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1.
Haemophilia ; 11(2): 133-41, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15810915

ABSTRACT

We report the results of genetic analysis on a series of 51 patients attending this Haemophilia Comprehensive Care Centre. The most common cause of severe haemophilia A--the factor VIII intron 22 inversion was detected in eight families and the factor VIII intron 1 inversion in three families. Mutation analysis was carried out on the remaining patients by nucleotide sequencing of genomic DNA after screening with conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC). A total of 27 different FVIII non-inversion mutations were detected. Severe haemophilia was associated with 12 null mutations (six nonsense, six frameshift) and four missense mutations. A further 11 different missense mutations were associated with moderate or mild disease. To our knowledge, six null mutations [1950del 4(tttg), 3270-75insA, 4416del 10, 6735-38delA, W1029X, Y1792X] and four missense mutations (E1682K, M1947V, P2048L, P2143L) have not been previously published. Each novel missense mutation occurred at a highly conserved residue, no other candidate mutation was detected on screening the entire coding region of the FVIII gene and they were not detected in a screen of individuals without haemophilia A. The genotype-phenotype correlations of the FVIII mutations detected will be discussed.


Subject(s)
Hemophilia A/genetics , Mutation/genetics , Base Sequence , Codon, Nonsense/genetics , DNA/genetics , DNA Mutational Analysis/methods , Factor VIII/antagonists & inhibitors , Factor VIII/genetics , Frameshift Mutation/genetics , Genotype , Humans , Inteins/genetics , Male , Mutation, Missense/genetics , Phenotype
3.
Thromb Haemost ; 85(2): 265-9, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11246545

ABSTRACT

We report two novel mutations in the Factor X gene which result in a bleeding tendency in two unrelated Caucasian families. Although the mutations occur at adjacent codons in exon 8 and result in reduced functional activity with normal antigen levels, the patterns of inheritance appear to be quite distinct. Factor X Nottingham (alanine 404 threonine) appears to be associated with an autosomal recessive pattern of inheritance. In contrast, Factor X Taunton (arginine 405 glycine) results in a mode of inheritance consistent with an autosomal dominant pattern, all five of the heterozygotes in this family being clinically affected. Molecular modelling studies suggest that, in the case of Factor X Nottingham, a drastic conformational change causes major unfolding of the protein. For Factor X Taunton, less extreme conformational changes occur causing loss of functional activity such that substrate binding sites might be maintained. It is proposed that competition with wild type for substrate binding could occur leading to a dominant negative effect.


Subject(s)
Factor X/genetics , Mutation , Adolescent , Adult , Blood Coagulation Tests , DNA Mutational Analysis , England , Factor X/chemistry , Family Health , Female , Genotype , Humans , Menorrhagia/etiology , Menorrhagia/genetics , Models, Molecular , Pedigree , Protein Conformation , Protein Denaturation/genetics
6.
Biochem Biophys Res Commun ; 221(1): 59-61, 1996 Apr 05.
Article in English | MEDLINE | ID: mdl-8660343

ABSTRACT

Der p1, a cysteine proteinase derived from the house dust mite (HDM) Dermatophagoides pteronyssinus, is a major component of the allergic immune response in HDM atopic individuals. Recent evidence suggests that cysteine proteinase activity is important in the disease process as it increases the permeability of the allergen in the respiratory tract and disrupts the regulation of IgE synthesis. Der p1 is found in high concentrations in the faecal pellets of mites which are aerosolised and inhaled via the respiratory tract. The serine proteinase inhibitor, alpha 1-antitrypsin, protects the lower respiratory tract against damage by proteinases released in the lung during inflammation. Der p1 catalytically inactivates alpha 1-antitrypsin by a thiol-dependent mechanism involving specific cleavage of the reactive centre loop and we propose that this mechanism may be important in the pathogenesis of asthma.


Subject(s)
Asthma/metabolism , Bronchial Hyperreactivity/metabolism , Glycoproteins/physiology , Inflammation/metabolism , alpha 1-Antitrypsin/metabolism , Allergens/physiology , Amino Acid Sequence , Animals , Antigens, Dermatophagoides , Asthma/physiopathology , Bronchial Hyperreactivity/physiopathology , Catalysis , Hydrolysis , Inflammation/physiopathology , Mites/immunology , Molecular Sequence Data
8.
Biochem Pharmacol ; 41(12): 2019-22, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-2039550

ABSTRACT

Porphyria cutanea tarda and the analogous hepatic uroporphyria produced in rodents by aromatic hydrocarbons result from inactivation of hepatic uroporphyrinogen decarboxylase (UROD). Inactivation appears to be iron-dependent and may require induction of cytochromes of the P450IA subfamily. To investigate the hypothesis that the mechanism of inactivation involves an intermediate of haem biosynthesis, we administered iron and the haem precursor, 5-aminolevulinate (ALA), to mice. Iron-overloaded male mice of the Ah-responsive C57BL/6 strain, given ALA solution as their only drink, developed severe uroporphyria after 49 days. ALA did not produce uroporphyria in iron-overloaded male mice of the Ah-nonresponsive DBA/2 strain. Iron or ALA alone did not produce porphyria in either strain. Hepatic iron concentrations and rates of ethoxyresorufin deethylation (an indicator of cytochrome P450IA-mediated activity) were similar in both strains. These experiments show that a haem precursor is involved in iron-dependent inactivation of UROD. They emphasize the importance of inherited factors in determining susceptibility to this type of porphyria, even in the absence of administration of compounds that act through the Ah locus to induce cytochromes of the P450IA subfamily.


Subject(s)
Aminolevulinic Acid/toxicity , Iron/toxicity , Porphyrias/chemically induced , Administration, Oral , Aminolevulinic Acid/administration & dosage , Animals , Chemical and Drug Induced Liver Injury , Cytochrome P-450 CYP1A1 , Cytochrome P-450 Enzyme System/metabolism , Disease Models, Animal , Iron/administration & dosage , Iron/metabolism , Liver/enzymology , Liver/metabolism , Liver Diseases/enzymology , Liver Diseases/metabolism , Male , Mice , Mice, Inbred Strains , Oxidoreductases/metabolism , Porphyrias/enzymology , Porphyrias/metabolism , Porphyrins/metabolism , Uroporphyrinogen Decarboxylase/metabolism
10.
J Clin Pathol ; 41(7): 726-9, 1988 Jul.
Article in English | MEDLINE | ID: mdl-3410967

ABSTRACT

A study programme was set up in Wales and the West Midlands to evaluate serum immunoreactive trypsin screening for cystic fibrosis in neonates using blood spots collected for metabolic screening. By screening half the blood spots from each area, it was hoped to generate two comparable groups of fibrocystic children; those detected by screening and those not screened who would be diagnosed clinically. Over almost three years, more than 120,000 specimens were screened and 37 infants detected with cystic fibrosis. Four additional fibrocystic patients were missed on screening: two had negative immunoreactive trypsin values, of which one had meconium ileus, and two, although giving initial positive tests, were negative on follow up. Excluding infants known to be at risk, comparison of the numbers of children detected in the screened and unscreened groups showed more than a two-fold difference in favour of the screened group. There may be a large number of undiagnosed fibrocystic patients in the general population.


Subject(s)
Clinical Enzyme Tests , Cystic Fibrosis/diagnosis , Mass Screening , Trypsin/blood , Cystic Fibrosis/epidemiology , England , Evaluation Studies as Topic , False Negative Reactions , False Positive Reactions , Humans , Infant, Newborn , Mass Screening/standards , Wales
11.
Wien Klin Wochenschr ; 100(2): 55-7, 1988 Jan 22.
Article in English | MEDLINE | ID: mdl-3279709

ABSTRACT

An enzyme immunoassay method for the assay of serum immunoreactive trypsin (IRT) is described. The method is a two site binding assay carried out on microtitre plates as the solid phase. Wells were coated with affinity purified anti-human trypsin and biotinylated anti-trypsin and avidin-beta-galactosidase were used as the second antibody and detection system respectively. The assay was sensitive enough to determine IRT concentrations in either serum or dried blood spots. A good correlation was obtained when the method was compared with the Hoechst radioimmunoassay method.


Subject(s)
Blood Stains , Immunoenzyme Techniques , Trypsin/blood , Humans , Radioimmunoassay
12.
Prostaglandins Leukot Med ; 24(1): 1-14, 1986 Sep.
Article in English | MEDLINE | ID: mdl-3464023

ABSTRACT

Antisera have been raised to the following prostaglandins as their methyl oximes; PGE2, PGD2, 13-14-dihydro-15-oxo PGE2, 13,14-dihydro-15-oxo PGF2 alpha, 6-oxo PGF1 alpha, 6-oxo PGE1 and thromboxane B2. These antisera have good specificity and sensitivity and their use allows the immediate treatment of biological fluids with oximating solution which prevents sample decomposition during storage. A methyl oximating reagent is described which gives greater than 95% conversion of PGs to their methyl oximes by treating samples at 20 degrees C overnight. The use of this reagent allows easy and reliable sample derivatisation prior to assay with the above antisera. Since the antisera (with the exception of that for thromboxane B2) do not recognise the underivatised PG or the oxime (=NOH) form, oxime formation can be carried out in parallel with methyl oxime formation and the oximated portion of the sample can act as a "reference" for the methyl oximated portion, which will allow non specific interference to be recognised.


Subject(s)
Prostaglandins, Synthetic/immunology , Prostaglandins/analysis , Antibody Specificity , Chemical Phenomena , Chemistry , Cross Reactions , Female , Humans , Isomerism , Ovarian Follicle/analysis , Oximes , Prostaglandins/immunology , Radioimmunoassay/methods
13.
Aust J Biol Sci ; 39(3): 283-93, 1986.
Article in English | MEDLINE | ID: mdl-3827742

ABSTRACT

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.


Subject(s)
Blastocyst/metabolism , Prostaglandins/metabolism , Steroids/biosynthesis , Swine/embryology , Androstenedione/biosynthesis , Animals , Estradiol/biosynthesis , Estrone/biosynthesis , Female , Pregnancy , Pregnenolone/biosynthesis , Progesterone/biosynthesis , Testosterone/biosynthesis
14.
J Clin Endocrinol Metab ; 60(4): 651-7, 1985 Apr.
Article in English | MEDLINE | ID: mdl-3156144

ABSTRACT

Vertebral mineral density, measured by computerized axial tomography, radiocalcium absorption, serum dehydroepiandrosterone (DHA), and serum cortisol (C) were measured in 98 postmenopausal women aged 56-70 yr. On the basis of spine radiographs and fracture history, the women were classified into 49 normal subjects (mean age, 60.5 yr) and 49 with osteoporosis (mean age, 63.1 yr). Vertebral mineral density (VMD), radiocalcium absorption (alpha), serum DHA, and the ratio of DHA to cortisol (DHA/C) were all significantly lower in the osteoporotic than in the normal subjects. DHA was significantly related to C in both groups but the regression was significantly flatter in the osteoporotic than in the normal subjects. Calcium absorption did not fall significantly with age in either group. In the normal group VMD, DHA, and DHA/C fell with age but VMD was not related to alpha, DHA, or DHA/C. In the osteoporotic group, VMD did not fall significantly with age but was significantly related to alpha and DHA/C. Stepwise regression analysis showed that in the normal subjects, age was the only variable significantly related to VMD (P less than 0.05). In the osteoporotic group, calcium absorption was the main determinant of VMD, with age and DHA/C contributing much less to the variance. Discriminant function analysis showed a theoretical misclassification of 45% of cases using DHA, 39% using DHA/C, 32% using alpha, and 18% when alpha and DHA or DHA/C were both taken into account. It is concluded that malabsorption of calcium is a significant risk factor for postmenopausal osteoporosis, probably because of a secondary increase in bone resorption to maintain serum calcium. The severity of the osteoporosis is directly related to the severity of the calcium malabsorption. Low serum DHA appears to represent a further risk factor, either because of its role as estrogen precursor or (possibly) because it promotes bone formation. However, the severity of the osteoporosis was not related to the serum DHA level and only weakly to the DHA/C ratio.


Subject(s)
Bone and Bones/metabolism , Calcium/metabolism , Dehydroepiandrosterone/blood , Minerals/metabolism , Osteoporosis/metabolism , Aged , Female , Humans , Hydrocortisone/blood , Intestinal Absorption , Menopause , Middle Aged , Spine/metabolism
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