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Biomedical subjects

D N Cooper

Publications and source records attributed to D N Cooper.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1994

Xenopus laevis L-14 lectin is expressed in a typical pattern in the adult, but is absent from embryonic tissues.

Soluble, dimeric, lactose-binding lectins with subunit M(r) of approximately 14-16 x 10(3), here called L-14s, are expressed in multiple tissues in all vertebrates that have been examined. L-14s have particular affinity for polylactosamine chains on laminin, co-localize with laminin in some basement membranes, and influence adhesion to laminin and proliferation for some cultured cells. In previous studies of mammals and chickens, L-14s have been found at high levels in a variety of adult tissues, such as muscle and peripheral nerve, but at much higher levels in many embryonic tissues, suggesting a special role in development. To further explore possible roles of L-14 in embryogenesis, we have studied the expression of L-14 in embryonic and adult Xenopus laevis tissues. Xenopus skin, we find that Xenopus L-14 is expressed in the same general distribution as its mammalian homologues. However, we could detect no expression of L-14 in Xenopus embryos using either a sensitive immunoassay for the protein or a sensitive RNase protection assay for its mRNA. Furthermore, use of affinity chromatography to identify other lactose-binding lectins in embryonic tissue revealed only scarce proteins with higher subunit molecular weights. These results suggest that in X.laevis L-14 functions in adult tissues and is not involved in embryogenesis.

Animals↗
PubMed1994

Disruption of a binding site for hepatocyte nuclear factor 1 in the protein C gene promoter is associated with hereditary thrombophilia.

A heterozygous T-->C transition was detected in the putative promoter region of the protein C (PROC) gene in a patient with type I protein C deficiency and a history of recurrent venous thrombosis. This mutation occurred 14 bp upstream of the transcription initiation site and within a sequence strongly homologous to the consensus binding site for the liver-enriched transcription factor, hepatocyte nuclear factor 1 (HNF-1). Transfection experiments demonstrated that a CAT reporter gene construct containing 626 bp of the putative PROC gene promoter was capable of driving CAT expression in HepG2 hepatoma cells. Levels of CAT expression from constructs bearing the mutation were found to be drastically reduced by comparison with the wild-type, consistent with the reduced plasma protein C antigen levels observed in the patient. Gel retardation and cotransfection experiments demonstrated that the mutation abolished both the binding and the transactivating ability of HNF-1 observed with the wild-type PROC gene promoter. Further, the ability of the mutation to disrupt HNF-1 binding appears to be a function not only of the nature of the nucleotide substitution and its position within the recognition sequence, but also of the relative affinity of the wild-type binding site for HNF-1. This analysis is therefore indicative of a vital role for HNF-1 in the expression of the PROC gene in vivo. Taken together with the identification of a human hepatoma cell line which contains HNF-1 but which does not express protein C, these findings are consistent with the view that HNF-1 is necessary although not sufficient for PROC gene expression in the liver.

Adult↗
PubMed1994

Molecular genetics of familial venous thrombosis.

Mutations in a variety of human genes are now known to predispose to venous thrombosis. Study of these mutations has led to important advances in our understanding of the structure and function of haemostatic regulatory proteins and has facilitated accurate presymptomatic and antenatal diagnosis. However, owing to the probabilistic nature of thromboembolism, only a certain proportion of patients with a recognized gene defect in the heterozygous state will actually suffer from thrombotic episodes. Allelic heterogeneity, epistatic effects resulting from the influence of other loci and environmental insults of various kinds all play a role in determining whether a thrombotic event occurs in individuals already compromised by a gene defect. Nevertheless, characterization of the wide spectrum of gene mutations giving rise to thrombosis may allow us to relate specific gene lesions to the probability of thromboembolism as well as to the severity and frequency of thrombotic episodes.

Antithrombin III↗
PubMed1994

Combined factor VIII/factor XI deficiency may cause intra-familial clinical variability in haemophilia A among Ashkenazi Jews.

Heterozygous factor XI deficiency occurs very frequently among Ashkenazi Jews. To investigate the potential influence of a co-inherited factor XI deficiency state on the clinical phenotype of mild/moderate haemophilia A, 28 unrelated haemophiliacs of Jewish origin were screened for the two most common factor XI gene mutations. Gene lesions were identified in two out of 14 patients of Ashkenazi origin. In the one family analysed further, co-inheritance of both factor XI and factor VIII deficiencies was associated with a bleeding tendency that was more severe than that associated with either deficiency alone.

Base Sequence↗
PubMed1994

Screening for inversions in the factor VIII (F8) gene causing severe haemophilia A.

A total of 164 unrelated patients with severe haemophilia A were screened for partial inversions of the factor VIII (F8) gene resulting from recombination between the intronic F8A gene and one or other of two homologous upstream A gene sequences. Inversions were found in 69 (42%) patients. Most inversions (90%) involved the distal rather than the proximal A gene. This unique mutational mechanism is estimated to occur with a frequency of 7.2 x 10(-6) per gene per gamete per generation. Although two patients with an inversion possessed inhibitors (antibodies) against factor VIII, possession of inhibitors did not appear to be associated disproportionately with inversion-type mutations.

Blotting, Southern↗
PubMed1994

Rat olfactory neurons can utilize the endogenous lectin, L-14, in a novel adhesion mechanism.

L-14 is a divalent, lactosamine-binding lectin expressed in many vertebrate tissues. In the rat nervous system, L-14 expression has been observed previously in restricted neuronal subsets within the dorsal root ganglia and spinal cord. In this study we report that L-14 is expressed by nonneuronal cells in the rat olfactory nerve. We demonstrate that L-14 binds and co-localizes with two ligands in the rat olfactory system: a beta-lactosamine-containing glycolipid, and a putative member of the laminin family. The former is expressed on the surfaces of nascent olfactory axons originating from neuron cell bodies in the olfactory epithelium. The latter is present in the extracellular matrix of the axonal path leading to synaptic targets in the olfactory bulb. In vitro, we find that recombinant L-14 promotes primary olfactory neuron adhesion to two laminin family members, and promotes intercellular adhesion. Both activities are dose-dependent, and are independent of integrin-mediated mechanisms. We have thus found that L-14 can serve two distinct adhesive functions in vitro, and propose that L-14 in vivo can promote olfactory axon fasciculation by crosslinking adjacent axons and promote axonal adhesion to the extracellular matrix.

Animals↗
PubMed1994

Selective modulation of the interaction of alpha 7 beta 1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation.

The alpha 7 beta 1 integrin was originally identified and isolated from differentiating skeletal muscle and shown to be a laminin-binding protein (Song et al. (1992) J. Cell Biol. 117, 643-657). Expression of the alpha 7 gene and protein are developmentally regulated during skeletal muscle differentiation and have been used to identify cells at distinct stages of the myogenic lineage (George-Weinstein et al. (1993) Dev. Biol. 156, 209-229). The lactoside-binding protein L-14 exists as a dimer and has been localized on a variety of cells, in association with extracellular matrix. During myogenesis in vitro, L-14 is synthesized within replicating myoblasts but it is not secreted until these cells commence terminal differentiation and fusion into multinucleate fibers (Cooper and Barondes, J. Cell Biol. (1990) 110, 1681-1691). Addition of purified L-14 to myogenic cells plated on laminin inhibits myoblast spreading and fusion, suggesting that the L-14 lectin regulates muscle cell interactions with the extracellular matrix that are germane to myogenic development (Cooper et al. (1991) J. Cell Biol. 115, 1437-1448). We demonstrate here, using affinity chromatography and immunoblots, that alpha 7 beta 1 also binds to fibronectin and to the L-14 lectin. L-14 binds to both laminin and to the alpha 7 beta 1 integrin, and it can effectively inhibit the association of laminin and this integrin. Modulation of alpha 7 beta 1 interaction with its ligands by L-14 is selective: L-14 does not bind to fibronectin, nor does it interfere with the binding of fibronectin to alpha 7 beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Ectopic (illegitimate) transcription: new possibilities for the analysis and diagnosis of human genetic disease.

By means of the Polymerase Chain Reaction (PCR), 'ectopic' or 'illegitimate' transcripts from any gene may be amplified from any tissue or cell type. RNA transcript analysis is therefore no longer dependent upon possession of the often inaccessible 'expressing' tissue. We review here the applications of ectopic transcript analysis to mutation detection and characterization, analysis of RNA splicing and the study of the genotype-phenotype relationship.

Genetic Diseases, Inborn↗
PubMed1994

A homozygous deletion/insertion mutation in the protein C (PROC) gene causing neonatal Purpura fulminans: prenatal diagnosis in an at-risk pregnancy.

A novel homozygous mutation in the protein C (PROC) gene was detected in an individual with severe type I protein C deficiency who presented with neonatal Purpura fulminans. The deletion/insertion mutation found [3351del4, 3350insA] resulted in an Asn102-->Lys substitution and the removal of codon Gly103. First trimester prenatal diagnosis was performed in a subsequent pregnancy by chorionic villus sampling and PCR/direct sequencing; the foetus was shown to be heterozygous for the lesion. This diagnosis was confirmed phenotypically after the birth of a clinically healthy child.

Base Sequence↗
PubMed1994

Mapping the human genome.

The haploid human genome contains approximately 70,000 different genes in its 3200 megabases of chromosomal DNA. High resolution maps of all chromosomes are urgently required both for functional analysis of the genome as a whole and for the identification and characterization of its constituent gene sequences. To this end, a variety of markers including genes, anonymous DNA segments and sequence-tagged sites are being recruited as single copy landmarks which can be readily incorporated into both genetic and physical maps. Current progress toward the generation of physical, genetic and cytogenetic maps of human chromosomes and their integration is reviewed.

Chromosome Mapping↗
PubMed1993

Diagnosis of human genetic disease using recombinant DNA. Fourth edition.

Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means of diagnosing inherited disease at the DNA level. Direct detection and analysis of a wide range of genetic lesions are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis is not yet feasible. RFLPs associated with linked anonymous DNA segments may also be used not only to diagnose hitherto undetectable disease states, but also for the chromosomal localization of the loci responsible. We present here an update to our previous list of reports describing the direct and indirect analysis/diagnosis of human inherited disease. This compilation is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.

DNA, Recombinant↗