Biomedical subjects

G Tromp

Publications and source records attributed to G Tromp.

65 records · Page 4Linked to original sources

PubMed1989

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.

Adult↗

PubMed1989

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.

Arginine↗

PubMed1989

Type I procollagen: the gene-protein system that harbors most of the mutations causing osteogenesis imperfecta and probably more common heritable disorders of connective tissue.

Recent data from several laboratories have established that most variants of osteogenesis imperfecta (OI) are caused by mutations in the 2 structural genes for type I procollagen. There are 2 general reasons for the large number of mutations in type I procollagen in OI. One reason is that most of the structure of the procollagen monomer is essential for normal biological function of the protein. The second reason is that most of the mutations cause synthesis of structurally altered pro alpha chains of type I procollagen. The deleterious effects of the structurally altered pro alpha chains are then amplified by at least 3 mechanisms. One mechanism is a phenomenon referred to as "procollagen suicide" whereby altered pro alpha chains cause degradation of normal pro alpha chains synthesized by the same cell. Another mechanism involves the fact that many of the structurally altered pro alpha chains prevent normal processing of the N-propeptides of procollagen and persistence of the N-propeptide interferes with normal fibril assembly. A third mechanism is a recently discovered phenomenon in which a substitution of a bulkier amino acid for glycine can cause a kink in the triple helix of the molecule. The kinked collagen, in turn, causes formation of abnormally branched fibrils. Because the deleterious effects of abnormal pro alpha chains are amplified by these 3 mechanisms, most of the mutations are dominant and many are dominant lethal. The conclusion that most variants of OI are caused by mutations in the structural genes for type I procollagen has broad implications for other diseases that affect connective tissue, diseases such as chondrodystrophies, osteoarthritis, and osteoporosis.

Amino Acid Sequence↗

PubMed1988

A 19-base pair deletion in the pro-alpha 2(I) gene of type I procollagen that causes in-frame RNA splicing from exon 10 to exon 12 in a proband with atypical osteogenesis imperfecta and in his asymptomatic mother.

Previous observations established that fibroblasts from a proband with atypical osteogenesis imperfecta synthesized about equal amounts of normal pro-alpha 2(I) chains and shortened pro-alpha 2(I) chains of type I procollagen. The pro-alpha 2(I) chains were shortened because of an in-frame deletion of most or all of the 18 amino acids encoded by exon 11 of the pro-alpha 2(I) gene. Here it was demonstrated that one of the proband's alleles for the pro-alpha 2(I) gene contained a 19-base pair deletion at the junction of intervening sequence 10 and exon 11 that produced an RNA splicing defect. Probe protection experiments did not reveal any evidence for use of cryptic splice sites, and they suggested that the major species of abnormally spliced pro-alpha 2(I) mRNA in the proband's fibroblasts was completely spliced from exon 10 to 12. The defect in RNA splicing is unusual among RNA-splicing mutations in producing an abnormal polypeptide chain that is used for protomer assembly. Since the probe protection experiments showed the same defect in the mRNA from the fibroblasts of the asymptomatic mother, the mutation was inherited in an autosomal dominant manner but showed variable phenotypic expression in the proband's family.

Base Sequence↗

PubMed1988

Structure of a full-length cDNA clone for the prepro alpha 1(I) chain of human type I procollagen.

A full-length cDNA clone for the human prepro alpha 1(I) chain of type I procollagen was characterized. Nucleotide sequencing of the first 1500 nucleotide residues of the 5'-end of the cDNA clone provided 729 nucleotide residues and the codons for 243 amino acid residues not previously defined from any species. The data made it possible, for the first time, to compare completely codon usage for the human alpha 1(I) and alpha 2(I) chains.

Amino Acid Sequence↗

PubMed1988

Structure of a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation.

A cDNA clone from a human placental library was found to consist of an essentially full-length cDNA of 4.6 kb for the prepro alpha 2(I) chain of type I procollagen. Nucleotide sequencing of the 5'-end of the cDNA provided a sequence of 1617 nucleotide residues and codons for 539 amino acid residues not previously defined. Comparison of the complete structure of the prepro alpha 2(I) cDNA with previously reported sequences for the chicken pro alpha 2(I) gene indicated that 83% of 1366 total amino acid residues were conserved. In the alpha-chain domain 84% of 1014 amino acid residues were conserved. Also, there was conservation of the previously noted preference for U and C in the third position of codons for glycine, proline and alanine. One major difference between the human and the chicken prepro alpha 2(I) chain was that the human chain contained 21 fewer proline residues, an observation that probably explains why the triple helix of human type I procollagen unfolds at temperatures that are 1-2 degrees C lower. In parallel experiments, sequencing of intron-exon boundaries for nine exons of genomic subclones confirmed and extended previous observations that the pro alpha 2(I) gene, like other genes from fibrillar collagens, has an unusual 54-base pattern of exon sizes that is highly conserved through evolution.

Amino Acid Sequence↗

PubMed1988

Expression of type I procollagen genes.

All of the type I collagen in connective tissue is the product of one structural gene for the pro alpha 1(I) chain and another for the pro alpha 2(I) chain of type I procollagen. An intriguing question therefore is how the expression of the two genes differs in mineralizing and non-mineralizing tissues. One approach that our laboratory has pursued to answer this and related questions is to develop a new system whereby one can examine the self-assembly of collagen fibrils de novo by controlled enzymic cleavage of procollagen to collagen under physiological conditions. The system has made it possible for the first time to define thermodynamic parameters for the self-assembly process. We are now using the system to define the normal kinetics for fibril formation. The results should make it possible to study the effects of other components of extracellular matrix on fibril assembly, including the effects of bone-specific components that initiate mineralization. A second approach has been to define mutations in type I procollagen genes that cause increased brittleness of bone. Over a dozen mutations in type I procollagen genes have been found in probands with osteogenesis imperfecta. One of the surprises has been that at least 25% of the probands with lethal variants of osteogenesis imperfecta have mutations in type I procollagen genes. Another surprise has been the observation that a number of the mutations are tissue specific in terms of their phenotypic manifestations even though the same abnormal pro alpha chains are being synthesized in a variety of tissues.

Amino Acid Sequence↗

PubMed1988

Single base mutation in the pro alpha 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro alpha 2(I) chain.

Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro alpha 2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro alpha 2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro alpha 2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro alpha 2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro alpha 2(I) gene except that four subclones had a single base mutation at the 3' end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3' splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro alpha 2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro alpha 2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.

Base Sequence↗

PubMed1986

Use of R-loop mapping for the assessment of human collagen mutations.

R-loop mapping of DNA:RNA hybrids formed between mutant pro-alpha 2(I) mRNAs and appropriate human pro-alpha 2(I) genomic clones was employed to define the location of mutations which result in the synthesis of shortened pro-alpha 2(I) chains in skin fibroblasts from two variants of osteogenesis imperfecta. Hybridization of the genomic clone NJ-9 with pro-alpha 2(I) mRNA from a patient with a mild atypical form of the disease resulted in the identification of mutant pro-alpha 2(I) mRNA lacking the sequences which correspond to exon 11 of the pro-alpha 2(I) collagen gene. Exon 11, a 54-base pair exon, encodes amino acids 73 to 90 of the alpha 2(I) chain. Also, electron microscopy of R-loop structures formed between the genomic clone NJ-1 and mRNA from a variant with a perinatal lethal form of osteogenesis imperfecta visualized pro-alpha 2(I) mRNAs which did not hybridize to the sequences of exon 28, a 54-base pair exon coding for amino acids 448 to 465 of the alpha 2(I) chain. Moreover, nuclease S1 mapping of the variant's mutant pro-alpha 2(I) mRNA, employing the human pro-alpha 2(I) cDNA clone Hf-15, confirmed the location of the mismatch to the sequences corresponding to exon 28. Although the data do not determine the exact nature of the mutations, they illustrate the use of R-loop mapping as an alternative approach to S1 mapping analysis for the detection and localization of collagen mRNA deletions.

Base Sequence↗

PubMedDate not supplied

First-stage autosomal genome screen in extended pedigrees suggests genes predisposing to low bone mineral density on chromosomes 1p, 2p and 4q.

Osteoporosis is characterized by low bone density, and osteopenia is responsible for 1.5 million fractures in the United States annually. In order to identify regions of the genome which are likely to contain genes predisposing to osteopenia, we genotyped 149 members of seven large pedigrees having recurrence of low bone mineral density (BMD) with 330 DNA markers spread throughout the autosomal genome. Linkage analysis for this quantitative trait was carried out using spine and hip BMD values by the classical lod-score method using a genetic model with parameters estimated from the seven families. In addition, non-parametric analysis was performed using the traditional Haseman-Elston approach in 74 independent sib pairs from the same pedigrees. The maximum lod score obtained by parametric analysis in all families combined was +2.08 (theta = 0.05) for the marker CD3D on chromosome 11q. All other combined lod scores from the parametric analysis were less than +1.90, the threshold for suggestive linkage. Non-parametric analysis suggested linkage of low BMD to chromosomes 1p36 (Zmax = +3.51 for D1S450) and 2p23-24 (Zmax = +2.07 for D2S149). Maximum multi-point lod scores for these regions were +2.29 and +2.25, respectively. A third region with associated lod scores above the threshold of suggestive linkage in both single-point and multi-point non-parametric analysis was on chromosome 4qter (Zmax = +2.95 for D4S1539 and Zmax = +2.48 for D4S1554). Our data suggest the existence of multiple genes involved in controlling spine and hip BMD, and indicate several candidate regions for further screening in this and other independent samples.

Bone Density↗

PubMedDate not supplied

Fibulin-2 exhibits high degree of variability, but no structural changes concordant with abdominal aortic aneurysms.

We used conformation sensitive gel electrophoresis and direct sequencing of PCR products to screen for mutations in the cDNA for fibulin-2, an extracellular matrix protein, from 11 patients with abdominal aortic aneurysms and two controls. When compared with the published reference sequence, a total of 14 single-base sequence variations were detected. Seven of the changes were neutral in that they did not result in an amino acid substitution. There were five missense changes at sites not conserved between human and mouse, and two missense changes at sites conserved between human and mouse. All but two of the sequence variants studied were also present in an additional set of 102 control alleles analyzed. One of these two changes was a missense mutation, but it did not segregate with abdominal aortic aneurysms in the family, whilst the other change was neutral. In conclusion, fibulin-2 has a large number of sequence variations in comparison with our previous analyses of type III collagen, and these variations will be useful in association studies. There was an excellent overall agreement between direct sequencing of PCR-products and conformation sensitive gel electrophoresis.

Adult↗