Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10-7-10 × 10-15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10-7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

Department of Clinical Biochemistry University Hospital in Olomouc 1 P Pavlova 6 775 20 Olomouc Czech Republic

Department of Mathematical Analysis and Applications of Mathematics Palacký University Olomouc 17 listopadu 12 771 46 Olomouc Czech Republic

Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Ke Karlovu 455 2 128 08 Prague Czech Republic

Institute of Molecular and Translational Medicine Czech Advanced Technology and Research Institute Palacký University Olomouc Hněvotínská 1333 5 779 00 Olomouc Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University and University Hospital in Olomouc Hněvotínská 1333 5 779 00 Olomouc Czech Republic

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