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International audience; This prospective study aims to analyze the serum metabolome of female goat kids just before the first breeding in order to characterize the changes in metabolites during sexual maturation. A better knowledge of these changes could help optimizing the time for first breeding and improving the fertilization rates at first insemination. Weekly blood sampling was performed on twenty 6- to 7-month-old female goats from Alpine breed born in February during the 5 weeks preceding their first contact with bucks in September. Progesterone assays and metabolome analysis using 1H Nuclear Magnetic Resonance Spectroscopy were performed on the serum samples. No spontaneous ovulatory cycle was observed before breeding based on progesterone assays. All female goats had reached the pubertal stage of maturity at breeding since all got pregnant. Metabolome analysis allowed the identification of 109 spectral bins in sera. When the metabolic profiles were compared between the 5 weeks preceding the first contact with bucks, 6 metabolites were highlighted that could differentiate between week 5 and week 1, 2 and 3: glucose, citrate, creatinine, 3-hydroxy-isobutyrate and two unidentified bins. Citrate and 3-hydroxy-isobutyrate are of particular interest because their level significantly varied during sexual maturation, with a significant decrease of 3-hydroxy-isobutyrate at week 5 and a significant increase of citrate at week 4 and 5. However, the lack of variability in the fertility results did not allow us to conclude on a potential link between these metabolomics profile and reproduction performances. Further studies are necessary to ascertain the reliability of these metabolites as biomarkers of sexual precocity.

Objectives Trimethylaminuria, also known as Fish Odor Syndrome (FOS), is a condition characterized by the presence of high concentrations of trimethylamine (TMA) in urine, sweat and expired air of affected patients. Diagnosis of this benign but unpleasant disease is mainly based on clinical presentation and assessment of TMA and its metabolite, TMAO (trimethylamine-N-oxide), concentrations in urine of patients. Material and methods We here described the validation of an analytical method for measurement of TMA and TMAO in urine using nuclear magnetic resonance (NMR) according to the specifications of the ISO 15189 norm. We used a fast validation protocol, based exactitude profile method, enabling to determine accuracy, intra and inter-day precision from a limited number of samples. Results The linearity was established from 2.5 to 100 mg/L for TMA measurement and from 10 to 1000 mg/L for TMAO measurement, with good analytical performances i.e. accuracy, intra and inter-day precision. We also report a case diagnose for FOS from this method. Conclusions This method validation ensures the robustness of NMR in routine use for diagnosis of trimethylaminuria, as part of the reference center for inherited metabolic diseases at the Tours hospital.

The dihydropyrimidinase-like 3 (DPYSL3) or Collapsin Response Mediator Protein 4a (CRMP4a) expression is modified in neurodegeneration and is involved in several ALS-associated pathways including axonal transport, glutamate excitotoxicity, and oxidative stress. The objective of the study was to analyze CRMP4 as a risk factor for ALS. We analyzed the DPYSL3/CRMP4 gene in French ALS patients (n = 468) and matched-controls (n = 394). We subsequently examined a variant in a Swedish population (184 SALS, 186 controls), and evaluated its functional effects on axonal growth and survival in motor neuron cell culture. The rs147541241:A>G missense mutation occurred in higher frequency among French ALS patients (odds ratio = 2.99) but the association was not confirmed in the Swedish population. In vitro expression of mutated DPYSL3 in motor neurons reduced axonal growth and accelerated cell death compared with wild type protein. Thus, the association between the rs147541241 variant and ALS was limited to the French population, highlighting the geographic particularities of genetic influences (risks, contributors). The identified variant appears to shorten motor neuron survival through a detrimental effect on axonal growth and CRMP4 could act as a key unifier in transduction pathways leading to neurodegeneration through effects on early axon development.

Several studies have highlighted disturbance of redox homeostasis in patients with phenylketonuria (PKU) which may be associated with neurological disorders observed in patients, especially during adulthood when phenylalanine restrictive diets are not maintained. The aim of this study was to assess the antioxidant profile in a cohort of PKU patients in comparison to the controls and to evaluate its relation to biochemical parameters especially phenylalaninemia.We measured RNA expression of 22 antioxidant genes and reactive oxygen species (ROS) levels in white blood cells of 10 PKU patients and 10 age- and gender-matched controls. We also assessed plasma amino acids, vitamins, oligo-elements, and urinary organic acids concentrations. Then we evaluated the relationship between redox status and biochemical parameters.In addition to expected biochemical disturbances, we highlighted a significant global decrease of antioxidant genes expression in PKU patients in comparison to the controls. This global decrease of antioxidant genes expression, including various isoforms of peroxiredoxins, glutaredoxins, glutathione peroxidases, and superoxide dismutases, was significantly correlated to hyperphenylalaninemia.This study is the first to evaluate the expression of 22 antioxidant genes in white blood cells regarding biochemical parameters in PKU. These findings highlight the association of hyperphenylalaninemia with antioxidant genes expression. New experiments to specify the role of oxidative stress in PKU pathogenesis may be useful in suggesting new recommendations in PKU management and new therapeutic trials based on antioxidant defenses.

NMR spectroscopy of urine samples is able to diagnose many inborn errors of metabolism (IEM). However, urinary metabolites have a poor stability, requiring special care for routine analysis (storage of urine at -20 or -80 degrees C, fast transport). The aim of our study was to investigate the reliability of dried urine filter paper for urine storage and transport and to evaluate the ability of NMR to detect several IEM using this method. Urine samples from five healthy subjects were analyzed by (1)H NMR following different storage conditions (-20 vs 4 degrees C vs dried on filter paper) and at different time points (24 h, 48 h, 96 h, and 7 days). Urine pattern of fresh urine was considered as a reference. We analyzed the conservation of some amino acids and organic acids using Bland and Altman plot with intraclass correlation coefficient determination. Then, we evaluated the use of filter paper to detect four different IEM (methylmalonic and isovaleric acidurias, ornithine transcarbamylase deficiency, and cystinuria). Analysis of urine samples from healthy subjects revealed a high stability of studied molecules (ICC > 0.8) even after 7 days of storage on filter paper. Moreover, an excellent preservation of metabolites specifically accumulated in IEM was observed when analysis of dried urine filter paper was compared to fresh urine (coefficient of variation < 15%). This preliminary study demonstrates that storage of dried urine on filter paper is reliable for (1)H NMR spectroscopy analysis. Preservation of urine molecules over time using that method is convenient for routine clinical practice.

International audience; Ubiquitin inclusions represent a cytopathological hallmark of ALS. The ubiquitin-dependent protein degradation pathway may also be involved in the pathophysiology of SOD1 mutated ALS cases as demonstrated in transgenic animals. UBE2H is an ubiquitin conjugating enzyme known to act on histones and cytoskeletal proteins, both involved in the degenerative pathway of the motor neuron. We screened the whole coding sequence of the UBE2H gene in 24 sporadic ALS (SALS) patients using single strand conformation polymorphism (SSCP). All variants detected by SSCP were analysed by genomic DNA sequencing. We found one known polymorphism (rs12539800) and two new synonymous single nucleotide polymorphisms (SNP) (nG78A and nG501A). The allele distribution of the rs12539800 (A336G) SNP were tested for association in 252 SALS patients and 357 controls. The allele and genotype distributions were identical in the two groups. The UBE2H gene is not implicated in SALS; however, the ubiquitin pathway is worthy of further investigation in ALS.

Background: SMN1 gene deletions cause spinal muscular atrophy, and SMN2 gene deletions have been associated with sporadic lower motor neuron diseases. Objectives: To study the frequency of abnormal SMN1 gene copy numbers and to determine whether SMN2 gene modulates the risk of amyotrophic lateral sclerosis (ALS) or the duration of evolution. Method: The authors studied SMN1 and SMN2 genes in 600 patients with sporadic ALS and 621 controls using a quantitative PCR method. Results: The authors found an association of ALS with an abnormal copy number (one or three copies) of SMN1 gene ( p SMN2 copy numbers and no effect of SMN2 copies on the duration of evolution in ALS independently of SMN1 copy number. Conclusion: Abnormal SMN1 gene copy numbers are a genetic risk factor in sporadic amyotrophic lateral sclerosis. There was no modulator effect of the SMN2 gene.

Introduction Le stress oxydatif est implique dans la physiopathologie des complications de la phenylcetonurie (PCU) [1] . L’objectif de cette etude preliminaire etait d’evaluer la relation entre les changements metaboliques observes dans la PCU, le stress oxydatif intra-leucocytaire et l’expression des genes antioxydants. Patients et methodes Des echantillons de sang et d’urines ont ete collectes lors de l’inclusion de 9 patients adultes atteints de PCU non traites et 9 temoins sains apparies sur l’âge et le sexe. La concentration intra-leucocytaire d’especes reactives de l’oxygene (ROS) a ete mesuree par cytometrie en flux (marquage au DCFDA). L’expression des genes antioxydants majeurs a ete quantifiee par RT-qPCR (patent WO2012085188 A1-2012/06/28). Les concentrations d’acides amines plasmatiques ont ete mesurees par chromatographie. Les tests de Wilcoxon et les correlations de Pearson ont ete utilises pour les analyses statistiques. Resultats Les patients atteints de PCU avaient des concentrations plasmatiques de phenylalanine hautes et de tyrosine basses par rapport aux temoins (p Conclusion Cette etude preliminaire est la premiere a analyser le profil d’expression des genes antioxydants majeurs chez des patients PCU adultes et a etudier ce profil au regard des changements metaboliques observes dans cette pathologie. Ces resultats, obtenus chez des patients PCU non traites, mettent en evidence un arsenal antioxydant diminue associe a l’hyperphenylalaninemie, ce qui contribue au stress oxydatif observe chez les patients. Les perspectives sont d’etudier l’effet du controle metabolique de la PCU (par un regime controle en phenylalanine) sur le stress oxydatif.

Objectives: To develop a CSF metabolomics signature for motor neuron disease (MND) using 1 H-NMR spectroscopy and to evaluate the predictive value of the profile in a separate cohort. Methods: We collected CSF from patients with MND and controls and analyzed the samples using 1 H-NMR spectroscopy. We divided the total patient sample in a 4:1 ratio into a training cohort and a test cohort. First, a metabolomics signature was created by statistical modeling in the training cohort, and then the analyses tested the predictive value of the signature in the test cohort. We conducted 10 independent trials for each step. Finally, we identified the compounds that contributed most consistently to the metabolome profile. Results: Analysis of CSF from 95 patients and 86 controls identified a diagnostic profile for MND ( R 2 X > 22%, R 2 Y > 93%, Q 2 > 66%). The best model selected the correct diagnosis with mean probability of 99.31% in the training cohort. The profile discriminated between diagnostic groups with 78.9% sensitivity and 76.5% specificity in the test cohort. Metabolites linked to pathophysiologic pathways in MND (i.e., threonine, histidine, and molecules related to the metabolism of branched amino acids) were among the discriminant compounds. Conclusion: CSF metabolomics using 1 H-NMR spectroscopy can detect a reproducible metabolic signature for MND with reasonable performance. To our knowledge, this is the first metabolomics study that shows that a validation in separate cohorts is feasible. These data should be considered in future biomarker studies. Classification of evidence: This study provides Class III evidence that CSF metabolomics accurately distinguishes MNDs from other neurologic diseases.

Contexte Differents mecanismes physiopathologiques ont ete decrits dans la phenylcetonurie (PCU) mais les consequences metaboliques indirectes des concentrations elevees de phenylalanine (Phe) et tyrosine (Tyr) restent partiellement inconnues. Nous avons utilise une approche metabolomique multimodale pour evaluer la signature metabolique associee aux concentrations de Phe et de Tyr. Materiel et methode Nous avons prospectivement inclus 10 patients adultes PCU et 10 controles apparies en sexe et âge. Nous avons analyse le metabolome par (1) GC-MS (urine), (2) un analyseur d’acides amines (urine et plasma), (3) spectroscopie par resonance magnetique (RMN, urine). Nous avons realise une analyse statistique multivariee a partir du profil metabolique des differentes matrices biologiques (apres exclusion de la Phe, Tyr et des metabolites directement derives) afin d’expliquer les concentrations de Phe, Tyr et le diagnostic. Enfin, nous avons realise une analyse statistique univariee des metabolites les plus discriminants et nous avons identifie les voies metaboliques associees. Resultats Nous avons obtenu un profil metabolique comprenant 118 metabolites et nous avons construit d’excellents modeles multivaries pour expliquer les concentrations de Phe, de Tyr et le diagnostic. Des metabolites communs ont ete identifies par ces modeles : Gln, Arg, succinate, acide alpha aminobutyrique. Nous avons montre une correlation inverse entre Arg, acide alpha-aminobutyrique et Phe ainsi qu’une correlation positive entre Arg, succinate, Gln et Tyr ( p Discussion Nous avons obtenu une signature metabolique specifique liee aux concentrations de Phe et Tyr. Nous avons confirme l’implication de mecanismes physiopathologiques precedemment decrits dans la PCU tels que la synthese proteique, le metabolisme energetique et le stress oxydant. Nous avons mis en evidence l’interet de l’approche metabolomique pour explorer la pathogenese de la PCU.

Abnormal survival motor neuron 1 (SMN1)-copy number has been associated with an increased risk of amyotrophic lateral sclerosis (ALS) in French and Dutch population studies. The aim of this study was to determine whether SMN gene copy number increases the risk of ALS or modulates its phenotype in a cohort of Swedish sporadic ALS (SALS) patients. In all, 502 Swedes with SALS and 502 Swedish controls matched for gender and age were enrolled. SMN1 and SMN2 gene copy numbers were studied by a semi-quantitative PCR method. A genotype–phenotype comparison was performed in order to determine whether SMN genes modulate the phenotype of ALS. The results were also compared with our previously reported French cohort of ALS patients. There was no difference between Swedish patients and controls in the frequency of SMN1 and SMN2 copy numbers. The frequency of SMN1 gene copies differed significantly between the French and Swedish ALS populations. The duration of the disease was significantly longer in the Swedish cohort with homozygous deletions of SMN2 when compared with the French cohort. Abnormal SMN1 gene copy number cannot be considered as a universal genetic susceptibility factor for SALS and this result underlines the importance of reproducing association gene studies in groups from different origins. We also suggest that SMN2 gene copy number might have different effects on ALS progression in disparate human populations.