Vitamin B6 and Serotonin Metabolism in Neurological Disorders of Childhood

Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6 in man where it functions as a cofactor for more than 140 enzyme catalysed reactions. Several inherited diseases characterised by seizures have been described which result in an intracellular deficiency of PLP; laboratory measurement of B6 forms an important element in the diagnosis and monitoring of these disorders. A review of PLP measured by HPLC in CSF from patients with neurological disorders showed that variance is greater than indicated by previous studies and the age-related reference limit was revised. This thesis also describes the metabolic disorders that may lead to PLP depletion and examines the relationship of CSF PLP to sulphite accumulation, medications and seizures in patient groups. B6 exists as six different vitamers and is catabolised to 4-pyridoxic acid for urinary excretion. An LC-MS/MS method was developed which could measure all vitameric forms in plasma. Its application to children with B6 responsive seizure disorders showed that patients with inborn errors of metabolism have characteristic B6 profiles which allow them to be differentiated from each other and control populations. PLP is the cofactor for aromatic L-amino acid decarboxylase (AADC) which catalyses the final step in serotonin biosynthesis. This thesis tested the hypothesis that hyperserotonaemia observed in some patients with autism is related to an abnormality in this pathway by investigating the relationship between plasma B6 vitamers, AADC activity and whole blood serotonin in a group of patients and controls. Plasma AADC activity was significantly reduced in autistic subjects; this is considered in the context of current biochemical and molecular understanding and its possible relevance to disease mechanisms is discussed

Inborn errors of metabolism causing epilepsy.

Seizures may be the first and the major presenting feature of an inborn error of metabolism (IEM), for example in a neonate with pyridoxine-dependent epilepsy. In other IEMs, seizures may be preceded by other major symptoms: by a reduced level of consciousness in a child with an organic acidaemia or urea cycle defect; or by loss of skills, progressive weakness, ataxia, and upper motor signs in a child with a lysosomal storage disorder or peroxisomal leukodystrophy. This review concentrates on those IEMs for which specific treatment is available. The common metabolic causes of seizures vary according to the age at presentation. Features from the history, examination, imaging, and first line biochemical investigations can all provide clues to an inborn error. This review attempts to delineate these and to provide a guide to the specific tests that can be used to make the diagnosis of disorders with specific treatment

An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy

We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B6, affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena

Consensus guidelines for the diagnosis and management of pyridoxine-dependent epilepsy due to α-aminoadipic semialdehyde dehydrogenase deficiency

Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an autosomal recessive condition due to a deficiency of α-aminoadipic semialdehyde dehydrogenase, which is a key enzyme in lysine oxidation. PDE-ALDH7A1 is a developmental and epileptic encephalopathy that was historically and empirically treated with pharmacologic doses of pyridoxine. Despite adequate seizure control, most patients with PDE-ALDH7A1 were reported to have developmental delay and intellectual disability. To improve outcome, a lysine-restricted diet and competitive inhibition of lysine transport through the use of pharmacologic doses of arginine have been recommended as an adjunct therapy. These lysine-reduction therapies have resulted in improved biochemical parameters and cognitive development in many but not all patients. The goal of these consensus guidelines is to re-evaluate and update the two previously published recommendations for diagnosis, treatment, and follow-up of patients with PDE-ALDH7A1. Members of the International PDE Consortium initiated evidence and consensus-based process to review previous recommendations, new research findings, and relevant clinical aspects of PDE-ALDH7A1. The guideline development group included pediatric neurologists, biochemical geneticists, clinical geneticists, laboratory scientists, and metabolic dieticians representing 29 institutions from 16 countries. Consensus guidelines for the diagnosis and management of patients with PDE-ALDH7A1 are provided. This article is protected by copyright. All rights reserved

Epilepsy due to PNPO mutations: genotype, environment and treatment affect presentation and outcome.

The first described patients with pyridox(am)ine 5'-phosphate oxidase deficiency all had neonatal onset seizures that did not respond to treatment with pyridoxine but responded to treatment with pyridoxal 5'-phosphate. Our data suggest, however, that the clinical spectrum of pyridox(am)ine 5'-phosphate oxidase deficiency is much broader than has been reported in the literature. Sequencing of the PNPO gene was undertaken for a cohort of 82 individuals who had shown a reduction in frequency and severity of seizures in response to pyridoxine or pyridoxal 5'-phosphate. Novel sequence changes were studied using a new cell-free expression system and a mass spectrometry-based assay for pyridoxamine phosphate oxidase. Three groups of patients with PNPO mutations that had reduced enzyme activity were identified: (i) patients with neonatal onset seizures responding to pyridoxal 5'-phosphate (n = 6); (ii) a patient with infantile spasms (onset 5 months) responsive to pyridoxal 5'-phosphate (n = 1); and (iii) patients with seizures starting under 3 months of age responding to pyridoxine (n = 8). Data suggest that certain genotypes (R225H/C and D33V) are more likely to result in seizures that to respond to treatment with pyridoxine. Other mutations seem to be associated with infertility, miscarriage and prematurity. However, the situation is clearly complex with the same combination of mutations being seen in patients who responded and did not respond to pyridoxine. It is possible that pyridoxine responsiveness in PNPO deficiency is affected by prematurity and age at the time of the therapeutic trial. Other additional factors that are likely to influence treatment response and outcome include riboflavin status and how well the foetus has been supplied with vitamin B6 by the mother. For some patients there was a worsening of symptoms on changing from pyridoxine to pyridoxal 5'-phosphate. Many of the mutations in PNPO affected residues involved in binding flavin mononucleotide or pyridoxal 5'-phosphate and many of them showed residual enzyme activity. One sequence change (R116Q), predicted to affect flavin mononucleotide binding and binding of the two PNPO dimers, and with high residual activity was found in Groups (ii) and (iii). This sequence change has been reported in the 1000 Genomes project suggesting it could be a polymorphism but alternatively it could be a common mutation, perhaps responsible for the susceptibility locus for genetic generalized epilepsy on 17q21.32 (close to rs72823592). We believe the reduction in PNPO activity and B6-responsive epilepsy in the patients reported here indicates that it contributes to the pathogenesis of epilepsy

Natural Variation in an ABC Transporter Gene Associated with Seed Size Evolution in Tomato Species

Seed size is a key determinant of evolutionary fitness in plants and is a trait that often undergoes tremendous changes during crop domestication. Seed size is most often quantitatively inherited, and it has been shown that Sw4.1 is one of the most significant quantitative trait loci (QTLs) underlying the evolution of seed size in the genus Solanum—especially in species related to the cultivated tomato. Using a combination of genetic, developmental, molecular, and transgenic techniques, we have pinpointed the cause of the Sw4.1 QTL to a gene encoding an ABC transporter gene. This gene exerts its control on seed size, not through the maternal plant, but rather via gene expression in the developing zygote. Phenotypic effects of allelic variation at Sw4.1 are manifested early in seed development at stages corresponding to the rapid deposition of starch and lipids into the endospermic cells. Through synteny, we have identified the Arabidopsis Sw4.1 ortholog. Mutagenesis has revealed that this ortholog is associated with seed length variation and fatty acid deposition in seeds, raising the possibility that the ABC transporter may modulate seed size variation in other species. Transcription studies show that the ABC transporter gene is expressed not only in seeds, but also in other tissues (leaves and roots) and, thus, may perform functions in parts of the plants other than developing seeds. Cloning and characterization of the Sw4.1 QTL gives new insight into how plants change seed during evolution and may open future opportunities for modulating seed size in crop plants for human purposes

Cirrhosis associated with pyridoxal 5'-phosphate treatment of pyridoxamine 5'-phosphate oxidase deficiency

We report the case of an 8-year-old boy with pyridoxamine 5'-phosphate oxidase (PNPO) deficiency. He developed seizures at 24 h of age that were refractory to standard anticonvulsant therapy and a trial of pyridoxine but responded to pyridoxal phosphate (PLP) at 28 days of life. Genetic testing identified compound heterozygous mutations in the PNPO gene. Management of encephalopathic episodes required escalation of PLP dose to 100 mg/kg/day by 2 years of age. Routine blood tests at this time showed significantly deranged liver function tests (LFTs). A wedge liver biopsy showed early cirrhosis with marked elevation of pyridoxal and pyridoxic acid levels in the liver sample. Despite extensive investigation, no cause other than PLP therapy could be identified for the cirrhosis. The PLP dose was weaned to 50 mg/kg/day before episodes of encephalopathy recurred. Concurrent with the reduction of his PLP dose, LFTs showed improvement. However, at 8 years of age, there is persistent evidence of hepatic fibrosis and early portal hypertension. We hypothesise that hepatic toxicity due to PLP or its degradation products is the cause of cirrhosis in this boy. Until further evidence becomes available, we would suggest that people with PNPO deficiency are treated with the minimum dose of PLP required to prevent episodes of encephalopathy

The value of plasma vitamin B6 profiles in early onset epileptic encephalopathies

BACKGROUND: Recent decades have unravelled the molecular background of a number of inborn errors of metabolism (IEM) causing vitamin B6-dependent epilepsy. As these defects interfere with vitamin B6 metabolism by different mechanisms, the plasma vitamin B6 profile can give important clues for further molecular work-up. This has so far been investigated in only a small number of patients. METHODS: We evaluated the vitamin B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN) and the catabolite pyridoxic acid (PA) in the so far largest patient cohort: reference (n = 50); pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (n = 6); antiquitin (ATQ) deficiency (n = 21); tissue non-specific alkaline phosphatase (TNSALP) deficiency (n = 2) and epileptic encephalopathy (EE) of unknown etiology tested negative for ATQ and PNPO deficiency (n = 64). RESULTS: High plasma PM concentration was found in all patients with PNPO deficiency irrespective of vitamin B6 supplementation. Their PM concentration and the PM/PA ratio was significantly higher (p < 0.0001), compared to any other patients analysed. One patient with TNSALP deficiency and sampling prior to PN supplementation had markedly elevated plasma PLP concentration. On PN supplementation, patients with TNSALP deficiency, ATQ deficiency and patients of the EE cohort had similar plasma vitamin B6 profiles that merely reflect the intake of supra-physiological doses of vitamin B6. The interval of sampling to the last PN intake strongly affected the plasma concentrations of PN, PL and PA. CONCLUSIONS: PM concentrations and the PM/PA ratio clearly separated PNPO-deficient patients from the other cohorts. The plasma PM/PA ratio thus represents a robust biomarker for the selective screening of PNPO deficiency

Normal neurodevelopmental outcomes in PNPO deficiency: a case series and literature review

Pyridox(am)ine 5'-phosphate oxidase deficiency results in an early-onset neonatal encephalopathy that can be fatal if not detected and treated early. The condition is rare, can result in preterm delivery, and can mimic hypoxic ischemic encephalopathy. Thus, suspicion of the diagnosis, appropriate investigations, and therapeutic trials with pyridoxal-5'-phosphate are often delayed. In this paper we report four cases of pyridox(am)ine 5'-phosphate oxidase deficiency, two of whom are siblings. Three were treated with pyridoxal-5'-phosphate in the first few days of life and the fourth within the first month. One of the siblings was electively treated from birth until a diagnosis was secured. Our cases demonstrate that early diagnosis and treatment can be associated with normal neurodevelopment in childhood. We suggest that a low threshold for investigating for pyridox(am)ine 5'-phosphate oxidase deficiency and electively treating with pyridoxal-5'-phosphate is considered in any neonate with encephalopathy, including those with presumed hypoxic ischemic encephalopathy in whom the degree of encephalopathy is not expected from perinatal history, cord gases and/ or neuroimaging