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Biomedical subjects

J J Hopwood

Publications and source records attributed to J J Hopwood.

At least 19 recordsLinked to original sources
PubMed1992

Deletion of the Hunter gene and both DXS466 and DXS304 in a patient with mucopolysaccharidosis type II.

Hunter syndrome is an X-linked mucopolysaccharidosis due to deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). A cDNA clone containing the entire coding region of the human IDS gene, mapped in Xq28, has been used as molecular probe to study a patient with Hunter syndrome. A submicroscopic deletion has been detected that spans the IDS gene as well as DXS466 and DXS304, 2 loci mapped probably not more than 900 kb from the IDS locus. A detailed clinical description of the patient is provided and his phenotype is compared to that of other patients with IDS deletion described recently. By following the segregation of a restriction fragment length polymorphism at the IDS locus in the patient's family, our data suggest that the deletion occurred in the germ cells of the patient's grandfather.

Blotting, Southern↗
PubMed1992

Correction of human mucopolysaccharidosis type-VI fibroblasts with recombinant N-acetylgalactosamine-4-sulphatase.

A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.

Alkaline Phosphatase↗
PubMed1992

A common mutation for mucopolysaccharidosis type I associated with a severe Hurler syndrome phenotype.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the glycosidase alpha-L-iduronidase which is required for the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Patients with MPS-I store these partially degraded glycosaminoglycans in their lysosomes. MPS-I patients have a wide range of clinical presentations, that makes it difficult to predict patient phenotype which is needed for genetic counselling and also impedes the selection and evaluation of patients undergoing therapy such as bone marrow transplantation. We report the presence of a common mutation accounting for 31% of MPS-I alleles in a study of 64 MPS-I patients. The mutation was originally detected by chemical cleavage and then direct PCR sequencing. The mutation is a single base substitution that introduces a stop codon at position 402 (W402X) of the alpha-L-iduronidase protein and is associated with an extremely severe clinical phenotype in homozygotes. Patients who are compound heterozygotes having one allele carrying the W402X mutation have a wide range of clinical phenotypes. Based on polymorphisms within the alpha-L-iduronidase gene, W402X is associated with three different haplotypes, implying that there is more than one origin for the mutation or that intragenic recombination has occurred. W402X introduces a MaeI restriction endonuclease site into MPS-I alleles enabling its simple detection, which should make possible the assessment of the efficacy of bone marrow transplantation in MPS-I patients homozygous for W402X.

Alleles↗
PubMed1992

alpha-L-iduronidase mutations (Q70X and P533R) associate with a severe Hurler phenotype.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the glycosidase alpha-L-iduronidase which is required for the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Patients with MPS-I store forms of these partially degraded glycosaminoglycans in their lysosomes. MPS-I patients present with a wide range of clinical phenotypes, which makes prognostic predictions and genetic counselling difficult, therefore impeding the selection and evaluation of patients undergoing experimental therapy, such as bone marrow transplantation. We report the presence of two mutations, one that introduces a stop codon at position 70 (Q70X), and the other that alters the proline at position 533 to an arginine (P533R) in the 653 amino acid alpha-L-iduronidase protein. These mutations were originally detected by chemical cleavage and then by direct PCR sequencing. Allele specific oligonucleotides were used to detect the mutations in a group of 73 MPS-I patients and Q70X was found to account for 15% of all MPS-I alleles and P533R for 3% of MPS-I alleles. Both mutations are associated with an extremely severe clinical phenotype in homozygotes. MPS-I patients heterozygous for either mutation may have a wide range of clinical phenotypes. We have now described three mutations, W402X (Scott et al., 1992c), Q70X, and P533R totalling 53% of MPS-I alleles which together define 28% of MPS-I genotypes.

Alleles↗
PubMed1992

An N-acetylgalactosamine-4-sulfatase mutation (delta G238) results in a severe Maroteaux-Lamy phenotype.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomally inherited lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (EC 3.1.6.1; 4-sulfatase). In order to determine the gene defect in a clinically severe MPS VI patient, polymerase chain reaction (PCR) products were generated from the patient's fibroblast mRNA and also from a 4-sulfatase cDNA clone and subjected to the chemical cleavage technique to detect mismatched bases, which were then identified by direct DNA sequencing of the PCR products. The patient was homozygous for an early frameshift mutation caused by the deletion of a G at position 238 (delta G238), which produces a truncated 4-sulfatase with an altered amino acid sequence from amino acid 80 to a premature stop codon at codon 113 relative to the normal 4-sulfatase reading frame of 533 amino acids. Since the mutation occurs only 40 amino acids past the signal peptidase cleavage site, it is most likely that this will result in a protein with no 4-sulfatase activity. This is consistent with the severe clinical presentation and the absence of 4-sulfatase enzyme activity or mutant 4-sulfatase protein in the patient. The patient was also found to be homozygous for two polymorphisms, i.e., a G to A transition at nucleotide 1072 resulting in a valine358 to methionine substitution (V358M) and a salient A to G transition in the third base of the proline397 codon at nucleotide 1191.

Amino Acid Sequence↗
PubMed1992

Structural gene aberrations in mucopolysaccharidosis II (Hunter).

A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.

Blotting, Southern↗
PubMed1992

PCR detection of two RFLPs in exon I of the alpha-L-iduronidase (IDUA) gene.

Two polymorphisms were detected within exon I of the alpha-L-iduronidase (IDUA) gene both of which create restriction endonuclease sites and one of which changes an amino acid. The polymorphisms may be detected by digesting the same 245-bp polymerase chain reaction product. The polymorphisms can be used diagnostically in families with IDUA deficiency (mucopolysaccharidosis type I) and Huntington disease, which is closely linked to the IDUA locus.

Base Sequence↗
PubMed1992

Molecular analysis of patients with Hunter syndrome: implication of a region prone to structural alterations within the IDS gene.

Hunter syndrome or mucopolysaccharidoses type II (MPS-II), is a lysosomal storage disorder caused by a deficiency in the activity of the enzyme iduronate-2-sulphatase (IDS). We have investigated the occurrence of rearrangements and deletions of the IDS gene in a Southern analysis of 46 unrelated MPS-II patients of different ethnic origins using a cDNA clone containing the entire IDS gene as a probe. Structural alterations of the IDS gene were found in DNA from 9 patients two of whom showed large deletions including all coding sequences of the gene. The distal and proximal breakpoint of these deletions were determined by hybridization of markers flanking the IDS gene. Seven of the observed alterations constitute major rearrangements of the gene. Interestingly, six of these rearrangements showed similar or identical patterns by Southern analysis suggestive for a region prone to structural alterations within the IDS gene. We also demonstrate the potential use of the IDS probe for carrier detection in families with a rearranged IDS gene. A contiguous gene deletion syndrome characterized by Hunter syndrome and epilepsy is also discussed.

Blotting, Southern↗
PubMed1992

Mutation analysis of the iduronate-2-sulfatase gene in patients with mucopolysaccharidosis type II (Hunter syndrome).

Iduronate-2-sulfatase (IDS) cDNA from fibroblasts of nine patients with Hunter syndrome (mucopolysaccharidosis type II) was screened for mutations using single strand conformation polymorphism analysis. Direct sequencing revealed a number of different mutations including missense or nonsense point mutations, deletions of one, two, or 60 base pairs, and a 22 base pair-insertion. Mutations of these types probably account for most IDS gene defects as only about 20% of Hunter patients have a complete deletion or gross structural alteration of their IDS gene. Thus the broad clinical variability amongst the Hunter patients may be due to the extensive genetic heterogeneity seen. The relationship between genotype and clinical phenotype is analysed in 12 Hunter patients.

Adolescent↗
PubMed1979

A fluorometric assay using 4-methylumbelliferyl alpha-L-iduronide for the estimation of alpha-L-iduronidase activity and the detection of Hurler and Scheie syndromes.

Incubation of 4-methylumbelliferyl alpha-L-iduronide with whole cell homogenates prepared from cultured skin fibroblasts and amniotic cells, and peripheral blood leukocytes gave 4-methylumbelliferone which was easily measured fluorometrically. This reaction, presumably due to the action of alpha-L-iduronidase, has a maximum hydrolytic activity at pH 3.25. The apparent KM value of alpha-L-iduronidase in leukocyte whole cell homogenates for this substrate was 179 mumol/l compared to 353, 41 and 166 mumol/l for other alpha-L-iduronidase substrates phenyl alpha-L-iduronide, iduronosyl anhydrol[1-3H]mannitol 6-sulfate and iduronosyl anhydro[1-3H]mannitol respectively; the corresponding Vmax values were 617, 394, 158 and 10 pmol/min/mg protein respectively. Incubation of the 4-methylumbelliferyl alpha-L-iduronide with whole cell homogenates prepared from cultured skin fibroblasts and leukocytes from a Hurler patient gave 4-methylumbelliferone at a rate more than 20 times less than found for control normal preparations. 4-Methylumbelliferyl alpha-L-iduronide is a sensitive, convenient and superior substrate to phenyl alpha-L-iduronide for the assay of alpha-L-iduronidase activity, but is not a suitable replacement for the radiolabelled substrate iduronosyl anhydro[1-3H]mannitol 6-sulfate.

Amniotic Fluid↗
PubMed1979

alpha-L-iduronidase, beta-D-glucuronidase, and 2-sulfo-L-iduronate 2-sulfatase: preparation and characterization of radioactive substrates from heparin.

Radioactive disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and 2-sulfo-L-iduronate 2-sulfatase have been prepared from heparin by deaminative cleavage followed by reduction with NaBT4. Six disaccharides were isolated from this reaction mixture and identified. Acid hydrolysis of the major disaccharide, O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 linked to 4)-(2,5-anhydro-D-mannitol-l-t 6-sulfate (IdAs--Ms), produced 48% of O-(alpha-L-idopyranosyluronic acid)-(1 linked to 4)-(2,5-anhydro-D-mannitol-l-t 6-sulfate) (IdA--Ms) and 25% of O-(alpha-L-idopyranosyluronic acid)-(1 linked to 4)-2,5-anhydro-D-mannitol-l-t. The most-sensitive substrate for determining alpha-L-iduronidase activity was IdA--Ms which, when incubated with leucocyte and skin-fibroblast homogenates prepared from patients having a deficiency of alpha-L-iduronidase (Mucopolysaccharidosis Type I; MPS-I), was hydrolysed to yield 2,5-anhydro-D-mannitol-l-t 6-sulfate at a rate 50-times less than that found for normal control-preparations. Similarly, O-(beta-D-glucopyranosyluronic acid)-(1 linked to 4)-(2,5-anhydro-D-mannitol-l-t 6-sulfate) was degraded by whole-cell homogenates prepared from beta-D-glucuronidase-deficient (Mucopolysaccharidosis, Type VII) fibroblasts, to yield 2,5-anhydro-D-mannitol-l-t 5-sulfate at a rate 60-times less that that found for MPS-I and normal control-preparations. IdAs--Ms was degraded by 2-sulfo-L-iduronate 2-sulfatase at a rate more than 45-times greater than that found for O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 linked to 4)-2,5-anhydro-D-mannitol-l-t. C-6 Sulfation of the anhydro-D-mannitol-l-t residue is an important structural determinant in the mechanism of action of both alpha-L-iduronidase and 2-sulfo-L-iduronate 2-sulfatase on disaccharide substrates.

Cells, Cultured↗
PubMed1979

Biochemical discrimination of Hurler and Scheie syndromes.

1. Homogenates of cultured skin fibroblasts derived from patients with alpha-L-iduronidase-deficiency disorders (Hurler and Scheie syndromes) were capable of hydrolysing iduronosyl anhydro-[1-3H]mannitol 6-sulphate although at considerably reduced rates compared with normal controls. 2. The Vmax. values of alpha-L-iduronidase from patients with Hurler or Scheie syndromes and from normal controls were 11, 12 and 833 pmol min-1 mg-1 of protein respectively; the corresponding apparent Km values were 656, 50 and 53 mumol/l respectively. The alpha-L-iduronidases from normal and Scheie fibroblast homogenates were shown to exhibit pH optima at 3.6 and 4.1 and were competitively inhibited by both chloride and sulphate ions: Hurler alpha-L-iduronidase activity exhibited the pH optimum at 3.8 and was also inhibited by chloride and to a lesser extent by sulphate ions. 3. The thermal stability of Hurler, Scheie and normal alpha-L-iduronidase activities at 55 degrees C gave half-lives of approximately 1.0, 2.5 and 1.0 h respectively. 4. These biochemical findings clearly demonstrate enzyme differences for these two clinically distinct phenotypes and provide biochemical evidence that the Hurler and Scheie syndromes result from different allelic mutations.

Child↗
PubMed1978

Report of a mucopolysaccharidosis occurring in Australian aborigines.

The first 2 reported cases of a mucopolysaccharidosis occurring in an Australian aboriginal family are presented. Though these children had the characteristic morphological features of the Hurler syndrome, enzyme assay of cultured fibroblasts showed normal levels of alpha-L-iduronidase and decreased activity of arylsulphatase B. Thus, they represented the Hurler syndrome clinically, while they had the enzyme defect of the Maroteaux-Lamy syndrome, and they may represent a new severe form of the Maroteaux-Lamy syndrome. The parents of these children were first cousins. Though the children were not full blood aborigines, examination of the pedigree indicates that the gene originated in the common aboriginal family.

Australia↗
PubMed1978

Glycosaminoglycan synthesis by Wilms' tumor.

Wilms' tumor contains approximately 1 mg hyaluronic acid and approximately 0.3 mg sulfated glycosaminoglycan per g tissue. Minced tumor and cells cultured from the tumor incorporate labeled acetate and glucosamine into hyaluronic acid and sulfated glycosaminoglycans. A particulate enzyme preparation derived from the tumor catalyzed the transfer of GlcUA or GlcNAc from UDP-GlcUA or UDP-GlcNAc at a rate of approximately 20 nmol/hr/mg protein to produce high molecular weight hyaluronic acid chains. The urine and plasma of a Wilms' tumor patient contained approximately 20 mg hyaluronic acid and 8 mg sulfated glycosaminoglycan/100 ml, respectively. It appears that this higher than normal level of circulating glycosaminoglycan is synthesized by the Wilms' tumor.

Cells, Cultured↗
PubMed1977

Glycosaminoglycan synthesis by cultured human skin fibroblasts after transformation with simian virus 40.

The synthesis of glycosaminoglycans by human skin fibroblasts derived from normal subjects, Hurler and Marfan patients before and after transformation by SV40 virus has been studied. Virus transformation results in a marked increase in hyaluronic acid synthesis in normal and Hurler fibroblasts and, to a lesser extent, in Marfan fibroblasts which show augmented synthesis of this polysaccharide before transformation. There is also an increase in heparan sulfate synthesis but a moderate decrease in dermatan sulfate synthesis on transformation. Incubation of transformed fibroblasts with 4-methylumbelliferyl-beta-D-xyloside results in a marked increase in synthesis of free chondroitin sulfate chains. The synthesis of hyaluronic acid, but not of dermatan sulfate, is inversely proportional to cell density in normal fibroblasts but not in transformed fibroblasts.

Acetates↗