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Biomedical subjects

K A High

Publications and source records attributed to K A High.

71 records · Page 4Linked to original sources
PubMed1991

Factor XSanto Domingo. Evidence that the severe clinical phenotype arises from a mutation blocking secretion.

Factor X (FX) is a vitamin K-dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation. FXSanto Domingo is a hereditary FX deficiency which is characterized clinically by a severe bleeding diathesis. The proposita has a FX activity of less than 1% and a FX antigen of less than 5%. We have determined the molecular basis of the defect in the FXSanto Domingo gene by amplification of all eight exons with polymerase chain reaction and subsequent sequence analysis. The patient is homozygous for a G----A transition in exon I at codon -20 (numbering the alanine at the NH2 terminus of the mature protein as +1), resulting in the substitution of arginine for glycine in the carboxy-terminal part of the signal peptide. This amino acid change occurs near the presumed cleavage site of the signal peptidase. We hypothesized that the mutation might prevent cleavage by the signal peptidase which in turn would impair proper secretion of the FX protein. To test this hypothesis, we compared the expression of wild type and mutant FX cDNA in a human kidney cell line. Wild type and mutant constructs in the expression vector pCMV4 were introduced into the human embryonic kidney cell line 293 by calcium phosphate transfection. FX antigen levels in the supernatant of the cells harboring the wild type construct were 2.4 micrograms/10(7) cells per 24 h, whereas antigen levels in media from cells containing the FXSanto Domingo construct were undetectable. No FX antigen was detected in the cell lysates of cells transfected with the mutant construct. To insure that the difference in protein levels was not due to a difference in steady state levels of mRNA, Northern analysis was performed on RNA from the cell lysates of both constructs. The results showed a transcript of the same size, present in roughly equal amounts, in both cases. Thus, the defect in the signal sequence of FXSanto Domingo exerts its effect posttranscriptionally. FXSanto Domingo is the first described example of a bleeding diathesis due to a mutation in the signal sequence.

Adolescent↗
PubMed1990

Molecular defect (Gla+14----Lys) and its functional consequences in a hereditary factor X deficiency (factor X "Vorarlberg").

Factor X (FX) "Vorarlberg" is a congenital FX deficiency characterized clinically by a mild bleeding tendency. Homozygous individuals have a FX activity of less than 10% in the extrinsic system and 25% in the intrinsic system. FX antigen is 20%. Using molecular techniques, two point mutations were detected in the coding sequence of the FX Vorarlberg gene: a G----A at base pair 160 in exon II resulting in a change of Gla14 (GAA) to Lys (AAA); a G----A at base pair 424 in exon V resulting in a change from Glu102 (GAG) to Lys (AAG). The mutations abolished a TaqI restriction site in exon II and an MnlI site in exon V. To determine whether these mutations are present on one or on both alleles, restriction analyses of amplified exon II and exon V fragments were performed. Analysis of the pedigree showed that the genotype for the mutation on exon II (homozygous versus heterozygous) correlates with the severity of the phenotypic coagulation defect. We therefore conclude that the mutation in exon II is responsible for the functional defect in FX Vorarlberg. We have also purified the mutant FX protein from patient plasma. Purified FX Vorarlberg is indistinguishable from normal FX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity is 15% of normal FX upon activation with factor VIIa/tissue factor, 75% upon activation with factor IXa/factor VIIIa, and 100% upon activation with RVV. Activation at varying Ca2+ concentrations shows that the affinity of FX Vorarlberg for Ca2+ is decreased. Factor Xa Vorarlberg is able to convert prothrombin at a normal rate but also shows decreased affinity for Ca2+ in this interaction. Upon addition of Ca2+, FX Vorarlberg does not undergo the same conformational change as normal FX. Our data show that FX Vorarlberg has a decreased affinity for Ca2+ which impedes a normal conformational change. This leads to a decreased rate of activation by factor VIIa/tissue factor and by factor IXa. The decrease is much more marked for the extrinsic than for the intrinsic pathway.

Amino Acid Sequence↗
PubMed1990

Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B.

We describe a novel point mutation in the fourth exon of human factor IX (encoding the first EGF-like domain) in which cytosine is substituted for adenosine at position 10,401, resulting in the substitution of proline for glutamine at position 50 in the polypeptide chain. Sequence analysis of all eight exons, all exon-intron junctions, 160 base pairs (bp) of DNA 5' to the proposed translation start site, and 60 bp 3' to the translation termination site shows no other difference from the normal factor IX gene, with the exception of a previously described benign polymorphism at position 148 in the protein (Ala----Thr). The affected subject has severe hemophilia B with no detectable factor IX activity despite normal factor IX antigen levels. We purified the abnormal factor IX by immunoaffinity chromatography and demonstrated that its activation by factor Xla is markedly delayed compared with normal factor lX. Once activated, the abnormal factor lX binds antithrombin III in a 1:1 molar ratio, and the activated protein demonstrates catalytic activity, suggesting an intact active site. The mutation creates a new Bst Yl restriction endonuclease cleavage site. Restriction with Bst Yl shows the mutation in maternal DNA and offers the possibility of direct carrier status analysis and prenatal diagnosis in kindreds with this mutation. We designate this new mutation factor lXNew London. This is the only reported mutation in the first EGF-like domain that causes severe hemophilia B.

Amino Acid Sequence↗
PubMed1989

Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo.

Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.

Amino Acid Sequence↗
PubMed1989

Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide.

A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

Amino Acid Sequence↗
PubMed1989

Canine hemophilia B resulting from a point mutation with unusual consequences.

We have used the polymerase chain reaction to amplify the entire coding region of canine factor IX from a hemophilia B animal. When the sequence was compared to that which codes for normal canine factor IX, a single missense mutation was identified. This mutation (G----A at nucleotide 1477) results in the substitution of glutamic acid for glycine-379 in the catalytic domain of the molecule. The mutation creates a new restriction site that allowed confirmation of the abnormal sequence in both hemophilic and carrier animals. Amino acid 379 in canine factor IX corresponds to position 381 in human factor IX, a location at which no human mutations have been described. Moreover, it occurs at one of the few amino acids that have been rigorously conserved among the trypsin-like serine proteases throughout evolution. The mutation responsible for canine hemophilia B results in a complete lack of circulating factor IX in the affected animals. As it is unusual for a missense mutation to result in a complete absence of protein product, structural modeling of the mutant and normal proteins was pursued. These studies suggest that the observed mutation would have major adverse effects on the tertiary structure of the aberrant factor IX molecule. The elucidation of this mutation sheds light on structure-function relationships in factor IX and should facilitate future experiments directed toward gene therapy of this disease.

Amino Acid Sequence↗
PubMed1989

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide.

The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G1/G0 and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G1 DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G1 to G0.

Blotting, Northern↗
PubMed1989

Molecular cloning of a cDNA encoding canine factor IX.

Factor IX (F.IX) is a vitamin K-dependent plasma protein, a deficiency of which results in hemophilia B. A canine model of hemophilia B exists; attempts to use this model for gene transfer experiments or characterization of the hemophilic defect require elucidation of normal canine F.IX structure. We report the isolation and characterization of the coding region for canine F.IX cDNA. Canine F.IX possesses 86% identity at the amino-acid level with human F.IX. The leader peptide, Gla domain, EGF domains, and the carboxy-terminal portion of the heavy chains show extensive sequence conservation between the canine and human. All Glu residues undergoing gamma-carboxylation in humans are conserved in canines. The complete coding sequence for canine F.IX has been determined, and the derived translation product has been analyzed. A similar approach should allow identification of the causative mutation in canine hemophilia B. Furthermore, this clone may prove a valuable resource in gene transfer experiments for this disease.

Amino Acid Sequence↗
PubMed1989

Molecular characterization of human factor XSan Antonio.

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.

Alleles↗
PubMed1988

Antithrombin III, protein C, and protein S. Naturally occurring anticoagulant proteins.

Protein C, protein S, and antithrombin III are naturally occurring anticoagulant proteins. Activated protein C and protein S act by inhibiting the action of the cofactors factor Va and factor VIIIa. Antithrombin III inhibits the serine proteases (factors II, IX, X, XI, and XII); its anticoagulant action is dramatically enhanced by heparin. Deficiencies of these proteins are associated with thromboembolic disease. Antigen and activity assays practical for the clinical laboratory are available for protein C and antithrombin III. Protein S antigen assays are also available. Diagnosis of an anticoagulant protein deficiency has serious implications for a patient and his kindred, and should therefore only be made on the basis of repeated abnormal determinations of a single protein in an individual patient, or documentation of the same protein deficiency in other family members.

Antithrombin III↗
PubMed1987

c-myc gene inactivation during induced maturation of HL-60 cells. Transcriptional repression and loss of a specific DNAse I hypersensitive site.

The c-myc proto-oncogene is amplified and expressed at high levels in HL-60 cells, a cell line derived from a patient with acute promyelocytic leukemia. Upon induction to terminal maturation, expression of c-myc is greatly reduced. We have studied the level of gene expression at which the change in c-myc expression is controlled and the changes in chromatin configuration that accompany the repression of myc expression. We report here that the repression of myc expression with induced maturation is controlled at the level of transcription, and that reduced expression is accompanied by the loss of a single DNAse I hypersensitive site 0.9 kilobase pair upstream from the gene.

Cell Differentiation↗
PubMed1985

Serous retinal detachments in thrombotic thrombocytopenic purpura.

Serous retinal detachments are a rare ocular complication of thrombotic thrombocytopenic purpura (TTP), with only six previous case reports in the literature. We have recently observed two patients with relapsing TTP who developed bilateral serous retinal detachments during acute exacerbations of their disease. In contrast to all but one of the previously described patients, both of our patients survived the episode during which retinal detachments occurred. Hypertension appears to contribute to the development of retinal detachments in TTP, and vigorous efforts at blood pressure control are indicated. Serous retinal detachments may be a more frequent component of chronic relapsing TTP than has been suspected.

Adult↗
PubMedDate not supplied

Culturally induced postpartum depression: a theoretical position.

A three-factor theory, based on an independently conducted survey of 49 primigravidas, is postulated to explain the potential that exists for cultural attitudes toward pregnancy to become precipitating agents in the postpartum depression syndrome. Changes in body proportions, public attitudes, and the social lives of expectant women are viewed primarily as negative experiences which result in a loss of self-esteem. A means of reducing cognitive dissonance by increasing interaction with those also pregnant is proposed.

Adolescent↗