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Background and aims: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). Materials and Methods: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. Results: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. Conclusion: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

Supplementary Figure 1 Examples of 1H NMR spectra for different genotypes A. Example of NMR spectrum of a sporadic tumor depicting the peak positions of lactic acid, acetic acid and catecholamines. B. Example of NMR spectrum of a SDHB tumor depicting peak position of succinic acid. C. Example of NMR spectrum of a RET tumor depicting ATP/ADP/AMP and adrenaline specific peaks. Supplementary Figure 2 Comparison of epinephrine and norepinephrine content in tumor tissues as determined by HPLC and 1H-NMR spectroscopy. Passing - Bablok regression plot to compare measurement of epinephrine and norepinephrine content in PGL tumor tissues by HPLC and 1HNMR spectroscopy expressed as μg per mg tissue in Log10 scale. Supplementary Figure 3: Activities of respiratory chain complex II and tumor tissue succinate content in SDH related tumors. Black bars represent complex II activity expressed in mU/mg protein and grey bars represent tumor tissue succinate content in mmol/mg tissue for each individual tumor. Represented on X axis is the hereditary mutation in the patient from whom the tumor was resected.

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Purpose: Pheochromocytomas and paragangliomas (PGL) are neuroendocrine tumors of sympathetic and parasympathetic paraganglia. This study investigated the relationships between genotype-specific differences in mitochondrial function and catecholamine content in PGL tumors.Experimental Design: Respiratory chain enzyme assays and 1H-nuclear magnetic resonance (NMR) spectroscopy at 500 MHz were conducted on homogenates of 35 sporadic PGLs and 59 PGLs from patients with hereditary mutations in succinate dehydrogenase subunits B and D (SDHB, SDHD), succinate dehydrogenase assembly factor 2, von Hippel-Lindau (VHL), rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and myc-associated factor X.Results: In SDHx-related PGLs, a significant decrease in complex II activity (P < 0.0001) and a significant increase in complex I, III, and IV enzyme activities were observed when compared to sporadic, RET, and NF1 tumors. Also, a significant increase in citrate synthase (P < 0.0001) enzyme activity was observed in SDHx-related PGLs when compared to sporadic-, VHL-, RET-, and NF1-related tumors. An increase in succinate accumulation (P < 0.001) and decrease in ATP/ADP/AMP accumulation (P < 0.001) was observed when compared to sporadic PGLs and PGLs of other genotypes. Positive correlations (P < 0.01) were observed between respiratory chain complex II activity and total catecholamine content and ATP/ADP/AMP and total catecholamine contents in tumor tissues.Conclusions: This study for the first time establishes a relationship between determinants of energy metabolism, like activity of respiratory chain enzyme complex II, ATP/ADP/AMP content, and catecholamine content in PGL tumors. Also, this study for the first time successfully uses NMR spectroscopy to detect catecholamines in PGL tumors and provides ex vivo evidence for the accumulation of succinate in PGL tumors with an SDHx mutation. Clin Cancer Res; 19(14); 3787–95. ©2013 AACR.

Infrared ion spectroscopy (IRIS) continues to see increasing use as an analytical tool for small-molecule identification in conjunction with mass spectrometry (MS). The IR spectrum of an m/z selected population of ions constitutes a unique fingerprint that is specific to the molecular structure. However, direct translation of an IR spectrum to a molecular structure remains challenging, as reference libraries of IR spectra of molecular ions largely do not exist. Quantum-chemically computed spectra can reliably be used as reference, but the challenge of selecting the candidate structures remains. Here we introduce an in silico library of vibrational spectra of common MS adducts of over 4500 compounds found in the human metabolome database (HMDB). In total, the library currently contains more than 75 000 spectra computed at the DFT level that can be queried with an experimental IR spectrum. Moreover, we introduce a database of 189 experimental IRIS spectra, which is employed to validate the automated spectral matching routines. This demonstrates that 75% of metabolites in the experimental dataset is correctly identified, based solely on their exact m/z and IRIS spectrum. Additionally, we demonstrate an approach for specifically identifying substructures by performing a search without m/z constraints to find structural analogues. Such an unsupervised search paves the way towards the de novo identification of unknowns that are absent in spectral libraries. We apply the in silico spectral library to identify an unknown in a plasma sample as 3-hydroyxhexanoic acid, highlighting the potential of the method.

Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5’-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII.

Traditionally, clinicians consider lactate as a waste product of anaerobic glycolysis. Interestingly, research has shown that lactate may serve as an alternative fuel for the brain to protect it against harm. The increasing scientific awareness of the potential beneficial side of lactate, however, is entering the clinic rather slowly. Following this, and realizing that the application of potential novel therapeutic strategies in pediatric populations often lags behind the development in adults, this review summarizes the key data on therapeutic use of intravenous infusion of sodium lactate in humans. PubMed and clinicaltrial.gov were searched up until November 2021 focusing on interventional studies in humans. Thirty-four articles were included in this review, with protocols of lactate infusion in adults with diabetes mellitus, traumatic brain injury, Alzheimer’s disease, and cardiac disease. One study on lactate infusion in children was also included. Results of our literature search show that sodium lactate can be safely administrated, without major side effects. Additionally, the present literature clearly shows the potential benefits of therapeutic lactate infusion under certain pathological circumstances, including rather common clinical conditions like traumatic brain injury.Conclusion: This review shows that lactate is a save, alternative energy source for the adult brain warranting studies on the potential therapeutic effects of sodium lactate infusion in children. What is Known:• Lactate is generally considered a waste product of anaerobic glycolysis. However, lactate also is an alternative fuel for different organs, including the brain.• Lactate infusion is not incorporated in standard care for any patient population. What is New:• Thirty-four studies investigated the therapeutic use of intravenous sodium lactate in different patient populations, all with different study protocols.• Literature shows that lactate infusion may have beneficial effects in case of hypoglycemia, traumatic brain injury, and cardiac failure without the risk of major side effects.

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic cerebrospinal fluid (CSF) profiles from 12 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3- glucose, and xylose-α1-3-xylose-α1-3- glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3- xylose-α1-3-glucose may originate from glycosylated proteins ; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O- glucosylation. Since many proteins are O- glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake.

Contains fulltext : 290351.pdf (Publisher’s version ) (Open Access)

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge" in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of alpha-aminoadipic semialdehyde (alpha-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While alpha-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic CSF profiles from 11 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from O-glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O-glucosylation. Since many proteins are O-glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake.

Contains fulltext : 229584.pdf (Publisher’s version ) (Open Access) Sjögren-Larsson syndrome (SLS) is a rare neurometabolic syndrome caused by deficient fatty aldehyde dehydrogenase. Patients exhibit intellectual disability, spastic paraplegia, and ichthyosis. The accumulation of fatty alcohols and fatty aldehydes has been demonstrated in plasma and skin but never in brain. Brain magnetic resonance imaging and spectroscopy studies, however, have shown an abundant lipid peak in the white matter of patients with SLS, suggesting lipid accumulation in the brain as well. Using histopathology, mass spectrometry imaging, and lipidomics, we studied the morphology and the lipidome of a postmortem brain of a 65-year-old female patient with genetically confirmed SLS and compared the results with a matched control brain. Histopathological analyses revealed structural white matter abnormalities with the presence of small lipid droplets, deficient myelin, and astrogliosis. Biochemically, severely disturbed lipid profiles were found in both white and gray matter of the SLS brain, with accumulation of fatty alcohols and ether lipids. Particularly, long-chain unsaturated ether lipid species accumulated, most prominently in white matter. Also, there was a striking accumulation of odd-chain fatty alcohols and odd-chain ether(phospho)lipids. Our results suggest that the central nervous system involvement in SLS is caused by the accumulation of fatty alcohols leading to a disbalance between ether lipid and glycero(phospho)lipid metabolism resulting in a profoundly disrupted brain lipidome. Our data show that SLS is not a pure leukoencephalopathy, but also a gray matter disease. Additionally, the histopathological abnormalities suggest that astrocytes and microglia might play a pivotal role in the underlying disease mechanism, possibly contributing to the impairment of myelin maintenance.

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4‐kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4‐phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl‐chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4‐kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3. Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

Contains fulltext : 225031.pdf (Publisher’s version ) (Open Access) 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.

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Acetyl-CoA transporter 1 (AT-1) is a transmembrane protein which regulates influx of acetyl-CoA from the cytosol to the lumen of the endoplasmic reticulum and is therefore important for the posttranslational modification of numerous proteins. Pathological variants in the SLC33A1 gene coding for AT-1 have been linked to a disorder called Huppke-Brendel syndrome, which is characterized by congenital cataracts, hearing loss, severe developmental delay and early death. It has been described in eight patients so far, who all had the abovementioned symptoms together with low serum copper and ceruloplasmin concentrations. The link between AT-1 and low ceruloplasmin concentrations is not clear, nor is the complex pathogenesis of the disease. Here we describe a further case of Huppke-Brendel syndrome with a novel and truncating homozygous gene variant and provide novel biochemical data on N- acetylated amino acids in cerebrospinal fluid (CSF) and plasma. Our results indicate that decreased levels of many N-acetylated amino acids in CSF are a typical metabolic fingerprint for AT- 1 deficiency and are potential biomarkers for the defect. As acetyl-CoA is an important substrate for protein acetylation, we performed N-terminal proteomics, but found only minor effects on this particular protein modification. The acetyl-CoA content in patient's fibroblasts was insignificantly decreased. Our data may help to better understand the mechanisms underlying the metabolic disturbances, the pathophysiology and the clinical phenotype of the disease.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5’-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.

International audience; Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing's syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies.

Contains fulltext : 282840.pdf (Publisher’s version ) (Open Access) Untargeted metabolomics (UM) allows for the simultaneous measurement of hundreds of metabolites in a single analytical run. The sheer amount of data generated in UM hampers its use in patient diagnostics because manual interpretation of all features is not feasible. Here, we describe the application of a pathway-based metabolite set enrichment analysis method to prioritise relevant biological pathways in UM data. We validate our method on a set of 55 patients with a diagnosed inherited metabolic disorder (IMD) and show that it complements feature-based prioritisation of biomarkers by placing the features in a biological context. In addition, we find that by taking enriched pathways shared across different IMDs, we can identify common drugs and compounds that could otherwise obscure genuine disease biomarkers in an enrichment method. Finally, we demonstrate the potential of this method to identify novel candidate biomarkers for known IMDs. Our results show the added value of pathway-based interpretation of UM data in IMD diagnostics context.

Contains fulltext : 282561.pdf (Publisher’s version ) (Open Access) Exome sequencing (ES) in the clinical setting of inborn metabolic diseases (IMDs) has created tremendous improvement in achieving an accurate and timely molecular diagnosis for a greater number of patients, but it still leaves the majority of patients without a diagnosis. In parallel, (personalized) treatment strategies are increasingly available, but this requires the availability of a molecular diagnosis. IMDs comprise an expanding field with the ongoing identification of novel disease genes and the recognition of multiple inheritance patterns, mosaicism, variable penetrance, and expressivity for known disease genes. The analysis of trio ES is preferred over singleton ES as information on the allelic origin (paternal, maternal, "de novo") reduces the number of variants that require interpretation. All ES data and interpretation strategies should be exploited including CNV and mitochondrial DNA analysis. The constant advancements in available techniques and knowledge necessitate the close exchange of clinicians and molecular geneticists about genotypes and phenotypes, as well as knowledge of the challenges and pitfalls of ES to initiate proper further diagnostic steps. Functional analyses (transcriptomics, proteomics, and metabolomics) can be applied to characterize and validate the impact of identified variants, or to guide the genomic search for a diagnosis in unsolved cases. Future diagnostic techniques (genome sequencing [GS], optical genome mapping, long-read sequencing, and epigenetic profiling) will further enhance the diagnostic yield. We provide an overview of the challenges and limitations inherent to ES followed by an outline of solutions and a clinical checklist, focused on establishing a diagnosis to eventually achieve (personalized) treatment.

Item does not contain fulltext BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessively inherited inborn error of metabolism. Neurological symptoms are considered to be a clinical hallmark of untreated adult patients. We describe a 'milder CTX phenotype', without neurological involvement. METHODS: We performed a retrospective patient file study in 79 genetically confirmed Dutch patients with CTX (55 patients aged ≥ 21 years) to study the clinical heterogeneity of CTX. We studied the frequency of adult patients with CTX without neurological involvement at diagnosis, in our Dutch cohort, and included a family from South Africa and patients from Italy, USA, Chile and Asia from the literature. RESULTS: In total, we describe 19 adult patients with CTX from 16 independent families, without neurological symptoms at diagnosis. A relatively small percentage (21%, n = 4) had a history of cataract. The majority, 84% (n = 16), presented with tendon xanthomas as the sole or predominant feature. The majority of patients showed increased plasma cholesterol levels. No correlation was found between this 'milder phenotype', the cholestanol levels and the CYP27A1 genotype. In addition, we describe three novel mutations in the CYP27A1 gene. CONCLUSIONS: This study shows the clinical heterogeneity of CTX, highlighting the existence of a 'milder phenotype', that is without neurological involvement at diagnosis. Adult patients with CTX may present with tendon xanthomas as the sole or predominant feature, mimicking familial hypercholesterolemia. It is important to realize that the absence of neurological symptoms does not rule out the development of future neurological symptoms. As CTX is a treatable disorder, early diagnosis and initiation of treatment when additional clinical signs occur is therefore essential.

Contains fulltext : 244271.pdf (Publisher’s version ) (Open Access) BACKGROUND: Treatment of animal models with ataxia telangiectasia (A-T) with nicotinamide riboside (NR) improved their neurological outcome and survival. OBJECTIVE: The aim of this study is to investigate the effects of NR in patients with A-T. METHODS: In this open-label, proof-of-concept study, 24 patients with A-T were treated with NR during four consecutive months. The effects of NR on ataxia, dysarthria, quality of life, and laboratory parameters were analyzed. RESULTS: During treatment, ataxia scores improved; mean total Scale for the Assessment and Rating of Ataxia and International Cooperative Ataxia Rating Scale scores decreased to 2.4 and 10.1 points, respectively. After NR withdrawal, ataxia scores worsened. In immunodeficient patients, the mean serum IgG concentration increased substantially until the end of the study period with 0.52 g/L. Untargeted metabolomics analysis revealed increased plasma levels of NR metabolites and purine nucleosides during treatment. Adverse effects did not occur. CONCLUSIONS: Treatment with NR is tolerated well and associated with improvement in ataxia and serum immunoglobulin concentrations in patients with A-T. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Contains fulltext : 237516.pdf (Publisher’s version ) (Open Access) BackgroundPyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5'-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available.MethodsWe combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening.ResultsWe identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system.ConclusionThis study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.FundingSociety for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC).

Liquid chromatography-mass spectrometry (LC-MS) is, due to its high sensitivity and selectivity, currently the method of choice in (bio)analytical studies involving the (comprehensive) profiling of metabolites in body fluids. However, as closely related isomers are often hard to distinguish on the basis of LC-MS(MS) and identification is often dependent on the availability of reference standards, the identification of the chemical structures of detected mass spectral features remains the primary limitation. Infrared ion spectroscopy (IRIS) aids identification of MS-detected ions by providing an infrared (IR) spectrum containing structural information for a detected MS-feature. Moreover, IR spectra can be routinely and reliably predicted for many types of molecular structures using quantum-chemical calculations, potentially avoiding the need for reference standards. In this work, we demonstrate a workflow for reference-free metabolite identification that combines experiments based on high-pressure liquid chromatography (HPLC), MS and IRIS with quantum-chemical calculations that efficiently generate IR spectra and give the potential to enable reference-standard free metabolite identification. Additionally, a scoring procedure is employed which shows the potential for automated structure assignment of unknowns. Via a simple, illustrative example where we identify lysine in the plasma of a hyperlysinemia patient, we show that this approach allows the efficient assignment of a database-derived molecular structure to an unknown.

Objective SLC13A3 encodes the plasma membrane Na+ /dicarboxylate cotransporter 3, which imports inside the cell 4 to 6 carbon dicarboxylates as well as N-acetylaspartate (NAA). SLC13A3 is mainly expressed in kidney, in astrocytes, and in the choroid plexus. We describe two unrelated patients presenting with acute, reversible (and recurrent in one) neurological deterioration during a febrile illness. Both patients exhibited a reversible leukoencephalopathy and a urinary excretion of α-ketoglutarate (αKG) that was markedly increased and persisted over time. In one patient, increased concentrations of cerebrospinal fluid NAA and dicarboxylates (including αKG) were observed. Extensive workup was unsuccessful, and a genetic cause was suspected. Methods Whole exome sequencing (WES) was performed. Our teams were connected through GeneMatcher. Results WES analysis revealed variants in SLC13A3. A homozygous missense mutation (p.Ala254Asp) was found in the first patient. The second patient was heterozygous for another missense mutation (p.Gly548Ser) and an intronic mutation affecting splicing as demonstrated by reverse transcriptase polymerase chain reaction performed in muscle tissue (c.1016 + 3A > G). Mutations and segregation were confirmed by Sanger sequencing. Functional studies performed on HEK293T cells transiently transfected with wild-type and mutant SLC13A3 indicated that the missense mutations caused a marked reduction in the capacity to transport αKG, succinate, and NAA. Interpretation SLC13A3 deficiency causes acute and reversible leukoencephalopathy with marked accumulation of αKG. Urine organic acids (especially αKG and NAA) and SLC13A3 mutations should be screened in patients presenting with unexplained reversible leukoencephalopathy, for which SLC13A3 deficiency is a novel differential diagnosis. ANN NEUROL 2019;85:385-395.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Rationale & Objective Lecithin-cholesterol acyltransferase (LCAT) catalyzes the maturation of high-density lipoprotein. Homozygosity for loss-of-function mutations causes familial LCAT deficiency (FLD), characterized by corneal opacities, anemia, and renal involvement. This study sought to characterize kidney biopsy findings and clinical outcomes in a family with FLD. Study Design Prospective observational study. Setting & Participants 2 (related) index patients with clinically apparent FLD were initially identified. 110 of 122 family members who consented to genetic analysis were also studied. Predictors Demographic and laboratory parameters (including lipid profiles and LCAT activity) and full sequence analysis of the LCAT gene. Kidney histologic examination was performed with samples from 6 participants. Outcomes Cardiovascular and renal events during a median follow-up of 12 years. Estimation of annual rate of decline in glomerular filtration rate. Analytical Approach Analysis of variance, linear regression analysis, and Fine-Gray competing-risk survival analysis. Results 9 homozygous, 57 heterozygous, and 44 unaffected family members were identified. In all affected individuals, full sequence analysis of the LCAT gene revealed a mutation (c.820C>T) predicted to cause a proline to serine substitution at amino acid 274 (P274S). Homozygosity caused a complete loss of LCAT activity. Kidney biopsy findings demonstrated lipid deposition causing glomerular basement membrane thickening, mesangial expansion, and "foam-cell" infiltration of kidney tissue. Tubular atrophy, glomerular sclerosis, and complement fixation were associated with worse kidney outcomes. Estimated glomerular filtration rate deteriorated among homozygous family members at an average annual rate of 3.56 mL/min/1.73 m2. The incidence of cardiovascular and renal complications was higher among homozygous family members compared with heterozygous and unaffected members. Mild thrombocytopenia was a common finding among homozygous participants. Limitations The presence of cardiovascular disease was mainly based on medical history. Conclusions The P274S LCAT mutation was found to cause FLD with renal involvement. Tubular atrophy, glomerular sclerosis, and complement fixation were associated with a worse renal prognosis.

Item does not contain fulltext Cerebrotendinous xanthomatosis (CTX) is an inborn error of cholesterol and bile acid metabolism, leading to neuropsychiatric and systemic manifestations. Movement disorders have rarely been reported in CTX, while a detailed appreciation of the full phenotypic spectrum is required in order to prevent underdiagnosis of this disease. This review focuses on the frequency of more unusual, non-ataxia and non-spasticity movement disorders reported in CTX. In total, 39 articles were reviewed, describing 55 CTX patients with a movement disorder. Additionally, we report on seven patients with parkinsonism out of our Dutch cohort of 79 (77 genetically proven) CTX patients. Mean age at onset of the movement disorder was 40+/-12 years (median 40, range 13-62 years). Movement disorders can be considered a late disease manifestation. Parkinsonism was the most frequently reported movement disorder, followed by dystonia, myoclonus and postural tremor. Movement disorders were found to be mixed in 23% of patients and were usually part of a complex clinical picture, rather than a prominent symptom. Still, in 18% of the cases, a movement disorder was the presenting symptom. Unusual movement disorders represent a rare clinical feature in CTX, but CTX should be considered in the differential diagnosis of these movement disorders, particularly in case of early onset, and when associated with other neurological features (especially cognitive impairment, pyramidal and cerebellar signs) and/or with systemic features (such as diarrhoea, cataract and tendon xanthomas). CTX is a treatable disorder, stressing the importance of considering CTX as a potential cause of movement disorders.

Bi-allelic pathogenic variants in ZBTB11 have been associated with intellectual developmental disorder, autosomal recessive 69 (MRT69; OMIM 618383). We report five patients from three families with novel, bi-allelic variants in ZBTB11. We have expanded the clinical phenotype of MRT69, documenting varied severity of atrophy affecting different brain regions and described combined malonic and methylmalonic aciduria as a biochemical manifestation. As ZBTB11 encodes for a transcriptional regulator, we performeded chromatin immunoprecipitation–sequencing targeting ZBTB11 in fibroblasts from patients and controls. Chromatin immunoprecipitation–sequencing revealed binding of wild-type ZBTB11 to promoters in 238 genes, among which genes encoding proteins involved in mitochondrial functions and RNA processing are over-represented. Mutated ZBTB11 showed reduced binding to 61 of the targeted genes, indicating that the variants act as loss of function. Most of these genes are related to mitochondrial functions. Transcriptome analysis of the patient fibroblasts revealed dysregulation of mitochondrial functions. In addition, we uncovered that reduced binding of the mutated ZBTB11 to ACSF3 leads to decreased ACSF3 transcript level, explaining combined malonic and methylmalonic aciduria. Collectively, these results expand the clinical spectrum of ZBTB11-related neurological disease and give insight into the pathophysiology in which the dysfunctional ZBTB11 affect mitochondrial functions and RNA processing contributing to the neurological and biochemical phenotypes.

Untargeted LC-MS based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification – confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids, and are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly rendered novel insights in the pathophysiology of PDE-ALDH7A1.

Peripheral decarboxylase inhibitors (PDIs) prevent conversion of levodopa to dopamine in the blood by the enzyme aromatic L-amino acid decarboxylase (AADC). Alterations in enzyme activity may contribute to the required higher dosages of levodopa observed in many patients with Parkinson’s disease. We evaluated the effect of levodopa/PDI use on serum AADC enzyme activity. Serum AADC enzyme activity was evaluated in three independent cohorts of patients with Parkinson’s disease or parkinsonism (n = 301) and compared between patients on levodopa/PDI vs. patients not on this medication. AADC enzyme activity was elevated in 62% of patients on levodopa/PDI treatment, compared to 19% of patients not on levodopa/PDI (median 90 mU/L vs. 50 mU/L, p

Monoamine oxidase A (MAO‐A) deficiency is a rare inborn error of metabolism with impaired degradation of biogenic amines including 5‐hydroxytryptamine (5‐HT), resulting in borderline intellectual disability and behavioral abnormalities. Genetic variants in MAOA need functional confirmation to enable a definite diagnosis. To this end, we developed an inexpensive, simple and nonradioactive MAO‐A activity assay based on the conversion of 5‐HT into 5‐hydroxyindoleacetic acid (5‐HIAA). Fibroblast cell lysates were incubated with 5‐HT and aldehyde dehydrogenase to allow 5‐HIAA production. 5‐HIAA was quantified using high‐performance liquid chromatography with fluorimetric detection. We optimized reaction mixture components, pH, and substrate concentration and tested linearity and specificity of the assay. We verified the functional validity of the enzyme assay using fibroblasts of controls, female mutation carriers and MAO‐A deficient patients. This included a newly described patient with a novel MAOA variant (c.1336G>A, p.(Glu446Lys)), who represents the fifth MAO‐A deficiency family so far. The optimized enzyme assay showed good linearity and specificity. Application to clinical samples showed a 100% differentiation of affected patients (with negligible MAO‐A enzyme activity) and controls or mutation carriers. In conclusion, the described MAO‐A activity assay is easy to implement and can readily be used to test the pathogenicity of variants in the MAOA gene in a clinical setting. Especially in this era of whole‐exome (and whole‐genome) sequencing, this functional assay fulfills a clinical need for functional confirmation of a suspected diagnosis of MAO‐A deficiency.

Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn error of lysine metabolism that presents with refractory epilepsy in newborns. Bi-allelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important co-factor pyridoxal-5’-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but despite this treatment, intellectual disability may occur. Early diagnosis and treatment, preferably based on newborn screening, potentially optimize long-term clinical outcome. However, the currently known diagnostic PDE-ALDH7A1 biomarkers are incompatible with newborn screening procedures. Using a combination of the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we have been able to discover novel biomarkers for PDE-ALDH7A1: 2S,6S-and 2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP). We demonstrate the applicability of 2-OPP as a PDE-ALDH7A1 biomarker in newborn screening. Additionally, we show that 2-OPP accumulates in brain tissue of patients and Aldh7a1 knock-out mice, and induces epilepsy-like behavior in a zebrafish model system. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.

Purpose Pheochromocytomas and Paragangliomas (PPGL) result in chronic catecholamine excess and serious health complications. A recent study obtained a metabolic signature in plasma from PPGL patients; however, its targeted nature may have generated an incomplete picture and a broader approach could provide additional insights. We aimed to characterize the plasma metabolome of PPGL patients before and after surgery, using an untargeted approach, and to broaden the scope of the investigated metabolic impact of these tumors. Design A cohort of 36 PPGL patients was investigated. Blood plasma samples were collected before and after surgical tumor removal, in association with clinical and tumor characteristics. Methods Plasma samples were analyzed using untargeted nuclear magnetic resonance (NMR) spectroscopy metabolomics. The data were evaluated using a combination of uni- and multi-variate statistical methods. Results Before surgery, patients with a nonadrenergic tumor could be distinguished from those with an adrenergic tumor based on their metabolic profiles. Tyrosine levels were significantly higher in patients with high compared to those with low BMI. Comparing subgroups of pre-operative samples with their post-operative counterparts, we found a metabolic signature that included ketone bodies, glucose, organic acids, methanol, dimethyl sulfone and amino acids. Three signals with unclear identities were found to be affected. Conclusions Our study suggests that the pathways of glucose and ketone body homeostasis are affected in PPGL patients. BMI-related metabolite levels were also found to be altered, potentially linking muscle atrophy to PPGL. At baseline, patient metabolomes could be discriminated based on their catecholamine phenotype.

The identification of disease biomarkers plays a crucial role in developing diagnostic strategies for inborn errors of metabolism and understanding their pathophysiology. A primary metabolite that accumulates in the inborn error phenylketonuria is phenylalanine, however its levels do not always directly correlate with clinical outcomes. Here we combine infrared ion spectroscopy and NMR spectroscopy to identify the Phe-glucose Amadori rearrangement product as a biomarker for phenylketonuria. Additionally, we find analogous amino acid-glucose metabolites formed in the body fluids of patients accumulating methionine, lysine, proline and citrulline. Amadori rearrangement products are well-known intermediates in the formation of advanced glycation end-products and have been associated with the pathophysiology of diabetes mellitus and ageing, but are now shown to also form under conditions of aminoacidemia. They represent a general class of metabolites for inborn errors of amino acid metabolism that show potential as biomarkers and may provide further insight in disease pathophysiology., Rianne van Outersterp et al. combine mass spectrometry, NMR, and infrared ion spectroscopy to identify amino acid-hexose conjugates in the blood plasma from patients with metabolic disorders such as phenylketonuria (PKU). These conjugates, or Amadori rearrangement products, are generally not detectable in blood samples from unaffected individuals, and may therefore represent disease biomarkers.

Contains fulltext : 234019.pdf (Publisher’s version ) (Open Access) Dopamine beta hydroxylase (DBH) deficiency is an extremely rare autosomal recessive disorder with severe orthostatic hypotension, that can be treated with L-threo-3,4-dihydroxyphenylserine (L-DOPS). We aimed to summarize clinical, biochemical, and genetic data of all world-wide reported patients with DBH-deficiency, and to present detailed new data on long-term follow-up of a relatively large Dutch cohort. We retrospectively describe 10 patients from a Dutch cohort and 15 additional patients from the literature. We identified 25 patients (15 females) from 20 families. Ten patients were diagnosed in the Netherlands. Duration of follow-up of Dutch patients ranged from 1 to 21 years (median 13 years). All patients had severe orthostatic hypotension. Severely decreased or absent (nor)epinephrine, and increased dopamine plasma concentrations were found in 24/25 patients. Impaired kidney function and anemia were present in all Dutch patients, hypomagnesaemia in 5 out of 10. Clinically, all patients responded very well to L-DOPS, with marked reduction of orthostatic complaints. However, orthostatic hypotension remained present, and kidney function, anemia, and hypomagnesaemia only partially improved. Plasma norepinephrine increased and became detectable, while epinephrine remained undetectable in most patients. We confirm the core clinical characteristics of DBH-deficiency and the pathognomonic profile of catecholamines in body fluids. Impaired renal function, anemia, and hypomagnesaemia can be part of the clinical presentation. The subjective response to L-DOPS treatment is excellent and sustained, although the neurotransmitter profile in plasma does not normalize completely. Furthermore, orthostatic hypotension as well as renal function, anemia, and hypomagnesaemia improve only partially.

Inborn errors of metabolism (IEM) are inherited conditions caused by genetic defects in enzymes or cofactors. These defects result in a specific metabolic fingerprint in patient body fluids, showing accumulation of substrate or lack of an end-product of the defective enzymatic step. Untargeted metabolomics has evolved as a high throughput methodology offering a comprehensive readout of this metabolic fingerprint. This makes it a promising tool for diagnostic screening of IEM patients. However, the size and complexity of metabolomics data have posed a challenge in translating this avalanche of information into knowledge, particularly for clinical application. We have previously established next-generation metabolic screening (NGMS) as a metabolomics-based diagnostic tool for analyzing plasma of individual IEM-suspected patients. To fully exploit the clinical potential of NGMS, we present a computational pipeline to streamline the analysis of untargeted metabolomics data. This pipeline allows for time-efficient and reproducible data analysis, compatible with ISO:15189 accredited clinical diagnostics. The pipeline implements a combination of tools embedded in a workflow environment for large-scale clinical metabolomics data analysis. The accompanying graphical user interface aids end-users from a diagnostic laboratory for efficient data interpretation and reporting. We also demonstrate the application of this pipeline with a case study and discuss future prospects.

Contains fulltext : 238254.pdf (Publisher’s version ) (Closed access) PURPOSE: To investigate monoallelic CLPB variants. Pathogenic variants in many genes cause congenital neutropenia. While most patients exhibit isolated hematological involvement, biallelic CLPB variants underlie a neurological phenotype ranging from nonprogressive intellectual disability to prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, 3-methylglutaconic aciduria, and neutropenia. CLPB was recently shown to be a mitochondrial refoldase; however, the exact function remains elusive. METHODS: We investigated six unrelated probands from four countries in three continents, with neutropenia and a phenotype dominated by epilepsy, developmental issues, and 3-methylglutaconic aciduria with next-generation sequencing. RESULTS: In each individual, we identified one of four different de novo monoallelic missense variants in CLPB. We show that these variants disturb refoldase and to a lesser extent ATPase activity of CLPB in a dominant-negative manner. Complexome profiling in fibroblasts showed CLPB at very high molecular mass comigrating with the prohibitins. In control fibroblasts, HAX1 migrated predominantly as monomer while in patient samples multiple HAX1 peaks were observed at higher molecular masses comigrating with CLPB thus suggesting a longer-lasting interaction between CLPB and HAX1. CONCLUSION: Both biallelic as well as specific monoallelic CLPB variants result in a phenotypic spectrum centered around neurodevelopmental delay, seizures, and neutropenia presumably mediated via HAX1.

Many inborn errors of metabolism (IEMs) are amenable to treatment, therefore early diagnosis is imperative. Whole-exome sequencing (WES) variant prioritization coupled with phenotype-guided clinical and bioinformatics expertise is typically used to identify disease-causing variants; however, it can be challenging to identify the causal candidate gene when a large number of rare and potentially pathogenic variants are detected. Here, we present a network-based approach, metPropagate, that uses untargeted metabolomics (UM) data from a single patient and a group of controls to prioritize candidate genes in patients with suspected IEMs. We validate metPropagate on 107 patients with IEMs diagnosed in Miller et al. (2015) and 11 patients with both CNS and metabolic abnormalities. The metPropagate method ranks candidate genes by label propagation, a graph-smoothing algorithm that considers each gene’s metabolic perturbation in addition to the network of interactions between neighbors. metPropagate was able to prioritize at least one causative gene in the top 20th percentile of candidate genes for 92% of patients with known IEMs. Applied to patients with suspected neurometabolic disease, metPropagate placed at least one causative gene in the top 20th percentile in 9/11 patients, and ranked the causative gene more highly than Exomiser’s phenotype-based ranking in 6/11 patients. Interestingly, ranking by a weighted combination of metPropagate and Exomiser scores resulted in improved prioritization. The results of this study indicate that network-based analysis of UM data can provide an additional mode of evidence to prioritize causal genes in patients with suspected IEMs.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality., Author summary Biallelic inactivating mutations in the progressive ankylosis gene (ANKH/Ank, humans/mice) cause a severe form of arthritis, primarily affecting joints of hands, feet and the spine. Low extracellular concentrations of the mineralization inhibitor pyrophosphate underlie the joint calcification in mice and humans that are devoid of functional Ank or ANKH, respectively. It was previously thought that ANKH mediates the release of pyrophosphate from the cytosol to the extracellular environment. We here show, however, that PPi is formed in the extracellular environment by Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1), from ATP released via ANKH. Untargeted metabolomics further revealed that ANKH provides a pathway for the release of nucleoside triphosphates in general and, unexpectedly, the Krebs-cycle intermediate citrate. Mice lacking Ank activity (Ankank/ank mice), have substantially reduced concentrations of citrate in their plasma and urine. Citrate was even undetectable in urine of a human patient lacking functional ANKH. Bones of Ankank/ank mice have reduced mechanical strength and their bone matrix contains dramatically reduced levels of PPi and citrate. Our data demonstrate that ANKH/Ank crucially contributes to the disposition of citrate and PPi in vivo and profoundly affects bone quality. Extracellular citrate is ubiquitous, and additional phenotypes are therefore expected to be associated with absence of Ank activity.

Following publication of the original article, the authors would like to correct a sentence in the paragraph “1H-NMR spectra were recorded at 298 K…” under the heading “NMR experiments”. The sentence currently reads: “The LED pulse sequence had the form -RD-901-G1- 1801-G1-901-G2-T-901-G1-1801-G1-901-G2-t-901-acquire FID, where RD is a relaxation delay, 901 is a 901 RF pulse, G1 is the pulsed-field gradient that is applied to allow editing, 1801 is a 1801 RF pulse, G2 is a spoil gradient applied to remove unwanted magnetization components. The diffusion delay Δ is the time during which the molecules are allowed to diffuse—this is the period (901-G1-1801-G1- 901-G2-T-); and t is a delay to allow the longitudinal eddy currents caused within the sample to decay (Beckonert et al. 2007).” The sentence should read: “The LED pulse sequence had the form -RD-90°-G1- 180°-G1-90°-G2-T-90°-G1-180°-G1-90°-G2-t-90°-acquire FID, where RD is a relaxation delay, 90° is a 90° RF pulse, G1 is the pulsed-field gradient that is applied to allow editing, 180° is a 180° RF pulse, G2 is a spoil gradient applied to remove unwanted magnetization components. The diffusion delay ? is the time during which the molecules are allowed to diffuse—this is the period (90°-G1-180°-G1-90°-G2 T-); and t is a delay to allow the longitudinal eddy currents caused within the sample to decay (Beckonert et al. 2007).” This has been corrected with this erratum.

Purpose BiallelicCADvariants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. Methods Retrospective case series of 20 patients. Results Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. Conclusion We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

Many inborn errors of metabolism (IEMs) are amenable to treatment, therefore early diagnosis before irreversible damage occurs is imperative. Despite recent advances, the genetic basis of many metabolic phenotypes remains unknown. For discovery purposes, Whole Exome Sequencing (WES) variant prioritization coupled with phenotype-guided clinical and bioinformatics expertise is currently the primary method used to identify novel disease-causing variants; however, it can be challenging to identify the causal candidate gene given the large number of plausible variants. Using untargeted metabolomics (UM) to prioritize metabolically relevant candidate genes is a promising approach to diagnosing known or novel IEMs in a single patient. Here, we present a network-based bioinformatics approach, metPropagate, that uses UM data from a single patient and a group of controls to prioritize candidate genes. We validate metProp on 107 patients with diagnosed IEMs and 11 patients with novel IEMs diagnosed through the TIDE gene discovery project at BC Children’s Hospital. The metPropagate method ranks candidate genes by considering the network of interactions between them. This is done by using a graph smoothing algorithm called label propagation to quantify the metabolic disruption in genes’ local neighbourhood. metPropagate was able to prioritize the causative gene in the top 20th percentile of candidate genes for 91% of patients with known IEM disorders. For novel IEMs, metPropagate placed the causative gene in the top 20th percentile in 9/11 patients. Using metPropagate, the causative gene was ranked higher than Exomiser’s phenotype-based ranking in 6/11 patients. The results of this study indicate that for diagnostic and gene discovery purposes, network-based analysis of metabolomics data can lend support to WES gene-discovery methods by providing an additional mode of evidence to help identify causal genes.

Timely diagnosis is essential for patients with neurometabolic disorders to enable targeted treatment. Next‐Generation Metabolic Screening (NGMS) allows for simultaneous screening of multiple diseases and yields a holistic view of disturbed metabolic pathways. We applied this technique to define a cerebrospinal fluid (CSF) reference metabolome and validated our approach with patients with known neurometabolic disorders. Samples were measured using ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry followed by (un)targeted analysis. For the reference metabolome, CSF samples from patients with normal general chemistry results and no neurometabolic diagnosis were selected and grouped based on sex and age (0‐2/2‐5/5‐10/10‐15 years). We checked the levels of known biomarkers in CSF from seven patients with five different neurometabolic disorders to confirm the suitability of our method for diagnosis. Untargeted analysis of 87 control CSF samples yielded 8036 features for semiquantitative analysis. No sex differences were found, but 1782 features (22%) were different between age groups (q

Introduction When analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr–Purcell–Meiboom–Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites. Objectives Our aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples. Methods We compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares–Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated. Results We found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation ( Conclusion Overall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.

Background: Overweight and obesity can lead to adipose tissue inflammation, which causes insulin resistance and on the long-term type 2 diabetes mellitus (T2D). The inflammatory changes of obese-adipose tissue are characterized by macrophage infiltration and activation, but validated circulating biomarkers for adipose tissue inflammation for clinical use are still lacking. One of the most secreted enzymes by activated macrophages is chitotriosidase (CHIT1). Objective: To test whether circulating CHIT1 enzymatic activity levels reflect adipose tissue inflammation. Methods: Plasma and adipose tissue samples of 105 subjects (35 lean, 37 overweight, and 33 T2D patients) were investigated. CHIT1 mRNA levels were determined in adipose tissue-resident innate immune cells. CHIT1 mRNA levels, protein abundance, and plasma enzymatic activity were subsequently measured in adipose tissue biopsies and plasma of control subjects with varying levels of obesity and adipose tissue inflammation as well as in T2D patients. Results: In adipose tissue, CHIT1 mRNA levels were higher in stromal vascular cells compared to adipocytes, and higher in adipose tissue-residing macrophages compared to circulating monocytes (p < 0.001). CHIT1 mRNA levels in adipose tissue were enhanced in overweightcompared to lean subjects and even more in T2D patients (p < 0.05). In contrast, plasma CHIT1 enzymatic activity did not differ between lean, overweight subjects and T2D patients. A mutation of the CHIT1 gene decreases plasma CHIT1 activity. Conclusions: CHIT1 is expressed by adipose tissue macrophages and expression is higher in overweight subjects and T2D patients, indicating its potential as tissue biomarker for adipose tissue inflammation. However, these differences do not translate into different plasma CHIT1 activity levels. Moreover, a common CHIT1 gene mutation causing loss of plasma CHIT1 activity interferes with its use as a biomarker of adipose tissue inflammation. These results indicate that plasma CHIT1 activity is of limited value as a circulating biomarker for adipose tissue inflammation in human subjects.

The membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of mineralization. Using global metabolite profiling, we identified citrate as the most prominent metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release of ATP and other nucleoside triphosphates.Ankank/ankmice, which lack functional ANK, had substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable in urine of a human patient lacking functional ANKH. Bone hydroxyapatite ofAnkank/ankmice also contained markedly reduced levels of citrate and PPi and displayed diminished strength. Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis that is essential for normal bone development.

Contains fulltext : 219662.pdf (Publisher’s version ) (Open Access)