3-Methoxytyramine: An independent prognostic biomarker that associates with high-risk disease and poor clinical outcome in neuroblastoma patients.

INTRODUCTION: Prognosis of neuroblastoma patients is very diverse, indicating the need for more accurate prognostic parameters. The excretion of catecholamine metabolites by most neuroblastomas is used for diagnostic purposes, but their correlation with prognosis has hardly been investigated. Therefore, we performed an in-depth analysis of a panel of elevated urinary catecholamine metabolites at diagnosis and their correlation with prognosis. PATIENTS AND METHODS: Retrospective study of eight urinary catecholamine metabolites in a test (n = 96) and validation (n = 205) cohort of patients with neuroblastoma (all stages) at diagnosis. RESULTS: Multivariate analyses, including risk factors such as stage and MYCN amplification, revealed that 3-methoxytyramine (3MT) was an independent risk factor for event-free survival (EFS) and overall survival (OS). Furthermore, only 3MT appeared to be an independent risk factor for both EFS and OS in high-risk patients, which was independent of modern high-risk therapy and immunotherapy. Among high-risk patients, those with elevated 3MT and older than 18 months had an extremely poor prognosis compared to patients with non-elevated 3MT and younger than 18 months (5-year EFS of 14.3% ± 4% and 66.7% ± 18%, respectively, p = 0.001; 5-year OS of 21.8% ± 5% and 87.5% ± 12%, respectively, p < 0.001). CONCLUSIONS: Elevated 3MT at diagnosis was associated with high-risk disease and poor prognosis. For high-risk patients, elevated 3MT at diagnosis was the only significant risk factor for EFS and OS. 3MT was also able to identify subgroups of high-risk patients with favourable and extremely poor prognosis.

Cerebral dopamine deficiency, plasma monoamine alterations and neurocognitive deficits in adults with phenylketonuria.

BACKGROUND: Phenylketonuria (PKU), a genetic metabolic disorder that is characterized by the inability to convert phenylalanine to tyrosine, leads to severe intellectual disability and other cerebral complications if left untreated. Dietary treatment, initiated soon after birth, prevents most brain-related complications. A leading hypothesis postulates that a shortage of brain monoamines may be associated with neurocognitive deficits that are observable even in early-treated PKU. However, there is a paucity of evidence as yet for this hypothesis. METHODS: We therefore assessed in vivo striatal dopamine D2/3 receptor (D2/3R) availability and plasma monoamine metabolite levels together with measures of impulsivity and executive functioning in 18 adults with PKU and average intellect (31.2 ± 7.4 years, nine females), most of whom were early and continuously treated. Comparison data from 12 healthy controls that did not differ in gender and age were available. RESULTS: Mean D2/3R availability was significantly higher (13%; p = 0.032) in the PKU group (n = 15) than in the controls, which may reflect reduced synaptic brain dopamine levels in PKU. The PKU group had lower plasma levels of homovanillic acid (p < 0.001) and 3-methoxy-4-hydroxy-phenylglycol (p < 0.0001), the predominant metabolites of dopamine and norepinephrine, respectively. Self-reported impulsivity levels were significantly higher in the PKU group compared with healthy controls (p = 0.033). Within the PKU group, D2/3R availability showed a positive correlation with both impulsivity (r = 0.72, p = 0.003) and the error rate during a cognitive flexibility task (r = 0.59, p = 0.020). CONCLUSIONS: These findings provide further support for the hypothesis that executive functioning deficits in treated adult PKU may be associated with cerebral dopamine deficiency.

Pyridoxine responsive epilepsy caused by a novel homozygous PNPO mutation.

We report a patient with anti-epileptic treatment refractory neonatal seizures responsive to pyridoxine. Biochemical analysis revealed normal markers for antiquitin deficiency and also mutation analysis of the ALDH7A1 (Antiquitin) gene was negative. Mutation analysis of the PNPO gene revealed a novel, homozygous, presumed pathogenic mutation (c.481C > T; p.(Arg161Cys)). Measurements of B6 vitamers in a CSF sample after pyridoxine administration revealed elevated pyridoxamine as the only metabolic marker for PNPO deficiency. With pyridoxine monotherapy the patient is seizure free and neurodevelopmental outcome at the age of 14 months is normal.

A Korean Case of ß-Ureidopropionase Deficiency Presenting with Intractable Seizure, Global Developmental Delay, and Microcephaly.

ß-Ureidopropionase deficiency (OMIM #613161) is a rare autosomal recessive inborn error of metabolism due to mutations in the UPB1 gene, which encodes the third enzyme involved in the pyrimidine degradation pathway. A total of 28 cases have been reported, mainly presenting with seizures, microcephaly, and intellectual disabilities. However, 11 of them were asymptomatic cases (Nakajima et al., J Inherit Metab Dis 37(5):801-812, 2014). We report on a 9-year-old female presenting with intractable epilepsy, microcephaly, and global developmental delay. She was homozygous for p.R326Q (c.977G>A) and heterozygous for p.G31S (c.91G>A) in the UPB1 gene, detected by targeted next-generation sequencing test and subsequently confirmed by biochemical analysis of urine, plasma, and cerebrospinal fluid (CSF) using reversed-phase HPLC, combined with electrospray tandem mass spectrometry. We report a first Korean female case with ß-ureidopropionase deficiency.

Identification of two novel mutations C79X and R235Q in the dihydropyrimidine dehydrogenase gene in a patient presenting with hematuria.

A patient with hematuria was shown to have thymine-uraciluria. The dihydropyrimidine dehydrogenase (DPD) activity in peripheral blood mononuclear cells was 0.16 nmol/mg/h; controls: 9.9 +/- 2.8 nmol/mg/h. Analysis of DPYD showed that the patient was compound heterozygous for the novel mutations 237C > A (C79X) in exon 4 and 704G > A (R235Q) in exon 7. The nonsense mutation (C79X) leads to premature termination of translation and thus to a non-functional protein. Analysis of the crystal structure of pig DPD suggested that the R235Q mutation might interfere with the binding of FAD and the electron flow between the NADPH and the pyrimidine substrate site of DPD.

Perioperative hyperinsulinaemic normoglycaemic clamp causes hypolipidaemia after coronary artery surgery.

BACKGROUND: Glucose-insulin-potassium (GIK) administration is advocated on the premise of preventing hyperglycaemia and hyperlipidaemia during reperfusion after cardiac interventions. Current research has focused on hyperglycaemia, largely ignoring lipids, or other substrates. The present study examines lipids and other substrates during and after on-pump coronary artery bypass grafting and how they are affected by a hyperinsulinaemic normoglycaemic clamp. METHODS: Forty-four patients were randomized to a control group (n=21) or to a GIK group (n=23) receiving a hyperinsulinaemic normoglycaemic clamp during 26 h. Plasma levels of free fatty acid (FFA), total and lipoprotein (VLDL, HDL, and LDL)-triglycerides (TG), ketone bodies, and lactate were determined. RESULTS: In the control group, mean FFA peaked at 0.76 (sem 0.05) mmol litre(-1) at early reperfusion and decreased to 0.3-0.5 mmol litre(-1) during the remaining part of the study. GIK decreased FFA levels to 0.38 (0.05) mmol litre(-1) at early reperfusion, and to low concentrations of 0.10 (0.01) mmol litre(-1) during the hyperinsulinaemic clamp. GIK reduced the area under the curve (AUC) for FFA by 75% and for TG by 53%. The reduction in total TG was reflected by a reduction in the VLDL (-54% AUC) and HDL (-42% AUC) fraction, but not in the LDL fraction. GIK prevented the increase in ketone bodies after reperfusion (-44 to -47% AUC), but was without effect on lactate levels. CONCLUSIONS: Mild hyperlipidaemia was only observed during early reperfusion (before heparin reversal) and the hyperinsulinaemic normoglycaemic clamp actually resulted in hypolipidaemia during the largest part of reperfusion after cardiac surgery.

Molecular and clinical characterization of a Moroccan Cog7 deficient patient.

Mutations in the N-linked glycosylation pathway cause rare autosomal recessive defects known as Congenital Disorders of Glycosylation (CDG). A previously reported mutation in the Conserved Oligomeric Golgi complex gene, COG7, defined a new subtype of CDG in a Tunisian family. The mutation disrupted the hetero-octomeric COG complex and altered both N- and O-linked glycosylation. Here we present clinical and biochemical data from a second family with the same mutation.

Genetic analysis of the first 4 patients with beta-ureidopropionase deficiency.

beta-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ss-aminoisobutyric acid or N-carbamyl-ss-alanine to beta-aminoisobutyric acid or ss-alanine, ammonia, and CO2. Analysis of the beta-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete beta-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic beta-ureidopropionase protein sequences.

A pivotal role for beta-aminoisobutyric acid and oxidative stress in dihydropyrimidine dehydrogenase deficiency?

Dihydropyrimidine dehydrogenase (DPD) constitutes the first step of the pyrimidine degradation pathway in which the pyrimidine bases uracil and thymine are catabolised to beta-alanine and beta-aminoisobutyric acid (beta-AIB), respectively. The mean concentration of beta-AIB was approximately 5- to 8-fold lower in urine of patients with a DPD deficiency, when compared to age-matched controls. Comparable levels of 8-hydroxydeoxyguanosine (8-OHdG) were present in urine from controls and DPD patients at the age <2 year. In contrast, slightly elevated levels of 8-OHdG were detected in urine from DPD patients with an age >2 year, suggesting the presence of increased oxidative stress.

Analysis of pyrimidine synthesis de novo intermediates in urine during crisis of a patient with ornithine transcarbamylase deficiency.

Analysis of pyrimidine synthesis de novo intermediates and pyrimidine degradation products in urine samples from a decompensated patient with an ornithine transcarbamylase deficiency showed a strikingly aberrant metabolic profile. Strongly elevated levels of N-carbamyl-aspartate, orotate and uracil were present whereas the concentration of uridine was only marginally increased. The level of pyrimidine excretion appeared to be independent of the ammonia levels in blood, which were only mildly increased.

A homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE) results in mild methylmalonic aciduria.

Methylmalonic aciduria (MMA-uria) is an autosomal recessive inborn error of amino acid metabolism, involving valine, threonine, isoleucine, and methionine. This organic aciduria may present in the neonatal period with life-threatening metabolic acidosis, hyperammonemia, feeding difficulties, pancytopenia, and coma. Most affected patients have mutations in the methylmalonyl-coenzyme A (methylmalonyl-CoA) mutase gene. Mildly affected patients may present in childhood with failure to thrive and recurrent attacks of metabolic acidosis. Both a higher residual activity of methylmalonyl-CoA mutase as well as the vitamin B12-responsive defects (cblA and cblB) may form the basis of the mild disorder. A few patients with moderate MMA-uria are known in whom no defect could be identified. Here we present a 16-year-old female patient with persisting moderate MMA-uria (approximately 50 mmol/mol creatinine). She was born to consanguineous Caucasian parents. Her fibroblast mutase activity was normal and no effect of vitamin B12 supplementation could be established. Reduced incorporation of 14C-propionate into macromolecules suggested a defect in the propionate-to-succinate pathway. We found a homozygous nonsense mutation (c.139C>T) in the methylmalonyl-CoA epimerase gene (MCEE), resulting in an early terminating signal (p.R47X). Both parents were heterozygous for this mutation; they were found to excrete normal amounts of methylmalonic acid (MMA). This is the first report of methylmalonyl-CoA epimerase deficiency, thereby unequivocally demonstrating the biochemical role of this enzyme in human metabolism.

Fatty acids and homocysteine levels in patients with recurrent depression: an explorative pilot study.

Major depressive disorders (MDD) and cardiovascular disease are mutually associated. They share signs and symptoms of the "metabolic syndrome". Two observations that may be causally related with the metabolic syndrome and therefore with both MDD and cardiovascular disease are a decrease in omega-3 polyunsaturated fatty acids (PUFAs) and a rise in plasma homocysteine (tHcy) levels. Both the rise in tHcy and the decrease in omega-3 PUFAs may be associated with enhanced lipid peroxidation. We exploratively studied 44 randomly chosen patients out of a cohort of 134 patients with the recurrent form of MDD (MDD-R). We measured tHcy levels together with saturated FAs, monounsaturated fatty acids (MUFAs) and PUFAs of the omega-3, omega-6 and omega-9 series in plasma and erythrocytes. Levels were compared with laboratory reference values. The main findings were a decrease in the erythrocytes of C22:5omega-3, C22:6omega-3, C24:1omega-9 and C20:3omega-9 and in the plasma a decrease in C24:1omega-9 and C20:3omega-9. The only significant association we found was between the total of omega-6 fatty acids and plasma tHcy. The FA alterations were found in patients although most of them were clinically recovered, suggesting that the alterations may represent a biological" trait" marker for recurrent depression.

Mild learning difficulties and offending behaviour--is there a link with monoamine oxidase A deficiency?

We have attempted to replicate the findings of Brunner et al., who described a large Dutch kindred where several males were of borderline intelligence and showed characteristically aggressive and sometimes dangerous or extremely antisocial behaviour. The genetic defect for this syndrome was assigned to the p11-p21 region of the X chromosome following linkage analysis in a single kindred. Subsequent sequencing of a candidate gene, monoamine oxidase A (MAO-A), at the position of maximum linkage revealed a causative mutation in the coding region of the MAO-A gene in position 936. In addition to identifying both the phenotype and the associated mutation found by Brunner et al., we also wished to test the hypothesis that mutations elsewhere in the MAO-A gene could cause the low intelligence quotient/personality disorder phenotype associated with low urinary catecholamine degradation products. Fifty-four male subjects similar in clinical characteristics to the affected males in the Dutch kindred were identified within secure mental health facilities in England and Wales. All were assessed using the antisocial personality disorder section of the SCID-II interview instrument, and information about their offending behaviour and family history was obtained from the medical notes. A blood and early-morning urine sample was obtained from each patient. Analysis of urinary excretion patterns of biogenic amines and their metabolites, represented as ratios of normetanefrine to vanillylmandelic acid, revealed two possible cases of MAO-A deficiency, which were found to be negative after resampling.

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?

Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological symptoms. Both sibs are homozygous for the E325K mutation, the same genotype that was found in the most severe form of alpha-NAGA deficiency presenting as infantile neuroaxonal dystrophy. Thus, at the age of 7 years the same genotype of alpha-NAGA may present as a 'non-disease' (present healthy case) and can be associated with the vegetative state (the first two patients described with alpha-NAGA deficiency). The clinical heterogeneity among the 11 known individuals with alpha-NAGA deficiency is extreme, with a 'non-disease' (two cases) and infantile neuroaxonal dystrophy (two cases) at the opposite sides of the clinical spectrum. The broad spectrum is completed by a very heterogeneous group of patients with various degrees of epilepsy/behavioural difficulties/psychomotor retardation (four patients) and a mild phenotype in adults without overt neurological manifestations who have angiokeratoma and clear vacuolisation in various cell types (three cases). These observations are difficult to reconcile with a straightforward genotype-phenotype correlation and suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the 11 patients with alpha-NAGA deficiency.

Prolidase deficiency diagnosed by 1H NMR spectroscopy of urine.

Three urine samples from two prolidase-deficient patients were analysed using 1H NMR spectroscopy. One-dimensional 1H NMR spectra showed a characteristic pattern of overlapping resonances of the proline and hydroxyproline protons of the imidodipeptides. The model compounds Ala-Pro, Gly-Pro, Phe-Pro, Leu-Pro, Val-Pro, Gly-Hyp and Pro-Hyp were measured as well. The non-proline resonances of Val-Pro, Ala-Pro and Gly-Pro could be assigned in the urine spectra. These resonances could then be used for quantification of the corresponding imidodipeptids. The presence of Leu-Pro in the patients' urine was demonstrated by the results of COSY experiments. However, this imidodipeptide could not be quantified owing to overlap of the resonaces in the one-dimensional 1H NMR spectrum of the patients' urine. Phe-Pro, Pro-Hyp and Gly-Hyp could not be assigned in the spectrum of the patient's urine. The characteristic resonances in the urine from a prolidase-deficient patient, i.e. Ala-Pro, Val-Pro, Gly-Pro, and resonances of the (hydroxy)proline part of the imidodipeptides can be used to diagnose this disease.

Tyrosine hydroxylase deficiency unresponsive to L-dopa treatment with unusual clinical and biochemical presentation.

Tyrosine hydroxylase (TH) deficiency is generally considered as a cause of the autosomal recessive form of dopa-responsive dystonia, also known as Segawa disease. Clinical hallmarks comprise parkinsonian and other extrapyramidal symptoms. Biochemically the defect leads to the defective synthesis of catecholamines, in particular dopamine. The diagnosis relies on a characteristic pattern of biogenic amine metabolites exclusively in the CSF and can be confirmed by establishing a mutation in the TH gene. Here we present a patient meeting all diagnostic criteria, including a new homozygous mutation (926T > C) with confirmed parental heterozygosity, extrapyramidal symptoms, but atypical other symptoms with periodic neurological episodes observed every 4 days and unresponsive to dopa treatment. The CSF biochemical abnormalities were severe. Uncharacteristically, a strongly abnormal urinary catecholamine metabolite pattern was also consistently observed. The atypical presentation of this patient shows that the clinical and metabolic phenotype of TH deficiency is more variable than formerly thought, and that the condition should no longer be considered as a treatable disorder per se.

Eniluracil treatment completely inactivates dihydropyrimidine dehydrogenase in colorectal tumors.

PURPOSE: To determine the effect of eniluracil on colorectal tumor dihydropyrimidine dehydrogenase (DPD) activity. PATIENTS AND METHODS: Patients who were to undergo primary colorectal tumor resection received oral eniluracil 10 mg/m(2) twice daily for 3 days before surgery. Mononuclear cells were obtained before the start of eniluracil and on the morning of surgery, to measure DPD activity, protein, and mRNA. Plasma uracil was also measured at these two time points to assess the effect of eniluracil on pyrimidine accumulation. DPD activity, protein, and mRNA were also assessed in colorectal tumors and adjacent normal mucosa of patients who received eniluracil and untreated control patients. RESULTS: DPD activity in tumors from 10 untreated patients ranged from 30 to 92 pmol/min/mg of protein. In contrast, there was no detectable tumor DPD activity in 10 patients who received eniluracil. A similar pattern was observed in mononuclear cells, where median pretherapy activity was 366.5 pmol/min/mg of protein (range, 265 to 494 pmol/min/mg of protein) and was undetectable immediately before surgery. Plasma uracil changed from a median less than 0.2 micromol/L before therapy to 27.76 micromol/L before surgery. No difference in DPD protein or mRNA was observed between pretherapy and presurgery mononuclear cell samples or between treated and untreated tumor samples. CONCLUSION: This study provides definitive evidence that eniluracil completely inactivates DPD activity in human solid tumors. The increased plasma uracil and decreased DPD activity are consistent with systemic inactivation of the enzyme. The mechanism of inactivation is at the catalytic level, because no changes in DPD protein or mRNA were observed. Treatment with eniluracil will eliminate DPD activity as a source of pharmacokinetic fluorouracil variability or resistance in human colorectal cancer.