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C T Baldwin

Publications and source records attributed to C T Baldwin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chromosome 13q deletion with Waardenburg syndrome: further evidence for a gene involved in neural crest function on 13q.

Waardenburg syndrome (WS) is an autosomal dominant disorder characterised by pigmentary abnormalities and sensorineural deafness. It is subcategorised into type 1 (WS1) and type 2 (WS2) on the basis of the presence (WS1) or absence (WS2) of dystopia canthorum. WS1 is always caused by mutations in the PAX3 gene, whereas WS2 is caused by mutations in the microphthalmia (MITF) gene in some but not all families. An association of WS symptoms with Hirschsprung disease (HSCR) has been reported in many families. We report here a patient with characteristics of WS2 and a de novo interstitial deletion of chromosome 13q. We also describe a family with two sibs who have both WS2 and HSCR. In this family, all possible genes for WS and HSCR, but not chromosome 13q, could be excluded. As an association between chromosome 13q and HSCR/WS has been reported previously, these data suggest that there is a gene on chromosome 13q that is responsible for WS or HSCR or both.

Animals↗
PubMed1995

Linkage of early-onset osteoarthritis and chondrocalcinosis to human chromosome 8q.

Calcium pyrophosphate-deposition disease (CPDD), also called "chondrocalcinosis" or "pseudogout," is a disorder characterized by the deposition of calcium-containing crystals in joint tissue, which leads to arthritis-like symptoms. The presence of these crystals in joint tissue is a common finding in the elderly, and, in this population, there is a poor correlation with joint pain. In contrast, early-onset CPDD has been described in several large families in which the disease progresses to severe degenerative osteoarthritis (OA). In these families, an autosomal dominant mode of inheritance is observed, with an age at onset between the 2d and 5th decades of life. In this report, we describe a large New England family with early-onset CPDD and severe degenerative OA. We found genetic linkage between the disease in this family and chromosome 8q, with a multipoint lod score of 4.06. These results suggest that a defective gene at this location causes the disease in this family.

Adult↗
PubMed1994

Mutations in PAX3 associated with Waardenburg syndrome type I.

Waardenburg syndrome (WS) types I, II, and III (McKusick #14882, #19351, and #19350) are related autosomal dominant disorders characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Disease causing PAX3 mutations have been identified in a few families from each of the three disease subtypes, WS-I, WS-II, and WS-III. In others, although the mutations have not been pinpointed, linkage with the PAX3 locus on chromosome 2q35 has been demonstrated. The PAX3 protein is a transcription factor that contains both a paired-domain and a homeodomain DNA binding motif and appears to play a key role during embryogenesis. In this report, we describe two mutations in the human PAX3 gene that cause WS type I. One mutation is a deletion/frameshift in the paired-domain of PAX3 and results in a protein without functional DNA binding domains. The second mutation is a single-base substitution and results in a premature termination codon in the homeodomain of PAX3. This is the first demonstration of a mutation in the homeodomain DNA binding motif in this protein resulting in WS and one of the few examples of a mutation in a homeodomain of any protein that results in human disease.

Amino Acid Sequence↗
PubMed1994

Locus heterogeneity for Waardenburg syndrome is predictive of clinical subtypes.

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between -2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.

Chromosome Mapping↗
PubMed1993

Amplification of cDNAs for human cartilage-specific types II, IX and XI collagens from chondrocytes and Epstein-Barr virus-transformed lymphocytes.

The identification of mutations in cartilage-specific collagen genes in inherited forms of osteoarthritis (OA) and other heritable cartilage diseases has been hampered by the difficulty in obtaining sufficient tissue to isolate RNA and by the loss of the cartilage phenotype during in vitro chondrocyte culture. To overcome these limitations we employed RNA Polymerase Chain Reaction (PCR) to amplify cDNAs for the cartilage-specific collagens types II, IX and XI from mRNA obtained from small numbers of chondrocytes. We also amplified cDNAs for these collagens from "illegitimate transcripts" from Epstein-Barr virus (EBV)-transformed lymphocytes. Total RNA was obtained from freshly isolated human fetal and adult chondrocytes and from long-term (90 days) chondrocyte cultures. The RNA was reverse transcribed to cDNA and the cDNA amplified in the same tube with oligonucleotide primers specific for types II, IX and XI collagens. The amplified double-stranded products were cloned and sequenced. Successful amplification of the entire 4.4 kb of the type II procollagen cDNA was obtained from as little as 6 ng of RNA from freshly isolated fetal human chondrocytes. Seven hundred and eighty base pairs of the alpha 1 (IX) collagen cDNA and the entire published sequence of alpha 2 (XI) collagen cDNA, were also amplified from these cells. We were also able to amplify cDNAs for the three cartilage-specific collagens from "illegitimate transcripts" from EBV-transformed lymphocytes. Thus, these methods will allow the identification of mutations in cartilage-specific collagen genes in patients with inherited OA and other heritable cartilage diseases from small amounts of cartilage or chondrocyte RNA or from non-cartilaginous sources.

Base Sequence↗
PubMed1993

Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I).

Waardenburg syndrome type I (WS-I) is an autosomal dominant disorder characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Klein-Waardenburg syndrome (WS-III) is a disorder with many of the same characteristics as WS-I and includes musculoskeletal abnormalities. We have recently reported the identification and characterization of one of the first gene defects, in the human PAX3 gene, which causes WS-I. PAX3 is a DNA-binding protein that contains a structural motif known as the paired domain and is believed to regulate the expression of other genes. In this report we describe two new mutations, in the human PAX3 gene, that are associated with WS. One mutation was found in a family with WS-I, while the other mutation was found in a family with WS-III. Both mutations were in the highly conserved paired domain of the human PAX3 gene and are similar to other mutations that cause WS. The results indicate that mutations in the PAX3 gene can cause both WS-I and WS-III.

Amino Acid Sequence↗
PubMed1992

An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome.

Here we report the identification and characterization of a gene defect causing Waardenburg's syndrome with hearing loss in a large Brazilian family. This demonstrates a mutation causing Waardenburg's syndrome as well as a mutation causing a form of congenital deafness. The mutation was found in the HuP2 gene, a member of the paired domain family of proteins that bind DNA and regulate gene expression. The mutation occurred in 100% of the cases with the disease in this family and was absent in a random sample of 50 unrelated control subjects. Identification of the Waardenburg's syndrome gene and future characterization of its gene product is likely to increase our understanding of the pathogenesis of this disorder and may allow prevention of deafness of this type.

Amino Acid Sequence↗
PubMed1992

Detection of sequence variants in the gene for human type II procollagen (COL2A1) by direct sequencing of polymerase chain reaction-amplified genomic DNA.

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.

Alleles↗
PubMed1991

Heterozygous mutation in the G+5 position of intron 33 of the pro-alpha 2(I) gene (COL1A2) that causes aberrant RNA splicing and lethal osteogenesis imperfecta. Use of carbodiimide methods that decrease the extent of DNA sequencing necessary to define an unusual mutation.

Cultured skin fibroblasts from a proband with osteogenesis imperfecta were found to synthesize normal and shortened alpha 2(I) chains of type I procollagen. A cDNA library was prepared using mRNA isolated from the proband's fibroblasts. Partial nucleotide sequencing of five clones demonstrated that two clones lacked the 54 base pairs (bp) of coding sequences found in exon 33 of the pro-alpha 2(I) gene (COL1A2). To reduce the amount of nucleotide sequencing required, heteroduplexes were prepared from two of the clones, one normal and the other lacking exon 33, and reacted with a water-soluble carbodiimide under conditions in which nonbase-paired G and T nucleotides are specifically modified by the reagent. Analysis of the heteroduplexes by immunoelectron microscopy suggested that the sequence variation near the codons of exon 33 was the only sequence difference in the cDNA clones. Amplification of cDNA from the proband by polymerase chain reaction gave products of two sizes, one of the expected size for the normal sequence and the other of the expected size for a product lacking the 54 bp in exon 33. To define the mutation in genomic DNA, a 1.6-kilobase region spanning exons 32 and 34 was amplified by the polymerase chain reaction and DNA heteroduplexes were prepared from the products. The heteroduplexes were treated with a water-soluble carbodiimide and then used as templates for primer extension under conditions in which extension terminates at the site of a carbodiimide-modified base. The results suggested a mismatch near the exon-intron boundary of exon 33 and a second mismatch near the 3' end of intron 33. Nucleotide sequencing of the polymerase chain reaction products revealed a single-base substitution in one allele that changed the moderately conserved G at position +5 of the 5' splice site of intron 33 to an A. In addition, there was an apparently neutral single-base substitution that placed both a G and T at position +661 of intron 33. The results provide only the third example of a mutation in the G at the +5 position of an intron that causes aberrant RNA splicing. Also, the results demonstrate that use of techniques involving carbodiimide modification of DNA heteroduplexes can reduce the amount of nucleotide sequencing necessary to define mutations in large and complex genes.

Adult↗
PubMed1990

Single base mutation in the type II procollagen gene (COL2A1) as a cause of primary osteoarthritis associated with a mild chondrodysplasia.

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.

Alleles↗
PubMed1989

A new epidermal growth factor-like domain in the human core protein for the large cartilage-specific proteoglycan. Evidence for alternative splicing of the domain.

A series of cDNA clones for the human core protein of the large cartilage-specific proteoglycan was isolated. Nucleotide sequencing of the clones provided over 2 kilobases of new coding sequences for the human protein. Comparison with published data for cDNA clones covering the same region in rat and chick indicated that domain 8, the lectin-like domain, is highly conserved among species. In contrast, domain 7 is poorly conserved among species. Some of the cDNA clones also contained an additional structural domain between domains 7 and 8 which was not described in the rat or chick sequences. The additional domain of 38 amino acids was highly homologous to epidermal growth factor (EGF)-like sequences seen in other proteins. Because some cDNA clones contained codons for the EGF-like domain and some did not, the results suggested that the EGF-like domain underwent alternative RNA splicing. To confirm alternative splicing of the EGF-like domain, RNA from cartilage cells was used as a template for the polymerase chain reaction. Products of two sizes were obtained. One had the size predicted for mRNA containing the domain and the other had the size predicted for mRNA not containing the domain. Alternative splicing of an EGF-like domain may provide a mechanism of feedback regulation for both the biosynthetic activity and the proliferation of cartilage cells.

Aggrecans↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

A single base mutation that converts glycine 907 of the alpha 2(I) chain of type I procollagen to aspartate in a lethal variant of osteogenesis imperfecta. The single amino acid substitution near the carboxyl terminus destabilizes the whole triple helix.

Type I procollagen was examined in cultured skin fibroblasts from a patient with a lethal variant of osteogenesis imperfecta. About half of the pro-alpha chains were post-translationally overmodified and had a decreased thermal stability. The vertebrate collagenase A fragment had a normal thermal stability, but the B fragment had a decreased thermal stability. Therefore, there was a change in primary structure in amino acids 776-1014 of either the alpha 1(I) or alpha 2(I) chain. Three of five cDNA clones for the alpha 2(I) chain contained a single-base substitution of an A for a G that converted the codon for glycine at amino acid position 907 to aspartate. Complete nucleotide sequencing of bases coding for amino acids 776 to 1014 of the alpha 2(I) chain was carried out in one cDNA clone that contained the mutation in the glycine codon and in one that did not. Also, nucleotide sequencing was performed of bases coding for amino acids 776-1014 of the alpha 1(I) chain in seven independent cDNA clones. No other mutations were found. Therefore, the single base substitution that converts glycine 907 in the alpha 2(I) chain to aspartate is solely responsible for the decreased thermal stability of the type I procollagen synthesized by the proband's fibroblasts. Also, glycine 907 of the alpha 2(I) chain is an important component of a cooperative block that determines the melting temperature of the whole molecule.

Amino Acid Sequence↗
PubMed1989

Increased proteoglycan synthesis following the differentiation of F9 embryonal carcinoma cells: formation of a differentiation-specific proteoheparan sulfate.

We have examined changes in proteoglycan synthesis by F9 embryonal carcinoma cells after the cells have been treated with retinoic acid or retinoic acid plus cholera toxin. Retinoic acid is known to stimulate the differentiation of this cell type to a primitive endoderm-like cell characterized by the production of basement membrane components such as type IV collagen, laminin and proteoglycans. We have now demonstrated that proteoglycan synthesis and secretion were further stimulated when cholera toxin was added in addition to retinoic acid. Moreover, media of these fully differentiated cells was found to contain a different species of proteoheparan sulfate not produced by stem cells or retinoic acid-treated cells. This proteoheparan sulfate had a high density upon CsCl gradient centrifugation. The protein core of this proteoheparan sulfate was estimated by SDS gel electrophoresis to be approximately 15,000 daltons.

Animals↗
PubMed1988

Undifferentiated F9 embryonal carcinoma cells produce a short-chain collagen molecule.

The undifferentiated F9 embryonal carcinoma cells produce a unique collagen that decreases in amount during retinoic acid-induced differentiation of F9 cells into basement-membrane parietal endoderm. A bacterial-collagenase-sensitive protein of approx. 60,000 Da was resolved on polyacrylamide-gel electrophoresis. After pepsin digestion, two pepsin-resistant fragments containing hydroxyproline were demonstrated, suggesting that a portion of the molecule has a stable triple helix. The mRNA from the undifferentiated F9 cells translates a collagenase-sensitive protein with a molecular mass consistent with the 60,000 Da collagenous protein produced by undifferentiated F8 cells.

Animals↗