Identification of novel biomarkers for pyridoxine-dependent epilepsy using untargeted metabolomics and infrared ion spectroscopy - biochemical insights and clinical implications

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. Using a combination of the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we have been able to discover novel biomarkers for PDE-ALDH7A1: 2S,6S-and 2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP). We demonstrate the applicability of 2-OPP as a PDE-ALDH7A1 biomarker in newborn screening. Additionally, we show that 2-OPP accumulates in brain tissue of patients and Aldh7a1 knock-out mice, and induces 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.

Cognitive and neurological outcome of patients in the Dutch pyridoxine-dependent epilepsy (PDE-ALDH7A1) cohort, a cross-sectional study

“…48,49 Also, we support the inclusion of this metabolic epilepsy in newborn screening panels; the discovery of reliable biomarkers measurable by tandem Mass Spectroscopy makes this feasible. 47 J o u r n a l P r e -p r o o f…”

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

“…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 1 . In short, LC-MS runs were preformed using an Agilent 1290 ultra-high pressure LC system coupled to a 6545 QTOF mass spectrometer.…”

“…Alignment and feature extraction was performed using the open access software package XCMS 42 and twosided t-tests were performed to identify significantly altered features between patients and controls. See Coene et al 30 and Engelke et al 1 for details regarding this procedure.…”

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