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V Gieselmann

Publications and source records attributed to V Gieselmann.

At least 73 records · Page 4Linked to original sources
PubMed1991

Restoration of arylsulphatase A activity in human-metachromatic-leucodystrophy fibroblasts via retroviral-vector-mediated gene transfer.

Metachromatic leukodystrophy is a lysosomal storage disease caused by the deficiency of arylsulphatase A (ASA). A human ASA cDNA was subcloned into the retroviral vector pXT1. Replication-defective retrovirus was generated by transfection of the vector into the amphotropic packaging cell line PA317. Human fibroblasts from a patient suffering from metachromatic leucodystrophy was infected with the recombinant retrovirus. Infected fibroblasts expressed ten times more ASA compared with control fibroblasts from a normal individual. The ASA encoded by the integrated provirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of cerebroside sulphate.

Cell Fractionation↗
PubMed1991

Molecular basis of different forms of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A. Three forms of the disease can be distinguished according to severity and the age at onset: late infantile (1 to 2 years), juvenile (3 to 16), and adult (greater than 16). METHODS AND RESULTS: To understand the molecular basis of the different forms of the disease, we analyzed arylsulfatase A alleles associated with metachromatic leukodystrophy. Two alleles (termed I and A) were identified and accounted for about half of all arylsulfatase A alleles among 68 patients with metachromatic leukodystrophy whom we examined. Sufficient information was available for 66 of the patients to allow classification of their disease. Of the six instances of homozygosity for allele I, all were associated with the late-infantile form of the disease; of the eight instances of homozygosity for allele A, five were associated with the adult form and three with the juvenile form. When both alleles were present, the juvenile form resulted (seven of seven instances). Heterozygosity for allele I (with the other allele unknown) is usually associated with late-infantile disease, and heterozygosity for allele A with a later onset of the disease. The clinical variability can be explained by the different levels of residual arylsulfatase A activity associated with these genotypes. CONCLUSIONS: Like many lysosomal storage disorders, metachromatic leukodystrophy shows clinical heterogeneity that seems to reflect genetic heterogeneity. One of the known alleles (allele I) is associated with earlier and more severe disease than the other (allele A).

Adolescent↗
PubMed1991

An assay for the rapid detection of the arylsulfatase A pseudodeficiency allele facilitates diagnosis and genetic counseling for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). A substantial ASA deficiency has also been described in clinically healthy persons, a condition for which the term pseudodeficiency was introduced. The discrimination of both kinds of deficiencies based on ASA activity determination is difficult and unreliable. This creates a serious problem in the genetic counseling and diagnosis of MLD. The mutations characteristic for the pseudodeficiency (PD) allele have recently been identified. A non-radioactive assay based on the polymerase chain reaction is described, which allows the rapid detection of the ASA pd allele. The assay utilizes pairs of primers that allow either the amplification of the ASA PD allele or of other ASA alleles, since their 3' residues match either the ASA PD allele or other ASA alleles.

Alleles↗
PubMed1991

An 11-bp deletion in the arylsulfatase A gene of a patient with late infantile metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Examination of the arylsulfatase A gene in a patient suffering from late infantile metachromatic leukodystrophy revealed an 11-bp deletion in exon 8. Although this allele produces normal amounts of ASA mRNA, no arylsulfatase A cross-reacting material could be detected in cultured fibroblasts from the patient. The patient was found to be a compound heterozygote, the other allele is also known to generate no ASA polypeptides. This patient is another example where absence of ASA polypeptides correlates with the severe late infantile form of metachromatic leukodystrophy.

Amino Acid Sequence↗
PubMed1991

Low arylsulphatase A activity and choreoathetotic syndrome in three siblings: differentiation of pseudodeficiency from metachromatic leukodystrophy.

We report on a family with a sibship of three children for whom the diagnosis of "an unusual form of metachromatic leukodystrophy (MLD)" had been suggested earlier. The patients had choreiform movements and dystonic posturing accompanied by dysarthria since childhood. The availability of the polymerase chain reaction enabled us to show that the three siblings have a pseudodeficiency genotype (ASAp/ASAp). There was no abnormal sulphatiduria, and we propose that the neurological disease and low arylsulphatase A activity are unrelated to one another in this family. A diagnosis of MLD carries very serious implications, and we recommend that gene amplification by polymerase chain reaction and hybridization with allele-specific oligonucleotide probes should be used to corroborate the diagnosis, especially when there is no abnormal sulphatiduria and when metachromatic material cannot be demonstrated in a sural nerve biopsy.

Adolescent↗
PubMed1991

Molecular genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA). The ASA cDNA as well as the gene has been cloned. The gene is about 3 kb long and consists of 8 exons. The two most frequent alleles causing MLD have been characterized and the distribution of these alleles among patients with different clinical forms of MLD has revealed a simple genotype-phenotype correlation. Some individuals have low ASA activities but are healthy. This condition has been called ASA pseudodeficiency. These individuals are homozygous for the ASA pseudodeficiency allele which only encodes 5-10% of the ASA activity compared to the normal allele. The mutations in the PD allele have been characterized. Based on the knowledge of these mutations diagnostic assays have been developed to differentiate ASA deficiencies associated with PD or MLD.

Alleles↗
PubMed1991

Phenotypic consequences of low arylsulfatase A genotypes (ASAp/ASAp and ASA-/ASAp): does there exist an association with multiple sclerosis?

Arylsulfatase A (ASA) pseudodeficiency does per definitionem not lead to metachromatic leukodystrophy. It is conceivable, however, that it may contribute to the susceptibility for more common, multifactorial disorders of the nervous system. In order to examine whether there is an association with multiple sclerosis (MS), the most common demyelinating disease, we screened 160 MS patients for ASA activity and looked for pseudodeficiency genotypes using polymerase chain reaction. Four homozygotes for the ASA pseudodeficiency allele were found among the MS patients, but only one in the control sample. Further studies are necessary to validate whether ASA pseudodeficiency is associated with MS.

Alleles↗
PubMed1991

Mutations in the arylsulfatase A pseudodeficiency allele causing metachromatic leukodystrophy.

We identified a patient suffering from late infantile metachromatic leukodystrophy who genetically seemed to be homozygous for the mutations signifying the arylsulfatase A pseudodeficiency allele. Homozygosity for the pseudodeficiency allele is associated with low arylsulfatase A activity but does not cause a disease. Analysis of the arylsulfatase A gene in this patient revealed a C----T transition in exon 2, causing a Ser 96----Phe substitution in addition to the sequence alterations causing arylsulfatase A pseudodeficiency. Although this mutation was found only in 1 of 78 metachromatic leukodystrophy patients tested, five more patients were identified who seemed hetero- or homozygous for the pseudodeficiency allele. The existence of nonfunctional arylsulfatase A alleles derived from the pseudodeficiency allele calls for caution when the diagnosis of arylsulfatase A pseudodeficiency is based solely on the identification of the mutations characterizing the pseudodeficiency allele.

Alleles↗
PubMed1991

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.

Adolescent↗
PubMed1990

Structure of the arylsulfatase A gene.

A 14-kb genomic clone containing the entire gene of human lysosomal arylsulfatase A was isolated. The arylsulfatase A gene is about 3.2 kb long and has eight exons (103-320 nucleotides in size). All intron-exon splice junctions conformed to the GT/AG consensus sequence. S1 nuclease mapping shows multiple transcription initiation sites between nucleotides -367 and -387. A fragment encompassing 360 nucleotides of the flanking sequence upstream of the transcription initiation site shows promoter activity when it was transiently expressed in COS cells using the gene for bacterial chloramphenicol acetyltransferase as a reporter gene. This putative promoter region shows four potential Sp1 binding sites but lacks typical TATA and CAAT box sequences. Three different mRNA species of 2.1, 3.7 and 4.8 kb are transcribed from the gene and arise probably from the use of different polyadenylation signals.

Amino Acid Sequence↗
PubMed1990

Advances in the molecular genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (EC 3.1.6.1). This results in the intralysosomal storage of cerebroside sulphate, which leads to a progressive demyelination of the nervous system. The patients usually die within a few years from the onset of symptoms. Clinically, there are different forms of the disease and the molecular basis for this heterogeneity is unknown. The gene for arylsulphatase A has recently been cloned and provides a necessary tool for the exact description of the molecular defects occurring in the different forms of metachromatic leukodystrophy. Metachromatic leukodystrophy can also be caused by the deficiency of an arylsulphatase A activator protein (sphingolipid activator protein B). The cDNA for the precursor of this protein has been isolated and a mutant cDNA of one patient has been analysed. A substantial arylsulphatase A deficiency can also occur in healthy individuals, a phenotype termed pseudodeficiency. Two concurrent mutations have been identified in this low arylsulphatase A activity allele. This permitted the development of a rapid assay which allows the detection of the pseudodeficiency allele. Bone marrow transplantation has been tried in several metachromatic leukodystrophy patients and there is evidence that this treatment might slow or even halt the progression of the disease. A final conclusion as to whether bone marrow transplantation is a suitable therapy for metachromatic leukodystrophy cannot be drawn yet.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of human arylsulfatase A.

A full length cDNA for human arylsulfatase A was cloned and sequenced. The predicted amino acid sequence comprises 507 residues. A putative signal peptide of 18 residues is followed by the NH2-terminal sequence of placental arylsulfatase A. One of the arylsulfatase A peptides ends 3 residues ahead of the predicted COOH terminus. This indicates that proteolytic processing of arylsulfatase A is confined to the cleavage of the signal peptide. The predicted sequence contains three potential N-glycosylation sites, two of which are likely to be utilized. The sequence shows no homology to any of the known sequences of lysosomal enzymes but a 35% identity to human steroid sulfatase. Transfection of monkey and baby hamster kidney cells resulted in an up to 200-fold increase of the arylsulfatase A activity. The arylsulfatase A was located in lysosome-like structures and transported to dense lysosomes in a mannose 6-phosphate receptor-dependent manner. The arylsulfatase A cDNA hybridizes to 2.0- and 3.9-kilobase species in RNA from human fibroblasts and human liver. RNA species of similar size were detected in metachromatic leukodystrophy fibroblasts of two patients, in which synthesis of arylsulfatase A polypeptides was either detectable or absent.

Amino Acid Sequence↗
PubMed1989

Arylsulfatase A pseudodeficiency: loss of a polyadenylylation signal and N-glycosylation site.

Metachromatic leukodystrophy is a metabolic disorder caused by the deficiency of arylsulfatase A. Deficiency of this enzyme is also found in apparently healthy individuals, a condition for which the term pseudodeficiency was introduced. The arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase; EC 3.1.6.8) (ASA) encoding gene was isolated from an individual homozygous for the ASA pseudodeficiency allele. Sequence analysis revealed two A----G transitions. One changes Arg-350 to serine, which leads to the loss of a utilized N-glycosylation site. This loss explains the smaller size of ASA in ASA pseudodeficiency fibroblasts. The introduction of Ser-350 into normal ASA cDNA does not affect the rate of synthesis, the stability, or the catalytic properties of ASA in stably transfected baby hamster kidney cells. Therefore, the loss of the N-linked oligosaccharide does not contribute to the reduction of ASA activity in ASA pseudodeficiency. The other A----G transition changes the first polyadenylylation signal downstream of the stop codon from AATAAC to AGTAAC. The latter causes a severe deficiency of a 2.1-kilobase (kb) mRNA species. The deficiency of the 2.1-kb RNA species provides an explanation for the diminished synthesis of ASA seen in pseudodeficiency fibroblasts. Amplification of genomic DNA and hybridization with allele-specific oligonucleotides detected both mutations in four unrelated individuals with ASA pseudodeficiency.

Alleles↗
PubMed1988

Characterization of a third form of the human T cell receptor gamma/delta.

A subpopulation of the CD3+ peripheral T lymphocytes express the TCR-gamma/delta complex. Three distinct TCR-gamma forms that differ in size and in the ability to form a disulfide bridge with the TCR-delta subunit have been described. In this study we analyze the structural difference between the non-disulfide-linked 55-kD and 40-kD TCR-gamma chains. The 40-kD TCR-gamma form contains a smaller polypeptide backbone and carries less carbohydrate compared with the 55-kD TCR-gamma form. A cDNA clone corresponding to the 40-kD TCR-gamma subunit lacks one copy of the second exon of the constant region that is present in the other TCR-gamma subunit. This exon copy encodes part of the connector region that is located between the constant domain and the membrane spanning region. We show that the number of potential N-linked glycan attachment sites are the same for the two TCR-gamma forms. Since these attachment sites are located in the connector region we conclude that the connector region influences the amount of N-linked carbohydrates added to the core TCR-gamma polypeptide, probably by affecting the conformation of the protein. In contrast to the TCR-beta constant region usage, the TCR-gamma constant regions are unequally expressed. Virtually exclusive usage of disulfide-linked complexes were found in some individuals, while both the disulfide-linked and the 40-kD, non-disulfide-linked TCR-gamma forms were detected in other subjects. The ability to distinguish these TCR-gamma/delta forms now makes it possible to study the mechanisms that govern their selection and to determine if they correspond to functionally distinct isotypes.

Amino Acid Sequence↗
PubMed1985

Synthesis and transport of lysosomal acid phosphatase in normal and I-cell fibroblasts.

The biosynthesis, proteolytic processing, and transport of lysosomal acid phosphatase in normal and I-cell human skin fibroblasts was studied by metabolic labeling of the cells and isolation of acid phosphatase by immunoprecipitation. Several forms of the enzyme were identified in pulse-chase experiments. The largest precursor form had a Mr of 110,000. It was accompanied by several smaller polypeptides (Mr = 84,000-62,000), which were localized to light membranes containing the markers of endoplasmic reticulum and Golgi complex. These polypeptides were further processed to mature forms with apparent Mr of 57,000, 48,000, and 43,000 that accumulated in the cells and were associated with dense lysosomes. Less than 10% of newly synthesized acid phosphatase was secreted mainly as Mr = 112,000 and 74,000 forms. The processing of acid phosphatase was inhibited by NH4Cl and by a peptidyldiazomethyl ketone inhibitor of cysteine proteinases. The intracellular Mr = 110,000, 57,000, and 48,000 and the secreted Mr = 112,000 and 64,000 forms contained phosphorylated oligosaccharides cleavable by endo-beta-N-acetylglucosaminidase H. Transport of acid phosphatase into lysosomes was sensitive to NH4Cl and dependent on mannose 6-phosphate specific receptors by the following criteria: (i) inhibition of endocytosis of acid phosphatase by mannose 6-phosphate, (ii) enhancement of the secretion of acid phosphatase in the presence of antibodies to the mannose 6-phosphatase specific receptor, and (iii) secretion of about two-thirds of newly synthesized acid phosphatase in I-cell fibroblasts. Obviously, the mechanism of transport of acid phosphatase into lysosomes is indistinguishable from that operating for other lysosomal enzymes in fibroblasts. In contrast to other lysosomal enzymes, acid phosphatase appears to be subjected to an early proteolytic processing, presumably within the endoplasmic reticulum, which results in secretion of several processed forms of the enzyme.

Acid Phosphatase↗