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In metabolic diagnostics, there is an emerging need for a comprehensive test to acquire a complete view of metabolite status. Here, we describe a non-quantitative direct-infusion high-resolution mass spectrometry (DI-HRMS) based metabolomics method and evaluate the method for both dried blood spots (DBS) and plasma. 110 DBS of 42 patients harboring 23 different inborn errors of metabolism (IEM) and 86 plasma samples of 38 patients harboring 21 different IEM were analyzed using DI-HRMS. A peak calling pipeline developed in R programming language provided Z-scores for ~1875 mass peaks corresponding to ~3835 metabolite annotations (including isomers) per sample. Based on metabolite Z-scores, patients were assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients. Based on DBS sample analysis, 37/42 of the patients, corresponding to 22/23 IEM, could be correctly assigned a 'most probable diagnosis'. Plasma sample analysis, resulted in a correct 'most probable diagnosis' in 32/38 of the patients, corresponding to 19/21 IEM. The added clinical value of the method was illustrated by a case wherein DI-HRMS metabolomics aided interpretation of a variant of unknown significance (VUS) identified by whole-exome sequencing. In summary, non-quantitative DI-HRMS metabolomics in DBS and plasma is a very consistent, high-throughput and nonselective method for investigating the metabolome in genetic disease. [ABSTRACT FROM AUTHOR]

Corneal clouding, causing visual impairment, is seen in nearly all patients with mucopolysaccharidosis type 1 (MPS-1). Hematopoietic cell transplantation (HCT) is able to stabilize disease in many organs. Residual disease in several tissues is being increasingly recognized, however. Data on the effect of HCT on ocular disease in patients with MPS-1 are contradictory. With this study, we aim to clarify the long-term effects of HCT on ocular disease in these patients. Best corrected visual acuity (BCVA), refraction, intraocular pressure (IOP), and slit-lamp biomicroscopic and fundoscopic examinations, including corneal clouding, were collected prospectively from 24 patients with MPS-1 who underwent HCT successfully between 2003 and 2018 (92% with >95% chimerism and normal enzyme activity after HCT). The course of corneal clouding and BCVA after HCT were analyzed using a linear mixed model. Other parameters studied were clinical phenotype, age at time of transplantation, and hematologic enzyme activity after transplantation. Outcomes of additional ophthalmologic tests were described. In addition, IDUA and α-galactosidase A (AGAL) enzyme activity and glycosaminoglycan (GAG) concentration in tear fluid were determined. Corneal clouding stabilized in the first years after HCT but increased rapidly beyond 3 years (P < .0001). BCVA and IOP also worsened over time (P = .01 and P < .0001, respectively). IDUA activity in tear fluid remained very low (P < .0001). After initial stabilization in the cornea, ongoing ocular disease and low IDUA activity in tear fluid is seen in patients with MPS-1 despite treatment with HCT, unveiling a weak spot of current standard therapy. New therapies that overcome these shortcomings are needed to improve the late outcomes of patients., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Untargeted metabolomics may become a standard approach to address diagnostic requests, but, at present, data interpretation is very labor-intensive. To facilitate its implementation in metabolic diagnostic screening, we developed a method for automated data interpretation that preselects the most likely inborn errors of metabolism (IEM). The input parameters of the knowledge-based algorithm were (1) weight scores assigned to 268 unique metabolites for 119 different IEM based on literature and expert opinion, and (2) metabolite Z-scores and ranks based on direct-infusion high resolution mass spectrometry. The output was a ranked list of differential diagnoses (DD) per sample. The algorithm was first optimized using a training set of 110 dried blood spots (DBS) comprising 23 different IEM and 86 plasma samples comprising 21 different IEM. Further optimization was performed using a set of 96 DBS consisting of 53 different IEM. The diagnostic value was validated in a set of 115 plasma samples, which included 58 different IEM and resulted in the correct diagnosis being included in the DD of 72% of the samples, comprising 44 different IEM. The median length of the DD was 10 IEM, and the correct diagnosis ranked first in 37% of the samples. Here, we demonstrate the accuracy of the diagnostic algorithm in preselecting the most likely IEM, based on the untargeted metabolomics of a single sample. We show, as a proof of principle, that automated data interpretation has the potential to facilitate the implementation of untargeted metabolomics for metabolic diagnostic screening, and we provide suggestions for further optimization of the algorithm to improve diagnostic accuracy.

Introduction: Hartnup disorder is caused by a deficiency of the sodium dependent B 0 AT1 neutral amino acid transporter in the proximal kidney tubules and jejunum. Biochemically, Hartnup disorder is diagnosed via amino acid excretion patterns. However, these patterns can closely resemble amino acid excretion patterns of generalized aminoaciduria, which may induce a risk for misdiagnosis and preclusion from treatment. Here we explore whether calculating a diagnostic ratio could facilitate correct discrimination of Hartnup disorder from other aminoacidurias., Methods: 27 amino acid excretion patterns from 11 patients with genetically confirmed Hartnup disorder were compared to 68 samples of 16 patients with other aminoacidurias. Amino acid fold changes were calculated by dividing the quantified excretion values over the upper limit of the age-adjusted reference value., Results: Increased excretion of amino acids is not restricted to amino acids classically related to Hartnup disorder ("Hartnup amino acids", HAA), but also includes many other amino acids, not classically related to Hartnup disorder ("other amino acids", OAA). The fold change ratio of HAA over OAA was 6.1 (range: 2.4-9.6) in the Hartnup cohort, versus 0.2 (range: 0.0-1.6) in the aminoaciduria cohort ( p  < .0001), without any overlap observed between the cohorts., Discussion: Excretion values of amino acids not classically related to Hartnup disorder are frequently elevated in patients with Hartnup disorder, which may cause misdiagnosis as generalized aminoaciduria and preclusion from vitamin B3 treatment. Calculation of the HAA/OAA ratio improves diagnostic differentiation of Hartnup disorder from other aminoacidurias., Competing Interests: All authors state that they have no competing financial interests to declare. None of the authors accepted any reimbursements, fees or funds from any organization that may in any way gain or lose financially from the results of this review. The authors have not been employed by such an organization. The authors have not act as an expert witness on the subject of the review. The authors do not have any other competing financial interest., (© 2019 The Authors.)

Background: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently., Methods: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases., Results: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8-8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of -0.1 (range: -2.1-4.0) in DBS of healthy controls and patients with other diseases., Discussion: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: For inborn errors of metabolism (IEM), metabolomics is performed for three main purposes: 1) development of next generation metabolic screening platforms, 2) identification of new biomarkers in predefined patient cohorts and 3) for identification of new IEM. To date, plasma, urine and dried blood spots are used. We anticipate that cerebrospinal fluid (CSF) holds additional - valuable - information, especially for IEM with neurological involvement. To expand metabolomics to CSF, we here tested whether direct-infusion high-resolution mass spectrometry (DI-HRMS) based non-quantitative metabolomics could correctly capture the biochemical profile of patients with an IEM in CSF., Methods: Eleven patient samples, harboring eight different IEM, and thirty control samples were analyzed using DI-HRMS. First we assessed whether the biochemical profile of the control samples represented the expected profile in CSF. Next, each patient sample was assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients., Results: the biochemical profile identified using DI-HRMS in CSF samples resembled the known profile, with - among others - the highest median intensities for mass peaks annotated with glucose, lactic acid, citric acid and glutamine. Subsequent analysis of patient CSF profiles resulted in correct 'most probable diagnoses' for all eleven patients, including non-ketotic hyperglycinaemia, propionic aciduria, purine nucleoside phosphorylase deficiency, argininosuccinic aciduria, tyrosinaemia type I, hyperphenylalaninemia and hypermethioninaemia., Conclusion: We here demonstrate that DI-HRMS based non-quantitative metabolomics accurately captures the biochemical profile of this set of patients in CSF, opening new ways for using metabolomics in CSF in the metabolic diagnostic laboratory., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Diagnosis and treatment of inborn errors of metabolism (IEM) require the analysis of a variety of metabolites. These compounds are usually quantified by targeted platforms. High resolution mass spectrometry (HRMS) has the potential to detect hundreds to thousands of metabolites simultaneously. A chip-based nanoelectrospray source (chip-based nanoESI) enables the direct infusion of biological samples. Major advantages of this system include high sample throughput, no sample carryover, and low sample consumption. The combination, chip-based nanoESI-HRMS enables untargeted metabolomics of biological samples but its potential for quantification of metabolites has not been reported. We investigated whether chip-based nanoESI-HRMS is suitable for quantification of metabolites in dried blood spots (DBS). After addition of internal standards, metabolites were extracted with methanol. Aliquots of each extract were analysed by chip-based nanoESI-HRMS operating in both positive and negative mode with an m/z window of 70-600 and a resolution of 140,000. Total run time was 4.5 min per sample and a full report could be generated within 40 min. Concentrations of all 21 investigated diagnostic metabolites in DBS as quantified by chip-based nanoESI-HRMS correlated well with those obtained by targeted liquid chromatography-tandem mass spectrometry. We conclude that chip-based nanoESI-HRMS is suitable for quantification., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism., Methods: To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15 NH 4 Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice., Results: In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice., Conclusions: In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The conserved oligomeric Golgi (COG) complex consists of eight subunits and plays a crucial role in Golgi trafficking and positioning of glycosylation enzymes. Mutations in all COG subunits, except subunit 3, have been detected in patients with congenital disorders of glycosylation (CDG) of variable severity. So far, 3 families with a total of 10 individuals with biallelic COG6 mutations have been described, showing a broad clinical spectrum. Here we present 7 additional patients with 4 novel COG6 mutations. In spite of clinical variability, we delineate the core features of COG6-CDG i.e. liver involvement (9/10), microcephaly (8/10), developmental disability (8/10), recurrent infections (7/10), early lethality (6/10), and hypohidrosis predisposing for hyperthermia (6/10) and hyperkeratosis (4/10) as ectodermal signs. Regarding all COG6-related disorders a genotype-phenotype correlation can be discerned ranging from deep intronic mutations found in Shaheen syndrome as the mildest form to loss-of-function mutations leading to early lethal CDG phenotypes. A comparison with other COG deficiencies suggests ectodermal changes to be a hallmark of COG6-related disorders. Our findings aid clinical differentiation of this complex group of disorders and imply subtle functional differences between the COG complex subunits., (Copyright © 2015. Published by Elsevier Inc.)

Methylmalonic acid (MMA) and total homocysteine (tHCYS) concentrations are used to detect acquired and inborn errors of cobalamin (vitamin B12, Cbl) metabolism and to evaluate the effect of therapeutic interventions. Dried blood spot sampling offers a patient-friendly and easy alternative to plasma sampling. However, dried blood spot concentrations are not necessarily equal to plasma concentrations. Therefore, the objective of this work was to establish the relationship between MMA and tHYS dried blood spot and plasma concentrations to facilitate clinical implementation of dried blood spot sampling. MMA and tHCYS in both plasma and DBS were validated on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). While position of the punch (in DBS) did affect tHCYS concentration, no influence of hematocrit (Ht) and blood volume on both MMA and tHCYS concentrations was observed. The plasma assay performed better than the DBS assay by most criteria. However, the DBS matrix was superior for tHCYS stability. Paired plasma and DBS samples were obtained from patients suspected for Cbl deficiency and from patients with a known inborn error of metabolism affecting MMA or tHCYS concentration. Based on the strong correlation of tHCYS in both matrices (y=0.46±1.12 (r(2)=0.91)), determination of tHCYS in plasma can be replaced by tHCYS in DBS. However, for MMA, a correlation in the higher (pathological) range of MMA exist, but no correlation was observed in the lower ranges. Therefore the added value of MMA concentrations in DBS is currently unknown and should be further investigated., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To examine cognitive functioning in patients with tyrosinemia type I treated with nitisinone and a protein-restricted diet., Study Design: We performed a cross-sectional study to establish cognitive functioning in children with tyrosinemia type I compared with their unaffected siblings. Intelligence was measured using age-appropriate Wechsler Scales. To assess cognitive development over time, we retrieved sequential IQ scores in a single-center subset of patients. We also evaluated whether plasma phenylalanine and tyrosine levels during treatment was correlated with cognitive development., Results: Average total IQ score in 10 patients with tyrosinemia type I receiving nitisinone was significantly lower compared with their unaffected siblings (71 ± 13 vs 91 ± 13; P = .008). Both verbal and performance IQ subscores differed (77 ± 14 vs 95 ± 11; P < .05 and 70 ± 11 vs 87 ± 15; P < .05, respectively). Repeated IQ measurements in a single-center subset of 5 patients revealed a decline in average IQ score over time, from 96 ± 15 to 69 ± 11 (P < .001). No significant association was found between IQ score and either plasma tyrosine or phenylalanine concentration., Conclusion: Patients with tyrosinemia type I treated with nitisinone are at risk for impaired cognitive function despite a protein-restricted diet., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Objectives: Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to a defect in phenylalanine hydroxylase (PAH). Treatment principle is to reduce phenylalanine concentration sufficiently to prevent neuropathological effects. Dietary management is performed and the effect of treatment is monitored by regular analysis of phenylalanine and tyrosine. The aim of the study was to develop a rapid method to routinely measure both metabolites in minimal bloodspot volume (1.5 mm Ø, corresponding with a volume of 1.3 μL blood)., Method: Whole blood was spiked with phenylalanine and tyrosine at 24 different concentrations. Dried blood spots (DBS) were prepared, after which punches of 1.5 mm Ø and 6 mm Ø (corresponding with a volume of 12.4 μL) were taken. Additionally, punches of both sizes were prepared from DBS of PKU-patients (n=77). All samples were analyzed by tandem mass-spectrometry and results between both punches were compared., Results: A good correlation between concentrations of phenylalanine and tyrosine in 1.5 and 6 mm punches was found (r(2)=0.9917 and r(2)=0.9892, respectively). Analysis of phenylalanine and tyrosine in punches of PKU-patients (n=77) showed similar results and fitted within the procentual range of the between run variation., Conclusion: We developed an accurate and rapid method to analyze phenylalanine and tyrosine concentrations in a 1.5 mm Ø bloodspot punch with an estimated whole blood volume of 1.3 μL. This technical improvement does not only result in a 10 fold reduction in required patients' material, but also in a 30-60 min time saving in sample preparation., (Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)