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.

PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation.

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.

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.

Inherited disorders of transition metal metabolism: an update.

Elements with a biological role include six trace transition metals: manganese, iron, cobalt, copper, zinc and molybdenum. Transition metals participate in group transfer reactions such as glycosylation and phosphorylation and those that can transfer an electron by alternating between two redox states such as iron (3+/2+) and copper (2+/1+) are also very important in biological redox reactions including the reduction of molecular oxygen and the transport of oxygen. However, these trace metals are also potentially toxic, generating reactive oxygen species through Fenton chemistry. Recently, a role of trace metals in host defence ("nutritional immunity") has been recognized. The host can deprive the pathogen of a trace metal or poison it with a toxic concentration. Disorders leading to low concentrations of a trace metal can often be treated by supplementing that metal; disorders leading to excessively high concentrations can often be treated with chelating agents such as penicillamine and disodium calcium edetate. This update will address: i) the manganese/zinc transporters (because two new treatable disorders were described in 2016 - SLC39A8 deficiency and SLC39A14 deficiency); ii) copper transporter disorders because we need to improve the treatment of patients with neurological symptoms due to Wilson's disease; and iii) iron homeostasis because recent progress in research into the metabolism of iron and its regulation helps us better understand several inborn errors affecting these pathways.

Quality and stability of extemporaneous pyridoxal phosphate preparations used in the treatment of paediatric epilepsy.

OBJECTIVES: To assess the pyridoxal 5'-phosphate (PLP) content and stability of extemporaneous PLP liquids prepared from dietary supplements used for the treatment of vitamin B6 -dependent epilepsy. METHODS: Pyridoxal 5'-phosphate liquids were prepared in accordance with the guidelines given to patients from marketed 50 mg PLP dietary capsules and tablets. The PLP content and its stability were evaluated under conditions resembling the clinical setting using reverse phase HPLC and mass spectrometry. KEY FINDINGS: Pyridoxal 5'-phosphate content in most of the extemporaneously prepared liquids from dietary supplements was found to be different from the expected amount (~16-60 mg). Most of these PLP extemporaneous liquids were stable at room temperature (protected from light) after 24 h but unstable after 4 h when exposed to light. A key photodegradation product of PLP in water was confirmed as 4-pyridoxic acid 5'-phosphate (PAP). CONCLUSION: Pyridoxal 5'-phosphate tablets from Solgar® were found to be the most reliable product for the preparation of extemporaneous PLP liquids. This work highlighted the difference between the marketed PLP dietary supplements quality and the importance of proper storage of aqueous PLP. There is a need to develop pharmaceutical forms of PLP that ensure dose accuracy and avoid potentially unsafe impurities with the aim of enhancing safety and compliance.

The neuroendocrine sequelae of paediatric craniopharyngioma: a 40-year meta-data analysis of 185 cases from three UK centres.

OBJECTIVES: The management of paediatric craniopharyngiomas was traditionally complete resection (CR), with better reported tumour control compared to that by partial resection (PR) or limited surgery (LS). The subsequent shift towards hypothalamic sparing, conservative surgery with adjuvant radiotherapy (RT) to any residual tumour aimed at reducing neuroendocrine morbidity, has not been systematically studied. Hence, we reviewed the sequelae of differing management strategies in paediatric craniopharyngioma across three UK tertiary centres over four decades. METHODS: Meta-data was retrospectively reviewed over two periods before (1973-2000 (Group A: n = 100)) and after (1998-2011 (Group B: n = 85)) the introduction of the conservative strategy at each centre. RESULTS: Patients had CR (A: 34% and B: 19%), PR (A: 48% and B: 46%) or LS (A: 16% and B: 34%), with trends reflecting the change in surgical approach over time. Overall recurrence rates between the two periods did not change (A: 38% vs B: 32%). More patients received RT in B than A, but recurrence rates were similar: for A, 28% patients received RT with 9 recurrences (32%); for B, 62% received RT with 14 recurrences (26%). However, rates of diabetes insipidus (P = 0.04), gonadotrophin deficiency (P < 0.001) and panhypopituitarism (P = 0.001) were lower in B than those in A. In contrast, post-operative obesity (BMI SDS >+2.0) (P = 0.4) and hypothalamic (P = 0.1) and visual (P = 0.3) morbidity rates were unchanged. CONCLUSION: The shift towards more conservative surgery has reduced the prevalence of hormone deficiencies, including diabetes insipidus, which can be life threatening. However, it has not been associated with reduced hypothalamic and visual morbidities, which remain a significant challenge. More effective targeted therapies are necessary to improve outcomes.

Assessing the population impact of low rates of vitamin D supplementation on type 1 diabetes using a new statistical method.

Vitamin D supplementation for all children <5 is recommended by the UK Department of Health for its skeletal effects. Vitamin D is also linked with a number of extra-skeletal effects; one of them being protection against type 1 diabetes. With a rapid increase in the incidence of type 1 diabetes and the associated costs, measures of curtailing the rapid increase of type 1 diabetes are needed. In this review, we look at type 1 diabetes using a statistical method (PIN-ER-t) and published data in an attempt to quantify the impact on the population of babies born in 2012 of increasing vitamin D supplementation rates. Calculations show that for the population of 729,674 babies born in England and Wales in 2012, 374 cases of type 1 diabetes (out of 1357 total predicted) could be prevented over 18 years if all were supplemented with vitamin D. This could lead to savings in excess of £62 million for the cohort. This piece of work adds to the argument for studying the potential link between vitamin D supplementation and type 1 diabetes further.

Small 6q16.1 Deletions Encompassing POU3F2 Cause Susceptibility to Obesity and Variable Developmental Delay with Intellectual Disability.

Genetic studies of intellectual disability and identification of monogenic causes of obesity in humans have made immense contribution toward the understanding of the brain and control of body mass. The leptin > melanocortin > SIM1 pathway is dysregulated in multiple monogenic human obesity syndromes but its downstream targets are still unknown. In ten individuals from six families, with overlapping 6q16.1 deletions, we describe a disorder of variable developmental delay, intellectual disability, and susceptibility to obesity and hyperphagia. The 6q16.1 deletions segregated with the phenotype in multiplex families and were shown to be de novo in four families, and there was dramatic phenotypic overlap among affected individuals who were independently ascertained without bias from clinical features. Analysis of the deletions revealed a ∼350 kb critical region on chromosome 6q16.1 that encompasses a gene for proneuronal transcription factor POU3F2, which is important for hypothalamic development and function. Using morpholino and mutant zebrafish models, we show that POU3F2 lies downstream of SIM1 and controls oxytocin expression in the hypothalamic neuroendocrine preoptic area. We show that this finding is consistent with the expression patterns of POU3F2 and related genes in the human brain. Our work helps to further delineate the neuro-endocrine control of energy balance/body mass and demonstrates that this molecular pathway is conserved across multiple species.

Pyridoxamine and pyridoxal are more effective than pyridoxine in rescuing folding-defective variants of human alanine:glyoxylate aminotransferase causing primary hyperoxaluria type I.

Vitamin B6 in the form of pyridoxine (PN) is one of the most widespread pharmacological therapies for inherited diseases involving pyridoxal phosphate (PLP)-dependent enzymes, including primary hyperoxaluria type I (PH1). PH1 is caused by a deficiency of liver-peroxisomal alanine: glyoxylate aminotransferase (AGT), which allows glyoxylate oxidation to oxalate leading to the deposition of insoluble calcium oxalate in the kidney. Only a minority of PH1 patients, mostly bearing the F152I and G170R mutations, respond to PN, the only pharmacological treatment currently available. Moreover, excessive doses of PN reduce the specific activity of AGT in a PH1 cellular model. Nevertheless, the possible effect(s) of other B6 vitamers has not been investigated previously. Here, we compared the ability of PN in rescuing the effects of the F152I and G170R mutations with that of pyridoxamine (PM) and PL. We found that supplementation with PN raises the intracellular concentration of PN phosphate (PNP), which competes with PLP for apoenzyme binding leading to the formation of an inactive AGT-PNP complex. In contrast, PNP does not accumulate in the cell upon PM or PL supplementation, but higher levels of PLP and PM phosphate (PMP), the two active forms of the AGT coenzyme, are found. This leads to an increased ability of PM and PL to rescue the effects of the F152I and G170R mutations compared with PN. A similar effect was also observed for other folding-defective AGT variants. Thus, PM and PL should be investigated as matter of importance as therapeutics for PH1 patients bearing folding mutations.

Reduced Glycemic Variability in Diazoxide-Responsive Children with Congenital Hyperinsulinism Using Supplemental Omega-3-Polyunsaturated Fatty Acids; A Pilot Trial with MaxEPA(R.).

OBJECTIVE: Congenital hyperinsulinism (CHI) is a rare condition of hypoglycemia where therapeutic options are limited and often complicated by side-effects. Omega-3-polyunsaturated fatty acids (PUFA), which can suppress cardiac myocyte electrical activity, may also reduce ion channel activity in insulin-secreting cells. PUFA supplements in combination with standard medical treatment may improve glucose profile and may reduce glycemic variability in diazoxide-responsive CHI. DESIGN: Open label pilot trial with MaxEPA(R) liquid (eicosapentaenoic and docosahexaenoic acid) PUFA (3 ml/day for 21 days) in diazoxide-responsive CHI patients (https://eudract.ema.europa.eu/, EudraCT number 201100363333). METHODS: Glucose levels were monitored pre-treatment, end of treatment, and at follow-up by subcutaneous continuous glucose monitoring systems (CGMS) in 13 patients (7 girls) who received PUFA. Outcome measures were an improved glucose profile, reduced glycemic variability quantified by a reduction in the frequency of glucose levels <4 and >10 mmol/l, and safety of PUFA. All children were analyzed either as intention to treat (n = 13) or as per protocol (n = 7). RESULTS: Mean (%) CGMS glucose levels increased by 0.1 mmol/l (2%) in intention to treat and by 0.4 mmol/l (8%) in per protocol analysis (n = 7). The frequency of CGMS <4 mmol/l was significantly less at the end of treatment than in the pre-treatment period [556 (7%) vs. 749 (10%)]. Similarly, the frequency of CGMS >10 mmol/l, was also less at the end of treatment [27 (0.3%) vs. 49 (0.7%)]. Except for one child with increased LDL cholesterol, all safety parameters were normal. CONCLUSION: MaxEPA(R) was safe and reduced glycemic variability, but did not increase glucose profiles significantly in diazoxide-responsive CHI. The supplemental value of PUFA should be evaluated in a comprehensive clinical trial.

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2.

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Manganese and the brain.

Manganese (Mn) is an essential trace metal that is pivotal for normal cell function and metabolism. Its homeostasis is tightly regulated; however, the mechanisms of Mn homeostasis are poorly characterized. While a number of proteins such as the divalent metal transporter 1, the transferrin/transferrin receptor complex, the ZIP family metal transporters ZIP-8 and ZIP-14, the secretory pathway calcium ATPases SPCA1 and SPCA2, ATP13A2, and ferroportin have been suggested to play a role in Mn transport, the degree that each of them contributes to Mn homeostasis has still to be determined. The recent discovery of SLC30A10 as a crucial Mn transporter in humans has shed further light on our understanding of Mn transport across the cell. Although essential, Mn is toxic at high concentrations. Mn neurotoxicity has been attributed to impaired dopaminergic (DAergic), glutamatergic and GABAergic transmission, mitochondrial dysfunction, oxidative stress, and neuroinflammation. As a result of preferential accumulation of Mn in the DAergic cells of the basal ganglia, particularly the globus pallidus, Mn toxicity causes extrapyramidal motor dysfunction. Firstly described as "manganism" in miners during the nineteenth century, this movement disorder resembles Parkinson's disease characterized by hypokinesia and postural instability. To date, a variety of acquired causes of brain Mn accumulation can be distinguished from an autosomal recessively inherited disorder of Mn metabolism caused by mutations in the SLC30A10 gene. Both, acquired and inherited hypermanganesemia, lead to Mn deposition in the basal ganglia associated with pathognomonic magnetic resonance imaging appearances of hyperintense basal ganglia on T1-weighted images. Current treatment strategies for Mn toxicity combine chelation therapy to reduce the body Mn load and iron (Fe) supplementation to reduce Mn binding to proteins that interact with both Mn and Fe. This chapter summarizes our current understanding of Mn homeostasis and the mechanisms of Mn toxicity and highlights the clinical disorders associated with Mn neurotoxicity.

Dystonia with brain manganese accumulation resulting from SLC30A10 mutations: a new treatable disorder.

BACKGROUND: The first gene causing early-onset generalized dystonia with brain manganese accumulation has recently been identified. Mutations in the SLC30A10 gene, encoding a manganese transporter, cause a syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia. METHODS: We present 10-year longitudinal clinical features, MRI data, and treatment response to chelation therapy of the originally described patient with a proven homozygous mutation in SLC30A10. RESULTS: The patient presented with early-onset generalized dystonia and mild hyperbilirubinemia accompanied by elevated whole-blood manganese levels. T1-sequences in MRI showed hyperintensities in the basal ganglia and cerebellum, characteristic of manganese deposition. Treatment with intravenous disodium calcium edetate led to clinical improvement and reduction of hyperintensities in brain imaging. CONCLUSIONS: We wish to highlight this rare disorder, which, together with Wilson's disease, is the only potentially treatable inherited metal storage disorder to date, that otherwise can be fatal as a result of complications of cirrhosis. © 2012 Movement Disorder Society.

Pyridoxine dependent epilepsy and antiquitin deficiency: clinical and molecular characteristics and recommendations for diagnosis, treatment and follow-up.

Antiquitin (ATQ) deficiency is the main cause of pyridoxine dependent epilepsy characterized by early onset epileptic encephalopathy responsive to large dosages of pyridoxine. Despite seizure control most patients have intellectual disability. Folinic acid responsive seizures (FARS) are genetically identical to ATQ deficiency. ATQ functions as an aldehyde dehydrogenase (ALDH7A1) in the lysine degradation pathway. Its deficiency results in accumulation of α-aminoadipic semialdehyde (AASA), piperideine-6-carboxylate (P6C) and pipecolic acid, which serve as diagnostic markers in urine, plasma, and CSF. To interrupt seizures a dose of 100 mg of pyridoxine-HCl is given intravenously, or orally/enterally with 30 mg/kg/day. First administration may result in respiratory arrest in responders, and thus treatment should be performed with support of respiratory management. To make sure that late and masked response is not missed, treatment with oral/enteral pyridoxine should be continued until ATQ deficiency is excluded by negative biochemical or genetic testing. Long-term treatment dosages vary between 15 and 30 mg/kg/day in infants or up to 200 mg/day in neonates, and 500 mg/day in adults. Oral or enteral pyridoxal phosphate (PLP), up to 30 mg/kg/day can be given alternatively. Prenatal treatment with maternal pyridoxine supplementation possibly improves outcome. PDE is an organic aciduria caused by a deficiency in the catabolic breakdown of lysine. A lysine restricted diet might address the potential toxicity of accumulating αAASA, P6C and pipecolic acid. A multicenter study on long term outcomes is needed to document potential benefits of this additional treatment. The differential diagnosis of pyridoxine or PLP responsive seizure disorders includes PLP-responsive epileptic encephalopathy due to PNPO deficiency, neonatal/infantile hypophosphatasia (TNSALP deficiency), familial hyperphosphatasia (PIGV deficiency), as well as yet unidentified conditions and nutritional vitamin B6 deficiency. Commencing treatment with PLP will not delay treatment in patients with pyridox(am)ine phosphate oxidase (PNPO) deficiency who are responsive to PLP only.

Hepatic cirrhosis, dystonia, polycythaemia and hypermanganesaemia--a new metabolic disorder.

We report a new constellation of clinical features consisting of hypermanganesaemia, liver cirrhosis, an extrapyramidal motor disorder and polycythaemia in a 12 year-old girl born to consanguineous parents. Blood manganese levels were >3000 nmol/L (normal range <320 nmol/L) and MRI revealed signal abnormalities of the basal ganglia consistent with manganese deposition. An older brother with the same phenotype died at 18 years, suggesting a potentially lethal, autosomal recessive disease. This disorder is probably caused by a defect of manganese metabolism with the accumulation of manganese in the liver and the basal ganglia similar to the copper accumulation in Wilson disease. In order to assess the genetic basis of this syndrome we investigated two candidate genes: ATP2C2 and ATP2A3 encoding the manganese-transporting calcium-ATPases, SPCA2 and SERCA3, respectively. Genotyping of the patient and the family for microsatellite markers surrounding ATP2C2 and ATP2A3 excluded these genes. The patient was found to be heterozygous for both gene loci. Despite the unknown pathophysiology, we were able to develop a successful treatment regime. Chelation therapy with disodium calcium edetate combined with iron supplementation is the treatment of choice, lowering blood manganese levels significantly and improving clinical symptoms.

B6-responsive disorders: a model of vitamin dependency.

Pyridoxal phosphate is the cofactor for over 100 enzyme-catalysed reactions in the body, including many involved in the synthesis or catabolism of neurotransmitters. Inadequate levels of pyridoxal phosphate in the brain cause neurological dysfunction, particularly epilepsy. There are several different mechanisms that lead to an increased requirement for pyridoxine and/or pyridoxal phosphate. These include: (i) inborn errors affecting the pathways of B(6) vitamer metabolism; (ii) inborn errors that lead to accumulation of small molecules that react with pyridoxal phosphate and inactivate it; (iii) drugs that react with pyridoxal phosphate; (iv) coeliac disease, which is thought to lead to malabsorption of B(6) vitamers; (v) renal dialysis, which leads to increased losses of B(6) vitamers from the circulation; (vi) drugs that affect the metabolism of B(6) vitamers; and (vii) inborn errors affecting specific pyridoxal phosphate-dependent enzymes. The last show a very variable degree of pyridoxine responsiveness, from 90% in X-linked sideroblastic anaemia (delta-aminolevulinate synthase deficiency) through 50% in homocystinuria (cystathionine beta-synthase deficiency) to 5% in ornithinaemia with gyrate atrophy (ornithine delta-aminotransferase deficiency). The possible role of pyridoxal phosphate as a chaperone during folding of nascent enzymes is discussed. High-dose pyridoxine or pyridoxal phosphate may have deleterious side-effects (particularly peripheral neuropathy with pyridoxine) and this must be considered in treatment regimes. None the less, in some patients, particularly infants with intractable epilepsy, treatment with pyridoxine or pyridoxal phosphate can be life-saving, and in other infants with inborn errors of metabolism B(6) treatment can be extremely beneficial.