Mutational spectrum and linkage disequilibrium patterns at the ornithine transcarbamylase gene (OTC).

Ornithine transcarbamylase (OTC; EC 2.1.3.3) is a hepatic enzyme involved in ammonia elimination via the urea cycle. Since the sequence of the OTC gene was reported many types of mutations continue to be found in OTC deficiency patients, continuing to increase the already wide mutational spectrum known for this gene. In this study we present the clinical, biochemical and molecular features of thirteen late-onset OTC deficiency patients. Mutations were identified in all these patients, among which six were novel point substitutions (L59R, A137P, L148S, Y176L, L186P, and K210N) and one was a 2-bp deletion at exon 4 (341-342delAA). In addition, a de novo genomic deletion of maternal origin encompassing exons 1 to 5 was also identified by the analysis of LD patterns using intragenic polymorphic markers. This work exemplifies the potential value of population genetic studies for the detection of large deletions.

Misleading diagnosis of partial N-acetylglutamate synthase deficiency based on enzyme measurement corrected by mutation analysis.

UNLABELLED: N-acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder. Most of the patients present in the early neonatal period with severe hyperammonaemia and marked neurological impairment. We report on a Turkish family with an index patient, who died due to hyperammonemia, and another three siblings, who received a prophylactic treatment consisting of arginine hydrochloride, sodium benzoate and phenylbutyrate directly after birth. Enzyme measurement in a liver biopsy suggested a diagnosis of partial NAGS deficiency in all three siblings. Thereafter, N-carbamylglutamate was added to the treatment. None of the patients developed hyperammonaemia. After the human NAGS gene was identified, mutation analysis revealed that the consanguineous parents and two siblings were heterozygous for a private mutation (W484R), whereas the wild-type gene was found in the eldest sibling. Therapy was stopped without any deterioration of urea cycle function. CONCLUSION: Diagnosis of partial NAGS deficiency based on enzyme measurement may be misleading and should be completed by mutation analysis.

Gene structure of human carbamylphosphate synthetase 1 and novel mutations in patients with neonatal onset.

Carbamylphosphate synthetase 1 (E.C. 6.3.4.16) deficiency is a rare autosomal recessive disorder of the urea cycle that can result in severe neonatal hyperammonemia. Since the genomic structure of the CPS1 gene was not yet elucidated, mutation detection was performed by analysis of transcripts in the past. Here, we present the entire DNA sequence of the human CPS1 gene including all exon-intron boundaries. Moreover, mutation analysis was performed in six patients leading to the detection of 9 novel mutations including the missense mutations c.2528T>C and c.2623A>G, the nonsense mutations c.712C>T and c.2115ins35bp, the splice site mutations c.1263+5G>C, c.3558+1G>C and c.4101+2T>C, and a small deletion c.3036_3038delGGT. The mutations c.2528T>C and c.2623A>G were identified on a double mutated allele. New data on the genomic structure of the CPS1 gene provided in this study are useful to characterize the heterogenous molecular basis of the disease in patients deficient for carbamylphosphate 1 deficiency.

[Variants of inborn errors of metabolism with late onset but nevertheless life threatening course]. / Varianten angeborener Stoffwechselstörungen mit spätem Beginn, aber bedrohlichem Verlauf.

BACKGROUND: There is a growing number of inborn errors of metabolism (IEM) with late onset but nevertheless life threatening course. PATIENTS: Patients with late onset variants of urea cycle defects, fatty acid oxidation defects and organic acidurias are demonstrated. METHODS: Biochemical, enzymatic, molecular methods and especially tandem mass spectrometry (TMS) are used for diagnostic purposes. RESULTS: IEM variants with late onset are difficult to be detected. TMS has some advantages as the simple sampling of dried blood on filter paper cards and the simultaneous detection of a broad spectrum of disturbances in amino acids and acylcarnitines. This may facilitate a prompt diagnosis. Asymptomatic persons not only carry an unrecognized risk for severe metabolic decompensation but also pass on their mutation of IEM and the associated disease risk to the next generation (Non-disease). CONCLUSION: TMS, which is used in newborn screening centers is very convenient to establish a prompt diagnosis in some unexpected late onset metabolic crisis following surgeries, infections or other catabolic stress. Furthermore TMS may be a suitable and rapid adjunct method to improve transplantation management.

Multiorgan donation from a donor with unrecognized ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency, the most common inherited urea cycle disorder, shows a spectrum of severity ranging from severe neonatal hyperammonemic coma to no symptoms among adults. We report on the multiorgan procurement from a donor who died of cerebral edema due to unrecognized late-onset OTC deficiency. The donor's OTC deficiency was diagnosed retrospectively since the liver graft recipient developed cerebral edema postoperatively due to hyperammonemia. Plasma ammonia was extremely elevated (3793 micromol/l), but was not accompanied by general liver dysfunction. Post mortem, the diagnosis of OTC deficiency was established by enzyme and molecular analysis in a biopsy of the transplanted liver. In contrast to the fatal course of the liver graft recipient, the kidney, lung, and heart transplantations were successful. Ten months after transplantation these recipients were alive and showed good graft function. This case demonstrates the importance of careful donor evaluation, particularly if the donor's cause of death is obscure.

Genetic analysis of carbamoylphosphate synthetase I and ornithine transcarbamylase deficiency using fibroblasts.

UNLABELLED: Deficiencies of carbamoylphosphate synthetase or of ornithine transcarbamylase, two urea cycle enzymes located within mitochondria, often present as severe neonatal hyperammonaemic crises and have a poor prognosis. While genetic analysis of the X-chromosomal transmitted ornithine transcarbamylase deficiency (OTC) is performed by exon-wise mutation screening of genomic DNA in most cases, identification of mutations in the autosomal inherited carbamoylphosphate synthetase (CPS 1) deficiency requires analysis of transcripts due to the unknown genomic structure. We tested the hypothesis that CPS 1 and OTC are expressed at low levels in fibroblasts and indeed were able to amplify full-length cDNA from that source. Using a reverse transcriptase polymerase chain reaction based procedure we completely characterised the genetic background in five patients and identified three novel mutations and a novel polymorphism of the CPS 1 gene (deletion/insertion 2170delGCTCinsCCA, nonsense mutation 2359C > T, missense mutation 3161T > G and Thr1406Asn, respectively), as well as the missense mutations 482A > G and 994T > A of the OTC gene. CONCLUSION: Cultured fibroblasts are an easily accessible source for genetic analysis of inborn errors of urea cycle enzymes which are functionally expressed only in liver and gut.

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38.

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k(cat)/K(m,NADPH)) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k(cat)/K(m,NADPH) 1000-fold but had little effect on k(cat)/K(m,NADH). The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.

Late-onset ornithine transcarbamylase deficiency in two families with different mutations in the same codon.

We report on late-onset ornithine transcarbamylase (OTC) deficiency in two families with mutations in the same codon, but different base substitutions. Onset of symptoms showed great variation, and five hemizygotes finally died. Clinical diagnosis was late and difficult. In family A, 1 patient also developed the signs of Gilbert's disease. In family B, the index case came to attention as OTC deficiency, after the transplantation of his liver when the recipient died of cerebral edema and hyperammonemia. In family A, the hemizygote males died at the ages of 12 and 18 years; in family B, they died at the ages of 20, 26, and 30 years, respectively. Diagnosis was confirmed by reduced OTC activity in liver specimens. The residual activity in autopsy liver of the index patient in family A was less than the activity in the biopsy of the transplanted liver of the index patient in family B. The molecular investigations showed mutations in exon 2 at codon 40 in the OTC gene in both families. However, different bases were substituted. In family A, the single-base mutation was a cytosine-to-thymine transition (Arg 40 Cys); in family B, it was a guanine-to-adenine transition (Arg 40 His). Published data on in vitro expression studies of the recurrent OTC mutation Arg 40 His have shown little effect on the protein structure of the enzyme. These studies would fit well with our observation of higher OTC activity and later age of onset of symptoms in family B.

Antioxidant and thyroid hormone status in selenium-deficient phenylketonuric and hyperphenylalaninemic patients.

BACKGROUND: Subjects consuming protein-restricted diets, such as patients with phenylketonuria (PKU) or milder hyperphenylalaninemias (HPAs) are at risk of selenium deficiency. Selenium is a cofactor of the antioxidant enzyme glutathione peroxidase and of the thyroid hormone converting enzyme thyroxine deiodinase. OBJECTIVE: Our goal was to investigate the effects of low plasma selenium on antioxidant and thyroid hormone status. DESIGN: We assessed plasma selenium, plasma total antioxidant status and the individual components thereof, erythrocyte antioxidant status, and plasma thyroid hormones in 24 PKU and 10 HPA patients and in 42 age-matched control subjects. RESULTS: Selenium was significantly lower in both PKU and HPA patients than in control subjects and the PKU patients had lower values than did the HPA patients. Total antioxidant status was lower in both patient groups than in the control group, whereas alpha-tocopherol, albumin, and uric acid were not significantly different among groups. Plasma selenium correlated well (r = 0.76) with erythrocyte glutathione peroxidase. PKU patients had lower glutathione peroxidase activity than did HPA patients and control subjects and lower glutathione concentrations than did control subjects. Both patient groups had lower superoxide dismutase activity than did control subjects. Free triiodothyronine was higher in both patient groups than in control subjects, whereas free thyroxine was higher in the PKU patients only. Free thyroxine and reverse triiodothyronine were inversely correlated with selenium. CONCLUSION: Supplementation with selenium seems to be advisable for patients consuming diets low in natural protein.

Mutation of threonine-241 to proline eliminates autocatalytic modification of human carbonyl reductase.

Carbonyl reductase catalyses the reduction of steroids, prostaglandins and a variety of xenobiotics. An unusual property of human and rat carbonyl reductases is that they undergo modification at lysine-239 by an autocatalytic process involving 2-oxocarboxylic acids, such as pyruvate and 2-oxoglutarate. Comparison of human carbonyl reductase with the pig enzyme, which does not undergo autocatalytic modification, identified three sites, alanine-236, threonine-241 and glutamic acid-246, on human carbonyl reductase that could be important in the reaction of lysine-239 with 2-oxocarboxylic acids. Mutagenesis experiments show that replacement of threonine-241 with proline (T241P) in human carbonyl reductase eliminates the formation of carboxyethyl-lysine-239. In contrast, the T241A mutant has autocatalytic activity similar to wild-type carbonyl reductase. The T241P mutant retains catalytic activity towards menadione, although with one-fifth the catalytic efficiency of wild-type carbonyl reductase. Replacement of threonine-241 with proline is likely to disrupt the local structure near lysine-239. We propose that integrity of this local environment is essential for chemical modification of lysine-239, but not absolutely required for carbonyl reductase activity.

Aminoglycosides and renal magnesium homeostasis in humans.

BACKGROUND: The use of aminoglycosides has been linked with hypomagnesaemia in scattered reports. The objective of the study was to measure prospectively the effect of treatment with the aminoglycoside amikacin on renal magnesium homeostasis. METHODS: Twenty-four cystic fibrosis patients (aged 9-19 years) admitted because of exacerbation of pulmonary symptoms caused by Pseudomonas aeruginosa were treated with the aminoglycoside amikacin and the cephalosporin ceftazidime for 14 days. Renal values and plasma and urinary electrolytes were measured before and at the end of the systemic anti-pseudomonal therapy. RESULTS: In the patients with cystic fibrosis, treatment with amikacin and ceftazidime did not modify plasma creatinine or urea and plasma or urinary sodium, potassium and calcium. Treatment with amikacin and ceftazidime significantly decreased both plasma total magnesium (from 0.77 (0. 74-0.81) to 0.73 (0.71-75) mmol/l; median and interquartile range) and ionized magnesium (from 0.53 (0.50-0.55) to 0.50 (0.47-0.52) mmol/l) concentration and increased fractional urinary magnesium excretion (from 0.0568 (0.0494-0.0716) to 0.0721 (0.0630-0.111)) and total urinary magnesium excretion (from 30.7 (26.5-38.0) to 38.5 (31. 5-49.0) micromol/l glomerular filtration rate). CONCLUSIONS: The present study demonstrates that systemic therapy with amikacin plus ceftazidime causes mild hypomagnesaemia secondary to renal magnesium wasting even in the absence of a significant rise in circulating creatinine and urea.

Cloning and expression of succinic semialdehyde reductase from human brain. Identity with aflatoxin B1 aldehyde reductase.

The neuromodulator gamma-hydroxybutyrate is synthesized in vivo from gamma-aminobutyrate by transamination to succinic semialdehyde and subsequent reduction of the aldehyde group. In human brain, succinic semialdehyde reductase is thought to be responsible for the conversion of succinic semialdehyde to gamma-hydroxybutyrate. In the present work, we cloned the cDNA coding for succinic semialdehyde reductase and expressed it in Escherichia coli. A data bank search indicated that the enzyme is identical with aflatoxin B1-aldehyde reductase, an enzyme implicated in the detoxification of xenobiotic carbonyl compounds. Structurally, succinic semialdehyde reductase thus belongs to the aldo-keto reductase superfamily. The recombinant protein was indistinguishable from native human brain succinic semialdehyde reductase by SDS/PAGE. In addition to succinic semialdehyde, it readily catalyzed the reduction 9,10-phenanthrene quinone, phenylglyoxal and 4-nitrobenzaldehyde, typical substrates of aflatoxin B1 aldehyde reductase. The results suggest multiple functions of succinic semialdehyde reductase/aflatoxin B1 aldehyde reductase in the biosynthesis of gamma-hydroxybutyrate and the detoxification of xenobiotic carbonyl compounds, respectively.

Permanently reduced plasma ionized magnesium among renal transplant recipients on cyclosporine.

Hypomagnesemia is common after kidney transplantation. Until recently, only the determination of total plasma magnesium was possible, whereas the assessment of ionized magnesium has since become practicable. One hundred and nine renal transplant patients on cyclosporine with allografts functioning stably for more than 6 months and plasma creatinine levels of less than 200 micromol/l entered the study. Total and ionized circulating magnesium were assessed among these 109 patients, as well as among 15 renal transplant patients not on cyclosporine and 21 healthy volunteers. Cyclosporine patients showed significantly lower total and ionized circulating magnesium values than the two control groups. Plasma total and ionized magnesium levels were also significantly lower among cyclosporine patients treated concurrently with insulin or oral hypoglycemic agents than among those who were not. No correlation was noted between time after transplantation and plasma magnesium with respect to patients on cyclosporine. In conclusion, the study demonstrates that a large subset of renal transplant patients treated with cyclosporine have permanent deficiencies of ionized and total magnesium. The tendency towards hypomagnesemia is also more pronounced among patients with diabetes mellitus.

Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase.

Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m). NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione.

Effect of increasing dietary threonine intakes on amino acid metabolism of the central nervous system and peripheral tissues in growing rats.

The threonine content of most of the infant formulas currently on the market is approximately 20% higher than the threonine concentration in human milk. Due to this high threonine content the plasma threonine concentrations are up to twice as high in premature infants fed these formulas than in infants fed human milk. To study the effect of different threonine intakes on plasma and tissue amino acid concentrations, 24 young male Wistar rats were fed three experimental diets based on a mixture of bovine proteins with a whey protein/casein ratio of 60/40 with different threonine contents [group A, 0.86 g of threonine/100 g (n = 8); group B, 1.03 g of threonine/100 g (n = 8); group C, 2.21 g of threonine/100 g (n = 8)]. Eight animals were fed a typical rat diet based on bovine casein as controls. After a feeding period of 15 d, amino acids were measured in plasma and in homogenates of the cerebral cortex, brain stem, liver, and muscle. There was a significant correlation between threonine intake and plasma threonine levels (r = 0.687, p < 0.001). The plasma threonine concentration correlated significantly with the threonine concentration in the cortex (r = 0.821, p < 0.01) and the brain stem (r = 0.882, p < 0.01). There was a positive significant correlation between threonine and glycine concentrations in the cortex (r = 0.673, p < 0.01), and the brain stem (r = 0.575, p < 0.01), whereas the glycine concentration decreased with increasing threonine intakes in the liver and muscle. The presented data indicate that increasing the threonine in plasma leads to increasing brain glycine and thereby affects the neurotransmitter balance in the brain. This may have consequences for brain development during early postnatal life. Therefore, excessive threonine intake during infant feeding should be avoided.

Circulating ionized and total magnesium in end-stage kidney disease.

BACKGROUND: Circulating magnesium exists in the ionized state and in the undissociated form, either bound to albumin, or complexed to various anions. Until recently, only the measurement of total plasma magnesium has been possible. Now circulating ionized magnesium can be assessed as well. METHODS: Total and ionized plasma magnesium were determined in 43 patients on maintenance hemodialysis (dialysate composition: calcium 1.75 mmol/l, magnesium 0.75 mmol/l) before a dialysis session and in a group of 23 healthy subjects. RESULTS: The total (1.16 [1.03-1.31] versus 0.81 [0.78-0.89] mmol/l; median and interquartile range) and the ionized (0.71 [0.66-0.78] versus 0.54 [0.53-0.59] mmol/l) plasma magnesium levels were significantly higher (p < 0.01) and the ionized plasma magnesium fraction lower (0.61 [0.58-0.65] versus 0.67 [0.64-0.70]; p < 0.02) in patients than in controls. CONCLUSION: The determination of circulating ionized magnesium using selective electrodes is an attractive method to evaluate extracellular magnesium in kidney disease.

Partial N-acetylglutamate synthetase deficiency in a 13-year-old girl: diagnosis and response to treatment with N-carbamylglutamate.

UNLABELLED: We report on a now 13-year-old girl, who presented with recurrent episodes of vomiting, psychotic behaviour and confusion during puberty until the diagnosis of partial N-acetylglutamate synthetase deficiency was established. She had suffered one prior unclear episode of acute vomiting, lethargy and somnolence at the age of 13 months, and from childhood onward had aversion to high protein food. Treatment with a protein-restricted diet and administration of phenylbutyrate as well as L-arginine were sufficient to normalize ammonia levels but glutamine concentrations remained high. Supplementation with N-carbamylglutamate rapidly improved her protein tolerance and reduced the need for co-medication. To our knowledge, so far only seven patients with N-acetylglutamate synthetase deficiency have been reported. CONCLUSION: Partial N-carbamylglutamate deficiency has to be considered in the differential diagnosis of hyperammonaemia. If proven by enzyme determination in liver tissue, the disorder should be cautiously treated with N-carbamylglutamate.