2-Pyrrolidinone and Succinimide as Clinical Screening Biomarkers for GABA-Transaminase Deficiency: Anti-seizure Medications Impact Accurate Diagnosis

Adam D Kennedy,Seema R Lalani,Taraka Donti,Kirk L Pappan,Anne M Evans,William J Craigen,Marwan Shinawi,V Reid Sutton,Qin Sun,Lisa T Emrick,Mauricio R Delgado,Sarah H Elsea,Toni S Pearson

doi:10.3389/fnins.2019.00394

Abstract

Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-aminobutyrate aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥ 2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide or its ring-opened form, succinamic acid, in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinamic acid and 2-pyrrolidinone showed a high level of correlation (R = 0.72), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.

Highlights

Gamma-aminobutyric acid (GABA) transaminase deficiency (OMIM #613163), known as 4-ABAT deficiency is a rare genetic disorder that disrupts the degradation of GABA to succinic semialdehyde (Figure 1)
Global biochemical profiling of EDTA plasma was performed on six samples from four unique subjects with GABA-transaminase deficiency (Table 1) and several subjects without variants in ABAT taking medication that inhibits GABA-transaminase or otherwise may alter GABA metabolism (n = 93)
We applied an untargeted metabolomics profiling approach on plasma, urine, and cerebrospinal fluid (CSF) from patients with known or suspected GABA-transaminase deficiency and non-ABAT-related patients treated with medications that inhibit GABA metabolism with the objective of unraveling metabolic biomarkers to differentiate disorder from treatment (Figures 3, 4)

Summary

Introduction

Gamma-aminobutyric acid (GABA) transaminase deficiency (OMIM #613163), known as 4-ABAT deficiency is a rare genetic disorder that disrupts the degradation of GABA to succinic semialdehyde (Figure 1). This IEM is due to decreased or deficient activity of the enzyme 4-ABAT (ABAT, EC 2.6.1.19, OMIM #137150). The accumulation of GABA, either through enzymatic inactivity of ABAT or medical intervention, can result in elevated levels of 2-pyrrolidinone, due to cyclization of GABA (Callery et al, 1978). 2-pyrrolidinone can be converted to GABA when it is administered intravenously (Callery et al, 1979) or orally (Fasolato et al, 1988). 2-pyrrolidinone can be converted to GABA when it is administered intravenously (Callery et al, 1979) or orally (Fasolato et al, 1988). 2-pyrrolidinone can be converted to succinimide through a two-step reaction (Bandle et al, 1984), and hydrolytic ring opening of cyclic imides such as succinimide can occur through enzymatic (Maguire and Dudley, 1978) and non-enzymatic (Kurono et al, 2008; Lerner et al, 2013) routes

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Conclusion