De novo synthesis of purines (DNPS) is a biochemical pathway that provides the purine bases for synthesis of essential biomolecules such as nucleic acids, energy transfer molecules, signaling molecules and various cofactors. Inborn errors of DNPS enzymes present with a wide spectrum of neurodevelopmental and neuromuscular abnormalities and accumulation of characteristic metabolic intermediates of the DNPS in body fluids and tissues. In this study, we present the second case of PAICS deficiency due to bi-allelic variants of PAICS gene encoding for a missense p.Ser179Pro and truncated p.Arg403Ter forms of the PAICS proteins. Two affected individuals were born at term after an uncomplicated pregnancy and delivery and presented later in life with progressive cerebral atrophy, epileptic encephalopathy, psychomotor retardation, and retinopathy. Plasma and urinary concentrations of dephosphorylated substrates of PAICS, AIr and CAIr were elevated, though they remained undetectable in skin fibroblasts. Both variants affect structural domains in SAICARs catalytic site and the oligomerization interface. In silico modeling predicted negative effects on PAICS oligomerization, enzyme stability and enzymatic activity. Consistent with these findings, affected skin fibroblasts were devoid of PAICS protein and enzyme activity. This was accompanied by alterations in contents of other DNPS proteins, which had co-localized in granular structures that are characteristic of purinosome formation. Our observation expands the clinical spectrum of PAICS deficiency from recurrent abortions and fatal neonatal form to later onset neurodevelopmental disorders. The rarity of this condition may be based on poor clinical recognition and limited access to specialized laboratory tests diagnostic for PAICS deficiency.

• MeSH
• lidé MeSH
• missense mutace MeSH
• multienzymové komplexy * genetika   nedostatek   MeSH
• sourozenci MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• multienzymové komplexy * MeSH

Purine de novo purine synthesis involves 10 reactions catalysed by six enzymes, including phosphoribosylformyglycinamidine synthase (PFAS). To date, genetic defects of three of these enzymes, namely ATIC, ADSL and PAICS, have been characterised in humans. Here, we report for the first time two individuals with PFAS deficiency. Probands were identified through metabolic and genetic screening of neurologically impaired individuals. The pathogenicity of the variants was established by structural and functional studies. Probands C1 and C2 presented with prematurity, short stature, recurrent seizures and mild neurological impairment. C1 had elevated urinary levels of formylglycineamide riboside (FGAr) and bi-allelic PFAS variants encoding the NP_036525.1:p.Arg811Trp substitution and the NP_036525.1:p.Glu228_Ser230 in-frame deletion. C2 is a 20-year-old female with a homozygous NP_036525.1:p.Asn264Lys substitution. These amino acid changes are predicted to affect the structural stability of PFAS. Accordingly, C1 skin fibroblasts showed decreased PFAS content and activity, with impaired purinosome formation that was restored by transfection with pTagBFP_PFAS_wt. The enzymatic activities of the corresponding recombinant mutant PFAS proteins were also reduced, and none of them, after transfection, corrected the elevated FGAR/r levels in PFAS-deficient HeLa cells. While genetic defects in purine de novo synthesis are typically considered in patients with severe neurological impairment, these disorders, especially PFAS deficiency, should also be considered in milder phenotypes.

• Klíčová slova
• FGAR, PFAS deficiency, formylglycinamide riboside, metabolic disorder, purine de novo synthesis, purinosome,
• MeSH
• lidé MeSH
• ligasy tvořící vazby C-N s glutaminem jako amidovým donorem * genetika   nedostatek   metabolismus   MeSH
• mladý dospělý MeSH
• mutace MeSH
• poruchy metabolismu purinů a pyrimidinů * genetika   MeSH
• předškolní dítě MeSH
• puriny * biosyntéza   MeSH
• Check Tag
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• ligasy tvořící vazby C-N s glutaminem jako amidovým donorem * MeSH
• phosphoribosylformylglycinamidine synthetase MeSH Prohlížeč
• purine MeSH Prohlížeč
• puriny * MeSH

Cytotoxicity of de novo purine synthesis (DNPS) metabolites is critical to the pathogenesis of three known and one putative autosomal recessive disorder affecting DNPS. These rare disorders are caused by biallelic mutations in the DNPS genes phosphoribosylformylglycineamidine synthase (PFAS), phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS), adenylosuccinate lyase (ADSL), and aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) and are clinically characterized by developmental abnormalities, psychomotor retardation, and nonspecific neurological impairment. At a biochemical level, loss of function of specific mutated enzymes results in elevated levels of DNPS ribosides in body fluids. The main pathogenic effect is attributed to the accumulation of DNPS ribosides, which are postulated to be toxic to the organism. Therefore, we decided to characterize the uptake and flux of several DNPS metabolites in HeLa cells and the impact of DNPS metabolites to viability of cancer cell lines and primary skin fibroblasts. We treated cells with DNPS metabolites and followed their flux in purine synthesis and degradation. In this study, we show for the first time the transport of formylglycinamide ribotide (FGAR), aminoimidazole ribotide (AIR), succinylaminoimidazolecarboxamide ribotide (SAICAR), and aminoimidazolecarboxamide ribotide (AICAR) into cells and their flux in DNPS and the degradation pathway. We found diminished cell viability mostly in the presence of FGAR and AIR. Our results suggest that direct cellular toxicity of DNPS metabolites may not be the primary pathogenetic mechanism in these disorders.

• Klíčová slova
• ADSL, AICAR, AIR, ATIC, FGAR, PAICS, PFAS, SAICAR, cytotoxicity, purine synthesis,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• HeLa cells, mass spectrometry, metabolomics, purine de novo synthesis, rare metabolic disorders,
• Publikační typ
• časopisecké články MeSH

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

• MeSH
• GAR-transformylasa metabolismus   genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• metabolom * MeSH
• puriny * metabolismus   biosyntéza   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• GAR-transformylasa MeSH
• purine MeSH Prohlížeč
• puriny * MeSH