BackgroundThe SLC39A14, SLC30A10 and SLC39A8 are considered to be key genes involved in manganese (Mn) homeostasis in humans. Mn levels in plasma and urine are useful tools for early recognition of these disorders. We aimed to explore further biomarkers of Mn deposition in the central nervous system in two siblings presenting with acute dystonia and hypermanganesemia due to mutations in SLC39A14. These biomarkers may help clinicians to establish faster and accurate diagnosis and to monitor disease progression after chelation therapy is administered.ResultsA customized gene panel for movement disorders revealed a novel missense variant (c.311G > T; p.Ser104Ile) in SLC39A14 gene in two siblings presenting at the age of 10 months with acute dystonia and motor regression. Mn concentrations were analyzed using inductively coupled mass spectrometry in plasma and cerebrospinal fluid, disclosing elevated Mn levels in the index case compared to control patients. Surprisingly, Mn values were 3-fold higher in CSF than in plasma. We quantified the pallidal index, defined as the ratio between the signal intensity in the globus pallidus and the subcortical frontal white matter in axial T1-weighted MRI, and found significantly higher values in the SLC39A14 patient than in controls. These values increased over a period of 10 years, suggesting the relentless pallidal accumulation of Mn. Following genetic confirmation, a trial with the Mn chelator Na2CaEDTA led to a reduction in plasma Mn, zinc and selenium levels. However, parents reported worsening of cervical dystonia, irritability and sleep difficulties and chelation therapy was discontinued.ConclusionsOur study expands the very few descriptions of patients with SLC39A14 mutations. We report for the first time the elevation of Mn in CSF of SLC39A14 mutated patients, supporting the hypothesis that brain is an important organ of Mn deposition in SLC39A14-related disease. The pallidal index is an indirect and non-invasive method that can be used to rate disease progression on follow-up MRIs. Finally, we propose that patients with inherited defects of manganese transport should be initially treated with low doses of Na2CaEDTA followed by gradual dose escalation, together with a close monitoring of blood trace elements in order to avoid side effects.

The presence of monoamines and their cofactors (the pterins and vitamin B (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.

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Melatonin is synthesized from serotonin and it is excreted as sulphatoxymelatonin in urine. We aim to evaluate urinary sulphatoxymelatonin as a biomarker of brain serotonin status in a cohort of patients with mutations in genes related to serotonin biosynthesis. We analized urinary sulphatoxymelatonin from 65 healthy subjects and from 28 patients with genetic defects. A total of 18 patients were studied: 14 with autosomal dominant and recessive guanosine triphosphate cyclohydrolase-I deficiency; 3 with sepiapterin reductase deficiency; and 1 with aromatic L-amino acid decarboxylase deficiency. Further 11 patients were studied after receiving serotoninergic treatment (serotonin precursors, monoamine oxidase inhibitors, selective serotonin re-uptake inhibitors): 5 with aromatic L-amino acid decarboxylase deficiency; 1 with sepiapterin reductase deficiency; 3 with dihydropteridine reductase deficiency; and 2 with 6-pyruvoyltetrahydropterin synthase deficiency. Among the patients without therapy, 6 presented low urinary sulphatoxymelatonin values, while most of the patients with guanosine triphosphate cyclohydrolase-I deficiency showed normal values. 5 of 11 patients under treatment presented low urine sulphatoxymelatonin values. Thus, decreased excretion of sulphatoxymelatonin is frequently observed in cases with severe genetic disorders affecting serotonin biosynthesis. In conclusion, sulphatoxymelatonin can be a good biomarker to estimate serotonin status in the brain, especially for treatment monitoring purposes.

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Mitochondrial diseases are a group of genetic disorders leading to the dysfunction of mitochondrial energy metabolism pathways. We aimed to assess the clinical phenotype and the biochemical cerebrospinal fluid (CSF) biogenic amine profiles of patients with different diagnoses of genetic mitochondrial diseases. We recruited 29 patients with genetically confirmed mitochondrial diseases harboring mutations in either nuclear or mitochondrial DNA (mtDNA) genes. Signs and symptoms of impaired neurotransmission and neuroradiological data were recorded. CSF monoamines, pterins, and 5-methyltetrahydrofolate (5MTHF) concentrations were analyzed using high-performance liquid chromatography with electrochemical and fluorescence detection procedures. The mtDNA mutations were studied by Sanger sequencing, Southern blot, and real-time PCR, and nuclear DNA was assessed either by Sanger or next-generation sequencing. Five out of 29 cases showed predominant dopaminergic signs not attributable to basal ganglia involvement, harboring mutations in different nuclear genes. A chi-square test showed a statistically significant association between high homovanillic acid (HVA) values and low CSF 5-MTHF values (chi-square = 10.916; p = 0.001). Seven out of the eight patients with high CSF HVA values showed cerebral folate deficiency. Five of them harbored mtDNA deletions associated with Kearns-Sayre syndrome (KSS), one had a mitochondrial point mutation at the mtDNA ATPase6 gene, and one had a POLG mutation. In conclusion, dopamine deficiency clinical signs were present in some patients with mitochondrial diseases with different genetic backgrounds. High CSF HVA values, together with a severe cerebral folate deficiency, were observed in KSS patients and in other mtDNA mutation syndromes.

BackgroundCerebrospinal fluid (CSF) metabolomic investigations are a powerful tool for studying neurometabolic diseases. We aimed to assess the effect of CSF contamination with blood on the concentrations of selected biomarkers.MethodsCSF samples were spiked in duplicate with increasing volumes of whole blood under two conditions: (A) pooled CSF spiked with fresh whole blood and frozen to cause red blood cell (RBC) lysis; (B) pooled CSF spiked with fresh blood and centrifuged (the supernatant with no RBCs was frozen until the moment of analysis). CSF concentrations of amino acids, biogenic amines, pterins, and vitamins were analysed by HPLC coupled with tandem mass spectrometry, electrochemical and fluorescence detection.ResultsAspartate, glutamate, taurine, ornithine, glycine, citrulline, pyridoxal 5´-phosphate, 5-methyltetrahydrofolate, and thiamine showed higher values when RBCs were lysed when compared with those of CSF with no RBC, while arginine, 5-hydroxyindoleacetic and homovanillic acids showed lower values. When RBCs were removed from CSF, only some amino acids, thiamine and pyridoxal 5´-phosphate showed moderately higher values when compared with the non-spiked CSF sample.ConclusionsCSF-targeted metabolomic analysis is feasible even when substantial RBC contamination of CSF has occurred since CSF centrifugation to remove RBC prior to freezing eliminated most of the interferences observed.

Patients with inborn errors of amino acid metabolism frequently show neuropsychiatric symptoms despite accurate metabolic control. This study aimed to gain insight into the underlying mechanisms of neural dysfunction. Here we analyzed the expression of brain-derived neurotrophic factor (BDNF) and 10 genes required for correct brain functioning in plasma and blood of patients with Urea Cycle Disorders (UCD), Maple Syrup Urine Disease (MSUD) and controls. Receiver-operating characteristic (ROC) analysis was used to evaluate sensitivity and specificity of potential biomarkers. CACNA2D2 (α2δ2 subunit of voltage-gated calcium channels) and MECP2 (methyl-CpG binding protein 2) mRNA and protein showed an excellent neural function biomarker signature (AUC ≥ 0,925) for recognition of MSUD. THBS3 (thrombospondin 3) mRNA and AABA gave a very good biomarker signature (AUC 0,911) for executive-attention deficits. THBS3 , LIN28A mRNA, and alanine showed a perfect biomarker signature (AUC 1) for behavioral and mood disorders. Finally, a panel of BDNF protein and at least two large neural AAs showed a perfect biomarker signature (AUC 1) for recognition of psychomotor delay, pointing to excessive protein restriction as central causative of psychomotor delay. To conclude, our study has identified promising biomarker panels for neural function evaluation, providing a base for future studies with larger samples.