Vitamin B6 and Related Inborn Errors of Metabolism

Vitamin B6 (vitB6) is a generic term that comprises six interconvertible pyridine compounds. These vitB6 compounds (also called vitamers) are pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their 5′-phosphorylated forms pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate (PLP). VitB6 is an essential nutrient for all living organisms, but only microorganisms and plants can carry out de novo synthesis of this vitamin. Other organisms obtain vitB6 from dietary sources and interconvert its different forms according to their needs via a biochemical pathway known as the salvage pathway. PLP is the biologically active form of vitB6 which is important for maintaining the biochemical homeostasis of the body. In the human body, PLP serves as a cofactor for more than 140 enzymatic reactions, mainly associated with synthesis, degradation and interconversion of amino acids and neurotransmitter metabolism. PLP-dependent enzymes are also involved in various physiological processes, including biologically active amine biosynthesis, lipid metabolism, heme synthesis, nucleic acid synthesis, protein and polyamine synthesis and several other metabolic pathways. PLP is an important vitamer for normal brain function since it is required as a coenzyme for the synthesis of several neurotransmitters including D-serine, D-aspartate, L-glutamate, glycine, γ-aminobutyric acid (GABA), serotonin, epinephrine, norepinephrine, histamine and dopamine. Intracellular levels of PLP are tightly regulated and conditions that disrupt this homeostatic regulation can cause disease. In humans, genetic and dietary (intake of high doses of vitB6) conditions leading to increase in PLP levels is known to cause motor and sensory neuropathies. Deficiency of PLP in the cell is also implicated in several diseases, the most notable example of which are the vitB6-dependent epileptic encephalopathies. VitB6-dependent epileptic encephalopathies (B6EEs) are a clinically and genetically heterogeneous group of rare inherited metabolic disorders. These debilitating conditions are characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but are well-controlled by the administration of PN or PLP. In addition to seizures, children affected with B6EEs may also suffer from developmental and/or intellectual disabilities, along with structural brain abnormalities. Five main types of B6EEs are known to date, these are: PN-dependent epilepsy due to ALDH7A1 (antiquitin) deficiency (PDE-ALDH7A1) (MIM: 266100), hyperprolinemia type 2 (MIM: 239500), PLP-dependent epilepsy due to PNPO deficiency (MIM: 610090), hypophosphatasia (MIM: 241500) and PLPBP deficiency (MIM: 617290). This chapter provides a review of vitB6 and its different vitamers, their absorption and metabolic pathways in the human body, the diverse physiological roles of vitB6, PLP homeostasis and its importance for human health. Finally, the chapter reviews the inherited neurological disorders affecting PLP homeostasis with a special focus on vitB6-dependent epileptic encephalopathies (B6EEs), their different subtypes, the pathophysiological mechanism underlying each type, clinical and biochemical features and current treatment strategies

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including δ1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics

Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy

Background. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/ antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5′-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available. Methods. We combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening. Results. We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system. Conclusion. This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients

Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy

Background. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/ antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5′-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available. Methods. We combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening. Results. We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system. Conclusion. This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients

PLPHP deficiency:clinical, genetic, biochemical, and mechanistic insights

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery