Two intronic polymorphisms of the human alpha subunit of the olfactory G-protein (G(olf)) are des... more Two intronic polymorphisms of the human alpha subunit of the olfactory G-protein (G(olf)) are described. They were detected with single-stranded conformational polymorphism (SSCP) methods and confirmed by sequencing both strands. These single base pair (bp) substitutions occur in introns 3 (an A/G at 35 bp 3' from the exon 3/intron 3 5' splice site) and 10 (an T/G at 7 bp 5' from the 3' splice site). Both polymorphisms are relatively common, with minor allele frequencies of 31% (intron 3) and 16% (intron 10). The intron 3 variant shows no linkage disequilibrium with an intron 5 (CA)n microsatellite located approximately 50 kb 3' from the intron 3 variant, among a small group of German individuals with schizophrenia. The intron 3 variant is interesting because it may create an 'in-frame' cryptic splice site which, if activated, would add 12 residues to exon 3. The intron 10 variant is interesting because a purine is substituted for a pyrimidine in the 'polypyrimidine' tract of the 3' splice site, a single base substitution of the type which has been associated with aberrant splicing in the androgen receptor gene.
Proceedings of the National Academy of Sciences, 1990
A cosmid clone was isolated that contained an allele for the type II procollagen gene previously ... more A cosmid clone was isolated that contained an allele for the type II procollagen gene previously shown to be coinherited with primary generalized osteoarthritis in a large family. Affected members of the family had evidence of a mild chondrodysplasia, but they developed progressive osteoarthritic changes in many joints that had no epiphyseal deformities. The clone contained 52 of the 54 exons of the gene. Nucleotide sequencing of greater than 20,000 base pairs from the clone demonstrated that all the coding sequences and all the intron-exon boundaries were normal except for a single base mutation that converted the codon for arginine at position 519 of the alpha 1(II) chain to a codon for cysteine, an amino acid not found in type II collagen from humans or a variety of other species. The mutation was found in all affected members of the family but not in unaffected members or in 57 unrelated individuals.
Proceedings of the National Academy of Sciences, 1993
Several techniques have recently been developed to detect single-base mismatches in DNA heterodup... more Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.
Proceedings of the National Academy of Sciences, 1991
Linkage analysis with restriction fragment length polymorphisms for the gene for type II procolla... more Linkage analysis with restriction fragment length polymorphisms for the gene for type II procollagen (COL2A1) was carried out in a family with the Stickler syndrome, or arthro-ophthalmopathy, an autosomal dominant disorder that affects the eyes, ears, joints, and skeleton. The analysis demonstrated linkage of the disease and COL2A1 with a logarithm-of-odds score of 1.51 at zero recombination. A newly developed procedure for preparing cosmid clones was employed to isolate the allele for type II procollagen that was linked to the disease. Analysis of over 7000 nucleotides of the gene revealed a single base mutation that altered a CG dinucleotide and converted the codon CGA for arginine at amino acid position alpha 1-732 to TGA, a stop codon. From previous work on procollagen biosynthesis, it is apparent that the truncated polypeptide synthesized from an allele with a stop codon at alpha 1-732 cannot participate in the assembly of type II procollagen, and therefore that the mutation would decrease synthesis of type II procollagen. It was not apparent, however, why the mutation produced marked changes in the eye, which contains only small amounts of type II collagen, but relatively mild effects on the many cartilaginous structures of the body that are rich in the same protein.
Multiple epiphyseal dysplasia (MED) is an osteochondrodysplasia characterized clinically by mild ... more Multiple epiphyseal dysplasia (MED) is an osteochondrodysplasia characterized clinically by mild short stature and early-onset degenerative joint disease and radiographically by epiphyseal hypoplasia/dysplasia. MED is genetically heterogeneous, with autosomal dominant cases resulting from mutations in at least three genes: the cartilage oligomeric matrix protein (COMP) gene (EDM1) and the COL9A2 (EDM2) and COL9A3 (EDM3) genes of type IX procollagen. We present here a comparison of the radiographic phenotypes of MED patients with type IX collagen gene mutations and those with COMP gene mutations. We reviewed radiographs from two patients with MED produced by COMP mutations, two families with COL9A2 mutations, and one family with a mutation in COL9A3. The data demonstrated that the patients with type IX collagen defects had more severe joint involvement at the knees and relative hip sparing, while the patients with COMP mutations had significant involvement at the capital femoral epiphyses and irregular acetabuli. This pattern of joint involvement was consistent regardless of overall degree of severity of the phenotype.
The etiology of spontaneous cervical artery dissection (sCAD) is unknown. An underlying connectiv... more The etiology of spontaneous cervical artery dissection (sCAD) is unknown. An underlying connective tissue disorder has been suggested. As a collagen disease is conceivable several genes encoding fibrillar collagens have been condsidered as candidate genes for sCAD. We analysed the COL3A1 gene in patients with spontaneous cervical artery dissection (sCAD) and in healthy controls, using three different genetic methods. 1) The promoter region, the 5' and 3' untranscribed regions and the N- and C- peptide encoding regions were studied by direct sequencing analysis of DNA from 12 patients. 2) A possible association of sCAD and the COL3A1 gene was tested for with 5 different DNA polymorphisms in 45 patients and 50 healthy control subjects. 3) DNA samples from a father and his two daughters, all suffering from spontaneous dissections of a cervical artery, were analysed with CA-repeat markers that flank the COL3A1 locus. No disease-causing mutations were found in an extended sequence analysis of the COL3A1 gene in patients with sCAD. However, we identified a single nucleotid polymorphism (SNP) in the promotor region in 2 patients and a 2 bp deletion in the 3' UTR in 7 patients. These sequence variants were also found among 50 healthy subjects. An analysis of multiple DNA polymorphisms of the COL3A1 locus in patients and healthy control persons did not indicate a significant association of sCAD with COL3A1. A deletion polymorphism in the 3' UTR was, however, found more often amongst patients with sCAD. The possible linkage of a hypothetical disease mutation with the COL3A1 locus was tested in a small family with three affected patients. As the affected daughters did not inherit the same COL3A1 allele from their affected father (LOD < - 2.3) COL3A1 was excluded as a disease gene in this family. This study confirms and extends earlier work which suggests that COL3A1 mutations are not a major cause for isolated sCAD.
Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1... more Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis.
The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reacti... more The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.
Two intronic polymorphisms of the human alpha subunit of the olfactory G-protein (G(olf)) are des... more Two intronic polymorphisms of the human alpha subunit of the olfactory G-protein (G(olf)) are described. They were detected with single-stranded conformational polymorphism (SSCP) methods and confirmed by sequencing both strands. These single base pair (bp) substitutions occur in introns 3 (an A/G at 35 bp 3' from the exon 3/intron 3 5' splice site) and 10 (an T/G at 7 bp 5' from the 3' splice site). Both polymorphisms are relatively common, with minor allele frequencies of 31% (intron 3) and 16% (intron 10). The intron 3 variant shows no linkage disequilibrium with an intron 5 (CA)n microsatellite located approximately 50 kb 3' from the intron 3 variant, among a small group of German individuals with schizophrenia. The intron 3 variant is interesting because it may create an 'in-frame' cryptic splice site which, if activated, would add 12 residues to exon 3. The intron 10 variant is interesting because a purine is substituted for a pyrimidine in the 'polypyrimidine' tract of the 3' splice site, a single base substitution of the type which has been associated with aberrant splicing in the androgen receptor gene.
Proceedings of the National Academy of Sciences, 1990
A cosmid clone was isolated that contained an allele for the type II procollagen gene previously ... more A cosmid clone was isolated that contained an allele for the type II procollagen gene previously shown to be coinherited with primary generalized osteoarthritis in a large family. Affected members of the family had evidence of a mild chondrodysplasia, but they developed progressive osteoarthritic changes in many joints that had no epiphyseal deformities. The clone contained 52 of the 54 exons of the gene. Nucleotide sequencing of greater than 20,000 base pairs from the clone demonstrated that all the coding sequences and all the intron-exon boundaries were normal except for a single base mutation that converted the codon for arginine at position 519 of the alpha 1(II) chain to a codon for cysteine, an amino acid not found in type II collagen from humans or a variety of other species. The mutation was found in all affected members of the family but not in unaffected members or in 57 unrelated individuals.
Proceedings of the National Academy of Sciences, 1993
Several techniques have recently been developed to detect single-base mismatches in DNA heterodup... more Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.
Proceedings of the National Academy of Sciences, 1991
Linkage analysis with restriction fragment length polymorphisms for the gene for type II procolla... more Linkage analysis with restriction fragment length polymorphisms for the gene for type II procollagen (COL2A1) was carried out in a family with the Stickler syndrome, or arthro-ophthalmopathy, an autosomal dominant disorder that affects the eyes, ears, joints, and skeleton. The analysis demonstrated linkage of the disease and COL2A1 with a logarithm-of-odds score of 1.51 at zero recombination. A newly developed procedure for preparing cosmid clones was employed to isolate the allele for type II procollagen that was linked to the disease. Analysis of over 7000 nucleotides of the gene revealed a single base mutation that altered a CG dinucleotide and converted the codon CGA for arginine at amino acid position alpha 1-732 to TGA, a stop codon. From previous work on procollagen biosynthesis, it is apparent that the truncated polypeptide synthesized from an allele with a stop codon at alpha 1-732 cannot participate in the assembly of type II procollagen, and therefore that the mutation would decrease synthesis of type II procollagen. It was not apparent, however, why the mutation produced marked changes in the eye, which contains only small amounts of type II collagen, but relatively mild effects on the many cartilaginous structures of the body that are rich in the same protein.
Multiple epiphyseal dysplasia (MED) is an osteochondrodysplasia characterized clinically by mild ... more Multiple epiphyseal dysplasia (MED) is an osteochondrodysplasia characterized clinically by mild short stature and early-onset degenerative joint disease and radiographically by epiphyseal hypoplasia/dysplasia. MED is genetically heterogeneous, with autosomal dominant cases resulting from mutations in at least three genes: the cartilage oligomeric matrix protein (COMP) gene (EDM1) and the COL9A2 (EDM2) and COL9A3 (EDM3) genes of type IX procollagen. We present here a comparison of the radiographic phenotypes of MED patients with type IX collagen gene mutations and those with COMP gene mutations. We reviewed radiographs from two patients with MED produced by COMP mutations, two families with COL9A2 mutations, and one family with a mutation in COL9A3. The data demonstrated that the patients with type IX collagen defects had more severe joint involvement at the knees and relative hip sparing, while the patients with COMP mutations had significant involvement at the capital femoral epiphyses and irregular acetabuli. This pattern of joint involvement was consistent regardless of overall degree of severity of the phenotype.
The etiology of spontaneous cervical artery dissection (sCAD) is unknown. An underlying connectiv... more The etiology of spontaneous cervical artery dissection (sCAD) is unknown. An underlying connective tissue disorder has been suggested. As a collagen disease is conceivable several genes encoding fibrillar collagens have been condsidered as candidate genes for sCAD. We analysed the COL3A1 gene in patients with spontaneous cervical artery dissection (sCAD) and in healthy controls, using three different genetic methods. 1) The promoter region, the 5' and 3' untranscribed regions and the N- and C- peptide encoding regions were studied by direct sequencing analysis of DNA from 12 patients. 2) A possible association of sCAD and the COL3A1 gene was tested for with 5 different DNA polymorphisms in 45 patients and 50 healthy control subjects. 3) DNA samples from a father and his two daughters, all suffering from spontaneous dissections of a cervical artery, were analysed with CA-repeat markers that flank the COL3A1 locus. No disease-causing mutations were found in an extended sequence analysis of the COL3A1 gene in patients with sCAD. However, we identified a single nucleotid polymorphism (SNP) in the promotor region in 2 patients and a 2 bp deletion in the 3' UTR in 7 patients. These sequence variants were also found among 50 healthy subjects. An analysis of multiple DNA polymorphisms of the COL3A1 locus in patients and healthy control persons did not indicate a significant association of sCAD with COL3A1. A deletion polymorphism in the 3' UTR was, however, found more often amongst patients with sCAD. The possible linkage of a hypothetical disease mutation with the COL3A1 locus was tested in a small family with three affected patients. As the affected daughters did not inherit the same COL3A1 allele from their affected father (LOD < - 2.3) COL3A1 was excluded as a disease gene in this family. This study confirms and extends earlier work which suggests that COL3A1 mutations are not a major cause for isolated sCAD.
Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1... more Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis.
The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reacti... more The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.
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