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The implementation of whole-exome sequencing in clinical diagnostics has generated a need for functional evaluation of genetic variants. In the field of inborn errors of metabolism (IEM), a diverse spectrum of targeted biochemical assays is employed to analyze a limited amount of metabolites. We now present a single-platform, high-resolution liquid chromatography quadrupole time of flight (LC-QTOF) method that can be applied for holistic metabolic profiling in plasma of individual IEM-suspected patients. This method, which we termed “next-generation metabolic screening” (NGMS), can detect >10,000 features in each sample. In the NGMS workflow, features identified in patient and control samples are aligned using the “various forms of chromatography mass spectrometry (XCMS)” software package. Subsequently, all features are annotated using the Human Metabolome Database, and statistical testing is performed to identify significantly perturbed metabolite concentrations in a patient sample compared with controls. We propose three main modalities to analyze complex, untargeted metabolomics data. First, a targeted evaluation can be done based on identified genetic variants of uncertain significance in metabolic pathways. Second, we developed a panel of IEM-related metabolites to filter untargeted metabolomics data. Based on this IEM-panel approach, we provided the correct diagnosis for 42 of 46 IEMs. As a last modality, metabolomics data can be analyzed in an untargeted setting, which we term “open the metabolome” analysis. This approach identifies potential novel biomarkers in known IEMs and leads to identification of biomarkers for as yet unknown IEMs. We are convinced that NGMS is the way forward in laboratory diagnostics of IEMs. Electronic supplementary material The online version of this article (10.1007/s10545-017-0131-6) contains supplementary material, which is available to authorized users.

Selenium-binding protein 1 (SELENBP1) has been associated with several cancers, although its exact role is unknown. We show that SELENBP1 is a methanethiol oxidase (MTO), related to the MTO in methylotrophic bacteria, that converts methanethiol to H2O2, formaldehyde, and H2S, an activity not previously known to exist in humans. We identified mutations in SELENBP1 in five patients with cabbage-like breath odor. The malodor was attributable to high levels of methanethiol and dimethylsulfide, the main odorous compounds in their breath. Elevated urinary excretion of dimethylsulfoxide was associated with MTO deficiency. Patient fibroblasts had low SELENBP1 protein levels and were deficient in MTO enzymatic activity; these effects were reversed by lentivirus-mediated expression of wild-type SELENBP1. Selenbp1-knockout mice showed biochemical characteristics similar to those in humans. Our data reveal a potentially frequent inborn error of metabolism that results from MTO deficiency and leads to a malodor syndrome. info:eu-repo/semantics/publishedVersion

Metabolomics is the discipline where endogenous and exogenous metabolites are assessed, identified and quantified in different biological samples. Metabolites are crucial components of biological system and highly informative about its functional state, due to their closeness to functional endpoints and to the organism's phenotypes. Nuclear Magnetic Resonance (NMR) spectroscopy, next to Mass Spectrometry (MS), is one of the main metabolomics analytical platforms. The technological developments in the field of NMR spectroscopy have enabled the identification and quantitative measurement of the many metabolites in a single sample of biofluids in a non-targeted and non-destructive manner. Combination of NMR spectra of biofluids and pattern recognition methods has driven forward the application of metabolomics in the field of biomarker discovery. The importance of metabolomics in diagnostics, e.g. in identifying biomarkers or defining pathological status, has been growing exponentially as evidenced by the number of published papers. In this review, we describe the developments in data acquisition and multivariate analysis of NMR-based metabolomics data, with particular emphasis on the metabolomics of Cerebrospinal Fluid (CSF) and biomarker discovery in Multiple Sclerosis (MScl).

In solids spinning at the magic angle, the indirect detection of single-quantum (SQ) and double-quantum (DQ) N-14 spectra (l = 1) via spy nuclei S = 1/2 such as protons can be achieved in the manner of heteronuclear single- or multiple-quantum correlation (HSQC or HMQC) spectroscopy. The HMQC method relies on the excitation of two-spin coherences of the type T-11(l) T-11(S) and T-21(l) T-11(S) at the beginning of the evolution interval t(1). The spectra obtained by Fourier transformation from t(1) to omega(1) may be broadened by the homogenous decay of the transverse terms of the spy nuclei S. This broadening is mostly due to homonuclear dipolar S-S' interactions between the proton spy nuclei. In this work we have investigated the possibility of inserting rotor-synchronized symmetry-based C or R sequences and decoupling schemes such as Phase-Modulated Lee-Goldburg (PMLG) sequences in the evolution period. These schemes reduce the homonuclear proton-proton interactions and lead to all enhancement of the resolution of both SQ and DQ proton-detected N-14 HMQC spectra. In addition, we have investigated the combination of HSQC with symmetry-based sequences and PMLG and shown that the highest resolution in the N-14 dimension is achieved by using HSQC in combination with symmetry-based sequences of the R-type. We show improvements in resolution in samples of L-alanine and the tripeptide ala-ala-gly (AAG). In particular, for L-alanine the width of the N-14 SQ peak is reduced from 2 to 1.2 kHz, in agreement with simulations. We report accurate measurements of quadrupolar coupling constants and asymmetry parameters for amide N-14 in AAG peptide bonds. (C) 2009 Elsevier Inc. All rights reserved.

This thesis describes new methods for the detection of 14N nuclei by solid-state nuclear magnetic resonance. So far, the low natural abundance (0.4 %) 15N isotope has been widely used to study nitrogen-containing samples because of its spin-1/2 nature. The limited use of the spin-1 isotope 14N (natural abundance 99.6 %) in NMR is due to its strong quadrupolar coupling constant, which leads to very broad spectra that are difficult to excite uniformly and equally difficult to detect, requiring broad receiver bandwidths. The new methods presented in this work result from two main projects covering the indirect detection of 14N via carbon and via protons. The indirect detection of 14N can be achieved by heteronuclear multiple- or single-quantum correlation (HMQC or HSQC) experiments, which rely on the transfer of coherence from a neighboring spin S = 1/2 (13C or 1H) to single- (SQ) or double-quantum (DQ) transitions of 14N nuclei. These methods allow one to observe 14N powder patterns with enhanced sensitivity. These spectra depend on the quadrupolar coupling constant CQ (typically a few MHz), the asymmetry parameter ηQ, and on the orientation of the internuclear vector rIS between the I (14N) and S (13C or 1H) nuclei with respect to the quadrupolar tensor. These parameters reveal valuable information about the structure and dynamics of nitrogen-containing solids. The indirect detection methods are discussed in detail, covering the optimization of experimental parameters; subsequently, applications to the study of amino acids at different magnetic fields are demonstrated.