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Succinate-CoA ligase (SUCL) is a heterodimer consisting of an alpha subunit encoded by SUCLG1, and a beta subunit encoded by either SUCLA2 or SUCLG2 catalyzing an ATP- or GTP-forming reaction, respectively, in the mitochondrial matrix. The deficiency of this enzyme represents an encephalomyopathic form of mtDNA depletion syndromes. We describe the fatal clinical course of a female patient with a pathogenic mutation in SUCLG1 (c.626C > A, p.Ala209Glu) heterozygous at the genomic DNA level, but homozygous at the transcriptional level. The patient exhibited early-onset neurometabolic abnormality culminating in severe brain atrophy and dystonia leading to death by the age of 3.5 years. Urine and plasma metabolite profiling was consistent with SUCL deficiency which was confirmed by enzyme analysis and lack of mitochondrial substrate-level phosphorylation (mSLP) in skin fibroblasts. Oxygen consumption- but not extracellular acidification rates were altered only when using glutamine as a substrate, and this was associated with mild mtDNA depletion and no changes in ETC activities. Immunoblot analysis revealed no detectable levels of SUCLG1, while SUCLA2 and SUCLG2 protein expressions were largely reduced. Confocal imaging of triple immunocytochemistry of skin fibroblasts showed that SUCLG2 co-localized only partially with the mitochondrial network which otherwise exhibited an increase in fragmentation compared to control cells. Our results outline the catastrophic consequences of the mutated SUCLG1 leading to strongly reduced SUCL activity, mSLP impairment, mislocalization of SUCLG2, morphological alterations in mitochondria and clinically to a severe neurometabolic disease, but in the absence of changes in mtDNA levels or respiratory complex activities. ispartof: MOLECULAR GENETICS AND METABOLISM vol:126 issue:1 pages:43-52 ispartof: location:United States status: published

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.

Metabolomics studies of diseases associated with chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways. Moreover, the holistic approach of such studies gives insights into the pathophysiological risk factors associated with chronic alcohol-induced disability, morbidity and mortality. Here, we report on a GC–MS-based organic acid profiling study on acute alcohol consumption. Our investigation — involving 12 healthy, moderate-drinking young men — simulated a single binge drinking event, and indicated its metabolic consequences. We generated time-dependent data that predicted the metabolic pathophysiology of the alcohol intervention. Multivariate statistical modelling was applied to the longitudinal data of 120 biologically relevant organic acids, of which 13 provided statistical evidence of the alcohol effect. The known alcohol-induced increased NADH:NAD+ ratio in the cytosol of hepatocytes contributed to the global dysregulation of several metabolic reactions of glycolysis, ketogenesis, the Krebs cycle and gluconeogenesis. The significant presence of 2-hydroxyisobutyric acid supports the emerging paradigm that this compound is an important endogenous metabolite. Its metabolic origin remains elusive, but recent evidence indicated 2-hydroxyisobutyrylation as a novel regulatory modifier of histones. Metabolomics has thus opened an avenue for further research on the reprogramming of metabolic pathways and epigenetic networks in relation to the severe effects of alcohol consumption.

International audience; Background In 2009, untargeted metabolomics led to the delineation of a new clinico-biological entity called cerebellar ataxia with elevated cerebrospinal free sialic acid, or CAFSA. In order to elucidate CAFSA, we applied sequentially targeted and untargeted omic approaches. Methods and results First, we studied five of the six CAFSA patients initially described. Besides increased CSF free sialic acid concentrations, three patients presented with markedly decreased 5-methyltetrahydrofolate (5-MTHF) CSF concentrations. Exome sequencing identified a homozygous POLG mutation in two affected sisters, but failed to identify a causative gene in the three sporadic patients with high sialic acid but low 5-MTHF. Using targeted mass spectrometry, we confirmed that free sialic acid was increased in the CSF of a third known POLG-mutated patient. We then pursued pathophysiological analyses of CAFSA using mass spectrometry-based metabolomics on CSF from two sporadic CAFSA patients as well as 95 patients with an unexplained encephalopathy and 39 controls. This led to the identification of a common metabotype between the two initial CAFSA patients and three additional patients, including one patient with Kearns-Sayre syndrome. Metabolites of the CSF metabotype were positioned in a reconstruction of the human metabolic network, which highlighted the proximity of the metabotype with acetyl-CoA and carnitine, two key metabolites regulating mitochondrial energy homeostasis. Conclusion Our genetic and metabolomics analyses suggest that CAFSA is a heterogeneous entity related to mitochondrial DNA alterations either through POLG mutations or a mechanism similar to what is observed in Kearns-Sayre syndrome.

Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of cholesterol in embryogenesis and development. FDFT1 encodes for an evolutionarily conserved enzyme, squalene synthase (SS, farnesyl-pyrophosphate farnesyl-transferase 1), which catalyzes the first committed step in cholesterol biosynthesis. We report three individuals with profound developmental delay, brain abnormalities, 2-3 syndactyly of the toes, and facial dysmorphisms, resembling Smith-Lemli-Opitz syndrome, the most common cholesterol biogenesis defect. The metabolite profile in plasma and urine suggested that their defect was at the level of squalene synthase. Whole-exome sequencing was used to identify recessive disease-causing variants in FDFT1. Functional characterization of one variant demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis/uptake in steady state.

The authors regret that in the Abstract and in the Molecular Analysis paragraphs of the above paper, the following was incorrect: “a homozygous c.131T > G (p.Leu44Pro) substitution”. This should read “a homozygous c.131T > C (p.Leu44Pro) substitution”. The authors would like to apologise for any inconvenience caused. They would like to thank Sally Heywood, Research Assistant, Human Gene Mutation Database, Institute of Medical Genetics, Cardiff University for bringing this error to their attention.

Metabolomics studies of disease conditions related to chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways underlying the pathophysiology of alcoholism. The objective of the present study was to utilize proton nuclear magnetic resonance (1H-NMR) spectroscopy metabolomics to study the holistic metabolic consequences of acute alcohol consumption in humans. The experimental design was a cross-over intervention study which included a number of substances to be consumed—alcohol, a nicotinamide adenine dinucleotide (NAD) supplement, and a benzoic acid-containing flavoured water vehicle. The experimental subjects—24 healthy, moderate-drinking young men—each provided six hourly-collected urine samples for analysis. Complete data sets were obtained from 20 of the subjects and used for data generation, analysis and interpretation. The results from the NMR approach produced complex spectral data, which could be resolved sufficiently through the application of a combination of univariate and multivariate methods of statistical analysis. The metabolite profiles resulting from acute alcohol consumption indicated that alcohol-induced NAD+ depletion, and the production of an excessive amount of reducing equivalents, greatly perturbed the hepatocyte redox homeostasis, resulting in essentially three major metabolic disturbances—up-regulated lactic acid metabolism, down-regulated purine catabolism and osmoregulation. Of these, the urinary excretion of the osmolyte sorbitol proved to be novel, and suggests hepatocyte swelling due to ethanol influx following acute alcohol consumption. Time-dependent metabolomics investigations, using designed interventions, provide a way of interpreting the variation induced by the different factors of a designed experiment, thereby also giving methodological significance to this study. The outcomes of this approach have the potential to significantly advance our understanding of the serious impact of the pathophysiological perturbations which arise from the consumption of a single, large dose of alcohol—a simulation of a widespread, and mostly naive, social practice.

Contains fulltext : 200371.pdf (Publisher’s version ) (Open Access)

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

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.

Rationale: Orthostatic hypotension is a common clinical problem, but the underlying mechanisms have not been fully delineated. Objective: We describe 2 families, with 4 patients in total, experiencing severe life-threatening orthostatic hypotension because of a novel cause. Methods and Results: As in dopamine β-hydroxylase deficiency, concentrations of norepinephrine and epinephrine in the patients were low. Plasma dopamine β-hydroxylase activity, however, was normal, and the DBH gene had no mutations. Molecular genetic analysis was performed to determine the underlying genetic cause. Homozygosity mapping and exome and Sanger sequencing revealed pathogenic homozygous mutations in the gene encoding cytochrome b561 ( CYB561 ); a missense variant c.262G>A, p.Gly88Arg in exon 3 in the Dutch family and a nonsense mutation (c.131G>A, p.Trp44*) in exon 2 in the American family. Expression of CYB561 was investigated using RNA from different human adult and fetal tissues, transcription of RNA into cDNA, and real-time quantitative polymerase chain reaction. The CYB561 gene was found to be expressed in many human tissues, in particular the brain. The CYB561 protein defect leads to a shortage of ascorbate inside the catecholamine secretory vesicles leading to a functional dopamine β-hydroxylase deficiency. The concentration of the catecholamines and downstream metabolites was measured in brain and adrenal tissue of 6 CYB561 knockout mice (reporter-tagged deletion allele [post-Cre], genetic background C57BL/6NTac). The concentration of norepinephrine and normetanephrine was decreased in whole-brain homogenates of the CYB561 (− /− ) mice compared with wild-type mice ( P P l -dihydroxyphenylserine, which can be converted directly to norepinephrine. Conclusions: This study is the first to implicate cytochrome b561 in disease by showing that pathogenic mutations in CYB561 cause an as yet unknown disease in neurotransmitter metabolism causing orthostatic hypotension.

Objective: To identify the underlying genetic defect in 5 patients from a consanguineous family with a Walker-Warburg phenotype, together with intracranial calcifications. Methods: Homozygosity mapping and exome sequencing, followed by Sanger sequencing of the obtained candidate gene, was performed. Expression of the candidate gene was tested by reverse transcription PCR. Patient fibroblasts were converted to myotubes, and the expression and function of dystroglycan was tested by Western blotting. Results: We detected a homozygous loss-of-function frameshift mutation in the DAG1 gene and showed that this mutation results in a complete absence of both α- and β-dystroglycan. Conclusions: A loss-of-function mutation in DAG1 can result in Walker-Warburg syndrome and is not embryonic lethal.

Contains fulltext : 155252.pdf (Publisher’s version ) (Closed access) Pediatric movement disorders are still a diagnostic challenge, as many patients remain without a (genetic) diagnosis. Magnetic resonance imaging (MRI) pattern recognition can lead to the diagnosis. MEGDEL syndrome (3-MethylGlutaconic aciduria, Deafness, Encephalopathy, Leigh-like syndrome MIM #614739) is a clinically and biochemically highly distinctive dystonia deafness syndrome accompanied by 3-methylglutaconic aciduria, severe developmental delay, and progressive spasticity. Mutations are found in SERAC1, encoding a phosphatidylglycerol remodeling enzyme essential for both mitochondrial function and intracellular cholesterol trafficking. Based on the homogenous phenotype, we hypothesized an accordingly characteristic MRI pattern. A total of 43 complete MRI studies of 30 patients were systematically reevaluated. All patients presented a distinctive brain MRI pattern with five characteristic disease stages affecting the basal ganglia, especially the putamen. In stage 1, T2 signal changes of the pallidum are present. In stage 2, swelling of the putamen and caudate nucleus is seen. The dorsal putamen contains an "eye" that shows no signal alteration and (thus) seems to be spared during this stage of the disease. It later increases, reflecting progressive putaminal involvement. This "eye" was found in all patients with MEGDEL syndrome during a specific age range, and has not been reported in other disorders, making it pathognomonic for MEDGEL and allowing diagnosis based on MRI findings.

Context: Testicular adrenal rest tumors (TART) are one of the major long term complications in patients with congenital adrenal hyperplasia. Although several adrenal-like properties have been assigned to these benign lesions, the etiology has not been confirmed yet. Objective: The aim of this study was to describe TART in more detail by analyzing several (steroidogenic) characteristics that may be classified as adrenal cortex or Leydig cell specific. Methods: Gene expression analysis by qPCR was performed for 14 genes in TART tissue (n = 12) and compared with the expression in healthy control fibroblasts (nonsteroidogenic control). In addition, a comparison was made with the expression levels in testis tissue (n = 9) and adrenal tissue (n = 13). Results: Nearly all genes were highly expressed in TART tissue, including all genes that encode the key steroidogenic enzymes. TART expression levels are in the majority almost identical to those found in adrenal tissue. The expression of adrenal cortex specific genes (CYP11B1, CYP11B2, and MC2R) in both TART and adrenal tissue is approximately 1000–10 000 times higher compared to that in testes samples. In addition, the Leydig cell markers INSL3 and HSD17B3 were not only found in testes, but also in TART, both at significantly higher levels than in the adrenal (p < 0.01). Conclusion: Our study shows for the first time that TART have multiple steroidogenic properties, which include not only the expression of adrenal cortex but also of Leydig cell markers. Therefore, the origin of these tumors might be a more totipotent embryonic cell type.

Item does not contain fulltext We studied a group of individuals with elevated urinary excretion of 3-methylglutaconic acid, neutropenia that can develop into leukemia, a neurological phenotype ranging from nonprogressive intellectual disability to a prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, and early death. Exome sequencing of two unrelated individuals and subsequent Sanger sequencing of 16 individuals with an overlapping phenotype identified a total of 14 rare, predicted deleterious alleles in CLPB in 14 individuals from 9 unrelated families. CLPB encodes caseinolytic peptidase B homolog ClpB, a member of the AAA+ protein family. To evaluate the relevance of CLPB in the pathogenesis of this syndrome, we developed a zebrafish model and an in vitro assay to measure ATPase activity. Suppression of clpb in zebrafish embryos induced a central nervous system phenotype that was consistent with cerebellar and cerebral atrophy that could be rescued by wild-type, but not mutant, human CLPB mRNA. Consistent with these data, the loss-of-function effect of one of the identified variants (c.1222A>G [p.Arg408Gly]) was supported further by in vitro evidence with the mutant peptides abolishing ATPase function. Additionally, we show that CLPB interacts biochemically with ATP2A2, known to be involved in apoptotic processes in severe congenital neutropenia (SCN) 3 (Kostmann disease [caused by HAX1 mutations]). Taken together, mutations in CLPB define a syndrome with intellectual disability, congenital neutropenia, progressive brain atrophy, movement disorder, cataracts, and 3-methylglutaconic aciduria.

Contains fulltext : 154354.pdf (Publisher’s version ) (Closed access) Premature aging syndromes are rare genetic disorders mimicking clinical and molecular features of aging. A recently identified group of premature aging syndromes is linked to mutation of the LMNA gene encoding lamins A and C, and is associated with nuclear deformation and dysfunction. Hutchinson-Gilford progeria syndrome (HGPS) was the first premature aging syndrome linked to LMNA mutation and its molecular bases have been deeply investigated. It is due to a recurrent de novo mutation leading to aberrant splicing and the production of a truncated and toxic nuclear lamin A precursor (prelamin ADelta50), also called progerin. In this work and based on the literature data, we propose to distinguish two main groups of premature aging laminopathies: (1) HGPS and HGP-like syndromes, which share clinical features due to hampered processing and intranuclear toxic accumulation of prelamin A isoforms; and (2) APS (atypical progeria syndromes), due to dominant or recessive missense mutations affecting lamins A and C. Among HGPS-like patients, several deleted prelamin A transcripts (prelamin ADelta50, ADelta35 and ADelta90) have been described. The purpose of this work was to characterize those transcripts in eight patients affected with HGP-like rare syndromes. When fibroblasts were available, the relationships between the presence and ratios of these transcripts and other parameters were studied, aiming to increase our understanding of genotype-phenotype relationships in HGPS-like patients. Altogether our results evidence that progerin accumulation is the major pathogenetic mechanism responsible for HGP-like syndromes due to mutations near the donor splice site of exon 11.

Item does not contain fulltext CONTEXT AND OBJECTIVE: Pheochromocytomas and paragangliomas (PGLs) are neuroendocrine tumors of sympathetic or parasympathetic paraganglia. Nearly 40% of PGLs are caused by germline mutations. The present study investigated the effect of genetic alterations on metabolic networks in PGLs. DESIGN: Homogenates of 32 sporadic PGLs and 48 PGLs from patients with mutations in SDHB, SDHD, SDHAF-2, VHL, RET, and NF-1 were subjected to proton ((1)H) nuclear magnetic resonance (NMR) spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling. RESULTS: (1)H NMR spectroscopy identified 28 metabolites in PGLs of which 12 showed genotype-specific differences. Part of these results published earlier reported low complex II activity (P < .0001) and low ATP/ADP/AMP content (P < .001) in SDH-related PGLs compared with sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA; P < .05) in SDH tumors and creatine (P < .05) in VHL tumors were observed compared with sporadics and other genotypes. Positive correlation was observed between NAA and ATP/ADP/AMP content (P < .001) and NAA and complex II activity (P < .0001) of PGLs. Targeted purine analysis in PGLs showed low adenine in cluster 1 compared with cluster 2 tumors (SDH P < .0001; VHL P < .05) whereas lower levels (P < .05) of guanosine and hypoxanthine were observed in RET tumors compared with SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes. CONCLUSIONS: The present study gives a comprehensive picture of alterations in energy metabolism in SDH- and VHL-related PGLs and establishes the interrelationship of energy metabolism and amino acid and purine metabolism in PGLs.

The 7th Santorini Conference was held in Santorini, Greece, and brought together 200 participants from 40 countries in several continents, including Europe, USA but also Japan, Korea, Brazil and South Africa. The attendees had the opportunity to: listen to 60 oral presentations; participate in two lunch symposia; look at 103 posters, which were divided in two groups (‘systems medicine and environment’ and ‘pharmacogenomics and cancer’) and attend a dedicated exhibition with six companies. The meeting was organized by the Institut National de la Santé et de la Recherche Médicale (INSERM) U1122; IGE-PCV and by ‘Biologie Prospective’ with the collaboration of the European Society of Pharmacogenomics and Theranostics (ESPT), under the auspices of international organizations (e.g., International Federation of Clinical Chemistry and Laboratory medicine [IFCC], European Federation of Clinical Chemistry and Laboratory Medicine [EFLM], European Diagnostic Manufacturers Association [EDMA], Federation of European Pharmacological Societies [EPHAR], European Science Foundation [ESF]). The 3 days of the conference stimulated intensive discussions on systems biology and the influence of omics technologies on personalized health. Sixty speakers were invited or selected from early abstracts and gave presentations on the following topics: From systems biology to systems medicine/pharmacology; Omics/translating pharmacogenomics/proteomic biomarkers/metabolomics; Human nutrition and health/personalized medicine. We are summarizing here the main topics and presentations, according to the successive sessions.

Contains fulltext : 153136.pdf (Publisher’s version ) (Closed access) Since the proposal to define a separate subgroup of inborn errors of metabolism involved in the biosynthesis and remodelling of phospholipids, sphingolipids and long chain fatty acids in 2013, this group is rapidly expanding. This review focuses on the disorders involved in the biosynthesis of phospholipids. Phospholipids are involved in uncountable cellular processes, e.g. as structural components of membranes, by taking part in vesicle and mitochondrial fusion and fission or signal transduction. Here we provide an overview on both pathophysiology and the extremely heterogeneous clinical presentations of the disorders reported so far (Sengers syndrome (due to mutations in AGK), MEGDEL syndrome (or SERAC defect, SERAC1), Barth syndrome (or TAZ defect, TAZ), congenital muscular dystrophy due to CHKB deficiency (CHKB). Boucher-Neuhauser/Gordon Holmes syndrome (PNPLA6), PHARC syndrome (ABHD12), hereditary spastic paraplegia type 28, 54 and 56 (HSP28, DDHD1; HSP54, DDHD2; HSP56, CYP2U1), Lenz Majewski syndrome (PTDSS1), spondylometaphyseal dysplasia with cone-rod dystrophy (PCYT1A), atypical haemolytic-uremic syndrome due to DGKE deficiency (DGKE).

Contains fulltext : 153789.pdf (Publisher’s version ) (Open Access) Serum free light chain (sFLC) assays are well established in the diagnosis and monitoring of plasma cell disorders. However, current FLC immunoassays are subject to several analytical issues, which results in a lack of harmonized results. To facilitate sFLC standardization, we investigated the strengths and limitations of mass spectrometry as a novel technological platform for sFLC quantification. Stable isotope labeled reference peptides are added to serum samples for quantitation by selected reaction monitoring (SRM). The use of redundant peptide sets allows for quality control measures during data analysis. Measurements on serum provide information on intact immunoglobulins, but depletion of these intact molecules from the sera during sample processing permits the quantitation of sFLC. sFLC concentrations measured with SRM were comparable to those obtained by nephelometry and showed excellent linearity (r(2) > 0.99). In samples with high levels of sFLC, SRM data was more consistent with serum protein electrophoresis than nephelometric data and SRM is unaffected by antigen excess. The lower limits of quantitation were 3.8 and 2.7 mg/L for kappa and lambda sFLC. Errors due to polymorphic sequences were prevented by comparison of redundant peptide pairs. The application of stable isotope labeling combined with SRM can overcome many of the current potential analytical issues of sFLC analysis. We describe which hurdles still need to be taken to make SRM a robust and more accurate method for sFLC measurements.

Since organic acid analysis in urine with gaschromatography-mass spectrometry (GC-MS) is a time-consuming technique, we developed a new liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) method to replace the classical analysis for diagnosis of inborn errors of metabolism (IEM). Sample preparation is simple and experimental time short. Targeted mass extraction and automatic calculation of z-scores generated profiles characteristic for the IEMs in our panel consisting of 71 biomarkers for defects in amino acids, neurotransmitters, fatty acids, purine, and pyrimidine metabolism as well as other disorders. In addition, four medication-related metabolites were included in the panel. The method was validated to meet Dutch NEN-EN-ISO 15189 standards. Cross validation of 24 organic acids from 28 urine samples of the ERNDIM scheme showed superiority of the UPLC-QTOF/MS method over the GC-MS method. We applied our method to 99 patient urine samples with 32 different IEMs, and 88 control samples. All IEMs were unambiguously established/diagnosed using this new QTOF method by evaluation of the panel of 71 biomarkers. In conclusion, we present a LC-QTOF/MS method for fast and accurate quantitative organic acid analysis which facilitates screening of patients for IEMs. Extension of the panel of metabolites is easy which makes this application a promising technique in metabolic diagnostics/laboratories. Electronic supplementary material The online version of this article (10.1007/s10545-017-0129-0) contains supplementary material, which is available to authorized users.

Corrigendum: NANS-mediated synthesis of sialic acid is required for brain and skeletal development

Defects of O-linked glycosylation of alpha-dystroglycan cause a wide spectrum of muscular dystrophies ranging from severe congenital muscular dystrophy associated with abnormal brain and eye development to mild limb girdle muscular dystrophy. We report a female patient who developed isolated pelvic girdle muscle weakness and wasting, which became symptomatic at age 42. Exome sequencing uncovered a homozygous c.131T > G (p.Leu44Pro) substitution in DPM3, encoding dolichol-P-mannose (DPM) synthase subunit 3, leading to a 50% reduction of enzymatic activity. Decreased availability of DPM as an essential donor substrate for protein O-mannosyltransferase (POMT) 1 and 2 explains defective skeletal muscle alpha-dystroglycan O-glycosylation. Our findings show that DPM3 mutations may lead to an isolated and mild limb girdle muscular dystrophy phenotype without cardiomyopathy.

Contains fulltext : 174067.pdf (Publisher’s version ) (Open Access) Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.

Contains fulltext : 174068.pdf (Publisher’s version ) (Open Access) Small molecule identification is a continually expanding field of research and represents the core challenge in various areas of (bio)analytical science, including metabolomics. Here, we unequivocally differentiate enantiomeric N-acetylhexosamines in body fluids using infrared ion spectroscopy, providing orthogonal identification of molecular structure unavailable by standard liquid chromatography/high-resolution tandem mass spectrometry. These results illustrate the potential of infrared ion spectroscopy for the identification of small molecules from complex mixtures.

Contains fulltext : 169910.pdf (Publisher’s version ) (Closed access) Unexplained global developmental delay and epilepsy in childhood pose a major socioeconomic burden. Progress in defining the molecular bases does not often translate into effective treatment. Notable exceptions include certain inborn errors of metabolism amenable to dietary intervention. CAD encodes a multifunctional enzyme involved in de novo pyrimidine biosynthesis. Alternatively, pyrimidines can be recycled from uridine. Exome sequencing in three families identified biallelic CAD mutations in four children with global developmental delay, epileptic encephalopathy, and anaemia with anisopoikilocytosis. Two died aged 4 and 5 years after a neurodegenerative disease course. Supplementation of the two surviving children with oral uridine led to immediate cessation of seizures in both. A 4-year-old female, previously in a minimally conscious state, began to communicate and walk with assistance after 9 weeks of treatment. A 3-year-old female likewise showed developmental progress. Blood smears normalized and anaemia resolved. We establish CAD as a gene confidently implicated in this neurometabolic disorder, characterized by co-occurrence of global developmental delay, dyserythropoietic anaemia and seizures. While the natural disease course can be lethal in early childhood, our findings support the efficacy of uridine supplementation, rendering CAD deficiency a treatable neurometabolic disorder and therefore a potential condition for future (genetic) newborn screening.

Contains fulltext : 174080.pdf (Publisher’s version ) (Closed access) Glucose transporter type 1 deficiency syndrome (GLUT1DS) is a neurometabolic disorder with a complex phenotypic spectrum but simple biomarkers in cerebrospinal fluid. The disorder is caused by impaired glucose transport into the brain resulting from variants in SCL2A1. In 10% of GLUT1DS patients, a genetic diagnosis can not be made. Using whole-genome sequencing, we identified a de novo 5'-UTR variant in SLC2A1, generating a novel translation initiation codon, severely compromising SLC2A1 function. This finding expands our understanding of the disease mechanisms underlying GLUT1DS and encourages further in-depth analysis of SLC2A1 non-coding regions in patients without variants in the coding region.

Contains fulltext : 174066.pdf (Publisher’s version ) (Open Access) BACKGROUND: Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. METHODS AND RESULTS: We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. CONCLUSIONS: We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

Contains fulltext : 169752.pdf (Publisher’s version ) (Closed access) Defects of the V-type proton (H+) ATPase (V-ATPase) impair acidification and intracellular trafficking of membrane-enclosed compartments, including secretory granules, endosomes, and lysosomes. Whole-exome sequencing in five families affected by mild to severe cutis laxa, dysmorphic facial features, and cardiopulmonary involvement identified biallelic missense mutations in ATP6V1E1 and ATP6V1A, which encode the E1 and A subunits, respectively, of the V1 domain of the heteromultimeric V-ATPase complex. Structural modeling indicated that all substitutions affect critical residues and inter- or intrasubunit interactions. Furthermore, complexome profiling, a method combining blue-native gel electrophoresis and liquid chromatography tandem mass spectrometry, showed that they disturb either the assembly or the stability of the V-ATPase complex. Protein glycosylation was variably affected. Abnormal vesicular trafficking was evidenced by delayed retrograde transport after brefeldin A treatment and abnormal swelling and fragmentation of the Golgi apparatus. In addition to showing reduced and fragmented elastic fibers, the histopathological hallmark of cutis laxa, transmission electron microscopy of the dermis also showed pronounced changes in the structure and organization of the collagen fibers. Our findings expand the clinical and molecular spectrum of metabolic cutis laxa syndromes and further link defective extracellular matrix assembly to faulty protein processing and cellular trafficking caused by genetic defects in the V-ATPase complex.

Contains fulltext : 137699.pdf (Publisher’s version ) (Open Access) Pheochromocytomas and paragangliomas (PPGLs) can be localized by (18)F-FDG PET. The uptake is particularly high in tumors with an underlying succinate dehydrogenase (SDH) mutation. SDHx-related PPGLs are characterized by compromised oxidative phosphorylation and a pseudohypoxic response, which mediates an increase in aerobic glycolysis, also known as the Warburg effect. The aim of this study was to explore the hypothesis that increased uptake of (18)F-FDG in SDHx-related PPGLs is reflective of increased glycolytic activity and is correlated with expression of different proteins involved in glucose uptake and metabolism through the glycolytic pathway.Twenty-seven PPGLs collected from patients with hereditary mutations in SDHB (n = 2), SDHD (n = 3), RET (n = 5), neurofibromatosis 1 (n = 1), and myc-associated factor X (n = 1) and sporadic patients (n = 15) were investigated. Preoperative (18)F-FDG PET/CT studies were analyzed; mean and maximum standardized uptake values (SUVs) in manually drawn regions of interest were calculated. The expression of proteins involved in glucose uptake (glucose transporters types 1 and 3 [GLUT-1 and -3, respectively]), phosphorylation (hexokinases 1, 2, and 3 [HK-1, -2, and -3, respectively]), glycolysis (monocarboxylate transporter type 4 [MCT-4]), and angiogenesis (vascular endothelial growth factor [VEGF], CD34) were examined in paraffin-embedded tumor tissues using immunohistochemical staining with peroxidase-catalyzed polymerization of diaminobenzidine as a read-out. The expression was correlated with corresponding SUVs.Both maximum and mean SUVs for SDHx-related tumors were significantly higher than those for sporadic and other hereditary tumors (P < 0.01). The expression of HK-2 and HK-3 was significantly higher in SDHx-related PPGLs than in sporadic PPGLs (P = 0.022 and 0.025, respectively). The expression of HK-2 and VEGF was significantly higher in SDHx-related PPGLs than in other hereditary PPGLs (P = 0.039 and 0.008, respectively). No statistical differences in the expression were observed for GLUT-1, GLUT-3, and MCT-4. The percentage anti-CD 34 staining and mean vessel perimeter were significantly higher in SDHx-related PPGLs than in sporadic tumors (P = 0.050 and 0.010, respectively). Mean SUVs significantly correlated with the expression of HK-2 (P = 0.027), HK-3 (P = 0.013), VEGF (P = 0.049), and MCT-4 (P = 0.020).The activation of aerobic glycolysis in SDHx-related PPGLs is associated with increased (18)F-FDG accumulation due to accelerated glucose phosphorylation by hexokinases rather than increased expression of glucose transporters.

Contains fulltext : 137474.pdf (Publisher’s version ) (Closed access) Alpha-fetoprotein (AFP) is present in fetal serum in concentrations up to 5,000,000 mug/l. After birth, AFP gene expression is turned down with a subsequent fall of the serum concentrations of this albumin-like protein to 'adult values' of circa 0.5-15 mug/l from the age of 2 years onwards. Irrespective of its assumed important functions, individuals with AFP deficiency appear fully healthy. The other way around, the presence of AFP in the circulation after the first years of life doesn't seem to harm, since individuals with 'hereditary persistence of AFP' are also without clinical abnormalities. During pregnancy, AFP (in maternal serum) has long been recognized as a marker for congenital anomalies of the fetus. Equally well known is AFP as biomarker for hepatocellular carcinoma and some other malignancies. There are at least four neurodegenerative disorders, all inherited as autosomal recessive traits and characterized by the presence of cerebellar ataxia, abnormal ocular movements, and neuropathy, for which an elevated concentration of serum AFP is an important diagnostic biomarker. The availability of a reliable biomarker is not only important during screening or diagnostic processes, but is also relevant for objective follow-up during (future) therapeutic interventions.

Contains fulltext : 137727.pdf (Publisher’s version ) (Closed access) Autosomal recessive cutis laxa (ARCL) is a connective tissue disorder characterized by wrinkled, inelastic skin, frequently associated with a neurologic involvement and multisystem disease. Next generation sequencing was performed in genetically unsolved patients with progeroid features, neurological and eye involvement to assess the underlying etiology. We describe an 6 month old child, diagnosed with a novel, homozygous nonsense mutation c.2339T>C in exon 18 of the ALDH18A1 gene, and reviewed all reported P5CS patients. So far 10 patients were described with mutations in ALDH18A1. Features of our patient that have been described in literature included cutis laxa on hands and feet, visible veins on thorax and abdomen, joint laxity, failure to thrive, short stature, microcephaly, and severe developmental and speech delay. Furthermore, abnormal fat distribution, retinal abnormalities, undescended testis, and retinitis pigmentosa have never been described in ALDH18A1. Some features described as unique in ALDH18A1 have been observed in PYCR1 patients, thus suggesting that the phenotypic overlap is higher than previously shown. In conclusion, the clinical phenotype caused by ALDH18A1 mutations is diverse, with variable degree of progeria in children, but always in association with neurologic disease. We suggest genetic testing for possible ALDH18A1 mutations in all patients with progeroid features, like wrinkled or parchment-like skin, abnormal growth, especially with central nervous system involvement and microcephaly.

Contains fulltext : 202680.pdf (Publisher’s version ) (Closed access) Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Contains fulltext : 119120.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To identify the underlying genetic defect in a patient with intellectual disability, seizures, ataxia, macrothrombocytopenia, renal and cardiac involvement, and abnormal protein glycosylation. METHODS: Genetic studies involved homozygosity mapping by 250K single nucleotide polymorphism array and SLC35A1 sequencing. Functional studies included biochemical assays for N-glycosylation and mucin-type O-glycosylation and SLC35A1-encoded cytidine 5'-monophosphosialic acid (CMP-sialic acid) transport after heterologous expression in yeast. RESULTS: We performed biochemical analysis and found combined N- and O-glycosylation abnormalities and specific reduction in sialylation in this patient. Homozygosity mapping revealed homozygosity for the CMP-sialic acid transporter SLC35A1. Mutation analysis identified a homozygous c.303G>C (p.Gln101His) missense mutation that was heterozygous in both parents. Functional analysis of mutant SLC35A1 showed normal Golgi localization but 50% reduction in transport activity of CMP-sialic acid in vitro. CONCLUSION: We confirm an autosomal recessive, generalized sialylation defect due to mutations in SLC35A1. The primary neurologic presentation consisting of ataxia, intellectual disability, and seizures, in combination with bleeding diathesis and proteinuria, is discriminative from a previous case described with deficient sialic acid transporter. Our study underlines the importance of sialylation for normal CNS development and regular organ function.

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.

The diagnosis of respiratory chain deficiencies (RCDs) is complicated and the need for a diagnostic biomarker or biosignature has been widely expressed. In this study, the metabolic profile of a selected group of 29 RCD patients, with a predominantly muscle disease phenotype, and 22 controls were investigated using targeted and untargeted analyses of three sub-sections of the human metabolome, including urinary organic acids and amino acids [measured by gas chromatography–mass spectrometry (GC–MS)], as well as acylcarnitines (measured by electrospray ionization tandem MS). Although MS technologies are highly sensitive and selective, they are restrictive by being applied only to sub-sections of the metabolome; an untargeted nuclear magnetic resonance (NMR) spectroscopy approach was therefore also included. After data reduction and pre-treatment, a biosignature comprising six organic acids (lactic, succinic, 2-hydroxyglutaric, 3-hydroxyisobutyric, 3-hydroxyisovaleric and 3-hydroxy-3-methylglutaric acids), six amino acids (alanine, glycine, glutamic acid, serine, tyrosine and α-aminoadipic acid) and creatine, was constructed from uni- and multivariate statistical analyses and verified by cross-validation. The results presented here provide the first proof-of-concept that the metabolomics approach is capable of defining a biosignature for RCDs. We postulate that the composite of organic acids ≈ amino acids > creatine > betaine > carnitines represents the basic biosignature for RCDs. Validated through a prospective study, this could offer an improved ability to assign individual patients to a group with defined RCD characteristics and improve case selection for biopsy procedures, especially in infants and children.

Contains fulltext : 108770.pdf (Publisher’s version ) (Closed access) We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (

Contains fulltext : 167862.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Alpha-1,3-glucosyltransferase congenital disorder of glycosylation (ALG6-CDG) is a congenital disorder of glycosylation. The original patients were described with hypotonia, developmental disability, epilepsy, and increased bleeding tendency. METHODS: Based on Euroglycan database registration, we approached referring clinicians and collected comprehensive data on 41 patients. RESULTS: We found hypotonia and developmental delay in all ALG6-CDG patients and epilepsy, ataxia, proximal muscle weakness, and, in the majority of cases, failure to thrive. Nine patients developed intractable seizures. Coagulation anomalies were present in

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1 and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP+ to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1R314C, which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1R314C lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP+ and differs from the IDH1R132 mutants in that it does not produce D-2-HG. Because IDH1R314C is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite.

Tuberculous meningitis (TBM) is a severe manifestation of tuberculosis, presenting with high morbidity and mortality in children. Existing diagnostic methods for TBM are invasive and time-consuming and the need for highly sensitive and selective diagnosis remains high on the TBM agenda. Our aim was to exploit metabolomics as an approach to identify metabolites as potential diagnostic predictors for children with TBM through a non-invasive means. Urine samples selected for this study were from three paediatric groups: patients with confirmed TBM (n = 12), patients clinically suspected with TBM but later confirmed to be negative (n = 19) and age-matched controls (n = 29). Metabolomics data were generated through gas chromatography–mass spectrometry analysis and important metabolites were identified according to standard statistical procedures used for metabolomics data. A global metabolite profile that characterized TBM was developed from the data, reflecting the host and microbial responses. Nine different logistic regression models were fitted to selected metabolites for the best combination as predictors for TBM. Four metabolites—methylcitric, 2-ketoglutaric, quinolinic and 4-hydroxyhippuric acids—showed excellent diagnostic ability and provided prognostic insight into our TBM patients. This study is the first to illustrate holistically the metabolic complexity of TBM and provided proof-of-concept that a biosignature of urinary metabolites can be defined for non-invasive diagnosis and prognosis of paediatric TBM patients. The biosignature should be developed and validated through future prospective studies to generate a medical algorithm for diagnosis in the initial stages of the disease and for monitoring of treatment strategies.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level.METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes.RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%).CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function., Here, Dirk Lefeber and colleagues identify functional mutations in ATP6AP1 encoding Ac45. The authors show that Ac45 is the functional ortholog of yeast V-ATPase assembly factor Voa1 and provide evidence for tissue-specific Ac45 processing, associated with the clinical phenotype of immunodeficiency, hepatopathy, and neurocognitive abnormalities.

Volatile organic compound (VOC) analysis in exhaled breath is proposed as a non-invasive method to detect respiratory infections in cystic fibrosis patients. Since polymicrobial infections are common, we assessed whether we could distinguish Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures using the VOC emissions. We took headspace samples of P. aeruginosa, A. fumigatus and co-cultures at 16, 24 and 48 h after inoculation, in which VOCs were identified by thermal desorption combined with gas chromatography – mass spectrometry. Using multivariate analysis by Partial Least Squares Discriminant Analysis we found distinct VOC biomarker combinations for mono- and co-cultures at each sampling time point, showing that there is an interaction between the two pathogens, with P. aeruginosa dominating the co-culture at 48 h. Furthermore, time-independent VOC biomarker combinations were also obtained to predict correct identification of P. aeruginosa and A. fumigatus in mono-culture and in co-culture. This study shows that the VOC combinations in P. aeruginosa and A. fumigatus co-microbial environment are different from those released by these pathogens in mono-culture. Using advanced data analysis techniques such as PLS-DA, time-independent pathogen specific biomarker combinations can be generated that may help to detect mixed respiratory infections in exhaled breath of cystic fibrosis patients.

Introduction Antiretroviral therapy (ART) for HIV-infected pregnant women is highly effective in preventing mother-to-child transmission (PMTCT) of the virus, but deleterious metabolic and mitochondrial observations in infants born to HIV-infected women treated with ART during pregnancy are periodically reported. Objectives This study addresses the concern of HIV-ART-induced metabolic perturbations through a metabolomics study of cord blood collected during transitional neonatal hypoglycaemia following birth from newborns either exposed or unexposed to fetal HIV-ART. Methods Proton magnetic resonance spectra from cord blood of 11 in utero HIV-ART-exposed and 14 unexposed newborns, as well as serum from 8 control infants, generated 114 spectral bins which were used to identify significant metabolites by means of univariate and multivariate statistical analyses. Results The metabolite profiles differed significantly between that from the unexposed newborns and that from infants-interpreted to characterize the state of transitional neonatal hypoglycaemia (low glucose and high lactic acid and ketone bodies). Quantitative analysis of potential ATP generation showed no meaningful difference in the global metabolite profiles of HIV-ART-exposed and unexposed neonates, but Volcano plot analysis, affirmed by odds ratios, indicated that exposure to HIV-ART affected the plasma 3-hydroxybutyric acid and hypoxanthine concentrations. Conclusions The metabolite profile for transitional neonatal hypoglycaemia indicated that HIV-ART did not compromise the exposed neonates to the energy stress of allostasis experienced at birth. Increased hypoxanthine and 3-hydroxybutyric acid indicates metabolic stress at birth in some of the newborns exposed to HIV-ART and raises a concern about unrecognized prolonged allostasis with potential neurological consequences for these infants

Contains fulltext : 167798.pdf (Publisher’s version ) (Closed access) Optimal glycosylation of immunoglobulins is essential in the generation of therapeutic biologicals with respect to efficacy, pharmacokinetics and immunogenic properties. This challenge in the field of biopharmaceuticals requires technologies for fast, robust and quantitative analysis of glycosylation. Current analyses of monoclonal antibody glycosylation are proteolysis-based mass spectrometry methods, which provide detailed structural information, but suffer a number of drawbacks such as lengthy sample preparation with the possibility to introduce artifacts. Here, we describe a fast, robust and high-resolution nanoLC-chip-QTOF method for quantitative analysis of intact monoclonal IgG glycosylation profiling. The method is able to detect hypoglycosylation, i.e. the lack of whole glycans, which is an important advantage over the well-established methods for free N-glycan or glycopeptide analysis. Moreover, the method is highly amenable to automation and because no digestion steps are involved, it provides direct relative quantitative information of both glycans on each IgG attachment site. We demonstrate that the ease and robustness make this technique ideally suited for quality control of the production process of mAb biopharmaceuticals, and provides new opportunities to study the clinical impact of mAb-glycosylation in patients with monoclonal gammopathies.

Contains fulltext : 168285.pdf (Publisher’s version ) (Open Access) is missing (Short communication).

Hydrogen cyanide is produced by S. aureus in vitro and in vivo and is not an exclusive biomarker for P. aeruginosa http://ow.ly/4nsh7y

We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic and metabolic investigations for acute liver failure failed to yield a diagnosis. Whole exome sequencing revealed a homozygous 12-bp deletion in PCK1 (MIM 614168) encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK); enzymatic studies subsequently confirmed its pathogenic nature. We propose that PEPCK deficiency should be considered in the young child with unexplained liver failure, especially where there are marked, accumulations of TCA cycle metabolites on urine organic acid analysis and/or an amino acid profile with hyperammonaemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-)physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes. (C) 2016 Elsevier Inc. All rights reserved

Background Lactate is traditionally seen as a marker of ischemia and a waste product of anaerobic glycolysis. In the last thirty years a more beneficial side of lactate as an alternative 'glucose sparing' fuel has been demonstrated. However, the translation of these growing insights to clinical practice seems to appear with great delay. Methods A review of the literature was performed, focusing on glucose and lactate in relation to cerebral energy metabolism, in the context of four typical clinical situations, namely (transient states of) low glucose availability for the brain due to hypoglycemia, combined with high blood lactate concentrations; permanent neuroglycopenia; lactic acidosis in mitochondrial disorders; and ischemic as well as traumatic brain injury. Results Lactate is thought to be an alternative fuel in the brain of patients with glucose transporter type 1 deficiency syndrome and glycogen storage disease, and it has been demonstrated that lactate might have a protective role in ischemic and traumatic brain injury. Conclusion Lactate has an apparently largely ignored, but potential beneficial role in the clinical management of several neurologic disorders.