D A Applegarth

University of British Columbia - Vancouver, Vancouver, British Columbia, Canada

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Publications (164)944.78 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The characteristic elevation of plasma glycine concentrations observed in propionic acidaemia (PA) and other 'ketotic hyperglycinaemias' has been attributed to secondary inhibition of the hepatic glycine cleavage system (GCS) by accumulating CoA derivatives of branched-chain amino acid metabolites. In nonketotic hyperglycinaemia (NKH), cerebrospinal fluid (CSF) and plasma glycine levels and their ratio are increased due to primary deficiency of central nervous system (CNS) as well as hepatic GCS. Whether the GCS in the CNS is also inhibited in PA is unclear, as there are scant data available on CSF glycine levels in this disorder. We studied the relation of CSF and plasma glycine levels in 6 paired samples from 4 PA patients, including one PA patient with bacterial meningitis who underwent ventriculoperitoneal shunting and multiple CSF analyses (n = 26). In contrast to the CSF glycine levels which were generally elevated in all four PA patients, the CSF/plasma glycine concentration ratios in paired samples were normal (0.016-0.029), with the exception of a single sample (0.132) with extremely high CSF protein concentration (2010 mg/L) during the course of meningitis indicating a disturbed blood-brain barrier. This finding of normal CSF/plasma glycine ratio in PA suggests that the observed elevations of CSF glycine levels are a reflection of the concurrent hyperglycinaemia resulting from secondary inhibition of hepatic GCS, but that brain GCS is not affected, in contrast to the situation in NKH. The neurological sequelae in PA are therefore unlikely to be related to disturbed glycine metabolism.
    Journal of Inherited Metabolic Disease 07/2008; 31(3):395-8. · 4.07 Impact Factor
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    ABSTRACT: To test for specific mutations in the alanine:glyoxylate aminotransferase (AGT) gene, in order to diagnose primary hyperoxaluria type 1 (PH1). Samples of liver and/or DNA from 81 patients were submitted to our laboratory for diagnostic testing for PH1. Using a panel of selected mutations, DNA was examined in 64 cases, of which 36 had the diagnosis of PH1 confirmed by liver AGT assay. DNA sequencing was employed if mutation testing revealed only one mutation. Identification of 100% of the mutations in the AGT-confirmed samples led to the development of a focused testing panel currently involving 4 common mutations, 7 mutations recurring at lower frequency and 5 with apparent ethnic associations. This mutation panel alone would have identified the two causative mutations in 64% of the PH1 samples.
    Clinical biochemistry 06/2008; 41(7-8):598-602. · 2.02 Impact Factor
  • Derek A Applegarth, Jennifer R Toone
    American Journal of Medical Genetics Part A 02/2006; 140(2):186-8. · 2.30 Impact Factor
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    ABSTRACT: We describe nine novel mutations and polymorphisms occurring on the major allele of the human alanine:glyoxylate aminotransferase gene in patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT; EC The PH1 mutations include two small frameshift mutations, 327delG and 117_118insCA, a large deletion spanning exon 9 and portions of the flanking introns, a splice junction mutation, IVS6+5G>C, and two missense mutations, G161R and S218L. Expression studies of the two missense mutations indicated very little enzymatic activity associated with either of them. Three polymorphisms in the coding sequence were also identified, I279T, A280V, and T235T. Expression studies of I279T and A280V suggested essentially normal AGT activity. I279T, found in two cases, was located on a 33_34insC allele. A280V and T235T were both located on the same allele as IVS6+5G>C. We have also identified recurrences of previously reported rare mutations, 33delC, IVS7-1G>C, and IVS4-1G>A. Five of the six novel PH1 mutations occurred in a compound heterozygous state with either of two common PH1 mutations, G170R or 33_34insC. S218L was apparently homozygous in two individuals. These findings contribute to our overall picture of heterogeneity of mutations in PH1 and the AGT major allele.
    Molecular Genetics and Metabolism 01/2005; 86(1-2):172-8. · 2.83 Impact Factor
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    ABSTRACT: Glycine encephalopathy, also known as nonketotic hyperglycinemia (NKH), is an autosomal recessive disorder caused by a defect in the glycine cleavage system. NKH is classically associated with neonatal apnea, lethargy, hypotonia, and seizures, followed by severe psychomotor retardation in those who survive. To determine the natural history of NKH, the authors mailed a 44-question survey to 170 households in the International NKH Family Network. Data for 65 patients (36 boys, 29 girls) were collected from 58 families. One-third of the subjects died; 8 girls died during the neonatal period, and 14 patients died thereafter (2 girls, 12 boys). Median age of death for boys was 2.6 years vs <1 month for girls (p = 0.02). Mean birth weight and length, occipitofrontal circumference, and gestation duration were normal. Two-thirds of infants were ventilated during the neonatal period; of these, 40% died. Ninety percent had confirmed seizures, 75% during the first month of life. Interestingly, three NKH patients never developed seizures. An abnormal corpus callosum and/or hydrocephalus were associated with especially poor gross motor and speech development. Of 25 patients living > or =3 years, 10 were able to walk and say/sign words; all were boys. In six families with more than one affected child, disease course and mortality were similar within each family. This study reveals a striking and unexpected gender difference in mortality and developmental progress. Of the two-thirds of nonketotic hyperglycinemia patients surviving the newborn period, up to 20% (mostly boys) may learn to walk and communicate by saying or signing words.
    Neurology 11/2004; 63(10):1847-53. · 8.25 Impact Factor
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    ABSTRACT: We describe 7 novel mutations occurring on the major allele of the human AGT gene in patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT; EC These mutations include 3 small deletions, 570delG, 744delC, and 983_988del, two splice junction mutations, IVS7-1G-->C and IVS8+1G-->T, and two nonsense mutations, R111X and W251X. We have also identified recurrences of previously identified reported mutations, 679-(IVS6+2)delAAgt, IVS8-3C-->G and 33insC. Deletion mutation 679-(IVS6+2)delAAgt has now been identified in a second Chinese patient and may be specific to that population. In contrast, 33insC has been found in patients of varying ethnic and racial backgrounds; a single vs multiple origin for this mutation is thus an intriguing question. It also appears to occur at a high frequency on the major allele. Five of the novel mutations were detected in patients who were compound heterozygotes for one of the common mis-targeting mutation, G170R or F152I, while the other two mutations occurred in the same patient.
    Molecular Genetics and Metabolism 05/2004; 82(1):64-8. · 2.83 Impact Factor
  • D A Applegarth, J R Toone
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    ABSTRACT: This article summarizes data and issues covered in the workshop on Glycine Encephalopathy using headings that cover important topics in our present knowledge of this disease.
    Journal of Inherited Metabolic Disease 02/2004; 27(3):417-22. · 4.07 Impact Factor
  • J R Toone, D A Applegarth, G Laliberte
    Human Genetics 11/2003; 113(5):465. · 4.63 Impact Factor
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    ABSTRACT: A defect in the P-protein component of the glycine cleavage system has been the most frequent abnormality found in patients with glycine encephalopathy (NKH). In a retrospective study of a more specific group of NKH patients, however, we found that >50% had T-protein mutations. The patients studied had one or more of the following unusual biochemical findings: residual glycine cleavage system activity in liver assayed by the standard method or a newly developed micromethod, residual glycine cleavage system activity in lymphoblasts, and/or increased amniotic fluid glycine/serine ratio with a normal amniotic fluid glycine level in prenatal diagnosis. The selected patients had a much higher incidence of T-protein defects than expected in the general NKH patient population. We report, here, three novel mutations and five polymorphisms in the T-protein gene, PCR/restriction enzyme methods for one mutation (R296H) and two polymorphisms (E211K and R318R), and an estimation of their frequency in normal controls. The co-occurrence of the polymorphism E211K with the mutation R320H in patients with a severe phenotype is discussed.
    Molecular Genetics and Metabolism 08/2003; 79(4):272-80. · 2.83 Impact Factor
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    ABSTRACT: Multiple carboxylase deficiency (MCD, MIM:253270) is a common organic aciduria and caused by deficiency of either biotinidase or holocarboxylase synthetase (HLCS; EC Patients commonly present during early infancy with acute metabolic derangements and severe metabolic acidosis. Recently, a late onset form of HLCS deficiency was also described. The different phenotypes (early and late presenting) may be related to a spectrum of mutations in HLCS gene. Applications of mutation analysis in HLCS had been limited previously by the requirement of cDNA from living tissue for study. We described here a genomic approach for molecular diagnosis of HLCS deficiency which we have used to detect mutations in Chinese patients who had the late-onset form of HLCS deficiency. In addition, a fibroblast cell line with MCD from Coriell Cell repositories was also studied. Three Chinese patients with late onset HLCS deficiency were studied. The genomic sequence of HLCS was retrieved and newly designed primers were used to cover all coding sequences of the gene. PCR products were analyzed by direct sequencing. Population allelic frequencies of mutations detected were determined by genotyping of control samples by restriction fragment length polymorphism. We found a recurrent mutation, R508W, in the three unrelated Chinese patients. Two were homozygous for this mutation. The other patient was a compound heterozygote of R508W and a novel mutation, D634N. The results suggest that R508W may be an important and relatively prevalent disease-causing mutation in Chinese MCD patients. A fibroblast cell-line from an African patient revealed an additional novel mutation, R565X and a known mutation, V550M. R508W is a recurrent mutation in Chinese MCD patients which is associated with the late onset phenotype. This new genomic approach for mutation analysis of HLCS gene provides new opportunities in studies of MCD.
    Clinical Biochemistry 04/2003; 36(2):145-9. · 2.45 Impact Factor
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    ABSTRACT: We describe a novel missense mutation (A112D) and polymorphism (V326I) in the human AGT gene in two black African patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT; EC V326I was found in DNA from normal control Blacks with an allele frequency of 3%. Expression studies confirmed that A112D reduced AGT enzyme activity by 95% while V326I had no effect. Both A112D and V326I were homozygous in both patients and lie on a variant of the minor allele of the AGT gene. This variant haplotype, Mi(A), includes an intron 1 duplication and intron 4 VNTR (38 repeat) but lacks the P11L and I340M normally associated with the minor allele in Caucasians. Among the South African Blacks tested, the Mi(A) haplotype had an allele frequency of 12% compared to 3 % for the Caucasian-type minor allele haplotype.
    Molecular Genetics and Metabolism 02/2003; 78(1):44-50. · 2.83 Impact Factor
  • Mary Anna Carbone, Derek A Applegarth, Brian H Robinson
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    ABSTRACT: This paper describes the molecular characterization of two male siblings displaying the complex (Type B) form of pyruvate carboxylase (PC) deficiency in which severe neonatal lactic acidosis and redox abnormalities results in death within the first few weeks of life. The two male siblings were found to be compound heterozygous for a TAGG deletion at the exon15/intron15 splice site (IVS15+2-5delTAGG) and a dinucleotide deletion in exon 16 (2491-2492delGT) of the PC gene. We also demonstrate through RT-PCR and sequencing of aberrant transcripts that the IVS15+2-5delTAGG results in the retention of intron 15 during pre-mRNA splicing. In addition, both deletions are predicted to result in a frameshift to generate a premature termination codon such that the encoded mRNA could be subject to nonsense mediated decay.
    Human Mutation 08/2002; 20(1):48-56. · 5.21 Impact Factor
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    ABSTRACT: Eight novel mutations were found in the P-protein (glycine decarboxylase) gene (GLDC) of the glycine cleavage system (EC by screening five exons of the gene in patients with glycine encephalopathy (NKH). The mutations identified were of eight single base changes: a one-base deletion 1054del A, a splice site mutation IVS18-2A-->G and six amino acid substitutions A283P, A313P, P329T, R410K, P700A, and G762R.
    Molecular Genetics and Metabolism 07/2002; 76(3):243-9. · 2.83 Impact Factor
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    ABSTRACT: Metachromatic leukodystrophy (MLD) is a genetic neurodegenerative disorder resulting from a deficiency of arylsulfatase A. Late onset forms are relatively rare. Central nervous system (CNS) involvement is characteristic at all ages. A patient in her late 40s with peripheral neuropathy was assessed by EEG, evoked potentials, CT and nerve conduction studies. Nerve and muscle biopsy samples were investigated by electron microscopy. Arylsulfatase A activity in leukocytes and excreted cerebroside sulfate were determined. The arylsulfatase A gene was investigated for mutations using polymerase chain reaction (PCR) and DNA sequencing. The identified mutation was expressed transiently in African green monkey kidney (COS) cells to determine the effect of the mutation on arylsulfatase A activity. Central nervous system functions were normal. Nerve conduction velocities were decreased. Sural nerve biopsy showed inclusions typical of MLD. Arylsulfatase A was less than 5% of normal. A homozygous mutation thr286pro was identified in the arylsulfatase A gene and demonstrated to be deleterious through transient expression studies. Our patient has a progressive peripheral neuropathy but has apparently intact CNS function at her present age of 57 years. Biochemical, physiological and pathological findings are consistent with a diagnosis of MLD. A homozygous mutation, thr286pro, found in her arylsulfatase A gene, decreased enzyme activity to a level consistent with a late onset form of MLD.
    The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques 06/2002; 29(2):159-63. · 1.33 Impact Factor
  • Prenatal Diagnosis 04/2002; 22(3):266-7. · 2.68 Impact Factor
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    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 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.
    Molecular Genetics and Metabolism 12/2001; 74(3):314-21. · 2.83 Impact Factor
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    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.
    Molecular Genetics and Metabolism 04/2001; 72(4):322-5. · 2.83 Impact Factor
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    ABSTRACT: Leber's hereditary optic neuropathy (LHON) is a common cause of bilateral optic nerve disease. The majority of LHON patients harbour one of three point mutations of the mitochondrial DNA (mtDNA) complex I, or NADH:ubiquinone oxidoreductase (ND) genes (G11778A in ND4, G3460A in ND1, T14484C in ND6). As a consequence, screening for these mutations has become part of the routine clinical investigation of young adults who present with bilateral optic neuropathy, and the absence of these mutations is interpreted as indicating there is a low likelihood that an optic neuropathy is LHON. However, there are many individuals who develop the clinical features of LHON but who do not harbour one of these primary LHON mutations. We describe two LHON pedigrees that harbour the same novel point mutation within the mtDNA ND6 gene (A14495G). This mutation was heteroplasmic in both families, and sequencing of the mitochondrial genome confirmed that the mutation arose on two independent occasions. This is the seventh mutation in the ND6 gene that causes optic neuropathy, indicating that this gene is a hot spot for LHON mutations. Protein modelling studies indicate that all of these pathogenic mutations lie within close proximity to one another in a hydrophobic cleft or pocket. This is the first evidence for a relationship between a specific disease phenotype and a specific structural domain within a mitochondrial respiratory chain subunit. These findings suggest that the mtDNA ND6 gene should be sequenced in all patients with LHON who do not harbour one of the three common LHON mutations.
    Brain 02/2001; 124(Pt 1):209-18. · 9.92 Impact Factor
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    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.
    Human Mutation 02/2001; 17(1):76. · 5.21 Impact Factor
  • D A Applegarth, J R Toone
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    ABSTRACT: Nonketotic hyperglycinemia (NKH) is an autosomal recessive disorder of glycine metabolism caused by a defect in the glycine cleavage enzyme complex (GCS). GCS is a complex of four proteins encoded on four different chromosomes. In classical neonatal NKH, levels of cerebrospinal fluid (CSF) glycine and CSF/plasma glycine ratio are very high but the CSF results, in particular, may be more difficult to interpret in later-onset, milder, or otherwise atypical NKH. Enzymatic confirmation of NKH requires a liver sample. Delineation of which protein of the complex is defective is necessary to screen for mutations in the appropriate gene. Except for Finnish NKH patients, few recurrent mutations have yet been found, although analysis of the P-protein gene (the site of the defect in the majority of patients) is at an early stage. Prenatal diagnosis by GCS assay in chorionic villus biopsies is not completely reliable and will be replaced by molecular analysis in families where the mutations are known.
    Molecular Genetics and Metabolism 01/2001; 74(1-2):139-46. · 2.83 Impact Factor

Publication Stats

1k Citations
944.78 Total Impact Points


  • 1968–2006
    • University of British Columbia - Vancouver
      • • Department of Pediatrics
      • • Department of Medical Genetics
      • • Faculty of Medicine
      • • Faculty of Pharmaceutical Sciences
      Vancouver, British Columbia, Canada
  • 2000
    • University of Victoria
      • Department of Biology
      Victoria, British Columbia, Canada
  • 1999
    • Baystate Medical Center
      • Department of Pediatrics
      Springfield, Massachusetts, United States
  • 1996
    • University of Washington Seattle
      • Department of Pediatrics
      Seattle, WA, United States
  • 1977–1996
    • SickKids
      Toronto, Ontario, Canada
  • 1994
    • University of Alberta
      • Department of Laboratory Medicine and Pathology
      Edmonton, Alberta, Canada
  • 1977–1994
    • BC Children's Hospital
      Vancouver, British Columbia, Canada
  • 1991
    • McGill University
      • Department of Pediatrics
      Montréal, Quebec, Canada
    • Duke University Medical Center
      • Department of Biochemistry
      Durham, NC, United States
  • 1984–1986
    • Childrens Hospital of Pittsburgh
      Pittsburgh, Pennsylvania, United States
  • 1975
    • Vancouver General Hospital
      Vancouver, British Columbia, Canada