V metabolických laboratořích je každoročně vyšetřeno velké množství pacientů s nespecifickým neurologickým postižením, u kterých se nepodaří stanovit diagnózu. Je velmi pravděpodobné, že u části z nich by se mohlo jednat o dosud nevyšetřované geneticky podmíněné poruchy de novo syntézy purinů (DNPS). Cílem tohoto projektu je studium prevalence těchto poruch v populaci dětských pacientů s nespecifickým neurologickým onemocněním stanovením substrátů pro enzymy DNPS metodou LC-MS/MS v močích a suchých krevních kapkách. Na základě naší zkušenosti se stanovením SAICA-ribosidu, jednoho ze substrátů enzymů DNPS, kdy se podařilo diagnostikovat 40% všech celosvětově popsaných pacientů s deficitem adenylosukcinátlyázy, předpokládáme, že analýzy dalších substrátů DNPS povedou k definici nových poruch de novo syntézy purinů a stanovení diagnózy alespoň u části postižených pacientů.; Many patients are examined in the Institute of Inherited Metabolic Disorders with a diagnostic efficiency of approximately 2%. High numbers of them suffer with neurological symptoms that do not fit into any currently known metabolic disorder. Therefore, it is highly probable that some of them can suffer from defects of de novo purine synthesis (DNPS). To date, there is no sufficient screening method for the detection of such defects. The aim of this project is to study the prevalence of DNPS disorders in a population of patients with neurological impairment that has not yet been diagnosed. Screening will be performed by determining the levels of DNPS intermediates by LC-MS/MS in the body fluids. Based on our experiences having established a detection method for SAICA-riboside, one of the DNPS intermediates, that provided a diagnosis in 40% of all the patients worldwide diagnosed with adenylosuccinate lyase deficiency, we suggest that the screening of the other intermediates can help to define new types of DNPS defects, as well as establish the diagnosis in some patients.

• MeSH
• adenylsukcinátlyasa MeSH
• analýza moči MeSH
• biochemická analýza krve MeSH
• dítě MeSH
• genetická predispozice k nemoci MeSH
• genetické testování MeSH
• nemoci nervového systému diagnóza   MeSH
• poruchy metabolismu purinů a pyrimidinů MeSH
• puriny biosyntéza   MeSH
• Check Tag
• dítě MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• neurologie
• biochemie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

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.

• 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

Dominantly inherited mutations in COMP gene encoding cartilage oligomeric matrix protein may cause two dwarfing skeletal dysplasias, milder multiple epiphyseal dysplasia (MED) and more severe pseudoachondroplasia (PSACH). We studied the phenotype and X-rays of 11 patients from 5 unrelated families with different COMP mutations. Whole exome and/or Sangers sequencing were used for molecular analyses. Four to ten X-ray images of hands hips, knees or spine were available for each patient for retrospective analyses. Eight patients with MED have mutation c.1220G>A and 3 children with PSACH mutations c.1359C>A, c.1336G>A, or the novel mutation c.1126G>T in COMP. Progressive failure in growth developed in all patients from early childhood and resulted in short stature < 3rd percentile in 7 patients and very short stature < 1st percentile in four. Most patients had joint pain since childhood, severe stiffness in shoulders and elbows but increased mobility in wrists. Six children had bowlegs and two had knock knees. In all patients, X-rays of hands, hips and knees showed progressive, age-dependent skeletal involvement more pronounced in the epiphyses of long rather than short tubular bones. Anterior elongation and biconvex configuration of vertebral bodies were more conspicuous for kids. Six children had correction of knees and two adults had hip replacement. Skeletal and joint impairment in patients with MED and PSACH due to COMP mutation start in early childhood. Although the clinical severity is mutation and age dependent, many symptoms represent a continuous phenotypic spectrum between both diseases. Most patients may benefit from orthopaedic surgeries.

• MeSH
• achondroplazie MeSH
• chrupavkový oligomerní matrixový protein * genetika   MeSH
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• matriliny genetika   MeSH
• mutace * MeSH
• osteochondrodysplazie * diagnostické zobrazování   genetika   MeSH
• předškolní dítě MeSH
• retrospektivní studie MeSH
• stupeň závažnosti nemoci MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH

Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.

• MeSH
• adenosin metabolismus   MeSH
• adenylsukcinátlyasa genetika   metabolismus   MeSH
• karcinogeneze MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• proliferace buněk MeSH
• prolyl-4-hydroxylasy HIF genetika   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• triple-negativní karcinom prsu enzymologie   genetika   patofyziologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The cellular pool of purines is maintained by de novo purine synthesis (DNPS), recycling and degradation. Mutations in genes encoding DNPS enzymes cause their substrates to accumulate, which has detrimental effects on cellular division and organism development, potentially leading to neurological impairments. Unspecified neurological symptoms observed in many patients could not be elucidated even by modern techniques. It is presumable that some of these problems are induced by dysfunctions in DNPS enzymes. Therefore, we determined the concentrations of dephosphorylated DNPS intermediates by LC-MS/MS as markers of yet unpublished mutations in PFAS and PAICS genes connected with dysfunctions of carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) or phosphoribosylformylglycinamidine synthase (PFAS). We determined the criteria for normal values of metabolites and investigated 1,447 samples of urine and 365 dried blood spots of patients suffering from various forms of neurological impairment. We detected slightly elevated aminoimidazole riboside (AIr) concentrations in three urine samples and a highly elevated 5-formamidoimidazole-4-carboxamide riboside (FGAr) concentration in one urine sample. The accumulation of AIr or FGAr in body fluids can indicate PAICS or PFAS deficiency, respectively, which would be new disorders of DNPS caused by mutations in the appropriate genes. Measurement of DNPS intermediates in patients with neurological symptoms can uncover the cause of serious cellular and functional impairments that are otherwise inaccessible to detection. Further genetic and molecular analysis of these patients should establish the causal mutations for prenatal diagnosis, genetic consultation, and reinforce the DNPS pathway as a therapeutic target.

• MeSH
• lidé MeSH
• limita detekce MeSH
• metabolom MeSH
• metabolomika metody   MeSH
• moč MeSH
• mutace genetika   MeSH
• nemoci nervového systému genetika   metabolismus   MeSH
• puriny biosyntéza   krev   chemie   moč   MeSH
• test suché kapky krve MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.

• MeSH
• chromatografie kapalinová MeSH
• CRISPR-Cas systémy MeSH
• DNA biosyntéza   chemie   MeSH
• editace genu MeSH
• HeLa buňky MeSH
• lidé MeSH
• puriny biosyntéza   chemie   MeSH
• RNA biosyntéza   chemie   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The enzymes involved in de novo purine synthesis (DNPS), one of the basic processes in eukaryotic cells, transiently and reversibly form a dynamic multienzyme complex called the purinosome in the cytoplasm. The purinosome has been observed in a broad spectrum of cells, but some studies claim that it is an artefact of the constructs used for visualization or stress granules resulting from the exposure of cells to nutrient-reduced growth media. Both may be true depending on the method of observation. To clarify this point, we combined two previously used methods, transfection and immunofluorescence, to detect purinosomes in purinosome-free cells deficient in particular DNPS steps (CR-DNPS cells) and in cells deficient in the salvage pathway, which resulted in construction of the purinosome regardless of purine level (CR-HGPRT cells). METHODS AND FINDINGS: To restore or disrupt purinosome formation, we transiently transfected CR-DNPS and CR-HGPRT cells with vectors encoding BFP-labelled wild-type (wt) proteins and observed the normalization of purinosome formation. The cells also ceased to accumulate the substrate(s) of the defective enzyme. The CR-DNPS cell line transfected with a DNA plasmid encoding an enzyme with zero activity served as a negative control for purinosome formation. No purinosome formation was observed in these cells regardless of the purine level in the growth medium. CONCLUSION: In conclusion, both methods are useful for the detection of purinosomes in HeLa cells. Moreover, the cell-based models prepared represent a unique system for the study of purinosome assembly with deficiencies in DNPS or in the salvage pathway as well as for the study of purinosome formation under the action of DNPS inhibitors. This approach is a promising step toward the treatment of purine disorders and can also provide targets for anticancer therapy.

• MeSH
• biologické modely * MeSH
• HeLa buňky MeSH
• lidé MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• puriny biosyntéza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Objectives: Phosphoribosylpyrophosphate synthetase (PRPS1) superactivity is an X-linked disorder characterized by urate overproduction Online Mendelian Inheritance in Man (OMIM) gene reference 300661. This condition is thought to rarely affect women, and when it does, the clinical presentation is mild. We describe a 16-year-old African American female who developed progressive tophi, nephrolithiasis and acute kidney failure due to urate overproduction. Family history included a mother with tophaceous gout who developed end-stage kidney disease due to nephrolithiasis and an affected sister with polyarticular gout. The main aim of this study was to describe the clinical manifestations of PRPS1 superactivity in women. Methods: Whole exome sequencing was performed in affected females and their fathers. Results: Mutational analysis revealed a new c.520 G > A (p.G174R) mutation in the PRPS1 gene. The mutation resulted in decreased PRPS1 inhibition by ADP. Conclusion: Clinical findings in previously reported females with PRPS1 superactivity showed a high clinical penetrance of this disorder with a mean serum urate level of 8.5 (4.1) mg/dl [506 (247) μmol/l] and a high prevalence of gout. These findings indicate that all women in families with PRPS1 superactivity should be genetically screened for a mutation (for clinical management and genetic counselling). In addition, women with tophaceous gout, gout presenting in childhood, or a strong family history of severe gout should be considered for PRPS1 mutational analysis.

• MeSH
• dnavá artritida etiologie   genetika   MeSH
• dospělí MeSH
• genetické nemoci vázané na chromozom X diagnóza   genetika   MeSH
• lidé MeSH
• mladiství MeSH
• molekulární struktura MeSH
• mutace MeSH
• nefrolitiáza etiologie   genetika   MeSH
• poruchy metabolismu purinů a pyrimidinů komplikace   diagnóza   genetika   MeSH
• ribosafosfátpyrofosfokinasa genetika   metabolismus   MeSH
• rodokmen MeSH
• sekvenování celého genomu metody   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakty MeSH