Combined Genome, Transcriptome and Metabolome Analysis in the Diagnosis of Childhood Cerebellar Ataxia

Supplementary Materials: Supplementary Materials can be found at https://www.mdpi.com/1422 -0067/22/6/2990/s1.The project was approved by the Biomedical Research Ethics Committee of the Andalusian Public Health System in Granada, Spain on Oct. 29, 2015, (project identification code AP163052016). Data recording, sample collection and all in vitro experiments were conducted in accordance with ethical guidelines following the Nuremberg Code, Belmont Report, and the Declaration of Helsinki.Acknowledgments: We are grateful for the support and cooperation of the subjects, as well as their families.Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient’s amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.Fundación Mutua Madrileña, Spain XIII Call on Research Grants 2016, reference number AP16305201

Piecewise linear analysis of an influence diagram

To each causal diagram, and the structure that it represents, a dynamical system can be associated. From its qualitative análisis, the behaviours associated to the structure can be deduced. This paper introduces a piecewise linear dynamical system associated to a causal diagram. Some interesting results on the qualitative behaviourof the system can be obtained from this dynamical system. In this paper a method is proposed to implement automatically the construction of a piecewise linear dynamical system to each causal diagram. The study of its equilibria and its stability. This allows us to obtain, automatically, the behaviour modes associated to a causal diagram

Qualitative modelling and simulations by piecewise linear analysis

Since applications of expert systems were typical in domains with no well defined models, qualitative methods for modelling and reasoning were soon developed. Most current qualitative reasoning programs derive the qualitative behaviour of a system by simulating a hand-crafted qualitative version of the differential equation that caracterizes the model of a system. This paper describes a method to construct a family of piecewise linear dynamical systems from the qualitative information contained in a model. We apply the dynamical system theory to deduce results about the behaviour of the family of dynamical systems constructed as a consequence of the qualitative model

Effect of the side of presentation in the visual field on phase-locked and nonphase-locked alpha and gamma responses

Recent studies have suggested that nonphase-locked activity can reveal cognitive mechanisms that cannot be observed in phase-locked activity. In fact, we describe a concomitant decrease in nonphase-locked alpha activity (desynchronization) when stimuli were processed (alpha phase-locked modulation). This desynchronization may represent a reduction in “background activity” in the visual cortex that facilitates stimulus processing. Alternatively, nonphase-locked gamma activity has been hypothesized to be an index of shifts in attentional focus. In this study, our main aim was to confrm these potential roles for nonphase-locked alpha and gamma activities with a lateralized Go/NoGo paradigm. The results showed that nonphase-locked alpha modulation is bilaterally represented in the scalp compared to the contralateral distribution of the phase-locked response. This fnding suggests that the decrease in background activity is not limited to neural areas directly involved in the visual processing of stimuli. Additionally, gamma activity showed a higher desynchronization of nonphase-locked activity in the ipsilateral hemisphere, where the phase-locked activity reached the minimum amplitude. This fnding suggests that the possible functions of nonphase-locked gamma activity extend beyond shifts in attentional focus and could represent an attentional flter reducing the gamma representation in the visual area irrelevant to the task

Spanish multicenter study: hyperammonemia not associated with inborn errors of metabolism in children

Introduction: The hyperammonemic encephalopathy induced by causes different from inborn errors of metabolism is a relatively uncommon but severe complication.Objectives: To study the characteristics of a secondary hyperammonemia episode to discern the triggering causes to get to the diagnosis, and the development in the therapeutic intervention.Methods: A multicenter retrospective study of children with hyperammonemia unrelated to inborn errors of metabolism, conducted in Spanish hospitals.Results: Nineteen patients were selected; hyperammonemia developed in infants under one year old in 47% of them, being diagnosed mostly with two or more symptoms. The most common clinical finding was an altered consciousness level similar to that of intoxication symptoms, followed by seizures. These clinical symptoms were present in 14 patients, with one of them or both. Twelve of the 19 patients were in treatment with more than two antiepileptic drugs routinely. All children were treated with protein restriction (n: 10), scavengers (n: 10) and/or carglumic acid (n: 12) for the treatment of hyperammonemia.Conclusions: This study suggests that secondary hyperammonemia could be underdiagnosed because it is only detected when severe symptoms appear. Risk seems to be higher in those patients receiving antiepileptic drugs or those critically ill with a restricted diet or incremented metabolism. The response to specific treatment is adequate but should be established earlier to avoid neurological sequelae of this entity

Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice

Interleukin-6 (IL-6) has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD) in wild-type (WT) and IL-6-deficient (IL-6−/−) mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6). Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1) and signal transducer and activator of transcription-3 (STAT3), increased AMP kinase phosphorylation (p-AMPK), and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β), FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis

CB1 blockade potentiates down-regulation of lipogenic gene expression in perirenal adipose tissue in high carbohydrate diet-induced obesity.

Journal Article; Research Support, Non-U.S. Gov't;De novo lipogenesis and hypercaloric diets are thought to contribute to increased fat mass, particularly in abdominal fat depots. CB1 is highly expressed in adipose tissue, and CB1-mediated signalling is associated with stimulation of lipogenesis and diet-induced obesity, though its contribution to increasing fat deposition in adipose tissue is controversial. Lipogenesis is regulated by transcription factors such as liver X receptor (LXR), sterol-response element binding protein (SREBP) and carbohydrate-responsive-element-binding protein (ChREBP). We evaluated the role of CB1 in the gene expression of these factors and their target genes in relation to lipogenesis in the perirenal adipose tissue (PrAT) of rats fed a high-carbohydrate diet (HCHD) or a high-fat diet (HFD). Both obesity models showed an up-regulated gene expression of CB1 and Lxrα in this adipose pad. The Srebf-1 and ChREBP gene expressions were down-regulated in HFD but not in HCHD. The expression of their target genes encoding for lipogenic enzymes showed a decrease in diet-induced obesity and was particularly dramatic in HFD. In HCHD, CB1 blockade by AM251 reduced the Srebf-1 and ChREBP expression and totally abrogated the remnant gene expression of their target lipogenic enzymes. The phosphorylated form of the extracellular signal-regulated kinase (ERK-p), which participates in the CB1-mediated signalling pathway, was markedly present in the PrAT of obese rats. ERK-p was drastically repressed by AM251 indicating that CB1 is actually functional in PrAT of obese animals, though its activation loses the ability to stimulate lipogenesis in PrAT of obese rats. Even so, the remnant expression levels of lipogenic transcription factors found in HCHD-fed rats are still dependent on CB1 activity. Hence, in HCHD-induced obesity, CB1 blockade may help to further potentiate the reduction of lipogenesis in PrAT by means of inducing down-regulation of the ChREBP and Srebf-1 gene expression, and consequently in the expression of lipogenic enzymes.This work was supported by Grants: from the Spanish Institute of Health ‘‘Carlos III (PI061109; CP12/03109; CIBERobn EU-ERDF-CB06/03/1008); from the Andalusian Ministry of Health (PI0552); from Andalusian Ministry of Economy, Innovation, Science and Employment EU-ERDF (CTS-8221 and CTS-433); from Spanish Ministry of Science and Innovation (SAF2010-20521); from European Union’s 7th Framework Programme (Health- F2-2008-223713, REPROBESITY). I3SNS Program of the Spanish National Health System of Spanish Ministry of Science and Innovation.Ye

Age-dependent nasal immune responses in non-hospitalized bronchiolitis children

Bronchiolitis in children is associated with significant rates of morbidity and mortality. Many studies have been performed using samples from hospitalized bronchiolitis patients, but little is known about the immunological responses from infants suffering from mild/moderate bronchiolitis that do not require hospitalization. We have studied a collection of nasal lavage fluid (NLF) samples from outpatient bronchiolitis children as a novel strategy to unravel local humoral and cellular responses, which are not fully characterized. The children were age-stratified in three groups, two of them (GI under 2-months, GII between 2-4 months) presenting a first episode of bronchiolitis, and GIII (between 4 months and 2 years) with recurrent respiratory infections. Here we show that elevated levels of pro-inflammatory cytokines (IL1b, IL6, TNFa, IL18, IL23), regulatory cytokines (IL10, IL17A) and IFNg were found in the three bronchiolitis cohorts. However, little or no change was observed for IL33 and MCP1, at difference to previous results from bronchiolitis hospitalized patients. Furthermore, our results show a tendency to IL1b, IL6, IL18 and TNFa increased levels in children with mild pattern of symptomseverity and in those in which non RSV respiratory virus were detected compared to RSV+ samples. By contrast, no such differences were found based on gender distribution. Bronchiolitis NLFs contained more IgM, IgG1, IgG3 IgG4 and IgA than NLF from their age-matched healthy controls. NLF from bronchiolitis children predominantly contained neutrophils, and also low frequency of monocytes and few CD4+ and CD8+ T cells. NLF from infants older than 4-months contained more intermediate monocytes and B cell subsets, including naïve and memory cells. BCR repertoire analysis of NLF samples showed a biased VH1 usage in IgM repertoires, with low levels of somatic hypermutation. Strikingly, algorithmic studies of the mutation profiles, denoted antigenic selection on IgA-NLF repertoires. Our results support the use of NLF samples to analyze immune responses and may have therapeutic implications.This work was supported by grants from the Ministerio de Ciencia e Innovación SAF 2015-70880-R and RTI 2018-099114-B-100.S

Urinary sulphatoxymelatonin as a biomarker of serotonin status in biogenic amine-deficient patients

Abstract 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