Inherited metabolic diseases (IMDs) are the largest subgroup of rare diseases constituting serious health problems. They are typically caused by reduced enzyme activity or protein transporter function resulting in changes in metabolite levels. These abnormalities cause clinical symptoms and are key to diagnosis. The identification of novel biomarkers improves the efficiency of the diagnostic process. This is especially true for population-based screening programs where selectivity and specificity are the key issues. Identification of these biomarkers by traditional methods can be either random or based on biochemical principles, however, it is always difficult. In previous studies, we successfully applied tools for untargeted metabolomics to describe novel biomarkers of several IMDs in broad international collaboration. The aim of this project is discovery, structural elucidation, and validation of new biomarkers of IMDs by advanced mass spectrometry techniques. Biomarkers discovered within the project will ultimately improve diagnostics and screening programs of the diseases.

Dědičné metabolické poruchy (DMP) tvoří největší skupinu vzácných onemocnění a představují závažný zdravotnický problém. Jsou typicky způsobeny sníženou aktivitou enzymu či abnormální funkcí proteinového transportéru vedoucí ke změnám v hladinách metabolitů. Tyto biochemické odchylky způsobují klinické příznaky a jsou klíčem k diagnostice. Identifikace nových biomarkerů zvyšuje efektivitu diagnostického procesu, což je rozhodující zejména pro populační screeningové programy, kde selektivita a specificita jsou klíčovými pojmy. Nalezení těchto biomarkerů tradičními metodami je experimentálně obtížné a ve většině případů založené na náhodě. V předchozích experimentech jsme ve spolupráci s několika zahraničními pracovišti úspěšně použili metody necílené metabolomiky k popsání nových biomarkerů DMP. Cílem předkládaného projektu je detekce, strukturní elucidace a validace nových biomarkerů DMP pokročilými technikami hmotnostní spektrometrie. Nové biomarkery v konečném důsledku zlepší diagnostiku a screening DMP jak v České republice tak celosvětově.

• Klíčová slova
• biomarkery, biomarkers, Metabolomika, Dědičné metabolické poruchy, Hmotnostní spektrometrie, Metabolomics, Inborn Errors of Metabolism, Mass Spectrometry, novorozenecký screening, Mnohorozměrná statistická analýza, Vysoce dimenzionální data, Newborn screening, Multivariate statistical analysis, High-dimensional data,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

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.

• Publikační typ
• časopisecké články MeSH

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS (p < 0.0001). The genotype-phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

• MeSH
• 3-hydroxyacyl-CoA-dehydrogenasy nedostatek   MeSH
• acyl-CoA-dehydrogenasa nedostatek   MeSH
• dítě MeSH
• hodnocení výsledků zdravotní péče MeSH
• incidence MeSH
• kardiomyopatie diagnóza   dietoterapie   epidemiologie   MeSH
• karnitin analogy a deriváty   krev   MeSH
• kojenec MeSH
• lidé MeSH
• mitochondriální myopatie diagnóza   dietoterapie   epidemiologie   MeSH
• mitochondriální trifunkční protein nedostatek   MeSH
• nemoci nervového systému diagnóza   dietoterapie   epidemiologie   MeSH
• novorozenec MeSH
• novorozenecký screening metody   MeSH
• předškolní dítě MeSH
• retrospektivní studie MeSH
• rhabdomyolýza diagnóza   dietoterapie   epidemiologie   MeSH
• stupeň závažnosti nemoci MeSH
• vrozené poruchy metabolismu tuků diagnóza   dietoterapie   epidemiologie   MeSH
• vrozené poruchy metabolismu diagnóza   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

We designed a concept of 3D-printed attachment with porous glass filter disks-SLIDE (Sweat sampLIng DevicE) for easy sampling of apocrine sweat. By applying advanced mass spectrometry coupled with the liquid chromatography technique, the complex lipid profiles were measured to evaluate the reproducibility and robustness of this novel approach. Moreover, our in-depth statistical evaluation of the data provided an insight into the potential use of apocrine sweat as a novel and diagnostically relevant biofluid for clinical analyses. Data transformation using probabilistic quotient normalization (PQN) significantly improved the analytical characteristics and overcame the 'sample dilution issue' of the sampling. The lipidomic content of apocrine sweat from healthy subjects was described in terms of identification and quantitation. A total of 240 lipids across 15 classes were identified. The lipid concentrations varied from 10-10 to 10-4 mol/L. The most numerous class of lipids were ceramides (n = 61), while the free fatty acids were the most abundant ones (average concentrations of 10-5 mol/L). The main advantages of apocrine sweat microsampling include: (a) the non-invasiveness of the procedure and (b) the unique feature of apocrine sweat, reflecting metabolome and lipidome of the intracellular space and plasmatic membranes. The SLIDE application as a sampling technique of apocrine sweat brings a promising alternative, including various possibilities in modern clinical practice.

• MeSH
• lidé MeSH
• lipidomika metody   MeSH
• lipidy analýza   MeSH
• metabolomika metody   MeSH
• odběr biologického vzorku * MeSH
• pot chemie   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH