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

Abstract

Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn error of lysine metabolism that presents with refractory epilepsy in newborns. Bi-allelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important co-factor pyridoxal-5'phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but despite this treatment, intellectual disability may occur. Early diagnosis and treatment, preferably based on newborn screening, potentially optimize long-term clinical outcome. However, the currently known diagnostic PDE-ALDH7A1 biomarkers are incompatible with newborn screening procedures. Combining the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we were able to discover a novel biomarker for PDE-ALDH7A1, 2S,6S-and 2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP), and confirmed 6oxopiperidine-2-carboxylic acid (6-oxoPIP) as biomarker. We demonstrated the applicability of 2-OPP as a PDE-ALDH7A1 biomarker in newborn screening. Additionally, we showed that 2-OPP accumulates in brain tissue of patients and aldh7a1 knock-out mice, and induced epilepsy-like behavior in a zebrafish model system. 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.

Metabolite Identification Using Infrared Ion Spectroscopy─Novel Biomarkers for Pyridoxine-Dependent Epilepsy

“…In a separate study, we focus on the implications of structures 1 and 2 on the pathophysiological understanding and diagnosis of PDE-ALDH7A1 . In that study, we explore the (bio)­chemical origins of structures 1 and 2 by conducting incubation experiments of P6C under physiological conditions.…”

“…This logically connects these metabolites to known biochemical routes of l -lysine metabolism, but also suggests a disease-specific expansion of this metabolic pathway in PDE-ALDH7A1. The identification of the molecular structures of the biomarkers allows us to deduce their likely biochemical origins, which may lead to a more complete understanding of the chemical basis for the pathophysiology of PDE-ALDH7A1 and to new treatment targets …”

“…A full description of the untargeted metabolic screening procedure and statistical analysis leading to the detection of the biomarkers discussed in this work are described separately . In short, LC–MS runs were preformed using an Agilent 1290 UHPLC system coupled to a 6545 QTOF mass spectrometer.…”

“…Alignment and feature extraction were performed using the open access software package XCMS, and two-sided t-tests were performed to identify significantly altered features between patients and controls. See Coene et al and Engelke et al for details regarding this procedure.…”

Novel CSF biomarkers of GLUT1 deficiency syndrome: implications beyond the brain’s energy deficit

“…Furthermore, we used Agilent MassHunter Qualitative Analysis 7.0.0 to predict a fitting molecular formula based on mass, isotope distance and relative isotope intensities, using the same adducts. Based on the putative annotations, ion-pair liquid chromatography tandem mass spectrometry (IP-LC-MS/MS) and hydrophilic interaction liquid chromatography infrared ion spectroscopy (HILIC-IRIS) 17 were applied to confirm the identity of the metabolites. More details on these methods are desribed in the Supplementary Material.…”