A resource of lipidomics and metabolomics data from individuals with undiagnosed diseases.

Every year individuals experience symptoms that remain undiagnosed by healthcare providers. In the United States, these rare diseases are defined as a condition that affects fewer than 200,000 individuals. However, there are an estimated 7000 rare diseases, and there are an estimated 25-30 million Americans in total (7.6-9.2% of the population as of 2018) affected by such disorders. The NIH Common Fund Undiagnosed Diseases Network (UDN) seeks to provide diagnoses for individuals with undiagnosed disease. Mass spectrometry-based metabolomics and lipidomics analyses could advance the collective understanding of individual symptoms and advance diagnoses for individuals with heretofore undiagnosed disease. Here, we report the mass spectrometry-based metabolomics and lipidomics analyses of blood plasma, urine, and cerebrospinal fluid from 148 patients within the UDN and their families, as well as from a reference population of over 100 individuals with no known metabolic diseases. The raw and processed data are available to the research community so that they might be useful in the diagnoses of current or future patients suffering from undiagnosed disorders.

5,10-methenyltetrahydrofolate synthetase deficiency causes a neurometabolic disorder associated with microcephaly, epilepsy, and cerebral hypomyelination.

Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4 month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite.

Secondary Abnormal CSF Neurotransmitter Metabolite Profiles in a Pediatric Tertiary Care Centre.

BACKGROUND: Secondary neurotransmitter deficiencies have been reported in several reviews. Our primary aim was to assess the relationship among epilepsy, antiseizure medications, and specific neurotransmitter abnormalities. We also evaluated movement disorders and brain abnormalities via magnetic resonance imaging scans in patients with secondary neurotransmitter defects. METHODS: This is a retrospective case series of 376 patients who underwent neurotransmitter analysis at BC Children's Hospital between 2009 and 2013, for a variety of neurological presentations. The biochemical genetics laboratory database was interrogated for results of cerebrospinal fluid neurotransmitter analyses. Clinical data for patients with abnormal results were collected from the hospital charts. Statistical analysis included one-way analysis of variance, chi-square, and a two-way contingency table. RESULTS: Abnormal neurotransmitter values were identified in 67 (17.8%) patients, two (0.53%) of which were attributable to a congenital neurotransmitter disorder and 11 (16.9%) secondary to other genetic diagnoses. Of 64 patients with secondary abnormal neurotransmitter values, 38 (59%) presented with epilepsy and 20 (31%) with movement disorders. A combination of epilepsy and movement disorder was less frequent. DISCUSSION: Acknowledging the limitations of this retrospective chart review, we conclude that, in our cohort, in addition to patients with movement disorders, a considerable number of patients with epilepsy and epileptic encephalopathy also showed secondary neurotransmitter mono-amine abnormalities. There is no clear relation, however, between clinical phenotype and type of neurotransmitter affected. In addition, no association was identified between the type of antiseizure medications and affected neurotransmitter type. We outline the need for prospective studies to further enrich our understanding of the relation between epilepsy and neurotransmitters with a focus on improving treatments and patient outcomes.

Evaluation of Metabolic and Synaptic Dysfunction Hypotheses of Alzheimer's Disease (AD): A Meta-Analysis of CSF Markers.

BACKGROUND: Alzheimer's disease (AD) is currently incurable and a majority of investigational drugs have failed clinical trials. One explanation for this failure may be the invalidity of hypotheses focusing on amyloid to explain AD pathogenesis. Recently, hypotheses which are centered on synaptic and metabolic dysfunction are increasingly implicated in AD. OBJECTIVE: Evaluate AD hypotheses by comparing neurotransmitter and metabolite marker concentrations in normal versus AD CSF. METHODS: Meta-analysis allows for statistical comparison of pooled, existing cerebrospinal fluid (CSF) marker data extracted from multiple publications, to obtain a more reliable estimate of concentrations. This method also provides a unique opportunity to rapidly validate AD hypotheses using the resulting CSF concentration data. Hubmed, Pubmed and Google Scholar were comprehensively searched for published English articles, without date restrictions, for the keywords "AD", "CSF", and "human" plus markers selected for synaptic and metabolic pathways. Synaptic markers were acetylcholine, gamma-aminobutyric acid (GABA), glutamine, and glycine. Metabolic markers were glutathione, glucose, lactate, pyruvate, and 8 other amino acids. Only studies that measured markers in AD and controls (Ctl), provided means, standard errors/deviation, and subject numbers were included. Data were extracted by six authors and reviewed by two others for accuracy. Data were pooled using ratio of means (RoM of AD/Ctl) and random effects meta-analysis using Cochrane Collaboration's Review Manager software. RESULTS: Of the 435 identified publications, after exclusion and removal of duplicates, 35 articles were included comprising a total of 605 AD patients and 585 controls. The following markers of synaptic and metabolic pathways were significantly changed in AD/controls: acetylcholine (RoM 0.36, 95% CI 0.24-0.53, p<0.00001), GABA (0.74, 0.58-0.94, p<0.01), pyruvate (0.48, 0.24-0.94, p=0.03), glutathione (1.11, 1.01- 1.21, p=0.03), alanine (1.10, 0.98-1.23, p=0.09), and lower levels of significance for lactate (1.2, 1.00-1.47, p=0.05). Of note, CSF glucose and glutamate levels in AD were not significantly different than that of the controls. CONCLUSION: This study provides proof of concept for the use of meta-analysis validation of AD hypotheses, specifically via robust evidence for the cholinergic hypothesis of AD. Our data disagree with the other synaptic hypotheses of glutamate excitotoxicity and GABAergic resistance to neurodegeneration, given observed unchanged glutamate levels and decreased GABA levels. With regards to metabolic hypotheses, the data supported upregulation of anaerobic glycolysis, pentose phosphate pathway (glutathione), and anaplerosis of the tricarboxylic acid cycle using glutamate. Future applications of meta-analysis indicate the possibility of further in silico evaluation and generation of novel hypotheses in the AD field.

Metabolic network failures in Alzheimer's disease: A biochemical road map.

INTRODUCTION: The Alzheimer's Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our understanding of Alzheimer's disease (AD) and propel the development of critically needed therapies. In response to their recommendations, big data at multiple levels are being generated and integrated to study network failures in disease. We used metabolomics as a global biochemical approach to identify peripheral metabolic changes in AD patients and correlate them to cerebrospinal fluid pathology markers, imaging features, and cognitive performance. METHODS: Fasting serum samples from the Alzheimer's Disease Neuroimaging Initiative (199 control, 356 mild cognitive impairment, and 175 AD participants) were analyzed using the AbsoluteIDQ-p180 kit. Performance was validated in blinded replicates, and values were medication adjusted. RESULTS: Multivariable-adjusted analyses showed that sphingomyelins and ether-containing phosphatidylcholines were altered in preclinical biomarker-defined AD stages, whereas acylcarnitines and several amines, including the branched-chain amino acid valine and α-aminoadipic acid, changed in symptomatic stages. Several of the analytes showed consistent associations in the Rotterdam, Erasmus Rucphen Family, and Indiana Memory and Aging Studies. Partial correlation networks constructed for Aß1-42, tau, imaging, and cognitive changes provided initial biochemical insights for disease-related processes. Coexpression networks interconnected key metabolic effectors of disease. DISCUSSION: Metabolomics identified key disease-related metabolic changes and disease-progression-related changes. Defining metabolic changes during AD disease trajectory and its relationship to clinical phenotypes provides a powerful roadmap for drug and biomarker discovery.

Simultaneous determination of all forms of biopterin and neopterin in cerebrospinal fluid.

In humans, genetic defects of the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor in hydroxylation reactions, are associated with severe neurological disorders. The diagnosis of these conditions relies on the determination of BH4, dihydrobiopterin (BH2), and dihydroneopterin (NH2) in cerebrospinal fluid (CSF). As MS/MS is less sensitive than fluorescence detection (FD) for this purpose, the most widely used method since 1980 involves two HPLC runs including two differential off-line chemical oxidation procedures aiming to transform the reduced pterins into their fully oxidized fluorescent counterparts, biopterin (B) and neopterin (N). However, this tedious and time-consuming two-step indirect method underestimates BH4, BH2, and NH2 concentrations. Direct quantification of BH4 is essential for studying its metabolism and for monitoring the efficacy of BH4 supplementation in patients with genetic defects. Here we describe a single step method to simultaneously measure BH4, BH2, B, NH2, and N in CSF by HPLC coupled to FD after postcolumn coulometric oxidation. All target pterins were quantified in CSF with a small volume (100 µL), and a single filtration step for sample preparation and analysis. As compared to the most widely used method in more than 100 CSF samples, this new assay is the easiest route for accurately determining in a single run BH4, BH2, and NH2 in CSF in deficit situations as well as for monitoring the efficacy of the treatment.

Glut1 deficiency syndrome and erythrocyte glucose uptake assay.

OBJECTIVE: The Glut1 deficiency syndrome (Glut1 DS) phenotype has expanded dramatically since first described in 1991. Hypoglycorrhachia and decreased erythrocyte 3-OMG uptake are confirmatory laboratory biomarkers. The objective is to expand previous observations regarding the diagnostic value of the uptake assay. METHODS: One hundred and nine suspected cases of Glut-1 DS were studied. All cases had a consistent clinical picture and hypoglycorrhachia. The uptake assay was decreased in 74 cases (group 1) and normal in 35 cases (group 2). We identified disease-causing mutations in 70 group 1 patients (95%) and one group 2 patient (3%). RESULTS: The cut-off for an abnormally low uptake value was increased from 60% to 74% with a corresponding sensitivity of 99% and specificity of 100%. The correlation between the uptake values for the time-curve and the kinetic concentration curve were strongly positive (R(2) = 0.85). Significant group differences were found in CSF glucose and lactate values, tone abnormalities, and degree of microcephaly. Group 2 patients were less affected in all domains. We also noted a significant correlation between the mean erythrocyte 3-OMG uptake and clinical severity (R(2) = 0.94). INTERPRETATION: These findings validate the erythrocyte glucose uptake assay as a confirmatory functional test for Glut1 DS and as a surrogate marker for GLUT1 haploinsufficiency.

Analysis of synaptic proteins in the cerebrospinal fluid as a new tool in the study of inborn errors of neurotransmission.

In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samples from 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman's correlation and Student's t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases.

Tyrosine hydroxylase deficiency in three Greek patients with a common ancestral mutation.

We present the clinical, biochemical, and molecular findings of three Greek patients with tyrosine hydroxylase (TH) deficiency. All patients presented with a severe clinical phenotype characterized by prominent motor delay, infantile parkinsonism, oculogyric crises, and signs of autonomic dysfunction. Cerebrospinal fluid analysis disclosed reduced dopamine metabolites and normal pterins. Response to levodopa was favorable though not dramatic. All patients were homozygous for a previously reported mutation (p.L236P). SNP haplotype analysis was consistent with a common ancestral mutation, thus indicating a founder effect in Greek patients with TH deficiency.

Differential diagnosis of hyperintense cerebrospinal fluid on fluid-attenuated inversion recovery images of the brain. Part I: pathological conditions.

The fluid-attenuated inversion recovery imaging sequence is a widely used MRI sequence of the brain. It is an inversion recovery pulse sequence, designed to suppress signals from the cerebrospinal fluid. It is highly sensitive in detection of lesions adjacent to or within the cerebrospinal fluid associated with T(2) prolongation or T(1) shortening. The term "hyperintense cerebrospinal fluid" is used to describe failed suppression, or hyperintensity, of cerebrospinal fluid on fluid-attenuated inversion recovery imaging of the brain. It is often encountered in many important pathological conditions, including subarachnoid haemorrhage, meningitis and leptomeningeal metastasis. However, certain non-pathological states in which there is no definite cerebrospinal fluid abnormality can also present with hyperintense cerebrospinal fluid. Correct interpretation of abnormalities is important to arrive at an appropriate diagnosis. This pictorial review provides fluid-attenuated inversion recovery images of hyperintense cerebrospinal fluid of the brain and describes distinguishing features. Part I features pathological conditions whereas Part II focuses on non-pathological conditions.

Biochemical diagnosis of dopaminergic disturbances in paediatric patients: analysis of cerebrospinal fluid homovanillic acid and other biogenic amines.

Homovanillic acid (HVA) is a major catabolite of dopamine. Its concentration in cerebrospinal fluid (CSF) provides insight into the turnover of dopamine. Our main purpose in this review was to analyze the role played by HVA determination in CSF as a diagnostic and prognostic tool in diseases that directly or indirectly affect the dopaminergic pathway in paediatric patients. There are several rare genetic diseases related with dopamine metabolism disturbances, both in the biosynthesis and catabolism of this neurotransmitter, so that diagnosis is often a major challenge. Decreased concentrations of CSF HVA, together with defects in other biogenic amine metabolites, are the hallmark of dopamine deficiency, and they may provide not only a clue for diagnosis but also information about prognosis and treatment monitoring. Concerning secondary deficiencies, genetic and non-genetic conditions have been identified as the cause of low CSF HVA concentrations, and the variability of clinical presentation and pathophysiological mechanisms is wide. As to CSF HVA analysis, lumbar puncture following a strict protocol has been applied for diagnosis of paediatric neurotransmitter diseases. Among laboratory methods developed for the analysis of CSF HVA and other biogenic amines, high pressure liquid chromatography with electrochemical detection is the most reliable procedure for clinical laboratories. Reference values should be established in each laboratory since there is a strong association between age and biogenic amine concentrations in CSF.

Cerebrospinal fluid: the role of biochemical analysis.

Biochemical analysis of cerebrospinal fluid may provide answers to important clinical questions. This review summarizes these questions and outlines the value and limitations of cerebrospinal fluid analysis.

Dimethyl sulfone in human cerebrospinal fluid and blood plasma confirmed by one-dimensional (1)H and two-dimensional (1)H-(13)C NMR.

(1)H-NMR spectroscopy at 500 MHz was used to confirm that a previously unidentified singlet resonance at 3.14 ppm in the spectra of cerebrospinal fluid and plasma samples corresponds to dimethyl sulfone (DMSO(2)). A triple resonance inverse cryogenic NMR probe, with pre-amplifier and the RF-coils cooled to low temperature, was used to obtain an (1)H-(13)C HSQC spectrum of CSF containing 8 microM (753 ng/ml) DMSO(2). The (1)H-(13)C correlation signal for DMSO(2) was assigned by comparison with the spectrum from an authentic reference sample. In plasma and CSF from healthy controls, the concentration of DMSO(2) ranged between 0 and 25 micromol/l. The concentration of DMSO(2) in plasma from three of four patients with severe methionine adenosyltransferase I/III (MAT I/III) deficiency was about twice the maximum observed for controls. Thus, DMSO(2) occurs as a regular metabolite at low micromolar concentrations in cerebrospinal fluid and plasma. It derives from dietary sources, from intestinal bacterial metabolism and from human endogenous methanethiol metabolism.

Assessment of amyloid beta protein in cerebrospinal fluid as an aid in the diagnosis of Alzheimer's disease.

The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39-42-amino-acid protein, amyloid beta protein (A beta). This study examined whether the measurement of A beta levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and A beta concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean A beta value for the AD group (15.9 +/- 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 +/- 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. A beta values did not correlate with age (r = -0.05, p = 0.59), severity of cognitive impairment (r = 0.22, p = 0.21), or duration of AD symptoms (r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of A beta in CSF. A beta immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of A beta reactivity. In conclusion, the total CSF A beta level is not a useful marker for current diagnosis of AD.

S-adenosylmethionine levels in psychiatric and neurological disorders: a review.

INTRODUCTION: S-adenosylmethionine (SAMe) is an important methyl donor in over 35 methylation reactions involving DNA, proteins, phospholipids and catechol- and indole- amines. MATERIAL AND METHODS: This article reviews the studies that have examined brain and blood levels of SAMe in several psychological, neurological and metabolic disorders. RESULTS: Although studies have found no consistent changes in whole blood SAMe levels in psychiatric patients, other investigators have found low cerebrospinal fluid (CSF) SAMe levels in patients with neurological disorders such as Alzheimer's dementia, subacute combined degeneration of the spinal cord (SACD), and HIV-related neuropathies, as well as in patients with metabolic disorders such as 5, 10-CH2-H4 folate reductase deficiency. CONCLUSION: Intravenous or oral administration of SAMe thus represents a possible treatment for these neurological and metabolic disorders.

Beta-methylcrotonic aciduria associated with lactic acidosis.

A patient is described in whom lactic acidosis of very severe degree was found to coincide with the presence of beta-methylcrotonic acid and rho-hydroxyphenyllactic acid in urine in large amounts, while beta-hydroxyisovaleric acid was found to be a relatively minor excretion product. Beta-methylcrotonic acid is demonstrated, for the first time, to be present in blood and CSF. These findings are discussed in relation to the patients previously reported to have beta-methylcrotonylglycinuria and raise the possibility that our patient's organic aciduria may be secondary to acquired disease rather than to an inborn error of metabolism.

[Behavior of cerebrospinal fluid proteins in degenerative diseases]. / Das Verhalten der Liquorproteine bei degenerativen Erkrankungen

132 cerebrospinals fluids from patients with degenerative diseases of the central nervous system have been analyzed for protein distribution in agar gel electrophoresis. After subdivision into diagnostically well defined groups these patients were compared with 48 with metabolic and psychiatric diseases and with 79 normal controls. The majority of diagnostic groups showed a tendency to permeability impairment. Other outstanding deviations were not found, except for single cases which were not statistically typical of the groups as a whole. However, the "degenerative type" of proteinogram emphasized in the literature predominated not only in the degenerative groups but also in certain other diseases with destruction of central nervous tissue which do not belong to the degenerative diseases in the strict sense. On the other hand, there are some degenerative subgroups without this "typical" electrophoretic pattern. There would thus appear to be grounds for amending the term "degenerative" into "tissue destroying" or "atrophic type", to avoid misinterpretation.