Pyridox (am) ine 5'-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5'-phosphate (PLP), the biologically active form of VB6 and involved in the synthesis of neurotransmitters including γ-aminobutyric acid (GABA), dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously, we identified a c.95 C>A missense mutation in sugarlethal (sgll)—the Drosophila homolog of human PNPO (hPNPO)—and found mutant (sgll⁹⁵) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here, we report the establishment of both sgll⁹⁵ and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll⁹⁵ and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with the GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

The active form of vitamin B6, pyridoxal phosphate (PLP), is essential for human metabolism. The brain is dependent on vitamin B6 for its neurotransmitter balance. To obtain insight into the genetic determinants of vitamin B6 homeostasis, we conducted a genome-wide association study (GWAS) of the B6 vitamers pyridoxal (PL), PLP and the degradation product of vitamin B6, pyridoxic acid (PA). We collected a unique sample set of cerebrospinal fluid (CSF) and plasma from the same healthy human subjects of Dutch ancestry (n = 493) and included concentrations and ratios in and between these body fluids in our analysis. Based on a multivariate joint analysis of all B6 vitamers and their ratios, we identified a genome-wide significant association at a locus on chromosome 1 containing the ALPL (alkaline phosphatase) gene (minimal p = 7.89 × 10⁻¹⁰, rs1106357, minor allele frequency (MAF) = 0.46), previously associated with vitamin B6 levels in blood. Subjects homozygous for the minor allele showed a 1.4-times-higher ratio between PLP and PL in plasma, and even a 1.6-times-higher ratio between PLP and PL in CSF than subjects homozygous for the major allele. In addition, we observed a suggestive association with the CSF:plasma ratio of PLP on chromosome 15 (minimal p = 7.93 × 10⁻⁷, and MAF = 0.06 for rs28789220). Even though this finding is not reaching genome-wide significance, it highlights the potential of our experimental setup for studying transport and metabolism across the blood–CSF barrier. This GWAS of B6 vitamers identifies alkaline phosphatase as a key regulator in human vitamin B6 metabolism in CSF as well as plasma. Furthermore, our results demonstrate the potential of genetic studies of metabolites in plasma and CSF to elucidate biological aspects underlying metabolite generation, transport and degradation.

Case presentation An 11-year-old girl, recently diagnosed with acute myeloid leukemia, was treated according to the NOPHO DBH AML 2012 protocol [1]. After the third chemotherapy course, consisting of cytarabine, mitoxantrone, and intrathecal methotrexate, she was admitted to the Department of Pediatric Oncology because of septic shock during febrile neutropenia. She was treated with meropenem and vancomycin and blood cultures were positive for Streptococcus mitis. Because of persistent fever, the central venous catheter was removed. Nevertheless, the fever persisted and a chest CT was performed, which revealed multiple abnormalities suggestive of pulmonary aspergillosis, which was confirmed by bronchoalveolar lavage (BAL). On day 5 of admission, she was started on AmBisome® (liposomal amphotericin B; 5 mg/kg in glucose 5%). Because of her persisting neutropenia, granulocyte colony-stimulating factor (G-CSF) was administered. Fever disappeared with neutrophil recovery, approximately 5 days after the start of G-CSF and AmBisome®. Repeated blood tests showed normal renal function (creatinine 35 μmol/L, urea 3.7 mmol/L). Potassium supplementation was started because of hypokalemia. While phosphate concentrations were low at 0.54 mmol/L on day 5, they rose spontaneously and from day 7 onwards laboratory tests showed progressive hyperphosphatemia, with a maximum of 2.28 mmol/L (Fig. 1). Question What is the most likely cause of the hyperphosphatemia observed in this patient?

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic var-iants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization and in severe cases from vitamin B6-responsive sei-zures. To study HPP we generated alpl-/- zebrafish line using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf) no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl-/- compared to alpl+/+ embryos. The survival of alpl-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl-/- embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl-/- embryos. Accumulation of d3-pyridoxal 5’-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl-/- embryos incu-bated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl-/- embryos. Metabolic profiling of alpl-/- larvae using direct-infusion high-resolution mass spectrome-try showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated first zebrafish model of HPP that shows multi-ple features of human disease and is suitable to study pathophysiology of HPP and to test novel treatments.

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B₆-responsive seizures. To study HPP, we generated alpl^-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl^-/- compared to alpl^+/+ embryos. The survival of alpl^-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl^-/- embryos. B₆ vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl^-/- embryos. Accumulation of d3-pyridoxal 5′-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl^-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B₆ metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl^-/- embryos. Metabolic profiling of alpl^-/- larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B₆-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.