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.

• Keywords
• HeLa cells, mass spectrometry, metabolomics, purine de novo synthesis, rare metabolic disorders,
• Publication type
• Journal Article MeSH

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

• Keywords
• ADSL, ADSLD, DNA damage, SAICAR, cell biology, chicken, cilia, developmental biology, human, microcephaly, zebrafish,
• MeSH
• Adenylosuccinate Lyase deficiency   metabolism   MeSH
• Aminoimidazole Carboxamide analogs & derivatives   metabolism   MeSH
• Autistic Disorder metabolism   MeSH
• Cell Line MeSH
• Cell Cycle MeSH
• Ciliopathies metabolism   MeSH
• Zebrafish metabolism   MeSH
• Phenotype MeSH
• Phosphoproteins metabolism   MeSH
• Chickens metabolism   MeSH
• Humans MeSH
• Microcephaly metabolism   MeSH
• Neurogenesis * MeSH
• Autism Spectrum Disorder metabolism   MeSH
• Purine-Pyrimidine Metabolism, Inborn Errors metabolism   MeSH
• DNA Damage MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Purines metabolism   MeSH
• Ribonucleotides metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Adenylosuccinate Lyase MeSH
• Aminoimidazole Carboxamide MeSH
• CCP110 protein, human MeSH Browser
• Phosphoproteins MeSH
• Microtubule-Associated Proteins MeSH
• Cell Cycle Proteins MeSH
• purine MeSH Browser
• Purines MeSH
• Ribonucleotides MeSH
• SAICAR MeSH Browser

Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.

• MeSH
• Models, Biological MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Electrochemical Techniques MeSH
• Carboxy-Lyases genetics   metabolism   MeSH
• Cricetinae MeSH
• Metabolomics * MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Mutation * MeSH
• Peptide Synthases genetics   metabolism   MeSH
• Purine-Pyrimidine Metabolism, Inborn Errors genetics   metabolism   MeSH
• Purines biosynthesis   MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Chromatography, High Pressure Liquid MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Carboxy-Lyases MeSH
• Peptide Synthases MeSH
• phosphoribosylaminoimidazole carboxylase MeSH Browser
• phosphoribosylaminoimidazole-succinocarboxamide synthetase MeSH Browser
• Purines MeSH