OBJECTIVE: The laboratory diagnosis of inherited metabolic disorders (IMD) has undergone significant development in recent decades, mainly due to the use of mass spectrometry, which allows rapid multicomponent analysis of a wide range of metabolites. Combined with advanced software tools, the diagnosis becomes more efficient as a benefit for both physicians and patients. METHODS: A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry assay for determination of urinary purines, pyrimidines, N-acylglycines, N-acetylated amino acids, sugars, sugar alcohols and other diagnostically important biomarkers was developed and validated. Evaluation of the results consisting of utilisation of robust scaling and advanced visualization tools is simple and even suitable for urgent requirements. RESULTS: The developed method, covering 65 biomarkers, provides a comprehensive diagnostic platform for 51 IMD. For most analytes, linearity with R2 > 0.99, intra and inter-day accuracy between 80 and 120 % and precision lower than 20 % were achieved. Diagnostic workflow was evaluated on 47 patients and External Quality Assurance samples involving a total of 24 different IMD. Over seven years, more than 2300 urine samples from patients suspected for IMD have been routinely analysed. CONCLUSIONS: This method offers the advantage of a broad coverage of intermediate metabolites of interest and therefore may be a potential alternative and simplification for clinical laboratories that use multiple methods for screening these markers.
Newborn screening (NBS) of inborn errors of metabolism (IEMs) is based on the reference ranges established on a healthy newborn population using quantile statistics of molar concentrations of biomarkers and their ratios. The aim of this paper is to investigate whether multivariate independent component analysis (ICA) is a useful tool for the analysis of NBS data, and also to address the structure of the calculated ICA scores. NBS data were obtained from a routine NBS program performed between 2013 and 2022. ICA was tested on 10,213/150 free-diseased controls and 77/20 patients (9/3 different IEMs) in the discovery/validation phases, respectively. The same model computed during the discovery phase was used in the validation phase to confirm its validity. The plots of ICA scores were constructed, and the results were evaluated based on 5sd levels. Patient samples from 7/3 different diseases were clearly identified as 5sd-outlying from control groups in both phases of the study. Two IEMs containing only one patient each were separated at the 3sd level in the discovery phase. Moreover, in one latent variable, the effect of neonatal birth weight was evident. The results strongly suggest that ICA, together with an interpretation derived from values of the "average member of the score structure", is generally applicable and has the potential to be included in the decision process in the NBS program.
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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.
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SUMMARY: Untargeted liquid chromatography-high-resolution mass spectrometry analysis produces a large number of features which correspond to the potential compounds in the sample that is analyzed. During the data processing, it is necessary to merge features associated with one compound to prevent multiplicities in the data and possible misidentification. The processing tools that are currently employed use complex algorithms to detect abundances, such as adducts or isotopes. However, most of them are not able to deal with unpredictable adducts and in-source fragments. We introduce a simple open-source R-script CROP based on Pearson pairwise correlations and retention time together with a graphical representation of the correlation network to remove these redundant features. AVAILABILITY AND IMPLEMENTATION: The CROP R-script is available online at www.github.com/rendju/CROP under GNU GPL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed-phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age-matched controls, we found 3-methylglutaconic acid and 3-hydroxy-3-methylglutaric acid, together with 3-hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first-tier NBS analysis to screen for elevated 3-hydroxyisovalerylcarnitine. In a positive case, a second-tier analysis of 3-hydroxy-3-methylglutaric acid and 3-methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second-tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.
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