Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

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.

4-Hydroxyproline metabolism and glyoxylate production: A target for substrate depletion in primary hyperoxaluria?

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.

Consequences of missense mutations for dimerization and turnover of alanine:glyoxylate aminotransferase: study of a spectrum of mutations.

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.

Three novel deletions in the alanine:glyoxylate aminotransferase gene of three patients with type 1 hyperoxaluria.

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.

Recurrent mutations in P- and T-proteins of the glycine cleavage complex and a novel T-protein mutation (N145I): a strategy for the molecular investigation of patients with nonketotic hyperglycinemia (NKH).

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.

Identification of the first reported splice site mutation (IVS7-1G-->A) in the aminomethyltransferase (T-protein) gene (AMT) of the glycine cleavage complex in 3 unrelated families with nonketotic hyperglycinemia.

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.

Variable onset of metachromatic leukodystrophy in a Vietnamese family.

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.

Biochemical and molecular investigations of patients with nonketotic hyperglycinemia.

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.

Identification of 6 new mutations in the iduronate sulfatase gene. Mutation in brief no. 233. Online.

Mucopolysaccharidosis type II (Hunter syndrome) is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme iduronate-2-sulfatase. We sequenced genomic DNA and RT-PCR products in the iduronate sulfatase (IDS) gene in 6 unrelated patients with Hunter syndrome to assess genotype/phenotype relationships and offer carrier testing where required. Six novel mutations were identified: four missense mutations, one four-base pair deletion (596-599delAACA) and a cryptic splice site mutation. Three of the missense mutations were significant amino acid substitutions (S143F, S491F, E341K) of which the latter two involve amino acids conserved amongst sulfatase enzymes. The patients identified with these mutations all had a severe clinical phenotype. One missense mutation with a minimal amino acid substitution (H342Y), in a non-conserved region of the gene, was associated with a mild clinical phenotype. We identified a novel cryptic splice site (IVS5+934G>A) with some normal (wild type) mRNA processing. We predict that the normal mRNA product confered some residual functional enzyme, resulting in a mild phenotype associated with the absence of overt central nervous system disease.

Aspartylglucosaminuria in a Canadian family.

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.

A protocol for detection of mitochondrial DNA deletions: characterization of a novel deletion.

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.

Metachromatic leucodystrophy in three families from Nova Scotia, Canada: a recurring mutation in the arylsulphatase A gene.

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.

DNA-based diagnosis of arylsulfatase A deficiencies as a supplement to enzyme assay: a case in point.

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.

Two new polymorphisms in the arylsulfatase A gene and their haplotype associations with normal, metachromatic leukodystrophy and pseudodeficiency alleles.

Linkage disequilibrium exists between metachromatic leukodystrophy (MLD) and pseudodeficiency mutations and selected polymorphisms within the arylsulfatase A gene. We have identified 2 new polymorphic NlaIII sites, NlaIII1 and NlaIII2, in the gene that, when used in combination with the known BsrI and BamHI polymorphisms, extends the haplotype associations of the pseudodeficiency and the most common infantile onset MLD alleles. Fixed haplotypes have also been established for 3 other recurring MLD mutations, ala212val, pro426leu, and thr274met. The NlaIII2 site is relatively rare and was found only in association with the pseudodeficiency variant carrying the glycosylation site mutation alone.