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1.
Mol Genet Metab ; 94(3): 368-74, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18448374

ABSTRACT

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.


Subject(s)
Molecular Chaperones/physiology , Mutation, Missense , Protein Folding , Transaminases/genetics , Transaminases/metabolism , Trypsin/metabolism , Amino Acid Sequence , Cell-Free System/chemistry , Cell-Free System/metabolism , Enzyme Activation/genetics , Humans , Ligands , Molecular Mimicry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense/physiology , Peptide Hydrolases/metabolism , Protein Processing, Post-Translational/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Transaminases/chemistry
2.
Kidney Int ; 70(11): 1891-3, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17130820

ABSTRACT

The primary hyperoxalurias are diseases of overproduction of oxalate. The immediate precursor of oxalate is glyoxylate. Metabolism of hydroxyproline, derived from collagen turnover or the diet, appears to be a major source of glyoxylate, and a potential target for a therapeutic strategy of substrate depletion.


Subject(s)
Glyoxylates/metabolism , Hydroxyproline/metabolism , Hyperoxaluria, Primary/therapy , Adult , Female , Humans , Hyperoxaluria, Primary/metabolism , Male , Oxalates/metabolism
3.
Mol Genet Metab ; 89(4): 349-59, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16971151

ABSTRACT

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme, deficiency of which results in primary hyperoxaluria type 1 (PH1). More than 65 PH1-related mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have generated a spectrum of 15 missense changes including the most common PH1 mutation, G170R, and expressed them on the appropriate background of the major or minor allele, in an Escherichia coli overexpression system and in a rabbit reticulocyte transcription/translation system. We have investigated their effects on enzyme activity, dimerization, aggregation, and turnover. The effect of pyridoxal phosphate (PLP) on dimerization and stability was also investigated. Although all 15 mutant AGTs were expressed as intact proteins in E. coli, only three: G41R and G41V on the major allele, and the common mutation G170R, resulted in significant amounts of enzymatic activity. Dimerization failure was a frequent observation (13/15) except for G41V and D183N. Dimerization was poor with S187F but was substantially improved with PLP. Proteasome-mediated protein degradation was observed for all the mutations except G41R on the major allele, G41V, D183N, G170R, and S218L. Increases in the stability of the mutant enzymes in the presence of PLP were small; however, G41R on the minor allele showed a direct relationship between its half life and the concentration of PLP. The minor allele AGT product and many of the mutants were subject to a limited non-proteasomal proteolytic cleavage when ATP was depleted.


Subject(s)
Mutation, Missense , Proteasome Endopeptidase Complex/metabolism , Transaminases/metabolism , Amino Acid Substitution , Animals , Dimerization , Enzyme Stability , Escherichia coli , Humans , Pyridoxal Phosphate/pharmacology , Rabbits , Transaminases/drug effects , Transaminases/genetics
4.
Mol Genet Metab ; 74(3): 314-21, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11708860

ABSTRACT

We describe three novel deletions in the human AGT gene in three patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme, alanine glyoxylate aminotransferase (AGT; EC 2.6.1.44). A deletion of 4 nucleotides in the exon 6/intron 6 splice junction (679-IVS6+2delAAgt) is expected to cause missplicing. It would also code for a K227E missense alteration in any mRNA successfully spliced. A 2-bp deletion in exon 11 (1125-1126del CG, cDNA) results in a frameshift. A deletion of at least 5-6 kb, EX1 EX5del, spanned exons 1-5 and contiguous upstream sequence. All three deletions are heterozygous with previously documented missense mutations; the intron 6 deletion with F152I, the exon 11 deletion with G82E, and EX1 EX5del with the common mistargeting mutation, G170R.


Subject(s)
Hyperoxaluria, Primary/genetics , Transaminases/genetics , Amino Acid Sequence , Base Sequence , Child , Child, Preschool , DNA Mutational Analysis , Family Health , Female , Humans , Hyperoxaluria, Primary/enzymology , Infant , Male , Molecular Sequence Data , Mutation , Polymorphism, Genetic , Sequence Deletion , Transaminases/drug effects , Transaminases/metabolism
6.
Mol Genet Metab ; 72(4): 322-5, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11286506

ABSTRACT

Screening a DNA bank from 50 patients with enzymatic confirmation of their diagnosis of nonketotic hyperglycinemia gave allele frequencies of 5% for R515S of P-protein (glycine decarboxylase) and 7% for R320H of T-protein (aminomethyltransferase). In a previous report we found that 3% of the same patient alleles were positive for T-protein IVS7-1G>A. In total, testing for these three mutations identified 15% of alleles and positive results (one or two mutations) were found in 11 of the 50 patients. In addition, a novel point mutation in T-protein, N145I, was found in a single case and a PCR/restriction enzyme assay was developed for its detection.


Subject(s)
Amino Acid Oxidoreductases/genetics , Glycine/blood , Hydroxymethyl and Formyl Transferases/deficiency , Hydroxymethyl and Formyl Transferases/genetics , Hyperglycinemia, Nonketotic/genetics , Mutation , Aminomethyltransferase , DNA Primers/chemistry , Exons , Gene Frequency , Glycine/metabolism , Glycine Dehydrogenase (Decarboxylating) , Heterozygote , Humans , Hyperglycinemia, Nonketotic/enzymology , Imines , Infant, Newborn , Ketosis , Liver/enzymology , Polymerase Chain Reaction , Prenatal Diagnosis
7.
Hum Mutat ; 17(1): 76, 2001.
Article in English | MEDLINE | ID: mdl-11139253

ABSTRACT

A novel splice site mutation (IVS7-1G-->A) in the T-protein gene (aminomethyltransferase, or AMT) of the glycine cleavage enzyme complex was found in a patient with nonketotic hyperglycinemia (NKH). A PCR/restriction enzyme method to detect this mutation was used to screen 100 NKH alleles and identified the mutation in three unrelated families.


Subject(s)
Amino Acid Oxidoreductases/genetics , Carrier Proteins/genetics , Hydroxymethyl and Formyl Transferases/genetics , Hyperglycinemia, Nonketotic/genetics , Multienzyme Complexes/genetics , Mutation/genetics , RNA Splice Sites/genetics , Transferases/genetics , Aminomethyltransferase , Genetic Carrier Screening , Humans , Hyperglycinemia, Nonketotic/enzymology , Introns/genetics , Polymerase Chain Reaction
8.
Pediatr Neurol ; 23(2): 173-6, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11020646

ABSTRACT

We report two siblings with metachromatic leukodystrophy, one who presented at 7 years of age (juvenile onset) and his sister who presented at 22 years of age (adult onset). They are compound heterozygotes for two novel mutations in the arylsufatase A gene (ARSA). The responsible mutations in this Vietnamese family consist of a missense mutation with 5% enzyme activity (R143G) and a nonsense mutation (W318ter), from which no enzyme activity would be expected. These mutations in the ARSA gene have not been previously reported and may be useful when diagnosing metachromatic leukodystrophy in other affected Vietnamese individuals. The variability in presentation suggests that the genotype alone is not sufficient to determine the onset and course of the disease and modifying genetic and perhaps nongenetic factors likely contribute.


Subject(s)
Cerebroside-Sulfatase/genetics , Codon, Nonsense/genetics , Leukodystrophy, Metachromatic/genetics , Mutation, Missense/genetics , Adult , Child , Female , Humans , Leukodystrophy, Metachromatic/diagnosis , Male , Pedigree , Vietnam
9.
Mol Genet Metab ; 70(2): 116-21, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10873393

ABSTRACT

The investigation of 14 unrelated patients with nonketotic hyperglycinemia led to the identification of mutations in 4 cases. Patients were initially categorized into probable P- or T-protein defects of the glycine cleavage enzyme complex, by the use of the glycine exchange assay without supplemental H-protein, then screened for mutations in the P-protein and T-protein genes, respectively.


Subject(s)
DNA Mutational Analysis , Hyperglycinemia, Nonketotic/genetics , Methyltransferases/genetics , Amino Acid Sequence , DNA Primers/chemistry , Exons , Female , Glycine/metabolism , Homocysteine S-Methyltransferase , Humans , Hyperglycinemia, Nonketotic/enzymology , Infant, Newborn , Introns , Liver/enzymology , Male , Methyltransferases/metabolism , Molecular Sequence Data , Pedigree
11.
Clin Invest Med ; 21(3): 114-23, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9627765

ABSTRACT

Aspartylglucosaminuria (McKusick 208400) is a lysosomopathy associated with aspartylglucosaminidase (L-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) deficiency. It has been most frequently encountered in Finland, where the regional incidence may be as high as 1 in 3600 births. In North America it is very rare, having been reported in only 8 patients. We encountered 4 patients with aspartylglucosaminuria in a Canadian family of 12 siblings. The 4 siblings affected--2 brothers and 2 sisters--were apparently normal at birth; however, their developmental milestones, particularly speech, were slow, and they acquired only a simple vocabulary. Throughout life, there was a progressive coarsening of facial features; 3 had inguinal hernia and recurrent diarrhea; all became severely retarded and by the 4th decade showed evident deterioration of both cognitive and motor skills; 2 exhibited cyclical behavioural changes. Three of the siblings have died, at 33, 39 and 44 years of age. Two died of bronchopneumonia and 1 of asphyxiation following aspiration. In the urine of all 4 siblings, and in the 1 liver examined, we found 2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta-D-glucosamine (GlcNAc-Asn) and alpha-D-mannose-(1,6)-beta-D-mannose-(1,4)-2-acetamido- 2-deoxy-beta-D-glucose-(1,4)-2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta - D-glucosamine (Man2-GlcNAc2-Asn). Compared with the level of activity in controls, aspartylglucosaminidase activity was less than 2% in fibroblasts from 3 of the siblings, less than 0.5% in leukocytes from 1 sibling, and less than 1% in the liver of 1 sibling, whereas other acid hydrolase activities in these tissues were normal. Ultrastructural studies of skin showed that fibroblasts, endothelial cells and pericytes contained vacuoles with fine reticulo-floccular material. Glial and neuronal cells of the central nervous system showed similar inclusions as well as others composed of concentric or parallel membranous arrays intermingled with lipid droplets.


Subject(s)
Acetylglucosamine/analogs & derivatives , Aspartylglucosaminuria , Lysosomal Storage Diseases/genetics , Acetylglucosamine/urine , Adult , Aspartylglucosylaminase/genetics , Canada , Child , Female , Humans , Lysosomal Storage Diseases/urine , Male , Middle Aged , Pedigree
13.
Clin Biochem ; 31(8): 627-32, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9876894

ABSTRACT

OBJECTIVES: To develop a protocol capable of identifying deletions in mitochondrial DNA and use it to identify the breakpoints of a mtDNA deletion in a patient with chronic progressive external ophthalmoplegia (CPEO). DESIGN AND METHODS: Deletions in mtDNA were identified by a combination of long range PCR and Southern blotting. The precise breakpoints were determined by automated DNA sequencing. RESULTS: A series of DNA samples from patients with suspected mitochondrial disease was subjected to a protocol, which combines long range PCR and Southern blotting. We found a unique deletion in a patient with CPEO and we identified the precise location of this deletion through DNA sequencing. CONCLUSIONS: Long range PCR has the advantages of speed, minimal samples requirements, and sensitivity. Southern blotting is better able to evaluate heteroplasmy and detect duplications. We suggest a protocol that enables us to identify precisely the breakpoints in a unique mutation of mtDNA in a patient with CPEO.


Subject(s)
Blotting, Southern/methods , DNA, Mitochondrial/analysis , Ophthalmoplegia/genetics , Polymerase Chain Reaction/methods , Sequence Deletion , Adolescent , Blepharoptosis/genetics , Humans , Kearns-Sayre Syndrome/genetics , Male , Mitochondrial Encephalomyopathies/genetics , Ophthalmoplegia, Chronic Progressive External/genetics , Sensitivity and Specificity
14.
J Med Genet ; 34(6): 493-8, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9192271

ABSTRACT

Metachromatic leucodystrophy (MLD) is a lysosomal storage disease resulting from a deficiency of arylsulphatase A. We have identified a child with infantile onset MLD who is homozygous for an A212V mutation, a mutation previously reported but not further characterised. We have introduced this mutation into an arylsulphatase A expression vector by site directed mutagenesis. Transient expression of this mutant plasmid in COS cells yields very low levels of arylsulphatase A activity consistent with the patient's phenotype. The arylsulphatase A pseudodeficiency also segregates in this family causing difficulty in interpreting enzyme levels in the absence of DNA data. Two other patients from the same province, also carrying the A212V allele, have juvenile and adult onset MLD and are heterozygous for P426L ("A" allele) and I179S alleles respectively, known late onset alleles.


Subject(s)
Cerebroside-Sulfatase/genetics , Leukodystrophy, Metachromatic/enzymology , Leukodystrophy, Metachromatic/genetics , Mutation , Adult , Age of Onset , Alleles , Animals , Base Sequence , COS Cells , Cerebroside-Sulfatase/deficiency , Child , Child, Preschool , DNA Primers/genetics , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Mutagenesis, Site-Directed , Nova Scotia , Pedigree , Phenotype , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational
15.
Clin Biochem ; 30(1): 57-61, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9056111

ABSTRACT

OBJECTIVE: To identify the molecular basis of arylsulfatase A deficiency in a family at risk for metachromatic leukodystrophy (MLD) and determine the genetic risk in the offspring. METHODS: Mutations in the arylsulfatase A gene were identified by PCR amplification and restriction enzyme digestion. Individuals had previously been tested for arylsulfatase A activity. RESULTS: Assays of arylsulfatase A activity had resulted in ambiguous results for MLD carrier identification. DNA analysis clearly identified two MLD mutations in the family, and an unsuspected arylsulfatase A pseudodeficiency. The DNA information immediately clarified the MLD risk for the family and confirmed that a newborn with low arylsulfatase A activity was unaffected. CONCLUSIONS: The overlap between activities for various combinations of MLD and pseudodeficiency alleles and the variability inherent in the assay of arylsulfatase A complicate the interpretation of activity levels in families at risk for MLD. Use of simple molecular biological tests for pseudodeficiency and the common MLD mutations in combination with the enzyme data can facilitate carrier identification and prenatal diagnosis.


Subject(s)
Cerebroside-Sulfatase/deficiency , Cerebroside-Sulfatase/genetics , Leukodystrophy, Metachromatic/diagnosis , Leukodystrophy, Metachromatic/genetics , Alleles , Arylsulfatases/chemistry , Arylsulfatases/deficiency , Arylsulfatases/genetics , Arylsulfatases/metabolism , Cell Line , Cerebroside-Sulfatase/chemistry , Cerebroside-Sulfatase/metabolism , Child, Preschool , Female , Fibroblasts/enzymology , Genetic Carrier Screening , Humans , Leukocytes/enzymology , Leukodystrophy, Metachromatic/enzymology , Male , Pedigree , Polymerase Chain Reaction
19.
J Med Genet ; 30(5): 363-8, 1993 May.
Article in English | MEDLINE | ID: mdl-8320697

ABSTRACT

Neurofibromatosis type 1 (NF1) is a common, autosomal dominant genetic disorder with a variety of highly variable symptoms including cutaneous manifestations (such as café au lait spots), Lisch nodules, plexiform neurofibromas, skeletal abnormalities, an increased risk for malignancy, and the development of learning disabilities. The wide clinical variability of expression of the disease phenotype and high (spontaneous) mutation rate of the NF1 gene indicate that careful clinical examination of patients and family members is necessary to provide an accurate diagnosis of the disease. Since very few NF1 mutations have been identified, and with the apparent lack of a predominant mutation in this large, highly mutable gene, molecular diagnosis of NF1 will continue to be based on haplotypes using linkage analysis. Here we report our experiences while providing a molecular diagnostic service for NF1 in the ethnically diverse region of south-western Ontario. Molecular diagnoses with at least one informative probe/enzyme combination are reported for 19 families including two families requesting prenatal diagnosis for NF1. We have augmented the classical Southern based approach to linkage analysis with the use of PCR based assays for molecular linkage. Furthermore, criteria have been established in our laboratory for executing molecular linkage based on heterozygosity values, recombination fractions, and the use of intragenic probes/markers.


Subject(s)
Chorionic Villi Sampling , Chromosomes, Human, Pair 17 , Genes, Neurofibromatosis 1/genetics , Genetic Linkage , Neurofibromatosis 1/genetics , Alleles , Base Sequence , Blotting, Southern , DNA/analysis , DNA/chemistry , Female , Fetal Diseases/diagnosis , Fetal Diseases/genetics , Gene Frequency , Genetic Markers , Humans , Infant, Newborn , Male , Molecular Sequence Data , Neurofibromatosis 1/diagnosis , Ontario , Pedigree , Polymerase Chain Reaction , Pregnancy
20.
Hum Mol Genet ; 2(4): 439-44, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8504305

ABSTRACT

Modification of mammalian DNA by the methylation of cytosine in CpG dinucleotides is a complex phenomenon involved in a number of cellular and developmental processes. In particular, the characteristic hypermutability of CpGs may be a major contributor of point mutations leading to human genetic disease. We have hypothesized that DNA methylation contributes to mutations in the gene causing neurofibromatosis type 1 (NF1), one of the most common genetic disorders in humans and a disease where up to half of all cases are the result of sporadic germline mutations, usually in the paternally-derived allele. We have used two experimental approaches to analyze patterns of DNA methylation at CpG dinucleotides in the NF1 gene region. Southern analyses using isoschizomeric restriction pairs have revealed DNA methylation in areas flanking the NF1 gene region, while PCR-methylation assays have shown that methylation occurs both on genomic sequences flanking the NF1 gene and within the coding region of the gene itself. We suggest that methylated CpG dinucleotides within and around the highly mutable NF1 gene serve as a reservoir within which C-->T transitions contribute to the high frequency of spontaneous germline mutations associated with the disease.


Subject(s)
Chromosomes, Human, Pair 17 , DNA/chemistry , DNA/genetics , Genes, Neurofibromatosis 1 , Neurofibromatosis 1/genetics , Base Sequence , Genetic Linkage , Genetic Markers , Humans , Methylation , Molecular Sequence Data , Polymerase Chain Reaction , Restriction Mapping
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