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Biomedical subjects

D N Cooper

Publications and source records attributed to D N Cooper.

At least 19 recordsLinked to original sources
PubMed1995

Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy.

The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

Base Sequence↗
PubMed1995

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Blotting, Southern↗
PubMed1995

Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents.

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2'-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Evidence for a protein S receptor(s) on human vascular smooth muscle cells. Analysis of the binding characteristics and mitogenic properties of protein S on human vascular smooth muscle cells.

The presence of specific binding sites for the coagulation factor protein S (PS) on the surface of human vascular smooth muscle cells (HVSMC) is described. The binding characteristics of 125I-PS to HVSMC were studied and found to be saturable, reversible and, as described by the Hill equation, co-operative (h 1.74; Kd 0.33 nM). Autoradiographic analysis of detergent extracts of HVSMC chemically cross-linked with 125I-PS and separated by SDS/PAGE revealed radioactivity associated with two proteins with apparent molecular masses of 220 and 230 kDa respectively. The mitogenic activity of PS on HVSMC was also investigated. Protein S was shown to stimulate DNA synthesis of growth-arrested HVSMC and to support their proliferation under low-serum conditions in a sustained and dose-dependent manner.

Adolescent↗
PubMed1995

Galectin-1 modulates human melanoma cell adhesion to laminin.

Galectins constitute a gene family of beta-galactoside-specific lectins that show high homology in their carbohydrate-binding site. They have been postulated to be involved in many biological events, but their specific functions are not yet well defined. Galectin-1 is a laminin binding protein that recognizes poly-N-acetyllactosamine chains on this major basement membrane glycoprotein. In this study, we analyzed the possibility that galectin-1 could modulate interactions between human melanoma cells and laminin. We demonstrated that A375 and A2058 cell lines express galectin-1 both intracellularly and on the cell surface. In an in vitro assay, recombinant galectin-1 increased melanoma cell attachment to laminin in a dose-dependent manner. This effect was abolished by lactose. Anti-galectin-1 inhibited adhesion of melanoma cells to laminin in a dose-dependent fashion. However, neither galectin-1 nor anti-galectin-1 antibody affected melanoma cell spreading on laminin in vitro. These data indicate that galectin-1 might participate in melanoma cell adhesion to laminin and therefore could be a modulator of invasion and metastasis.

Cell Adhesion↗
PubMed1995

Core database.

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Databases, Factual↗
PubMed1995

Sequence and mapping of galectin-5, a beta-galactoside-binding lectin, found in rat erythrocytes.

A monomeric rat beta-galactoside-binding lectin previously purified from extracts of rat lung has been localized to erythrocytes, and the cDNA encoding it has been isolated from a rat reticulocyte cDNA library. The deduced amino acid sequence of the cDNA predicts a protein with a M(r) of 16,199, with no evidence of a signal peptide. The deduced sequence is identical to the sequences of seven proteolytic peptides derived from the purified lectin. Peptide analysis by mass spectrometry indicates that the N-terminal methionine is cleaved and that serine 2 is acetylated. The lectin shares all the strictly conserved amino acid residues of other members of the mammalian galectin family and is designated galectin-5 (GenBank accession number L36862). Galectin-5 is a weak agglutinin of rat erythrocytes, despite its monomeric structure. The gene encoding galectin-5 (LGALS5) has been mapped in mouse to chromosome 11, approximately 50 centimorgans from the centromere and 1.8 +/- 1.8 centimorgans from the polymorphic marker D11Mit34n, a region syntenic with human chromosome 17q11.

Amino Acid Sequence↗
PubMed1995

Somatic spectrum of cancer-associated single basepair substitutions in the TP53 gene is determined mainly by endogenous mechanisms of mutation and by selection.

The spectrum of somatic TP53 single basepair substitutions detected in 955 cancers was compared with that of 2,224 different germline mutations in 279 different human genes (other than TP53), reported as the cause of inherited disease. This comparison reveals that, disregarding a relatively small subset (12%) of TP53 mutations that probably result from the action of exogenous mutagens, both the relative rates and the nearest-neighbor spectra of single basepair substitutions are similar in the two datasets. This spectral resemblance suggests that a substantial proportion of cancer-associated somatic TP53 mutations result from endogenous cellular mechanisms. The likelihood of clinical observation of a particular mutation type differs, however, between tumors and genetic diseases, when the chemical properties of the resulting amino acid substitutions are considered. Together with a sixfold higher observation likelihood for mutations at evolutionarily conserved residues, this finding argues that selection is a critical factor in determining which TP53 mutations are found to be associated with human cancer.

Base Sequence↗
PubMed1995

The mutational demography of protein C deficiency.

The geographical distribution and prevalence of 256 single base-pair substitutions (105 of them being different) within the coding region of the human protein C (PROC) gene were correlated with their initial likelihoods of generation. A significant positive correlation was observed between these "mutational likelihoods" and the geographical dispersal of the PROC gene lesions within and between 16 different countries. This relationship could be attributed to the fact that, with few exceptions, high dispersal was only exhibited by CG-->TG and CG-->CA transitions, i.e. those substitutions that are known to arise de novo at the highest frequency. The statistical distribution of mutational likelihoods was as predicted on the basis of the PROC cDNA sequence alone, allowing however for the redundancy of the genetic code. These findings suggest (1) that genetic drift and lesion-specific selection have been of relatively minor importance in determining the mutational spectrum observed in the PROC gene and (2) that most multiple reports of particular substitutions in different geographical locations appear to reflect recurrent mutation rather than identity-by-descent.

Blood Coagulation Disorders↗
PubMed1995

Three novel mutations in the protein C (PROC) gene causing venous thrombosis.

Missense mutations, three of them novel (Gly47-->Cys, Arg178-->Pro, Ala259-->Thr), were found in the protein C genes of four patients with inherited protein C deficiency associated with venous thrombosis. Comparison with the phenotypic effects of mutations in the analogous residues of factor IX and the use of a molecular model of protein C provided explanations as to how these lesions might alter either the structure, function or stability of the protein C molecules encoded.

Adolescent↗
PubMed1995

Polymorphic variation in the human protein C (PROC) gene promoter can influence transcriptional efficiency in vitro.

The haplotype frequencies of two closely linked diallelic polymorphisms in the protein C(PROC) gene promoter were determined in the British population. In principle, differences in transcription efficiency between PROC promoter haplotypes could represent an additional risk factor in determining whether or not an individual already predisposed to venous thrombosis will come to clinical attention. In order to explore this postulate, transient transfection of human hepatoma cells with PROC promoter-luciferase reporter gene constructs was performed in vitro. In HepG2 cells, the T.....A haplotype exhibited at least a two-fold higher transcription efficiency than the C.....G haplotype. A sample of British protein C deficiency patients with recurrent venous thrombosis was then allelotyped by a combination of oligonucleotide discriminant hybridization and DNA sequencing. The frequency of the low expressing C.....G haplotype was found to be slightly yet not significantly higher in these patients (0.43) than in controls (0.35).

Base Sequence↗
PubMed1995

Population differences in the frequency of the factor V Leiden variant among people with clinically symptomatic protein C deficiency.

The factor V Leiden variant, responsible for the phenomenon of activated protein C resistance, was found to be less frequent among British (0.06) and Swedish/Danish (0.15) protein C deficiency patients than previously reported in a Dutch study (0.19). In the Swedish population, a significantly increased frequency of the factor V Leiden allele was apparent in protein C deficiency patients as compared to healthy controls. However, this was not found in the British population. Coinheritance of the factor V Leiden variant is therefore unlikely to be the sole determinant of whether a person with protein C deficiency will come to clinical attention. Nevertheless, when patient data were analysed by type of protein C deficiency, it was noted that the frequency of the factor V Leiden variant was 2.8-fold higher in type II patients compared to type I patients. A possible explanation of this disparity is discussed.

Alleles↗
PubMed1994

Determinants of the factor IX mutational spectrum in haemophilia B: an analysis of missense mutations using a multi-domain molecular model of the activated protein.

A multi-domain molecular model of factor IXa was constructed by comparative methods. The quaternary structure of the protein was assembled by docking individual domains through consideration of their shape complementarity, polaric properties and the location of cross-reacting material positive/negative (CRM+/-) variants on domain surfaces. Some 217 different missense mutations in the factor IX (F9) gene were then selected for study. Using maximum likelihood analysis, missense mutations affecting highly conserved amino acid residues of factor IX were shown to be 15-20 times more likely to result in haemophilia B than those affecting non-conserved residues. However, about one quarter of this increase in likelihood of clinical observation could be attributed to the magnitude of the amino acid exchange. Missense mutations in structurally conserved residues were found to be 2.1-fold more likely to come to clinical attention than those in structurally variable residues. Missense mutations in residues whose side chains were inwardly pointing were 3.6-fold more likely to be observed than those in surface residues. These observations imply a complex hierarchy of sequence/structure conservation in the protein. The severity of the clinical phenotype correlated with both the extent of the evolutionary sequence conservation of the residue at the site of mutation and the magnitude of the amino acid exchange. Further, the substitution of residues exhibiting minimal side chain solvent accessibility was associated disproportionately with severe haemophilia compared with that of surface residues. Clusters of CRM+ mutations were observed at factor IX-specific residues on the surface of the molecule. These clusters may reflect factor IX-specific docking interactions. The likelihood that a given factor IX mutation will come to clinical attention is therefore a complex function of the sequence characteristics of the F9 gene, the nature of the amino acid substitution, its precise location and immediate environment within the protein molecule, and its resulting effects on the structure and function of the protein.

Factor IX↗
PubMed1994

Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis.

The polymerase chain reaction and direct sequencing were used to determine the nature of the mutations in the antithrombin III (AT3) gene in seven unrelated patients with familial antithrombin III (ATIII) deficiency and recurrent venous thrombosis. Three novel mutations were found, two associated with a type I deficiency state (Pro80-->Thr and His120-->Tyr) manifesting reduced synthesis of ATIII. The other novel lesion (Met251-->Ile) was associated with a dysfunctional ATIII protein (type II ATIII deficiency) and is predicted to interfere either with a heparin-induced conformational change in the ATIII molecule or with docking to thrombin. A novel polymorphism (Tyr158-->Cys) was also found to occur in several individuals of Scandinavian origin.

Amino Acid Sequence↗
PubMed1994

The molecular genetics of familial venous thrombosis.

This study of naturally occurring mutations predisposing to venous thrombosis has led to a number of important advances in our understanding of protein structure and function relationships and the molecular basis of gene mutation. It has also potentiated the accurate and reliable presymptomatic and antenatal detection of predisposing gene lesions. Perhaps the major challenge facing us is the probabilistic nature of thromboembolism; only a certain proportion of patients with recognized gene defects predisposing to thrombosis will actually suffer from thrombotic episodes. Environmental insults of various kinds, and perhaps epistatic effects resulting from the influence of other loci, are likely to be contributory factors and will help to determine whether a thrombotic event occurs in individuals already compromised by a defect in a gene whose malfunction is known to predispose to thrombosis. Since molecular genetic techniques allow us to dissect the allelioheterogeneity of the different deficiency states by characterizing the wide spectrum of gene mutations giving rise to thrombosis, it may eventually prove possible to relate specific gene lesions to the probability of thromboembolism as well as to the severity and frequency of thrombotic episodes. The multifactorial nature of thrombosis demands a multidisciplinary approach to the analysis of its causation, early detection, treatment and prevention. The application of the new and powerful techniques of molecular genetics promises to make a substantial contribution to all aspects of thrombosis research.

Afibrinogenemia↗