3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed-phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age-matched controls, we found 3-methylglutaconic acid and 3-hydroxy-3-methylglutaric acid, together with 3-hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first-tier NBS analysis to screen for elevated 3-hydroxyisovalerylcarnitine. In a positive case, a second-tier analysis of 3-hydroxy-3-methylglutaric acid and 3-methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second-tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.

Banská Bystrica Children's University Hospital Banská Bystrica Slovakia

Department of Clinical Chemistry Hannover Medical School Hannover Germany

Department of General Pediatrics Adolescent Medicine and Neonatology Medical Center University of Freiburg Faculty of Medicine Freiburg Germany

Department of Mathematical Analysis and Applications of Mathematics Faculty of Science Palacký University Olomouc Olomouc Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Olomouc Czech Republic

Laboratory Genetic Metabolic Diseases Department of Clinical Chemistry Amsterdam Netherlands

Laboratory of Inherited Metabolic Disorders Department of Clinical Chemistry University Hospital in Olomouc Olomouc Czech Republic

Screening Labor Hannover Hannover Germany

Translational Metabolic Laboratory Department of Laboratory Medicine Radboud University Medical Centre GA Nijmegen Netherlands

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Metabolic Pathways of Acylcarnitine Synthesis