K. M. Gibson

4-Aminobutyrate aminotransferase (GABA-transaminase) deficiency

Abstract4-Aminobutyrate aminotransferase (GABA-transaminase, GABA-T, EC 2.6.1.19) deficiency (McKusick 137150), an inborn error of GABA degradation, has until now been documented in only a single Flemish child. Compared to the other defects of GABA degradation, succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.24) deficiency with >150 patients (McKusick 271980) and pyridoxine-dependent seizures with >100 patients (putativeglutamic acid decarboxylase (GAD, EC 4.1.1.15) deficiency; McKusick 266100), GABA-T deficiency is very rare. We present a summary of the clinical, biochemical, enzymatic and molecular findings on the index proband, and a recently identified second patient, with GABA-T deficiency. The phenotype in both included psychomotor retardation, hypotonia, hyperreflexia, lethargy, refractory seizures and electroencephalographic abnormalities. In an effort to elucidate the molecular basis of GABA-T deficiency, we isolated and characterized a 1.5kb cDNA encoding human GABA-T, in addition to a 41kb genomic clone which encompassed the GABA-T coding region. Standard methods of cloning and sequencing revealed an A-to-G transition at nucleotide 754 of the coding region in lymphoblast cDNAs derived from the index proband. This mutation resulted in substitution of an invariant arginine at amino acid 220 by lysine. Expression of the mutant in E. coli, followed by isolation and enzymatic characterization of the recombinant protein, revealed an enzyme whose Vmax was reduced to 25% of wild-type activity. The patient and father were heterozygous for this allele; the second allele in the patient remains unidentified. Genomic Southern analysis revealed that the second proband most likely harbours a deletion in the 3′region of the GABA-T gene.

Kinetic characterization of human hydroxyacid-oxoacid transhydrogenase: Relevance toD: -2-hydroxyglutaric and gamma-hydroxybutyric acidurias

SummaryWe investigated the presence of hydroxyacid–oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of γ-hydroxybutyrate (GHB) to succinic semialdehyde coupled to reduction of 2-ketoglutarate (2-KG) to D-2-hydroxyglutarate (D-2-HG), in human liver extracts employing [2H6]GHB and 2-KG as substrates. We measured incorporation of 2H in D-[2H]2-HG using GC-MS analyses, providing evidence for HOT activity in humans. Kinetic characterization of HOT was undertaken in forward and reverse directions. We employed [2H6]GHB and [2H4]2-KG as cosubstrates in order to develop a HOT activity assay in cultured human fibroblasts derived from patients with D-2-hydroxyglutaric aciduria. HOT activity was quantified in this system by the measurement of D-[2H5]2-HG production. Fibroblasts derived from patients with D-2-hydroxyglutaric aciduria showed normal HOT activities. Our results provide the first demonstration and preliminary kinetic characterization of HOT activity in human tissues.

Molecular characterization of methylmalonate semialdehyde dehydrogenase deficiency

Three patients have been reported with (putative) methylmalonic semialdehyde dehydrogenase (MMSDH) deficiency. The urine metabolic pattern was strikingly different in all, including β-alanine, 3-hydroxypropionic acid, both isomers of 3-amino- and 3-hydroxyisobutyric acids in one and 3-hydroxyisobutyric and lactic acids in a second, and mild methylmalonic aciduria in a third patient. In an effort to clarify these disparate metabolite patterns, we completed the cDNA structure, and characterized the genomic structure of human MMSDH gene in order to undertake molecular analysis. Only the first patient had alterations in the MMSDH coding region, revealing homozygosity for a 1336G>A transversion, which leads to substitution of arginine for highly conserved glycine at amino acid 446. No abnormalities of the MMSDH cDNA were detected in the other patients. These data provide the first molecular characterization of an inborn error of metabolism specific to the L-valine catabolic pathway.

Succinyl-CoA:3-ketoacid transferase (SCOT) deficiency in a new patient homozygous for an R217X mutation

Summary: SCOT deficiency presents with persistent excess of ketones leading to ketoacidosis. Here we report patient GS15, homozygous for a novel R217X mutation, who had the first apparent ketoacidotic crisis at 8 months of age. Before confirmation of diagnosis, daily dialysis was the only mechanism by which to normalize her persistent metabolic acidosis of unknown aetiology.

Determination of the GABA analogue succinic semialdehyde in urine and cerebrospinal fluid by dinitrophenylhydrazine derivatization and liquid chromatography-tandem mass spectrometry: application to SSADH deficiency.

SummarySuccinic semialdehyde (SSA) accumulates in the inborn error of meta- bolism succinic semialdehyde dehydrogenase deficiency owing to impaired enzymatic conversion to succinic acid. We developed a stable-isotope dilution liquid chromato- graphy–tandem mass spectrometry method for the determination of SSA in urine and cerebrospinal fluid samples. Stable-isotope-labelled [13C4]SSA, serving as internal standard, was prepared by reaction of ninhydrin with L-[13C5]glutamic acid. SSA in body fluids was converted to its dinitrophenylhydrazine (DNPH) derivative, without sample purification prior to the derivatization procedure. The DNPH derivative of SSA was injected onto a C18 analytical column and chromatography was performed by isocratic elution. Detection was accomplished by tandem mass spectrometry operating in the negative multiple-reaction monitoring mode. The limit of detection was 10 nmol/L and the calibration curves over the range 0–500 pmol of SSA showed good linearity (r2 > 0.99). The intra-day coefficient of variation (n = 10) for urine was 2.7% and inter-day coefficient of variation (n = 5) for urine was 8.5%. The average recoveries performed on two levels by enriching urine and cerebrospinal fluid samples ranged between 85 and 115%, with coefficients of variation < 8%. The method enabled the first determination of normal values for SSA in urine and pathological values of SSA in urine and cerebrospinal fluid samples derived from patients with succinic semialdehyde dehydrogenase deficiency.

3-Methylglutaconyl-CoA hydratase deficiency: a new patient with speech retardation as the leading sign.

3-Methylglutaconyl (3-MG)-CoA hydratase (EC 4.2.1.18) catalyses the fourth step in leucine catabolism by converting 3-MG-CoA into 3-hydroxy-3-methylglutaryl (3-HMG)-CoA. Deficiency of 3-MG-CoA hydratase has been defined as 3-methylglutaconic (3-MGC) aciduria type I (McKusick 250950) presenting with increased urinary excretion of 3-MGC, 3-methylglutaric (3-MGR) acid and 3-hydroxyisovaleric (3-HIV) acid and with normal amounts of 3-hydroxy-3-methylglutaric (3-HMG) acid. Additional types of 3-MGC aciduria with normal activities of 3-MG-CoA hydratase have been classified (Gibson et al 1993): type II seems to be of X-linked inheritance and is associated with cardiomyopathy, growth retardation and neutropenia (Kelley et al 1989). Type III has been described as the Costeff Iraqi-Jewish optic atrophy syndrome (Costeff et al 1989). Another type (type IV, unclassified) comprises a heterogeneous group of patients with progressive neurological symptoms. In contrast to 3-MGC aciduria type I, the origin of the specific organic aciduria in these entities is unknown. Since 3-HIV acid seems to be excreted only in patients with deficient 3-MG-CoA hydratase activity, it is an important parameter for differentiation between type I and the other types. Owing to the variability of the clinical picture of the nine patients with deficiency of 3-MG-CoA hydratase reported so far, a specific phenotype of 3-MGC aciduria type I has not yet been established. There seem to be a benign and a more severe form: in several patients the only symptoms were delayed speech development, mild psychomotor retardation and attention deficits (Duran et al 1982; Gibson et al 1992; Sewell et al 1999). Three of the reported patients, however, presented with severe neurological symptoms including spastic quadriplegia, dystonic movements, hypotonia and marked psychomotor retardation in two patients as well as coma and seizures during an episode of Reye syndrome in another patient with speech retardation (Gibson et al 1998; Hou and Wang 1995; Shoji et al 1999). We report on a new patient with a mild clinical presentation of 3-MGC aciduria type I.

Gamma-hydroxybutyric aciduria: A biochemist's education from a heritable disorder of GABA metabolism

SummaryIn keeping with the theme of the 41st Annual Symposium (Metabolic Encephalopathies), the current report provides an overview of a quarter century of research investigating a rare inborn error of GABA metabolism, succinic semialdehyde dehydrogenase (SSADH) deficiency (also referred to as gamma-hydroxybutyric aciduria). The clinical phenotype, treatment challenges, pathomechanistic concepts, as well as metabolic, enzymatic and molecular characteristics, of the disorder are summarized. In addition, important features of a recently developed murine model are presented, with discussion of how this model has broadened thinking about, and treatment approaches to, the clinical disease. In addition, a brief scientific history of the author is provided, and an account of how serendipitous circumstances brought him together with other colleagues to begin delineating the aetiological mechanisms in SSADH deficiency. The evolution of research on SSADH deficiency re-establishes the classical approach beginning at clinical characterization, evolving to enzyme and metabolite identification as well as molecular characterization, and eventually to model development and preclinical treatment approaches. This scientific evolution admirably supports the life and ideals of Dr George Komrower, in whose honour this lecture is named.

3-Methylglutaconic aciduria and hypermethioninaemia in a child with clinical and neuroradiological findings of Leigh disease.

We report on a child with a clinical and neuroradiological picture consistent with Leigh disease and an unusual association of isolated hypermethioninaemia and 3-methylglutaconic aciduria. A low-methionine diet normalized both plasma methionine and urine 3-methylglutaconic acid; a methionine-loading test led to significant increase of both metabolites. In the skin fibroblasts the activity of 3-methylglutaconyl-CoA hydratase was essentially normal. No explanation of this uncommon association of hypermethioninaemia and glutaconic aciduria is available. The possibility of a common transporter for 3-methylglutaconic acid and methionine is an attractive hypothesis.

Increased urine heparan and chondroitin sulphate excretion in patients with osteopetrosis.

Increased urine heparan and chondroitin sulphate excretion in patients with osteopetrosis R. D. Steiner1,2, M. P. W hyte3, E. Chang4, J. Hanks4, C. Mattes2, H. Senephansiri2 and K. M. Gibson2* Departments of 1Pediatrics and 2Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon ; 3Metabolic Research Unit, Shriners Hospital for Children and Division of Bone and Mineral Diseases, Washington University School of Medicine, St Louis, Missouri ; 4Department of Pediatrics, St LukeÏs Regional Medical Center, Boise, Idaho, USA * Correspondence : Biochemical Genetics Laboratory, Baird Hall 2029, Mail Code L-473, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098, USA. E-mail gibsonm=ohsu.edu

Prenatal diagnosis of succinate semialdehyde dehydrogenase deficiency in non-identical twins

Prenatal diagnosis was performed by both DNA and enzymatic analysis on non-identical twins conceived by in vitro fertilization and at risk of succinate semialdehyde dehydrogenase deficiency. One fetus was predicted to be affected and one unaffected and selective fetal reduction was performed.