. Normal and mutant amino-terminal cyanogen bromide peptides (pN-alpha 1(I) CB0,1 peptides) were purified from the medium of the patient's cultured fibroblasts. Amino acid sequencing of tryptic peptides derived from the mutant pN-alpha 1(I) CB0,1 peptide showed that an expected sequence of 24 amino acids (positions 136-159 of the normal pN-alpha 1(I) CB0,1 peptide) was deleted. The segment deleted from the mutant pro-alpha 1(I) chain contains the small globular region of the NH2-propeptide, the procollagen N-proteinase cleavage site, the NH2-telopeptide, and first triplet of the helix of the alpha I(I) collagen chain (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340). Loss of the procollagen N-proteinase cleavage site from the mutant pro-alpha 1(I) chain accounted for the persistence of its NH2-propeptide despite normal production of the N-proteinase by cultured mutant fibroblasts. Collagen production by mutant fibroblasts was doubled possibly due to reduced feedback inhibition by the NH2-propeptides. The child appeared to be heterozygous for the peptide deletion and, as the parents did not show any evidence of the deletion, it is likely that the child had a new mutation of one allele of the pro-alpha 1(I) gene. The deleted peptide corresponds precisely to the sequence coded by exon 46 of the normal pro-alpha 1(I) gene (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340).     

Haploinsufficiency for one COL3A1 allele of type III procollagen results in a phenotype similar to the vascular form of Ehlers-Danlos syndrome, Ehlers-Danlos syndrome type IV

Mutations in the COL3A1 gene that encodes the chains of type III procollagen result in the vascular form of Ehlers-Danlos syndrome (EDS), EDS type IV, if they alter the sequence in the triple-helical domain. Although other fibrillar collagen-gene mutations that lead to allele instability or failure to incorporate proalpha-chains into trimers-and that thus reduce the amount of mature molecules produced-result in clinically apparent phenotypes, no such mutations have been identified in COL3A1. Furthermore, mice heterozygous for Col3a1 "null" alleles have no identified phenotype. We have now found three frameshift mutations (1832delAA, 413delC, and 555delT) that lead to premature termination codons (PTCs) in exons 27, 6, and 9, respectively, and to allele-product instability. The mRNA from each mutant allele was transcribed efficiently but rapidly degraded, presumably by the mechanisms of nonsense-mediated decay. In a fourth patient, we identified a point mutation, in the final exon, that resulted in a PTC (4294C-->T [Arg1432Ter]). In this last instance, the mRNA was stable but led to synthesis of a truncated protein that was not incorporated into mature type III procollagen molecules. In all probands, the presenting feature was vascular aneurysm or rupture. Thus, in contrast to mutations in genes that encode the dominant protein of a tissue (e.g., COL1A1 and COL2A1), in which "null" mutations result in phenotypes milder than those caused by mutations that alter protein sequence, the phenotypes produced by these mutations in COL3A1 overlap with those of the vascular form of EDS. This suggests that the major effect of many of these dominant mutations in the "minor" collagen genes may be expressed through protein deficiency rather than through incorporation of structurally altered molecules into fibrils.     

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.     

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.     

Structure of cDNA clones coding for the entire prepro alpha 1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences.

Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.     

The gene for the alpha 1(IV) chain of human type IV procollagen: the exon structures do not coincide with the two structural subdomains in the globular carboxy-terminus of the protein.

Previous studies on the coding sequences of DNAs for the alpha 1(IV) chain of basement membrane collagen demonstrated a striking homology between the first 115 and the second 114 amino acids of the globular (NC1) domain of the protein. Also, alignment of the 12 cysteine residues indicated that the homology was particularly strong around three paired clusters of amino acids around cysteine residues. Here we have isolated a cosmid clone containing the 3'-end of the gene. Analysis of the clone and previously isolated lambda clones demonstrated that the intron--exon patterns of the gene does not reflect the homology in the protein. Therefore the homology cannot have arisen in any simple manner from gene duplications.     

Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved pro alpha 1(I) chain

The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partically purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30 degrees C indicated that, during the initial stages of the reaction, pro alpha 1(I) and pro alpha 2(I) chains were cleaved at about the same rate. As a result, all the pro alpha 2(I) chains were converted to pC alpha 2(I) chains well before all the pro alpha 1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37, and 40 degrees C. The data established that over two-thirds of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction, however, did not fit a simple model for precursor-product relationship among substrate, intermediate, and product. Examination of the reaction products with a two-step gel procedure demonstrated that the intermediate consisted of three polypeptide chains in which the N propeptide was cleaved from one pro alpha 1 chain and one pro alpha 2(I) chain but the N propeptide was still present on one of the pro alpha 1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro alpha 1(I) chains was partially cleaved by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ehlers-Danlos syndrome type IV: a multi-exon deletion in one of the two COL3A1 alleles affecting structure, stability, and processing of type III procollagen

We have studied a patient with severe, dominantly inherited Ehlers-Danlos syndrome type IV. The results indicate that this patient carries a deletion of 3.3 kilo-base pairs in the triple helical coding domain of one of the two alleles for the pro-alpha-chains of type III collagen (COL3A1). His cultured skin fibroblasts contain equal amounts of normal length mRNA and of mRNA shortened by approximately 600 bases, and synthesize both normal and shortened pro-alpha 1(III)-chains. In procollagen molecules containing one or more shortened chains, a triple helix is formed with a length of only about 780 amino acids. The mutant procollagen molecules have decreased thermal stability, are less efficiently secreted, and are not processed as their normal counterpart. The deletion in this family is the first mutation to be described in COL3A1.     

Structure of cDNA clones coding for human type II procollagen. The alpha 1(II) chain is more similar to the alpha 1(I) chain than two other alpha chains of fibrillar collagens.

Overlapping cDNA clones were isolated for human type II procollagen. Nucleotide sequencing of the clones provided over 2.5 kb of new coding sequences for the human pro alpha 1(II) gene and the first complete amino acid sequence of type II procollagen from any species. Comparison with published data for cDNA clones covering the entire lengths of the human type I and type III procollagens made it possible to compare in detail the coding sequences and primary structures of the three most abundant human fibrillar collagens. The results indicated that the marked preference in the third base codons for glycine, proline and alanine previously seen in other fibrillar collagens was maintained in type II procollagen. The domains of the pro alpha 1(II) chain are about the same size as the same domains of the pro alpha chains of type I and type III procollagens. However, the major triple-helical domain is 15 amino acid residues less than the triple-helical domain of type III procollagen. Comparison of hydropathy profiles indicated that the alpha chain domain of type II procollagen is more similar to the alpha chain domain of the pro alpha 1(I) chain than to the pro alpha 2(I) chain or the pro alpha 1(III) chain. The results therefore suggest that selective pressure in the evolution of the pro alpha 1(II) and pro alpha 1(I) genes is more similar than the selective pressure in the evolution of the pro alpha 2(I) and pro alpha 1(III) genes.     

The complete primary structure of the alpha 2 chain of human type IV collagen and comparison with the alpha 1(IV) chain

The complete primary structure of the human type IV collagen alpha 2(IV) chain has been determined by nucleotide sequencing of cDNA clones. The overlapping cDNA clones cover 6,257 base pairs with a 5'-untranslated region of 283 base pairs, the 5,136-base pair open reading frame, and the 3'-untranslated region of 838 base pairs. The predicted amino acid sequence demonstrates that the complete translation product consists of 1,712 residues corresponding in molecular weight to 167,560. The translated polypeptide has a signal peptide of 36 amino acids, an amino-terminal noncollagenous part of 21 residues, a 1,428-residue collagenous domain with 23 interruptions, and a carboxyl-terminal noncollagenous (NC) domain of 227 residues. The calculated molecular mass of the mature human alpha 2(IV) chain is 163,774 Da.     

Inheritance of an RNA splicing mutation (G+ 1 IVS20) in the type III procollagen gene (COL3A1) in a family having aortic aneurysms and easy bruisability: phenotypic overlap between familial arterial aneurysms and Ehlers-Danlos syndrome type IV.

Inheritance of a single base mutation in the type III procollagen gene (COL3A1) was studied in a family with aortic aneurysms and easy bruisability. The mutation was a substitution of A for G+ 1 of intron 20 of the gene and caused aberrant splicing of RNA transcribed from the mutated allele. The phenotype in the family included aortic aneurysms that ruptured and produced death. It also included easy bruisability, but it did not include other characteristic features of Ehlers-Danlos syndrome type IV, such as ecchymoses, abnormal scarring, or prominent subcutaneous blood vessels. The data from the family, together with a review of other probands with mutations in the type III procollagen gene, indicated that there is phenotypic overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any of the striking changes in skin originally cited as a characteristic feature of Ehlers-Danlos syndrome type IV. In addition, the results suggested that DNA tests for mutations in the type III procollagen gene may be useful to identify individuals predisposed to developing arterial aneurysms.     

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.     

G to T transversion at position +5 of a splice donor site causes skipping of the preceding exon in the type III procollagen transcripts of a patient with Ehlers-Danlos syndrome type IV.

We identified a splicing mutation in a patient with Ehlers-Danlos syndrome type IV, a heritable connective tissue disorder associated with dysfunctions of type III collagen. The mutation was first localized in the patient's type III procollagen mRNA by amplifying the reverse transcribed product in several overlapping fragments using the polymerase chain reaction. Amplified products spanning exon 24-26 sequences displayed two distinct fragments, one of normal size and the other lacking the 99 base pairs of exon 25. Sequencing of amplified genomic products identified a G to T transversion at position +5 of the splice donor site of intron 25 in one of the patient's procollagen III genes. Expression of allelic minigene constructs correlated the T for G substitution with skipping of exon 25 sequences. Like previously characterized splicing mutations in other collagen genes, lowering the temperature at which the patient's fibroblasts were incubated nearly abolished exon skipping. As a part of this study, we also identified a highly polymorphic, intronic DNA sequence whose different allelic forms can be detected easily by the polymerase chain reaction technique.     

Substitution of aspartate for glycine 1018 in the type III procollagen (COL3A1) gene causes type IV Ehlers-Danlos syndrome: the mutated allele is present in most blood leukocytes of the asymptomatic and mosaic mother.

A proband with arterial ruptures and skin changes characteristic of the type IV variant of Ehlers-Danlos syndrome was found to have a single-base mutation in the type III procollagen gene, which converted the codon for glycine at amino acid position 1018 to a codon for aspartate. (Amino acid positions are numbered by the standard convention in which the first glycine of the triple-helical domain of an alpha chain is number 1. The numbers of positions in the alpha 1(III) chains can be converted to positions in the human pro alpha(III) chain by adding 167.) Nucleotide sequencing of overlapping PCR products in which the two alleles were distinguished demonstrated that the mutation of glycine 1018 was the only mutation that changed the primary structure of type III procollagen. The glycine substitution markedly decreased the amount of type III procollagen secreted into the medium by cultured skin fibroblasts from the proband. It is surprising that the same mutation was found in about 94% of the peripheral blood leukocytes from the proband's asymptomatic 72-year-old mother. Other tissues from the mother contained the mutated allele; it was present in 0%-100% of different samples of hair cells and in about 40% of cells from the oral epithelium. Therefore, the mother was a mosaic for the mutation. Since the mutated allele was present in cells derived from all three germ layers, the results indicated that the mutation arose by the late blastocyst stage of development. The results also indicate that assays of blood leukocytes do not always reveal mosaicism or predict phenotypic involvement of tissues, such as blood vessels, that are derived from the same embryonic cells as are leukocytes.