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D A Wenger

Publications and source records attributed to D A Wenger.

At least 19 recordsLinked to original sources
PubMed1995

Structure and organization of the human galactocerebrosidase (GALC) gene.

The deficiency of galactocerebrosidase (GALC; EC 3.2.1.46) is responsible for globoid cell leukodystrophy (GLD, Krabbe disease) in humans and certain animals. This enzyme catalyzes the lysosomal hydrolysis of specific galactolipids including galactosylceramide (galactocerebroside) and galactosylsphingosine (psychosine), among others. Recently we cloned the full-length human GALC cDNA using amino acid sequence information obtained from GALC purified from human urine and brain. In this communication we describe the organization of the human GALC gene. The gene, of nearly 60 kb, consists of 17 exons, which, aside from the first and last, are relatively small, ranging from 39 to 181 nucleotides. The 16 introns range from 247 nucleotides to nearly 12 kb. The 5' untranslated region is GC-rich, containing no perfect CAAT or TATA sequences, similar to genes for other lysosomal proteins. This information will be useful for studies to identify mutations causing low GALC activity in all patients with GLD and to identify the homologous gene in the important animal models.

Base Sequence↗
PubMed1995

Metachromatic leukodystrophy among southern Alaskan Eskimos: molecular and genetic studies.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder resulting from the inability to metabolize sulphatide, an important component of myelin. Although there is significant clinical variability between patients, most have the late-infantile form. It is one of the most common lysosomal disorders involving mental deterioration and is found throughout the world. The great majority of the cases have a deficiency of arylsulphatase A activity. Accurate diagnosis of MLD is complicated by the presence of so-called pseudodeficiency alleles and the need to receive specimens for biochemical testing within 24-48 h of collection. We report the identification of the mutation (a g-to-a transition in the first nucleotide of intron 4) in the arylsulphatase A gene causing late-infantile MLD among the Eskimo population of southern Alaska. As all patients and family members from living and deceased patients had the same mutation, a mutation-based test was developed to identify patients and carriers that can be done on dried blood spots sent via regular mail service. A possible genetic link between this population and the Navajo Indians of the southwestern United States is proposed.

Alaska↗
PubMed1995

Four novel mutations in mucopolysaccharidosis type VII including a unique base substitution in exon 10 of the beta-glucuronidase gene that creates a novel 5'-splice site.

Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is a lysosomal storage disorder caused by a deficiency in the enzyme beta-glucuronidase. Various clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of human beta-glucuronidase cDNA and the genomic sequences facilitate analysis of molecular defects underlying the different phenotypes, and eight mutations in the beta-glucuronidase gene have been described. This report summarizes studies characterizing four new mutations in two Caucasian patients with a severe form of MPS VII. Three are point mutations, resulting in two missense and one nonsense change, and one is a 38 bp deletion. The first patient was a compound heterozygote having P148S and Y495C alleles. The second patient was a compound heterozygote of W507X and a 38 bp deletion at position 1642-1679 in exon 10(1642 delta 38nt). The 38 bp deletion was caused by a single base change mutation in exon 10 that generates a new, premature 5' splice site. Expression of mutant cDNAs encoding each of the four mutations showed that all four resulted in a severe reduction of beta-glucuronidase activity, indicating that these mutations are responsible for the reduced enzyme activity in patient cells. These four previously undescribed mutations provide further evidence for the broad molecular heterogeneity in Sly syndrome.

Base Sequence↗
PubMed1995

A large deletion together with a point mutation in the GALC gene is a common mutant allele in patients with infantile Krabbe disease.

Galactocerebrosidase (GALC) activity is deficient in all patients with globoid cell leukodystrophy (GLD). While most patients have the severe infantile form of this autosomal recessive disorder (Krabbe disease), patients up to 50 years of age have been diagnosed in this laboratory. With the cloning of the GALC cDNA and availability of information regarding the gene organization, patients can be evaluated for their disease-causing mutations. We now report that a large deletion, together with a polymorphic C to T transition at position 502 of cDNA (counting from the A of the initiation codon), is responsible for a large number of disease-causing alleles in patients with Krabbe disease. Of 48 patients evaluated, 10 were found to be homozygous for the 502/del allele, five patients were heterozygous for this allele, 21 patients were heterozygous for the 502 mutation (presence of the deletion could not be confirmed), and one infantile patient was homozygous for the 502 mutation but at least one allele was not deleted. No patient was found to have the deletion without the 502 polymorphism. The delineation of mutations causing infantile Krabbe disease will provide new information regarding structure-function relationships in this multi-subunit enzyme and will improve the identification of patients and carriers in some families.

Alleles↗
PubMed1994

Metachromatic leukodystrophy in the Navajo Indian population: a splice site mutation in intron 4 of the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder of myelin metabolism, resulting from the inability to properly degrade 3-sulfogalactosylceramide (sulfatide). This metabolic block is often due to defective functioning of the lysosomal enzyme arylsulfatase A (ARSA). Unmetabolized sulfatide accumulates in the white matter of the CNS and in the peripheral nerves, leading to progressive demyelination and death. Late infantile, juvenile and adult clinical variants of MLD have been described. A Navajo Indian child was diagnosed with late infantile MLD (LIMLD), and his ARSA gene was amplified in three overlapping regions by the PCR and sequenced. A single mutation was found: a G-->A transition in the first nucleotide of intron 4 (IVS4nt1), which abolishes the 5' splice site consensus sequence. Negligible amounts of ARSA mRNA were observed in Northern blots. However, PCR amplification and sequencing of the ARSA cDNA showed that all of the mRNA species from the patient have exon 4 deleted. A new reading frame is thus established which results in a premature stop codon within exon 5. A minority of transcripts had additional splicing errors. Both parents carry this mutation, and the father also carries the pseudodeficiency (PD) allele. Three additional unrelated Navajo LIMLD patients were found to be homozygous for the same MLD-causing mutation by allele-specific oligonucleotide (ASO) hybridization. This method could be used for carrier and patient identification in this population.

Adult↗
PubMed1994

Molecular genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disease. It can be caused by mutations in two different genes, the arylsulfatase A and the prosaposin gene. These genes encode two proteins that are needed for the proper degradation of cerebroside sulfate, a glycolipid mainly found in the myelin membranes. Deficiency of arylsulfatase A or of a proteolytic product of prosaposin leads to the accumulation of cerebroside sulfate, which causes a lethal progressive demyelination. Mutations in the arylsulfatase A gene are far more frequent than those of the prosaposin gene. So far 31 amino acid substitutions, one nonsense mutation, three small deletions, three splice donor site mutations, and one combined missense/splice donor site mutation have been identified in the arylsulfatase A gene. Two of these mutant alleles are frequent, accounting for about one-half of all mutant alleles, whereas the remainder are heterogeneous. Amino acid substitutions cluster in exons 2 and 3, a region that shows a high degree of conservation among sulfatases of different function and origin. Different mutations are associated with phenotypes of different severity, but there is a remarkable variability of severity when patients with identical genotypes are compared. Demonstration of an arylsulfatase A deficiency is not a proof of metachromatic leukodystrophy, since a substantial deficiency without any clinical consequences is frequent in the general population. This deficiency is caused by an arylsulfatase A allele, which due to certain mutations encodes greatly reduced amounts of functional enzyme. However, these amounts are sufficient to sustain a normal phenotype. In the diagnosis and genetic counseling, these deficiencies must be differentiated from those causing metachromatic leukodystrophy. So far only six patients with mutations in the prosaposin gene have been described, in which three defective alleles two with amino acid substitutions and one with a 33-bp insertion have been identified.

Cerebroside-Sulfatase↗
PubMed1994

Adult and infantile Gaucher disease in one family: mutational studies and clinical update.

Molecular analysis and clinical updates are provided on a previously reported mother and adult son with Gaucher disease; two other children died with acute neuronopathic (type 2) Gaucher disease. The mother and son have the identical genotype (370/444) but very different clinical manifestations. These findings illustrate the need for additional studies before families with newly diagnosed Gaucher disease undergo counseling.

Adult↗
PubMed1994

Regional mapping of the human galactocerebrosidase gene (GALC) to 14q31 by in situ hybridization.

The cDNA for human galactocerebrosidase (GALC) has recently been cloned and expressed. A portion of this cDNA was used for in situ hybridization, and the region of strongest signal corresponded to human chromosome region 14q31. This agrees with recent linkage studies that localized Krabbe disease (globoid cell leukodystrophy) to the same region. This information will be useful in future studies for mapping this gene in animal models of GALC deficiency.

Brain↗
PubMed1994

Mutations in the lysosomal beta-galactosidase gene that cause the adult form of GM1 gangliosidosis.

Three adult patients with acid beta-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C-->T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C-->T mutation. Expression studies showed that this mutation produced 3%-4% of beta-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C-->T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5' splice donor site which led to the use of a cryptic splice site. It appears that the C-->T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme.

Adult↗
PubMed1993

Cloning and expression of cDNA encoding human galactocerebrosidase, the enzyme deficient in globoid cell leukodystrophy.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galactosylceramide, a major lipid in myelin, kidney and epithelial cells of small intestine and colon. We describe the molecular cloning of human GALC cDNA and its expression in COS-1 cells. Degenerate PCR primers, derived from N-terminal amino acid sequence from the 51 kDa band from human brain, were used to amplify cat testes RNA, and the resulting product was used to screen human testes and brain libraries. Two overlapping clones contained the total protein coding region, while additional clones and PCR amplification were needed to obtain the complete 3' end of the cDNA. The 3795 bp obtained include 47 bp 5' to the initiation start site, 2007 bp of open reading frame (coding for 669 amino acids), and 1741 bp of 3' untranslated sequence. Modification of the sequence surrounding the initiation codon to one more favorable for expression, resulted in a 6-fold increase in GALC activity in transfected COS-1 cells. The isolation of this clone will permit investigations into the causes for GALC deficiency in humans and available animal models, development of more accurate tests for patient and carrier identification, and evaluation of methods for effectively treating GALC deficiency, initially using the animal models.

Amino Acid Sequence↗
PubMed1993

Localization of the Krabbe disease gene (GALC) on chromosome 14 by multipoint linkage analysis.

The gene responsible for Krabbe disease, an autosomal recessive disorder caused by deficiency of galactocerebrosidase (GALC), was localized by multipoint linkage analysis on chromosome 14. Eight mapped dinucleotide repeat polymorphisms were tested for linkage to GALC. Two-point linkage analysis demonstrated close linkage of GALC and D14S48, with Z = 13.69 at theta = 0. Multipoint analysis yielded strong support for this finding, with maximum likelihood for GALC located within 1 cM of D14S48. This analysis also identified markers that clearly flank the GALC locus, as the map order of D14S53-GALC-D14S45 is favored by odds greater than 10(6):1. Additional support for close linkage of GALC and D14S48 comes from the apparent linkage disequilibrium between these two loci in a consanguineous Druze community in Israel. These data localize GALC to 14q24.3-q32.1.

Chromosome Mapping↗
PubMed1992

Bone marrow transplantation for Niemann-Pick type IA disease.

Bone marrow transplantation has been undertaken with encouraging results as therapy for a wide variety of lysosomal storage diseases. We report a case of Niemann-Pick disease Type IA in which, despite the presence of only mild hypotonia with depressed reflexes, the clinical course of the disease appeared to be only slightly modified by this procedure, which was performed at the earliest practical opportunity. The patient was diagnosed early when asymptomatic, because of a family history of an affected sibling who died at 14 months. He received a bone marrow transplant from an HLA-identical, MLC non-reactive sibling donor, whose leukocyte sphingomyelinase activity was in the homozygote normal range. There was adequate engraftment as evidenced by persistently normal leukocyte sphingomyelinase activities, and there was no evidence of graft-versus-host disease. Visceral storage and neurological impairment were less rapidly progressive than in his untreated sibling but he eventually died at 30 months. Autopsy confirmed that this was essentially due to the effects of the underlying Niemann-Pick disease. We conclude that despite some success in other neurovisceral lysosomal storage disorders, bone marrow transplantation is not likely to be an adequate treatment for Niemann-Pick disease Type IA.

Bone Marrow Transplantation↗
PubMed1992

Correction of sulfatide metabolism after transfer of prosaposin cDNA to cultured cells from a patient with SAP-1 deficiency.

The lysosomal removal of the sulfate moiety from sulfatide requires the action of two proteins, arylsulfatase A and sphingolipid activator protein-1 (SAP-1). Recently, patients have been identified who have a variant form of metachromatic leukodystrophy which is characterized by mutations in the gene coding for SAP-1, which is also called "prosaposin." All of the mutations characterized in these patients result in (a) deficient mature SAP-1, as determined by immunoblotting after SDS-PAGE of tissue and cell extracts, and (b) decreased ability of cultured skin fibroblasts to metabolize endocytosed [14C]-sulfatide. We now report the insertion of the full-length prosaposin cDNA into the Moloney murine leukemia virus-derived retroviral vector, pLJ, and the infection of cultured skin fibroblasts from a newly diagnosed and molecularly characterized patient with SAP-1 deficiency. The cultured cells infected with the prosaposin cDNA construct now show both production of normal levels of mature SAP-1 and completely normal metabolism of endocytosed [14C]-sulfatide. These studies demonstrate that the virally transferred prosaposin cDNA is processed normally and is localized within lysosomes, where it is needed for interaction between sulfatide and arylsulfatase A. In addition, normal as well as mutant sequences can now be found by allele-specific oligonucleotide hybridization of PCR-amplified genomic DNA by using exonic sequences as primers.

Antisense Elements (Genetics)↗
PubMed1991

Leukocyte sonicates as a source for both enzyme assay and DNA amplification for mutational analysis of certain lysosomal disorders.

At present the identification of patients and carriers of most lysosomal disorders is accomplished by finding decreased activity of one enzyme in an easily obtained tissue sample such as leukocytes. As the genes for these enzymes are cloned and mutations identified, the use of molecular techniques to supplement enzyme testing will be warranted. To facilitate the implementation of such studies a simple method for isolating DNA from the remaining leukocyte sonicate, and using this DNA for polymerase chain reaction amplification of regions involved in three lysosomal disorders is described. The DNA from the sonicate was isolated without proteinase K digestion, was readily soluble in Tris-EDTA buffer and available for amplification almost immediately. The usefulness of the methods was confirmed by studies on patients and family members with three relatively common lysosomal disorders, metachromatic leukodystrophy. Gaucher disease and Tay-Sachs disease. This method allows immediate DNA analysis without the need for securing an additional blood sample.

Base Sequence↗
PubMed1991

The mechanism for a 33-nucleotide insertion in mRNA causing sphingolipid activator protein (SAP-1)-deficient metachromatic leukodystrophy.

Metachromatic leukodystrophy is a severe autosomal recessive disorder caused by accumulation of sulfatide resulting from deficient lysosomal degradation. While most patients have mutations in the lysosomal enzyme arylsulfatase A, some patients have mutations in a required heat stable sphingolipid activator protein, we call SAP-1. One patient with SAP-1 deficiency was previously demonstrated to have a 33-nucleotide insertion in her mRNA. This resulted in the production of mature SAP-1 with 11 extra amino acids, which was unstable during intracellular processing. In this manuscript we demonstrate that the 33 nucleotides are present near the middle of a 4-kb intron, and that a single base change, c to a, in the second position preceding the 33-nucleotide insertion, coupled with the presence of a string of pyrimidines immediately upstream from this change, creates a new 3' splice junction. The presence of a string of pyrimidines within the 33-nucleotide insertion, which has three cag trinucleotides near the 3' end, leads to alternative splicing in normal people as found in this laboratory and by others. The insertion region is followed by a gt dinucleotide that is spliced to a typical 3' consensus sequence. The single nucleotide change, c to a, was confirmed by identifying normal and mutant sequence in the consanguineous parents and a sister, previously identified as a carrier of this disorder.

Base Sequence↗
PubMed1991

Complementation studies in human and caprine beta-mannosidosis.

Cell fusions were performed to investigate the possible involvement of different gene mutations in five patients with isolated beta-mannosidosis and a patient with a combined deficiency of beta-mannosidase and heparin sulphate sulphamidase. In none of the combinations of cell lines was beta-mannosidase activity restored in the fused cell culture. Similarly, no complementation of sulphamidase activity was observed after fusion of cells with the combined deficiency and cells with isolated sulphamidase deficiency (mucopolysaccharidosis type IIIA). The absence of complementation suggests that the combined deficiency is not caused by a defect in one common factor affecting the two enzymes: The results rather indicate a rare coincidence of two independent mutations which are allelic with the mutation in the respective conditions with isolated enzyme deficiencies.

Animals↗