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Background and purpose The short-term evolution of pediatric migraine remains unclear. We aimed to describe the evolution of migraine before and after puberty and its relationship with lifestyle habits. Methods We prospectively selected prepuberal patients from a neuropediatric unit who had a migraine diagnosis. Their medical history, migraine characteristics and impact, and lifestyle habits were recorded at the baseline visit. After 2 years we performed a telephone follow-up assessment. Results Nineteen patients were recruited (age 10.2±2.9 years, mean±SD; 57.9% female), of whom 27.5% had migraine with aura. The accompanying symptoms had changed at the follow-up, with significantly higher prevalence rates of dizziness (44.4% vs. 88.9%), vertigo (11.1% vs. 66.7%), mood changes (38.9% vs 83.3%), confusion (5.6% vs. 77.8%), and allodynia (27.8% vs. 61.1%). Sleep disturbances (5.6% vs. 38.9%) and schedule changes (0% vs. 38.9%) increased significantly as triggers. Prodromal symptoms became more prevalent (16.7% vs. 50%), with a higher proportion of sleep disturbances reported (50.0% vs. 87.5%). Conclusions Prodromal symptoms increase in pediatric migraine after 2 years, and some trigger factors for migraine become more prevalent, including sleep disturbances. New accompanying symptoms are also identified. These changes provide information about how migraine changes during puberty along with physical and lifestyle changes, and represent a dynamic physiopathological process that deserves more research.

Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%). Supported by Generalitat de Catalunya through Departament de Salut (SLT002/16/00174 to URD-Cat consortium) and Departament d’Empresa i Coneixement and the CERCA Program; FP7 and H2020 EU projects RD-Connect, Solve-RD, and EJP-RD grants FP7 305444, H2020 779257, and H2020 825575 for CNAG-CRG; Spanish Ministry of Science and Innovation to the EMBL partnership and through the Instituto de Salud Carlos III grants PT13/0001/0044 and PT17/0009/0019 for CNAG-CRG and grants PI16/01048, PI19/01310, PI18/00451, PI18/00498, and PI21/00935 for IDIBAPS (Instituto Nacional de Bioinformática); ELIXIR Implementation Studies (CNAG-CRG); Centro de Investigaciones Biomédicas en Red de Enfermedades Raras; Centro de Excelencia Severo Ochoa grant SEV-2016-0571 (CNAG-CRG) and grant CEX 2018-000806-S (ISGLOBAL); and cofinancing with funds from the European Regional Development Fund by the Spanish Ministry of Science and Innovation corresponding to the Programa Operativo FEDER Plurirregional de España (POPE) 2014 to 2020 and by the Secretaria d’Universitats i Recerca, Departament d’Empresa i Coneixement of the Generalitat de Catalunya corresponding to the Programa Operatiu FEDER de Catalunya 2014 to 2020.

Seqüenciació de gens; Genòmica; Imatges per ressonància magnètica Secuenciación de genes; Genómica; Imágenes por resonancia magnética Gene sequencing; Genomics; Magnetic resonance imaging Chiari Malformation Type 1 (CM-1) is characterized by herniation of the cerebellar tonsils below the foramen magnum and the presence of headaches and other neurologic symptoms. Cranial bone constriction is suspected to be the most common biologic mechanism leading to CM-1. However, other mechanisms may also contribute, particularly in the presence of connective tissue disorders (CTDs), such as Ehlers Danlos Syndrome (EDS). Accumulating data suggest CM-1 with connective tissue disorders (CTD+) may have a different patho-mechanism and different genetic risk factors than CM-1 without CTDs (CTD-). To identify CM-1 genetic risk variants, we performed whole exome sequencing on a single large, multiplex family from Spain and targeted sequencing on a cohort of 186 unrelated adult, Caucasian females with CM-1. Targeted sequencing captured the coding regions of 21 CM-1 and EDS candidate genes, including two genes identified in the Spanish family. Using gene burden analysis, we compared the frequency of rare, functional variants detected in CM-1 cases versus publically available ethnically-matched controls from gnomAD. A secondary analysis compared the presence of rare variants in these genes between CTD+ and CTD- CM-1 cases. In the Spanish family, rare variants co-segregated with CM-1 in COL6A5, ADGRB3 and DST. A variant in COL7A1 was present in affected and unaffected family members. In the targeted sequencing analysis, rare variants in six genes (COL7A1, COL5A2, COL6A5, COL1A2, VEGFB, FLT1) were significantly more frequent in CM-1 cases compared to public controls. In total, 47% of CM-1 cases presented with rare variants in at least one of the four significant collagen genes and 10% of cases harbored variants in multiple significant collagen genes. Moreover, 26% of CM-1 cases presented with rare variants in the COL6A5 gene. We also identified two genes (COL7A1, COL3A1) for which the burden of rare variants differed significantly between CTD+ and CTD- CM-1 cases. A higher percentage of CTD+ patients had variants in COL7A1 compared to CTD+ patients, while CTD+ patients had fewer rare variants in COL3A1 than did CTD- patients. In summary, rare variants in several collagen genes are particularly frequent in CM-1 cases and those in COL6A5 co-segregated with CM-1 in a Spanish multiplex family. COL6A5 has been previously associated with musculoskeletal phenotypes, but this is the first association with CM-1. Our findings underscore the contribution of rare genetic variants in collagen genes to CM-1, and suggest that CM-1 in the presence and absence of CTD symptoms is driven by different genes. This work was supported by a grant from Conquer Chiari to AAK. Collection of the Chiari1000 study participants utilized in this study was supported by a grant from Conquer Chiari to FL at University of Akron. Collection of the Duke study participants utilized in this study was supported by a grant from the National Institutes of Health (NS063273). A.U. was the recipient of a Postdoctoral Fellowship from Fundación Ramón Areces (Spain). RL is the Executive Director of Conquer Chiari which provided some of the funding for this work. For the manuscript, he assisted with revising and editing the manuscript. The funders did have a role in study design, but had no role in data collection and analysis, decision to publish, or preparation of the manuscript.

While rare diseases (RDs) are by definition of low prevalence, the total number of patients suffering from an RD is high, and the majority of them have neurologic manifestations, involving central, peripheral nerve, and muscle. In 2017, 24 European Reference Networks (ERNs), each focusing on a specific group of rare or low-prevalence complex diseases, were formed to improve the care for patients with an RD. One major aim is to have "the knowledge travel instead of the patient," which has been put into practice by the implementation of the Clinical Patient Management System (CPMS) that enables clinicians to perform pan-European virtual consultations. The European Reference Network for Rare Neurological Diseases (ERN-RND) provides an infrastructure for knowledge sharing and care coordination for patients affected by a rare neurological disease (RND) involving the most common central nervous system pathological conditions. It covers the following disease groups: (i) Cerebellar Ataxias and Hereditary Spastic Paraplegias; (ii) Huntington's disease and Other Choreas; (iii) Frontotemporal dementia; (iv) Dystonia, (non-epileptic) paroxysmal disorders, and Neurodegeneration with Brain Iron Accumulation; (v) Leukoencephalopathies; and (vi) Atypical Parkinsonian Syndromes. At the moment, it unites 32 expert centers and 10 affiliated partners in 21 European countries, as well as patient representatives, but will soon cover nearly all countries of the European Union as a result of the ongoing expansion process. Disease expert groups developed and consented on diagnostic flowcharts and disease scales to assess the different aspects of RNDs. ERN-RND has started to discuss diagnostically unclear patients in the CPMS, is one of four ERNs that serve as foundation of Solve-RD, and has established an RND training and education program. The network will facilitate trial readiness through the establishment of an ERN-RND registry with a minimal data of all patients seen at the ERN-RND centers, thus providing a unique overview of existing genotype-based cohorts. The overall aim of the ERNs is to improve access for patients with RDs to quality diagnosis, care, and treatment. Based on this objective, ERNs are monitored by the European Commission on a regular basis to provide transparency and reassurance to the RD community and the general public

FBXO7 is implicated in the ubiquitin–proteasome system and parkin-mediated mitophagy. FBXO7defects cause a levodopa-responsive parkinsonian-pyramidal syndrome(PPS). Methods: We investigated the disease molecular bases in a child with PPS and brain iron accumulation. Results: A novel homozygous c.368C>G (p.S123*) FBXO7 mutation was identified in a child with spastic paraplegia, epilepsy, cerebellar degeneration, levodopa nonresponsive parkinsonism, and brain iron deposition. Patient’s fibroblasts assays demonstrated an absence of FBXO7 RNA expression leading to impaired proteasome degradation and accumulation of poly-ubiquitinated proteins. Conclusion: This novel FBXO7 phenotype associated with impaired proteasome activity overlaps with neurodegeneration with brain iron accumulation disorders., Fundacio La Marato de TV3 [Grants 20143130 to BPD, and 20143131 to CE], by the Instituto de Salud Carlos III (ISCIII) - Subdireccion General de Evaluacion y Fomento de la Investigacion within the framework of the National R + D+I Plan cofunded with ERDF funds [Grants PI18/01319 to BPD and PI18/00147 to CE], and by the Generalitat Valenciana [Grant PROMETEO/2018/ 135 to CE]. Part of the equipment employed in this work has been funded by Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). SFR had a contract funded by the Spanish Foundation Per Amor a l’Art (FPAA). PS had a FPU-PhD fellowship funded by the Spanish Ministry of Education, Culture and Sport Inmunoterapia

To report new sporadic cases and 1 family with epilepsy of infancy with migrating focal seizures (EIMFSs) due to KCNT1 gain-of-function and to assess therapies' efficacy including quinidine. We reviewed the clinical, EEG, and molecular data of 17 new patients with EIMFS and KCNT1 mutations, in collaboration with the network of the French reference center for rare epilepsies. The mean seizure onset age was 1 month (range: 1 hour to 4 months), and all children had focal motor seizures with autonomic signs and migrating ictal pattern on EEG. Three children also had infantile spasms and hypsarrhythmia. The identified KCNT1 variants clustered as "hot spots" on the C-terminal domain, and all mutations occurred de novo except the p.R398Q mutation inherited from the father with nocturnal frontal lobe epilepsy, present in 2 paternal uncles, one being asymptomatic and the other with single tonic-clonic seizure. In 1 patient with EIMFS, we identified the p.R1106Q mutation associated with Brugada syndrome and saw no abnormality in cardiac rhythm. Quinidine was well tolerated when administered to 2 and 4-year-old patients but did not reduce seizure frequency. The majority of the KCNT1 mutations appear to cluster in hot spots essential for the channel activity. A same mutation can be linked to a spectrum of conditions ranging from EMFSI to asymptomatic carrier, even in the same family. None of the antiepileptic therapies displayed clinical efficacy, including quinidine in 2 patients.

Altres ajuts: This work was supported by the Ministerio de Ciencia e Innovación , by the Ministerio de Economía y Competitividad and by Generalitat de Catalunya. Roslyn Boyd is supported by a NHMRC Research Fellowship. Olga Laporta received a research grant from Ministerio de educación, cultura y deporte of the government of Spain and an Endeavour research Fellowship by the Australian Government (grant code 5240). Lee Reid and Alex Pagnozzi each are supported by an Advance Queensland Research Fellowship. To characterise brain lesions in dyskinetic cerebral palsy (DCP) using the semi-quantitative scale for structural MRI (sqMRI) and to investigate their relationship with motor, communication and cognitive function. Thirty-nine participants (19 females, median age 21y) with DCP were assessed in terms of motor function, communication and a variety of cognitive domains. Whole-head magnetic resonance imaging (MRI) was performed including T1-MPRAGE, T2 turbo spin echo (axial plane), and fluid attenuated inversion recovery images (FLAIR). A child neurologist visually assessed images for brain lesions and scored these using the sqMRI. Ordinal, Poisson and binomial negative regression models identified which brain lesions accounted for clinical outcomes. Brain lesions were most frequently located in the ventral posterior lateral thalamus and the frontal lobe. Gross (B = 0.180, p < .001; B = 0.658, p < .001) and fine (B = 0.136, p = .003; B = 0.540, p < .001) motor function were associated with global sqMRI score and parietal involvement. Communication functioning was associated with putamen involvement (B = 0.747, p < .028). Intellectual functioning was associated with global sqMRI score and posterior thalamus involvement (B = −0.018, p < .001; B = −0.192, p < .001). Selective attention was associated with global sqMRI score (B = −0.035, p < .001), parietal (B = −0.063, p = .023), and corpus callosum involvement (B = −0.448, p < .001). Visuospatial and visuoperceptive abilities were associated with global sqMRI score (B = −0.078, p = .007) and medial dorsal thalamus involvement (B = −0.139, p < .012), respectively. Key clinical outcomes in DCP are associated with specific observable brain lesions as indexed by a simple lesion scoring system that relies only on standard clinical MRI.

Stroke-like episodes (SLE) occur in phosphomannomutase deficiency (PMM2-CDG), and may complicate the course of channelopathies related to Familial Hemiplegic Migraine (FHM) caused by mutations in CACNA1A (encoding CaV2.1 channel). The underlying pathomechanisms are unknown. We analyze clinical variables to detect risk factors for SLE in a series of 43 PMM2-CDG patients. We explore the hypothesis of abnormal CaV2.1 function due to aberrant N-glycosylation as a potential novel pathomechanism of SLE and ataxia in PMM2-CDG by using whole-cell patch-clamp, N-glycosylation blockade and mutagenesis. Nine SLE were identified. Neuroimages showed no signs of stroke. Comparison of characteristics between SLE positive versus negative patients' group showed no differences. Acute and chronic phenotypes of patients with PMM2-CDG or CACNA1A channelopathies show similarities. Hypoglycosylation of both CaV2.1 subunits (α1A and α2α) induced gain-of-function effects on channel gating that mirrored those reported for pathogenic CACNA1A mutations linked to FHM and ataxia. Unoccupied N-glycosylation site N283 at α1A contributes to a gain-of-function by lessening CaV2.1 inactivation. Hypoglycosylation of the α₂δ subunit also participates in the gain-of-function effect by promoting voltage-dependent opening of the CaV2.1 channel. CaV2.1 hypoglycosylation may cause ataxia and SLEs in PMM2-CDG patients. Aberrant CaV2.1 N-glycosylation as a novel pathomechanism in PMM2-CDG opens new therapeutic possibilities. This work was supported by national grant PI14/00021 and PI17/00101 from the National Plan on I+D+I, cofinanced by ISC-III (Subdirección General de Evaluación y Fomento de la Investigación Sanitaria), the Spanish Ministry of Economy and Competitiveness (Grants IPT-2012-0561-010000, SAF2015-69762-R, MDM-2014-0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), FEDER (Fondo Europeo de Desarrollo Regional), and the Migraine Research Foundation (New York, USA). Mercè Izquierdo-Serra holds a “Juan de la Cierva-Formación” Fellowship funded by the Spanish Ministry of Economy and Competitiveness.

Abnormal lingual movements are not uncommon. A rare lingual syndrome consisting of involuntary wave-like lingual movements has been labeled as “galloping tongue.”1 Herein, we report a patient with galloping tongue syndrome carrier of a mutation in the PRRT2 gene.

Mutations in the CACNA1A gene, encoding the pore-forming CaV2.1 (P/Q-type) channel α1A subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α1A domain III (ΔF1502). Functional studies of ΔF1502 CaV2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α1A mutation, detected by whole-exome sequencing, and analyze its functional consequences on CaV2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of CaV2.1 within a wide range of voltage depolarization. ΔF1502 also slowed CaV2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function CaV2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A-associated phenotypic spectrum. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2012-31089 to JMF-F; SEV-2012-0208 to Centre for Genomic Regulation, “Centro de Excelencia Severo Ochoa 2013-2017”; and MDM-2014-0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), FEDER Funds, Fondo de Investigación Sanitaria, Instituto Carlos III, Spain (RIC RD12/0042/0014, Red HERACLES, and Grant PI12/1005 to AM). AM-G is a predoctoral fellow supported by Vall d’Hebron Institut de Recerca, Barcelona, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Background: Phosphomannomutase deficiency (PMM2-CDG) is the most frequent congenital disorder of glycosylation. The cerebellum is nearly always affected in PMM2-CDG patients, a cerebellar atrophy progression is observed, and cerebellar dysfunction is their main daily functional limitation. Different therapeutic agents are under development, and clinical evaluation of drug candidates will require a standardized score of cerebellar dysfunction. We aim to assess the validity of the International Cooperative Ataxia Rating Scale (ICARS) in children and adolescents with genetically confirmed PMM2-CDG deficiency. We compare ICARS results with the Nijmegen Pediatric CDG Rating Scale (NPCRS), neuroimaging, intelligence quotient (IQ) and molecular data. Methods: Our observational study included 13 PMM2-CDG patients and 21 control subjects. Ethical permissions and informed consents were obtained. Three independent child neurologists rated PMM2-CDG patients and control subjects using the ICARS. A single clinician administered the NPCRS. All patients underwent brain MRI, and the relative diameter of the midsagittal vermis was measured. Psychometric evaluations were available in six patients. The Mann-Whitney U test was used to compare ICARS between patients and controls. To evaluate inter-observer agreement in patients' ICARS ratings, intraclass correlation coefficients (ICC) were calculated. ICARS internal consistency was evaluated using Cronbach's alpha. Spearman's rank correlation coefficient test was used to correlate ICARS with NPCRS, midsagittal vermis relative diameter and IQ. Results: ICARS and ICARS subscores differed between patients and controls (p < 0.001). Interobserver agreement of ICARS was "almost perfect" (ICC = 0.99), with a "good" internal reliability (Cronbach's alpha = 0.72). ICARS was significantly correlated with the total NPCRS score (rs 0.90, p < 0.001). However, there was no agreement regarding categories of severity. Regarding neuroimaging, inverse correlations between ICARS and midsagittal vermis relative diameter (rs -0.85, p = 0.003) and IQ (rs -0.94, p = 0.005) were found. Patients bearing p.E93A, p.C241S or p.R162W mutations presented a milder phenotype. Conclusions: ICARS is a reliable instrument for assessment of PMM2-CDG patients, without significant inter-rater variability. Despite our limited sample size, the results show a good correlation between functional cerebellar assessment, IQ and neuroimagingFor the first a correlation between ICARS, neuroimaging and IQ in PMM2-CDG patients has been demonstrated, The work was supported by national grants PI14/00021, PI11/01096, PI11/01250, and PI10/00455 from the National Plan on I+D+I, cofinanced by ISC-III (Subdirección General de Evaluación y Fomento de la Investigación Sanitaria) and FEDER (Fondo Europeo de Desarrollo Regional) and IPT-2012- 0561-010000 from MINECO. Three research groups (U-746, U-737 and U703) from the Centre for Biomedical Research on Rare Diseases (CIBER-ER), Instituto de Salud Carlos III, Spain, have worked together for the present study

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A -p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2 -p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing.

In 5 prospectively diagnosed patients with relapsing post-herpes simplex encephalitis (HSE), N-methyl-D-aspartate receptor (NMDAR) antibodies were identified. Antibody synthesis started 1 to 4 weeks after HSE, preceding the neurological relapse. Three of 5 patients improved postimmunotherapy, 1 spontaneously, and 1 has started to improve. Two additional patients with NMDAR antibodies, 9 with unknown neuronal surface antibodies, and 1 with NMDAR and unknown antibodies, were identified during retrospective assessment of 34 HSE patients; the frequency of autoantibodies increased over time (serum, p=0.004; cerebrospinal fluid, p=0.04). The 3 retrospectively identified NMDAR antibody-positive patients also had evidence of relapsing post-HSE. Overall, these findings indicate that HSE triggers NMDAR antibodies and potentially other brain autoimmunity.

Episodic ataxia is an autosomal dominant ion channel disorder characterized by episodes of imbalance and incoordination. The disease is genetically heterogeneous and is classified as episodic ataxia type 2 (EA2) when it is caused by a mutation in the CACNA1A gene, encoding the α1A subunit of the P/Q-type voltage-gated calcium channel Cav2.1. The vast majority of EA2 disease-causing variants are loss-of-function (LoF) point changes leading to decreased channel currents. CACNA1A exonic deletions have also been reported in EA2 using quantitative approaches. We performed a mutational screening of the CACNA1A gene, including the promoter and 3'UTR regions, in 49 unrelated patients diagnosed with episodic ataxia. When pathogenic variants were not found by sequencing, we performed a copy number variant (CNV) analysis to screen for duplications or deletions. Overall, sequencing screening allowed identification of six different point variants (three nonsense and three missense changes) and two coding indels, one of them found in two unrelated patients. Additionally, CNV analysis identified a deletion in a patient spanning exon 35 as a result of a recombination event between flanking intronic Alu sequences. This study allowed identification of potentially pathogenic alterations in our sample, five of them novel, which cover 20% of the patients (10/49). Our data suggest that most of these variants are disease-causing, although functional studies are required. The funding for this study was provided by the Spanish Ministerio de Economía y Competitividad (SAF2009-13182-C01, SAF2009-13182-C03), AGAUR (2014SGR-0932, 2009SGR-0078) and Fundació La Marató de TV3 (grant 100731). These institutions had no further role in study design, collection, analysis, interpretation of data or in the submission of this paper for publication. CS and OC were supported by Ministerio de Economía y Competitividad (BES-2007-16450 and BES-2010-033895, respectively) and NF-C by Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER, ISCIII). MV-P was supported by a predoctoral grant from VHIR, Barcelona (Spain). CT was supported by the European Union (Marie Curie, PIEF-GA-2009-254930). EC-L is the recipient of the Beatriu de Pinós programme scholarship (BP-DGR 2010).

Els trastorns genètics de la substància blanca cerebral (Genetic White Matter Disorders; GWMDs) afecten predominantment a la producció i manteniment de la mielina del sistema nerviós central. L'abordatge diagnòstic "clàssic", basat en estudis genètics dirigits a partir de la sospita clínica, permet el diagnòstic molecular en només la meitat dels pacients. L'estudi de seqüenciació de l'exoma i del genoma complets (WES i WGS respectivament), ha demostrat un alt rendiment diagnòstic en estudis realitzats en diverses patologies de base genètica, principalment en trios (analitzant conjuntament les dades genòmiques del pacient i els seus progenitors). Aquest treball pretén establir la utilitat clínica dels estudis WES/WGS mitjançant l'anàlisi únicament de casos índex (no trio) amb GWMDs, identificar nous gens associats a aquests trastorns i descriure nous fenotips clínics. Al laboratori de Malalties Neurometabòliques de l'Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) vam estudiar mitjançant WES/WGS pacients amb GMWDs no diagnosticats després d'un abordatge "clàssic". Els casos van ser remesos des de serveis de neurologia d'hospitals terciaris d'arreu d'Espanya des de gener de 2015 fins a desembre de 2019. Els estudis WES/WGS van ser analitzats mitjançant un algoritme computacional que prioritza les variants genètiques segons la informació clínica codificada en termes HPO (Human Phenotype Ontology) i informació de les interaccions físiques i funcionals de les proteïnes de l'organisme. Vam incloure 126 famílies i vam poder establir el diagnòstic molecular en 91 d'elles (72% dels casos). A més, vam identificar 7 gens candidats, fenotips nous i cinc famílies amb diagnòstic dual. Es va evidenciar la gran heterogeneïtat genètica d'aquest grup de trastorns (57 gens identificats entre els 91 casos diagnosticats), sent els més freqüents EIF2B5, POLR3A, RNASEH2B i PLP1. Vam identificar un nou GWMD, produït per variants bial·lèliques en el gen PI4KA i vam fer la validació funcional. Vam descriure deu pacients amb un espectre fenotípic que comprenia des d'una malaltia hipomielinitzant greu associada a alteracions estructurals del cervell (hipoplàsia del tronc de l'encèfal i del cerebel o polimicrogíria), fins a una paraparèsia espàstica pura. Alguns pacients presentaven trastorns immunològics, gastrointestinals o anomalies genitourinàries. A més, vam poder participar en la descripció del fenotip clínic associat a variants bial·lèliques en el gen PRORP, que es caracteritzava per una sordesa neurosensorial, insuficiència ovàrica primària, retard del desenvolupament i canvis de la substància blanca cerebral. També en la descripció d'un nou fenotip associat a POLR3A, amb predomini de clínica extrapiramidal i afectació estriatal, i en la caracterització fenotípica de variants en el gen HNRNPH1. Finalment, vam coordinar l'estudi de 53 pacients amb variants en heterozigosi el gen DLG4, que codifica per la proteïna postsinàptica PSD-95. El quadre clínic es caracteritzava per una discapacitat intel·lectual, un trastorn de l'espectre autista, epilèpsia i trastorns del moviment. Cal destacar que alguns d'aquests pacients presentaven anomalies de la substància blanca cerebral, en forma d'atròfia, retard de la mielinització o hiperintensitats periventriculars, i d'altres, una paraparèsia espàstica. Per tant, aquest treball mostra com els trastorns sinàptics també poden tenir una forma de presentació compatible amb l'espectre fenotípic de GWMD-paraparèsia espàstica hereditària. Aquesta tesi mostra la utilitat clínica dels estudis de WES/WGS realitzats en casos índex (no trio) amb GWMDs, i com possibiliten la identificació de nous gens candidats i nous fenotips clínics. Reforça a més la necessitat d'implantar-los en estadis inicials del procés diagnòstic, per poder oferir un assessorament genètic adequat i tractaments específics abans que l'afectació neurològica estigui ja massa avançada. Finalment, posa de manifest la importància de la col·laboració entre centres assistencials i d'investigació, i de la participació dels clínics en el procés diagnòstic basat en la NGS. Los trastornos genéticos de la sustancia blanca cerebral (Genetic White Matter Disorders; GWMDs) afectan predominantemente a la producción y mantenimiento de la mielina del sistema nervioso central. El abordaje diagnóstico "clásico", basado en estudios genéticos dirigidos a partir de la sospecha clínica, permite el diagnóstico molecular en la mitad de los pacientes. El estudio de secuenciación del exoma y del genoma completos (WES y WGS respectivamente), ha demostrado un alto rendimiento diagnóstico en estudios realizados en diversas patologías de base genética, principalmente en tríos (analizando conjuntamente los datos genómicos del paciente y sus progenitores). Este trabajo pretende establecer la utilidad clínica de los estudios WES/WGS mediante el análisis únicamente de casos índice (no trío) con GWMDs, identificar nuevos genes asociados a estos trastornos y describir nuevos fenotipos clínicos. En el laboratorio de Enfermedades Neurometabólicas del Instituto de Investigación Biomédica de Bellvitge (IDIBELL) estudiamos mediante WES/WGS a pacientes con GMWDs no filiados después de un abordaje diagnóstico "clásico". Estos pacientes fueron remitidos desde servicios de neurología de hospitales terciarios de toda España desde enero de 2015 hasta diciembre de 2019. Los estudios WES/WGS fueron analizados mediante un algoritmo computacional que priorizaba las variantes genéticas según la información clínica codificada en términos HPO (Human Phenotype Ontology) e información de las interacciones físicas y funcionales de las proteínas del organismo. Incluimos 126 familias y pudimos establecer el diagnóstico molecular en 91 de ellas (72% de los casos). Además, identificamos 7 genes candidatos, nuevos fenotipos y cinco familias con diagnóstico dual. Se evidenció la gran heterogeneidad genética de este grupo de trastornos (57 genes identificados entre los 91 casos diagnosticados), siendo los más frecuentes EIF2B5, POLR3A, RNASEH2B y PLP1. Identificamos un nuevo GWMD, producido por variantes bialélicas en el gen PI4KA y realizamos la validación funcional. Describimos diez pacientes que presentaban un espectro fenotípico que comprendía desde una enfermedad hipomielinizante grave con alteraciones estructurales del cerebro (hipoplasia del tronco del encéfalo y del cerebelo o polimicrogiria), hasta una paraparesia espástica pura. Algunos pacientes presentaban trastornos inmunológicos, gastrointestinales o anomalías genitourinarias. Además, pudimos participar en la descripción del fenotipo clínico asociado a variantes bialélicas en el gen PRORP, que se caracterizaba por una sordera neurosensorial, insuficiencia ovárica primaria, retraso del desarrollo y cambios de la sustancia blanca cerebral. También en la descripción de un nuevo fenotipo asociado a POLR3A, con predominio de clínica extrapiramidal y afectación estriatal, y en la caracterización fenotípica de variantes en el gen HNRNPH1. Finalmente, coordinamos el estudio de 53 pacientes con variantes en heterozigosis el gen DLG4, que codifica por la proteína postsináptica PSD-95. El cuadro clínico se caracterizaba por una discapacidad intelectual, un trastorno del espectro autista, epilepsia y trastornos del movimiento. Cabe destacar que algunos de estos pacientes presentaban anomalías de la sustancia blanca cerebral, en forma de atrofia, retraso de la mielinización o hiperintensidades periventriculares, y otros, una paraparesia espástica. Por tanto, este trabajo muestra cómo los trastornos sinápticos también pueden tener un modo de presentación compatible con el espectro fenotípico de GWMD-paraparesia espástica hereditaria. Esta tesis muestra la utilidad clínica de los estudios de WES/WGS realizados en casos índice (no trío) con GWMDs, y cómo posibilitan la identificación de nuevos genes candidatos y nuevos fenotipos clínicos. Refuerza además la necesidad de implantarlos en estadios iniciales del proceso diagnóstico, para poder ofrecer un asesoramiento genético adecuado y tratamientos específicos antes de que la afectación neurológica esté ya demasiado avanzada. Por último, pone de manifiesto la importancia de la colaboración entre centros asistenciales y de investigación, y de la participación de los clínicos en el proceso diagnóstico basado en la NGS. Genetic White Matter Disorders (GWMDs) predominantly affect the production or maintenance of myelin in the central nervous system. The "classic" diagnostic approach, based on targeted genetic studies, allows molecular diagnosis in only half of patients. The study of Whole-Exome and Whole-Genome Sequencing (WES and WGS respectively) showed a high diagnostic performance in hereditary neurological diseases in studies performed mainly in trios (analysing together the genomic data of the patient and his parents). This work aims to establish the clinical utility of WES/WGS studies by analysing only index cases (not trio) with GWMDs, while identifying new genes associated with these disorders, and describing new clinical phenotypes. In the Neurometabolic Diseases Laboratory of the Bellvitge Biomedical Research Institute (IDIBELL), we ran singleton WES/WGS to study patients with undiagnosed GMWDs after a "classic" diagnostic approach. These patients were referred from neurology services of tertiary Spanish hospitals from January 2015 to December 2019. The WES/WGS studies were analysed using a computational algorithm that prioritized genetic variants according to the clinical information encoded in HPO terms (Human Phenotype Ontology) and information on the physical and functional interactions of the body's proteins. We included 126 families and were able to establish the molecular diagnosis in 91 of them (72% of cases). In addition, we identified 7 candidate genes, new phenotypes, and five families with dual diagnosis. A high genetic heterogeneity was evidenced (57 genes identified among the 91 diagnosed cases), with the most frequent being EIF2B5, POLR3A, RNASEH2B and PLP1. We identified a new GWMD, produced by biallelic variants in the PI4KA gene, and we made a functional validation. We described ten patients with a phenotypic spectrum ranging from severe hypomyelinating disease, associated with structural brain abnormalities (brainstem and cerebellum hypoplasia or polymicrogyria), to a pure spastic paraparesis. In addition, some patients had immune disorders, gastrointestinal manifestations or genitourinary abnormalities. We were also able to participate in the description of the clinical phenotype associated with biallelic variants in the PRORP gene, which was characterized by sensorineural hearing loss, primary ovarian failure, developmental delay and cerebral white matter abnormalities. We also collaborated in the description of a new phenotype associated with POLR3A, with a predominance of extrapyramidal manifestations and striatal involvement on neuroimaging, and in the phenotypic characterization of variants in the HNRNPH1 gene. Finally, we coordinated the study of 53 patients with heterozygous variants in the DLG4 gene, which encodes the PSD-95 postsynaptic protein. The clinical picture was characterized by intellectual disability, autism spectrum disorder, epilepsy, and movement disorders. It should be noted that some of these patients had cerebral white matter atrophy, delayed myelination or periventricular hyperintensities on MRI, while others had spastic paraparesis. Therefore, this work shows how synaptic disorders can also have a clinical presentation compatible with GWMD-hereditary spastic paraparesis spectrum. This thesis shows the clinical utility of WES/WGS studies performed in index cases (not trio) with GWMDs, and how they enable the identification of new candidate genes and new clinical phenotypes. It also reinforces the need to introduce them in the initial stages of the diagnostic process, to offer adequate genetic counselling and specific treatments before the advancement of neurological involvement. Finally, it highlights the importance of collaboration between healthcare and research centres, and the participation of clinicians in the diagnostic process based on NGS.

Antecedents: els corticoides són potents antiinflamatoris però alhora tenen un gran nombre de potencials efectes adversos. En l'actualitat, pel que fa a patologia neurològica aguda, estan indicats en meningitis agudes bacterianes i en alguns casos d'status migranyós i epilèptic, entre d'altres. Objectiu: identificar les estratègies actuals de la patologia neurològica aguda pediàtrica i el paper dels corticoides en aquesta. Metodologia: s'ha realitzat una recerca sobre l'ús de corticoides en nens amb encefalopaties agudes a la base de dades "Pubmed". Discussió: hi ha pocs estudis i gran part de les recomanacions es fan basades en l'ús de corticoides en adults o en resultats obtinguts en sèries de casos. Conclusions: l'ús de corticoides sembla segur atès la revisió bibliogràfica i sembla que en alguns casos podrien modificar la història natural en patologia neurològica aguda, malgrat la manca de recomanacions basades en l'evidència.

Les encefalopaties epilèptiques són una condició on les anomalies epileptiformes es postulen com a contribuïdores d'una alteració progressiva de la funció cerebral. Aquest treball se centra específicament en les Encefalopaties Epilèptiques d'Inici Precoç (EIEE), les quals són un grup d'encefalopaties epilèptiques amb inici al primer any de vida. De forma majoritària, són causades per un defecte genètic, que pot afectar a un gran nombre de gens, i no responen a fàrmacs antiepilèptics. Aquest trastorn cobreix un grup de síndromes clínics caracteritzats per un solapament clínic, l'absència de biomarcadors i una alta variabilitat de fenotips. Tot i això, juntament amb l'heterogeneïtat genètica, converteixen el seu diagnòstic en un gran repte. Degut a la seva elevada variabilitat genètica, les noves tècniques de seqüenciació massiva (NGS) es proposen com una bona eina per a l'estudi d'aquest trastorn perquè permet l'anàlisi d'un gran nombre de gens a la vegada. L'objectiu d'aquesta tesi és explorar la utilitat de les NGS com a eines més eficients tant per al diagnòstic com per al descobriment de nous gens relacionats amb la malaltia. Aquest estudi recull una cohort de 80 pacients EIEE, els quals han estat seqüenciats per a obtenir-ne un diagnòstic genètic. Setanta sis d'ells s'han sotmès a seqüenciació de l'exoma complet (WES), junt amb els seus progenitors, en tres centres diferents: CNAG, UCL i CentoGene. Cada un dels centres ha utilitzat la seva pipeline pròpia de processament de les dades i l'anàlisi de variants s'ha realitzat al laboratori de Neurologia Pediàtrica en dos passos. En primer lloc s'han avaluat les variants en gens relacionats amb epilèpsia, seguit per l'anàlisi de la resta de variants de l'exoma complet. Un reanàlisi posterior ha tingut lloc per aquells pacients amb resultat negatiu. Totes les variants seleccionades han estat validades pel mètode Sanger, resultant en una taxa de diagnòstic del 50% entre els pacients WES, i del 52,5% considerant totes les tècniques utilitzades. El diagnòstic de 31 pacients implica variants en gens associats al fenotip: STXBP1, KCNQ2, KCNT1, SCN1A, SCN2A, SCN8A, SLC35A2, SLC13A5, GABRA1, GNAO1, SZT2, SPATA5, RARS2, GRIA2 i ALDH7A1. Tot i això, l'establiment d'una correlació genotip-fenotip entre els gens d'EIEE segueix sent una tasca complicada. L'anàlisi NGS també ha proporcionat variants en 11 nous gens candidats. Per a provar la seva contribució al fenotip s'han dissenyat diferents assaigs funcionals. Per una banda, s'ha intentat desenvolupar un model en peix zebra de sobreexpressió de RNA per a analitzar-ne el potencial efecte in vivo. D'altra banda, altres assaigs in vitro s'han utilitzat per a estudiar la implicació molecular d'una mutació específica sobre el receptor muscarínic d'acetilcolina tipus I. Els resultats estableixen que CHRM1 podria ser un nou gen contribuïdor al fenotip de les epilèpsies. El present treball ha comprovat la utilitat de les tècniques NGS, no només per a millorar el diagnòstic genètic de les EIEE, sinó que també per a identificar nous gens associats a la malaltia. El debut clínic precoç s’associa a una taxa diagnòstica superior. Alhora, l’acurada consideració de les variants en funció d’un profund fenotipat i l’estreta col·laboració entre especialistes clínics i investigadors incrementa la taxa de diagnòstic de les NGS. Epileptic encephalopathy is a condition in which epileptiform abnormalities are believed to contribute to the progressive disturbance in cerebral function. This work is focused specifically on Early Infantile Epileptic Encephalopathies (EIEE), which are a group of Epileptic Encephalopathies which start within the first year of life. They are mostly caused by a genetic defect, which can affect a great number of different genes, and they are refractory to antiepileptic drugs. This disorder covers a group of clinical syndromes which are characterized by a clinical overlap, an absence of biomarkers and a variability of phenotypes. All this, together with its genetic heterogeneity makes its diagnosis a big challenge. Due to this genetic variability, Next Generation Sequencing (NGS) technology is emerging as a good tool to study this disorder because it allows screening a huge number of genes at the same time. The aim of this thesis is to explore the usefulness NGS as a more efficient tool for both diagnosis and discovery of new genes related to this disease. A cohort of 80 EIEE patients was collected for this study and sequenced in order to get a genetic diagnosis. 76 of them underwent whole exome sequencing (WES) together with their parents, which was performed in three different centers: CNAG, UCL and CentoGene. Each center used its own data processing pipeline and a two-tiered analysis was done in Pediatric Neurology laboratory to filter variants of interest. First, the evaluation of variants placed in epilepsy-related-genes took place, followed by the ones selected from the whole exome. Later, a reanalysis was performed with those patients without a clear diagnosis. All those variants selected as causal or contributing to the disease were validated by Sanger Sequencing, resulting in a 50% of diagnostic rate within patients sequenced by WES, and 52,5% considering all the techniques used in this work. Diagnosis obtained for 31 of the patients consisted of mutations lying in genes already associated with the phenotype: STXBP1, KCNQ2, KCNT1, SCN1A, SCN2A, SCN8A, SLC35A2, SLC13A5, GABRA1, GNAO1, SZT2, SPATA5, RARS2, GRIA2 and ALDH7A1. However, the establishment of a genotype-phenotype correlation for genes involved in EIEE is still a difficult issue. NGS analysis has provided also variants in 11 new candidate genes. To prove their contribution to the phenotype, functional assays were designed. A zebra fish RNA overexpression model was developed in order to screen easily the potential effect of the variant in vivo. Other in vitro assays were used to study molecular involvement of a specific mutation at muscarinic acetylcholine receptor I. Results establish CHRM1 as a strong candidate as new epilepsy-related-gene. The present work examines the usefulness of NGS technologies, not only to improve the genetic diagnosis of EIEE patients, but also to identify new genes associated to EIEE. Early clinical presentation is associated with higher diagnostic rates. Likewise, careful consideration of variants based on deep phenotyping and close collaboration among clinical specialists and researchers does increase the diagnostic rate of NGS.

Se trata de un estudio clínico-radiológico detallado de casos de malformación de Chiari tipo 1 (MC-1) en edad pediátrica y de casos con agregación familiar. El estudio clínico consiste en una revisión retrospectiva de la clínica de 55 niños afectos de MC-1 (herniación de amígdalas cerebelosas por debajo del plano del foramen magno > 5 mm) y 14 afectos de ectopia amigdalar (herniación 3-4,9 mm), así como de los resultados de las exploraciones complementarias que se les ha realizado. Entre los hallazgos más importantes encontramos que el diagnóstico incidental de la MC-1 es muy frecuente en la edad pediátrica y la cefalea occipital es la forma de debut clínico más frecuente. En la revisión de los hallazgos de resonancia craneal, se ha detectado que la MC-1 puede asociarse a alteraciones de charnela ósea y anomalías vasculares de la circulación posterior. En cuanto al uso de exploraciones complementarias, los datos sugieren que es recomendable el estudio mediante RM medular, debido al riesgo de siringomielia, incluso en pacientes con grados mínimos de herniación amigdalar. Los potenciales evocados somatosensitivos (PESS) están afectados con mucha más frecuencia que los potenciales evocados auditivos de tronco (PEATs) en la edad pediátrica. La polisomnografía detecta no sólo alteraciones respiratorias sino también anomalías en la arquitectura del sueño. En una segunda parte del estudio se han realizado 20 medidas morfométricas de la fosa craneal posterior (FCP) en todos aquellos pacientes con resonancia craneal accesible a revisión, que han consistido en 40 pacientes con MC-1 y 12 con ectopia amigdalar. Se ha realizado una comparación con las medidas de 52 controles pareados por edad y sexo. En este estudio morfométrico se ha detectado que existen diferencias significativas entre afectos de MC-1 y controles en varias medidas de FCP, que determinan principalmente la altura de la FCP y el tamaño del hueso occipital. Algunas de dichas medidas, también son significativamente menores en pacientes con ectopia amigdalar. Una nueva medida valorada en este estudio, el área ósea occipital, ha mostrado diferencias significativas respecto a controles tanto en MC-1 como en ectopia amigdalar. Dicha medida ha mostrado el mayor tamaño de efecto. En cuanto al estudio de familias afectas de MC-1, se ha revisado la clínica y pedigree de 9 casos con agregación familiar, así como un estudio de imagen morfométrico de las 6 familias con imágenes accesibles a revisión. El estudio muestra que las familias afectas de MC-1 pueden compartir patrones morfométricos específicos que difieren mucho entre distintas familias y que probablemente reflejan la heterogeneidad genética del trastorno. En algunas familias, los pacientes adultos presentan una o varias características morfométricas alteradas o un mayor grado de afectación que no están presentes en los pacientes pediátricos, o no han aparecido todavía, por ejemplo, una FCP ósea pequeña., This is a detailed clinicoradiological study of Chiari 1 malformation (CM-1) in children and also in families affected by this malformation. The clinical study is a retrospective review of the clinical picture in 55 children with CM-1 (tonsillar herniation > 5 mm below the foramen magnum) and 14 children with tonsillar ectopia (herniation 3-4,9 mm). Also, the complementary tests performed in these patients were reviewed. The main results show that CM-1 is frequently diagnosed as an incidental finding in brain magnetic resonance (MR) studies. In symptomatic patients, occipital headache is the more frequent onset. In the MR review, vascular anomalies of the posterior circulation and bone anomalies of the occipitocervical junction were found. Data of this study suggest that spinal MR is advised in patients with tonsillar herniation, as syringomyelia can be found even within ectopia range. The SEP (somatosensory evoked potential) test is more frequently affected in children than the brainstem auditory evoked potential (BAEP) test. The polysomnography showed not only breathing anomalies but also disturbances in sleep architecture. In the second part of this study, 20 morphometric measures of the posterior fosa (PF) were performed in those patients with MR accessible for review. These were 40 patients with CM-1 and 12 patients with tonsillar ectopia. The results were compared to the measures of 52 age and sex-matched controls. Significant differences were found between CM-1 and controls in several measures, mainly affecting the height of the PF and the occipital bone dimensions. Some of these measures were also affected in patients with ectopia. A new measure tested in this study, the occipital bone area, showed significant differences in CM-1 and ectopia versus controls. This measure showed the highest size effect. In the study of families with CM-1, a retrospective review of the clinical picture and pedigree was performed in 9 families. Morphometric studies of the PF were performed in 6 of the families. Specific morphometric patterns were detected in these families that differed between families and probably correlates with the genetic heterogeneity of the disorder. In some families, the adult cases show one or more anomalies than those present in children, as, for example, a small PF.

Aquesta tesi es centra en l’anàlisi genètica dels següents trastorns neurològics paroxístics pediàtrics: el torticoli paroxístic benigne del lactant (TPBL), l’hemiplegia alternant de la infància (HAI), la discinèsia paroxística cinesigènica (DPC) i el síndrome de la deficiència del transportador de glucosa GLUT1 (GLUT1DS); i en l’anàlisi genètica i epigenètica de la migranya, un trastorn neurològic paroxístic majoritàriament de l’adult. Els trastorns neurològics paroxístics pediàtrics analitzats són trastorns rars, poc estudiats, en els quals la simptomatologia i el patró d’herència han portat a sospitar-ne una base monogènica i un vincle amb el grup de les canalopaties neuronals. La dificultat en el seu estudi deriva, majoritàriament, de la manca de sèries importants de pacients de les quals se’n puguin obtenir conclusions extrapolables. L’interès d’aprofundir en el seu coneixement, a banda del propi interès científic, rau en trobar les causes que els originen i, així, poder trobar un tractament contra aquests trastorns, millorar la qualitat de vida dels pacients que els sofreixen i/o poder oferir consell genètic als familiars dels individus afectes. - El cribratge mutacional en 2 pacients afectes de TPBL ha identificat la mutació p.Glu533Lys en el gen CACNA1A com a causant de la malaltia, junt amb els estudis funcionals que indiquen que aquesta mutació provoca una pèrdua de funció de la proteïna codificada. - El cribratge mutacional en una cohort de 10 pacients d’HAI ha identificat 3 mutacions en el gen ATP1A3 (p.Asp801Asn, p.Glu815Lys i p.Gly947Arg) en 5 dels pacients, remarcant l’existència d’una heterogeneïtat genètica major de l’esperada en aquest trastorn. - El cribratge mutacional en la DPC ha identificat 3 mutacions diferents en el gen PRRT2 en 8 dels 10 pacients (c.649dupC, c.649delC i c.219_220delGA) descrivint per primera vegada tant la mutació c.219_220delGA com la presència de la c.649dupC i la c.649delC en un mateix pacient. - El cribratge mutacional en la GLUT1DS va permetre trobar mutacions de novo en el gen SLC2A1 en 3 dels 5 pacients analitzats: la c.667C>T, la c.710_711delGA i una deleció de tot l’exó 1; remarcant l’interès en cercar delecions GLUT1DS. La migranya és un trastorn neurològic primari molt prevalent que es manifesta amb crisis episòdiques i recurrents de mal de cap incapacitant. Els criteris de la International Headache Society subclassifiquen la malaltia en diferents subtipus, incloent la migranya sense aura (MO), la migranya amb aura (MA) i la migranya hemiplègica (MH). - El cribratge mutacional de la MH va permetre identificar 4 mutacions en el gen CACNA1A (p.Ser218Leu, p.Thr501Met, p.Arg583Gln i p.Thr666Met), 2 mutacions en el gen ATP1A2 (p.Ala606Thr i p.Glu825Lys) i la mutació p.Phe1661Leu en el gen SCN1A. - L’estudi d’associació genètica a nivell genòmic (GWAS) de la MO va permetre la identificació de 2 SNPs associats a MO, un localitzat en el gen MEF2D i l’altre proper al gen TGFBR2. També es van trobar SNPs que suggerien una tendència a la replicació en el gens PHACTR1 i ASTN2. A més a més, es van replicar els resultats obtinguts en un GWAS anterior, trobant novament associats a migranya els gens TRPM8 i LRP1. Aquest estudi va permetre identificar el primer loci d’associació a susceptibilitat a patir MO. - L’estudi epigenètic en un model de MA va permetre trobar diferències de metilació de l’ADN degudes al tractament amb àcid valproic o topiramat o a l’efecte de la depressió cortical propagant (DCP), el fenomen subjacent de l’aura, en gens que podrien estar relacionats amb la susceptibilitat a patir migranya. Els resultats podrien indicar que ambdós tractaments protegeixen davant les DCPs degut al seu efecte sobre la metilació de l’ADN, emfatitzant la importància dels mecanismes epigenètics en la susceptibilitat de la migranya., This thesis is focused on the genetic analysis of the following neurological paediatric paroxysmal disorders: benign paroxysmal torticollis of infancy (BPTI), alternating hemiplegia of childhood (AHC), paroxysmal kinesigenic dyskinesia (PKD) and GLUT1 deficiency syndrome (GLUT1DS); and on the genetic and epigenetic analysis of migraine, a neurological paroxysmal disorder mainly found in adults. The neurological paediatric paroxysmal disorders analyzed are rare and present a simptomatology and an inheritance that suggest a monogenic origin and a link with the neuronal channelopathies. The lack of significant cohorts of patients from which obtain transferable conclusions makes it difficult to study them. The main interest of their study, besides the own scientific interest, is based on finding the underlying causes of the disorder which would be the first step to find the appropriate treatment, improve the quality of life of the patients and/or be able to offer genetic counselling to the patients relatives. The results of this study are resumed below: - The mutational screening in 2 patients of BPTI identified the p.Glu533Lys mutation in the CACNA1A gene as the genetic cause of this disorder, in line with the functional studies that indicate that this mutation induces a loss-of-function of the coding protein. - The mutational screening in a cohort of 10 patients of AHC identified 3 mutations in the ATP1A3 gene (p.Asp801Asn, p.Glu815Lys and p.Gly947Arg) in 5 patients, highlighting the existence of a greater genetic heterogeneity than expected in this disorder. - The mutational screening in PKD identified 3 different mutations in the PRRT2 gene in 8 out of 10 patients (c.649dupC, c.649delC and c.219_220delGA), describing for the first time the mutation c.219_220delGA and also the presence of both the duplication and the deletion in the c.649 position in the same patient. - The mutational screening in 5 patients of GLUT1DS identified de novo mutations in the SLC2A1 gene in 3 patients, specifically c.667C>T, c.710_711delGA and a deletion affecting the whole first exon, highlighting the interest in looking for deletions in GLUT1DS. Migraine is a common primary neurological disorder that presents with episodic and recurrent attacks of disabling headache. The criteria of the International Headache Society divide the disorder into different subclasses, including migraine without aura (MO), migraine with aura (MA) and hemiplegic migraine (HM). The results of the genetic and epigenetic studies of migraine are resumed below: - The mutational screening of HM identified 4 mutations in the CACNA1A gene (p.Ser218Leu, p.Thr501Met, p.Arg583Gln and p.Thr666Met), 2 mutations in the ATP1A2 gene (p.Ala606Thr i p.Glu825Lys) and the mutation p.Thr501Met in the SCN1A gene. - The genome wide association study (GWAS) of MO identified 2 SNPs associated with MO, one in the MEF2D gene and the other close to the TGFBR2 gene. There were SNPs that showed suggestive evidence of replication at PHACTR1 and ASTN2 genes. Moreover, previous GWAS findings were replicated, finding the genes TRPM8 and LRP1 associated with migraine. This study allowed the identification of the first loci associated with MO. - The epigenetic study in a MA rat model identified DNA methylation differences due to the administration of valproate and topiramate drugs and/or due to the cortical spreading depression (CSD) effects in genes that could be related to the migraine susceptibility. These results could indicate that both treatments protect against CSD due to their effects on DNA methylation, highlighting the importance of the epigenetic mechanisms in the migraine susceptibility.

Las alteraciones de la ecogenicidad periventricular en el recién nacido prematuro o a término han sido asociadas a un amplio espectro de alteraciones cognitivas, motoras y sensoriales. Mediante el análisis minucioso de los hallazgos de neuroimagen neonatal en un grupo suficientemente extenso de supervivientes de leucomlacia periventricular (LPV)y su correlación con los datos neurológicos y las puntuaciones en evaluaciones neuropsicológicas efectuadas durante seguimiento a largo plazo, así como con los hallazgos de resonancia craneal (RM) en la infancia, se establecierongrupos pronósticos asociados a distintos patrones de alteración ecográfica cerebral neonatal.El estudio incluyó a 120 recién nacidos con LPV y 70 controles con media de edad gestacional equivalente. A parte de recoger las variables demográficas, de seguimiento clínico y radiológicas, a todos los individuos se les administró el test NEPSY. La ecografía presentó buena correlación con la RM en lo relativo a secuelas graves, pues tanto el grupo de lesiones quísticas por ecografiacomo el grupo de lesión grave por RM mostraronaumento significativo de las mismas secuelas neurológicas. En cuanto a la clasificación neuromotora funcional, evaluada mediante la escala GFMCS, tanto las lesiones no quísticas (ecografía) como las lesiones leves (RM) se asociaron en más del 90% de los casos a la presencia degrados 0 y 1, o sea sinlimitaciones para las actividades de la vida diaria. Se detectó alguna discordancia entre ecografia neonatal y RM de seguimiento, pues un tercio de niños que presentaron LPV quística fueron luego clasificados con RM alteración leve, y casi una cuarta parte de niños con diagnóstico de lesión no quística por ecografia tenían RM con alteración grave. Además niños con ecografia normal, un 14% d elos casos, tuvieron diagnóstico retrospectivo de LPV por RM y a menudo presentaron retraso en el desarrollo, parálisis cerebral u otras secuelas neurológicas. En relación con la evaluación neuropsicológica mediante el test NEPSY, todos los grupos presentaronalteraciones. Fue llamativo que los dominios con peor puntuacion para las lesiones ecográficas no quísticas fueronlenguaje, visuoepacial y memoria mientras que para las quísticas fue el sensoriomotor. Si consideramos las lesiones graves por RM,más de la mitad de los casos presentó puntuaciones por debajo de la normalidaden los dominiosatención ejecución,lenguaje y sensoriomotor, mientras que sorprendentemente el déficit en el dominio visuoespacial apareció en solo un 40 %. Los resultados neurológicos y neuropsicológicos difirieron de de los hallados en prematuros de edad gestacional equivalente y ecografía neonatal normal pero debe destacarse que por lo menos una cuarta parte de los controles puntuaron por debajo de la media poblacional en los cinco dominios.Por ello nuestros resultados enfatizan la necesidad de utilizar controles de igual edad gestacional en la valoración a largo plazo de las secuelas cognitivas de lesiones cerebrales perinatales., The alterations of periventricular echogenicity in premature infants or term newborns have been associated with a wide spectrum of cognitive, motor and sensory disfunctions. Through careful analysis of the neonatal neuroimaging findings in a extensive sample of periventricular leukomalacia (PVL) survivors and their correlation with neurological data and scores on neuropsychological assessments made during long-term follow-up, as well as cranial MRI findings in childhood, prognostic groups were established associated with different patterns of altered neonatal brain ultrasound. The present study analyzed demographic, follow-up clinical and radiological data from 120 newborns with PVL and 70 controls matching mean gestational age. All individuals underwent neuropsychological assessment by means of the NEPSY test. The ultrasound scan showed good correlation with MRI in predictingsevereneurological sequelae as cystic group (ultrasound) and severe group (MRI) showed almost the same significant results. On the issue of functional neuromotor classification,both non- cystic lesions (ultrasound) as well mild injuries (MRI) were associated with over 90 % of cases within GFMCS grades 0 and 1, meaning no limitations for daily life. There was howeversome diagnostic disagreement between cranial ultrasound andmagnetic resonance imaging , since one third of children who had cystic PVL were classified as with mild impairment by MRI, and almost a quarter of children with non-cystic lesion diagnosed by ultrasound, had severe impairment with MRI. Also children with normal ultrasound, but an MRI diagnosis of PVL, are 14 % of all cases. Of those, half had developmental delay, 70 % had CP, and nearly a quarter of them had someother neurological sequelae. Regarding neuropsychological assessment, all groups showed suboptimal scores, but it is noteworthy that the domains with the worst punctuation for non-cystic lesions were language, visuoepacial and memory while for cystic group was the sensorimotor domain. When the group with severe MRI lesions is considered, we found scores below averageinseveral broad domains:attention and execution, language and sensorimotor. Over half of these childrenhad disabilities related to these dysfunctions. Surprisingly enough, the visuospatial domain, with a 40% of patients with low scores,was relatively less impaired in this group. Neurological and neuropsychological outcomes in the group of patients did differ from those found in equivalent gestational age preterm with normal neonatal ultrasound, but it is remarkable that at least a quarter of the control group scoredbelow average for all five domains. Therefore, the present study emphasizes the extreme importance of using gestational age-matched controls when assessing long- term cognitive sequelae of perinatal brain injury .

[cat] La malformació de Chiari de tipus I (MCI) s'ha definit tradicionalment com una alteració congènita caracteritzada pel descens de les amígdales cerebel.loses a travès del forat magne en 3 o més mm. Es creu que és conseqüència d'una fosa cranial posterior (FCP) hipoplàstica, amb un os occipital anormalment curt, que resulta d'un mesoderma paraxial insuficient. La simptomatologia descrita pels pacients és molt variada: mals de cap occipitals, cervicàlgies, marejos, vertígens, pèrdua de sensibilitat i de força a les extremitats, alteracions de la son i problemes en la deglució entre d'altres, atribuïts a la compressió de les estructures neurològiques contingudes en la FCP i a l'alteració de la circulació del líquid cefaloraquidi. S'ha estimat la seva prevalença en 1/1280 i, tot i que es desconeix la seva etiologia, diversos estudis apunten a una component genètica com la causa més probable. En la MCI molts dels casos són esporàdics, però s'han identificat famílies amb diversos individus afectats en diferents generacions. Per aquest motiu, aquest treball va tenir com a objectiu principal estudiar les bases genètiques de la patologia. Es van reclutar més de 530 pacients, pertanyents a 450 famílies, i es van desenvolupar dues estratègies: 1) estudi de les formes aparentment complexes de la patologia mitjançant un estudi d'associació genètica, en què es van avaluar variants de susceptibilitat en gens implicats en el desenvolupament del mesoderma paraxial, somites, endoteli vascular i os occipital; i 2) estudi de les formes aparentment monogèniques, mitjançant un estudi mutacional del gen SUZ12 que, en haploinsuficiència en un model murí, produeix fenotips similars als de les malformacions de Chiari tipus I i II. Es van seqüenciar també els exornes de dos pacients pertanyents a una mateixa família. Donat que el descens amigdalar cerebel.lós pot ser produït per diferents causes, es va realitzar també un estudi morfomètric de la FCP en imatges de ressonància magnètica amb la finalitat de definir un mètode de diagnòstic més específic que l'existent, i que permetés diferenciar els pacients amb MCI produïda per una FCP hipoplàstica de la produïda de forma secundària, per altres mecanismes. Es va caracteritzar també la cavitat orofaríngia dels pacients. Els resultats que es van obtenir demostren que és necessari establir un nou criteri diagnòstic que permeti diferenciar els pacients amb MCI en funció de la seva causa, la qual cosa facilitaria l'estudi de la base genètica d'aquesta malformació. S'ha trobat correlació entre certes alteracions a la cavitat orofaríngia i símptomes típics de MCI com les apnees de la son o problemes en la deglució. També s'han identificat variants de susceptibilitat en diferents gens implicats en la via de síntesi de l'àcid retinoic. Però els estudis de cribatge mutacional del gen candidat i de seqüenciació d'exornes en casos familiars no han permès identificar gens responsables de la MCI., [eng] Chiari malformation type I (CMI) is a congenital disease that has been traditionally defined as the chronic tonsillar herniation of at least 3 mm below the foramen magnum. It is characterized by an overcrowding of a normally developed hindbrain within a hypoplastic PCF, although both age at presentation and symptoms show high variability. The etiology of the disease remains unknown, but the most accepted hypothesis is that CMI is the result of an insufficient development of the paraxial mesoderm. Several evidences suggest that, at least in a subset of CMI patients, genetic factors may play an important role. This project focused on the identification of genetic risk factors for CMI, including genes responsible for the mendelian and also the complex forms of the disease. To achieve this goal, two different strategies were followed: 1) Identification of susceptibility genetic variants in CMI patients using a casecontrol association approach. 2) Genetic analyses of monogenic forms by a mutational screen of the candidate gene SUZ12 and by exome sequencing in two CMI patients ofthe same family. We recruited 530 CMI patients from 450 unrelated families. Given that tonsilar herniation can be caused by five distinct mechanisms, we developed a probability model to establish a diagnosis with 93% confidence in those patients with a hypoplastic PCF. This diagnostic tool allowed to define a clinical homogeneous group for further genetic studies. A cephalometric oropharynx morphometric study was also performed to identify the possible underlying anatomical anomalies leading to less known symptoms. The results of the genetics association study paint to a putative role for defective retinoic acid signalling and fetal vasculogenesis in causing hypoplasia of the basichondrocranium. However, when we studied the monogenic forms, we did not find any causal variant.

La Malformació de Chiari tipus I (MCI) ha estat definida tradicionalment com la herniació de les amígdales cerebel•loses d’almenys 5mm, a través del forat mange. En general, els símptomes es posen de manifest durant la segona o tercera dècada de vida, tot i que s’han descrit casos pediàtrics. Donada la complexitat del quadre clínic, per realitzar un diagnòstic adient es requereix avaluació clínica i estudi de neuroimatge. La tècnica de preferència és la ressonància magnètica d’imatge, considerant-se actualment com a pacients de MCI aquells que presenten un descens de les amígdales superior a 3mm per sota del forat magne. L'existència de casos asimptomàtics dificulta establir una prevalença concreta, però s’ha estimat que podria estar entre 1/1000 a 1/5000 sent major en dones que en homes (2:1 aproximadament). Fins el moment, es desconeix l’etiologia de la malaltia però la hipòtesi més acceptada és que MCI és deguda al desenvolupament insuficient del mesoderm paraxial. Diferents estudis realitzats fins el moment evidencien que almenys, un subgrup de pacients amb MCI són deguts a contribució genètica: 1) casos d’agregació familiar amb afectes en tres generacions; 2) estudis de bessons 3) associació amb síndromes genètics coneguts amb herència mendeliana produïts per anomalies óssies que donen suport a la hipòtesi de la insuficiència del mesoderm com a causa de MCI. Davant l’evidència clara d’un component genètic com a principal causant de l’etiologia de MCI, l’objectiu del projecte va ser la identificació de les bases genètiques de la MCI, tant en gens responsables de les formes mendelianes com en gens responsables de les formes complexes de MCI mitjançant dues estratègies: 1-Identificació de variants genètiques de susceptibilitat en pacients amb MCI mitjançant estudis d’associació de tipus cas-control. 2-Anàlisi genètic de formes monogèniques mitjançant l’anàlisi de lligament a marcardors polimòrfics i la seqüenciació del DNA a gran escala. Chiari malformation type I (CMI) has been traditionally defined as the chronic tonsillar herniation of at least 5 mm below the foramen magnum (FM). In general, the symptoms manifest during the second or third decade of life, however, several pediatric cases have been described. Given the complexity of this symptoms, to perform a proper diagnostic of CMI, a clinical evaluation and a neuroimage analysis is required. The preferred diagnostic method is the Magnetic Resonance image (MRI), considering as CMI those patients showing a tonsilar descent upper 3mm through the FM. The existence of asymptomatic cases of CMI difficult to establish its prevalence, however, it has been estimated to be in the range of 1/1000 to 1/5000 individuals, being higher in women than in men (2:1, approximately). The etiology of this disease remains unknown, but the most accepted hypothesis is that CMI is the result of an insufficient development of the paraxial mesoderm. Several evidences suggest that, at least in a subset of CMI cases, genetic factors may play an important role: 1) cases of family aggregation with individuals affected in at least three different generations; 2) twin studies were monozygotic twins reveal a higher degree of concordance; 3) cases were CMI associated as part of known Mendelian syndromes, some of which feature prominent bony abnormalities, supporting the mesodermal origin of the disorder. Considering the clear evidences for a genetic contribution in the origin of CMI, this project was focused on identifying the genetic basis underlying the CMI, detecting the genes responsible for either the mendelian and the complex forms of CMI. To achive this main goal, two different strategies were followed: 1- Identification of susceptibility genetic variants in CMI patients using a case-control association approach. 2- Genetic analyses of monogenic forms by linkage analysis with polymorphic markers and high-throughput DNA sequencing.

[cat] Aquesta tesi es centra en la genètica de la migranya. La migranya comuna és un trastorn neurològic caracteritzat per episodis recurrents de mal de cap. Els criteris de la IHS (International Headache Society) subclasifiquen la malaltia en migranya amb aura (MA) i migranya sense aura (MO). L'aura són símptomes neurològics transitoris que poden acompanyar el mal de cap. Les aures més freqüents són les aures visuals, tot i que també existeixen les aures sensorials essent l'aura hemiplègica la seva forma severa. La nostra investigació es va dividir en dues areas d'acord amb la base genètica dels trastorns, d'una banda, s'ha estudiat la genètica complexa de la migranya comuna, d'altra banda s'ha estudiat una forma rara de la migranya que presenta una herència mendeliana anomenada migranya hemiplègica familiar (FHM). Per entendre més la base genètica de la migranya comuna es va utilitzar un estudi d'associació tipus cas-control amb gens candidats. Amb aquesta finalitat es van seleccionar al voltant de 550 pacients amb migranya (MA i MO) i el seu corresponent grup de control. Per tal d'analitzar la seva implicació en la susceptibilitat genètica a la migranya, es van triar gens que codifiquen per als canals de la superfamília heterogeni de potencial receptor transitori (Transient Receptor Potential- TRP) que se sap que estan implicats en les vies nociceptives. Aquesta feina ha donat lloc a una publicació (Carreño et al. SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet. 2012). En el cas particular de les formes monogèniques de FHM es coneixen tres gens involucrats en la malatia (CACNA1A, ATP1A2 i SCN1A), les proteïnes codificades per aquests gens tenen un paper rellevant en la neurotransmissió del glutamat. L'anàlisi funcional de les mutacions que causen FHM han mostrat en última instància un augment de l'alliberament de la neurotransmissió. En el cas de mutacions al CACNA1A s'ha vist un efecte de guany de funció, a diferència de les mutacions al ATP1A2 que presenten un efecte de pèrdua de funció. En aquest treball s'ha fet un screening mutacional per identificar mutacions en pacients per seqüenciació directa. Quan les mutacions eren suficientment interessants s'han generat construccions en vectors d'expressió per subseqüents estudis funcionals en cèl·lules eucariotes. Aquesta feina ha donat lloc a tres publicacions. A la primera (Serra et al. A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis. Proc Natl Acad Sci. 2010) es va identificar un canvi que modula la funció del canal de CACNA1A. Aquest estudi ajuda a explicar la contribució genètica en la heterogeneïtat clínica d'una família i a entendre millor el mecanisme molecular dels canals de calci tipus P/Q. El segon (Carreño et al. Acute striatal necrosis in hemiplegic migraine with the novo CACNA1A mutation. Headache. 2011) és un informe d'un pacient que presenta una necrosi aguda stratial. Té una rellevància clínica a causa de l'aparició primerenca dels símptomes neurològics previs als atacs hemiplègics. El tercer i últim treball (Carreño et al. Screening of the ATP1A2 and CACNA1A genes in patients with hemiplegic migraine: clinical, genetic and functional studies. [work in progress]) recull l'screening mutacional al gens ATP1A2 i CACNA1A en 19 pacients amb FHM. Es van identificar 5 mutacions prèviament descrites i dues mutacions noves., [eng] This Thesis is focused in migraine genetics, migraine is a prevalent neurological disorder characterized by recurrent episodes of headache. This research was divided in two areas according to the genetic basis of the disorders; on the one hand we studied the common migraine with a complex genetics, on the other hand we studied the rare mendelian forms of familial hemiplegic migraine (FHM). To understand more the genetic basis of the common migraine a case-control association study approach was used with candidate genes. For that purpose, around 550 patients with migraine and their corresponding control group were selected. In order to analyze their involvement in the genetic susceptibility to migraine, we chose genes encoding for channels of the heterogeneous superfamily of Transient Receptor Potential (TRP) which are known to be involved in the nociceptive pathway. In the particular case of FHM, a monogenic form of the disorder, there are three genes known to be involved in the FHM (CACNA1A, ATP1A2 and SCN1A), whose encoded proteins are playing a relevant role in the neurotransmission of the glutamate. Functional analysis of the mutations causing FHM have shown ultimately an increased neurotransmission release. CACNA1A previous studies reveled a gain-of-function effect from FHM mutations, unlike mutations on ATP1A2 that present a loss-of-function effect. Our work consisted on identifying mutations in patients by direct sequencing. If the mutations were interesting enough vector constructions were generated for functional studies in eukaryotic cells. This work gave rise to three publications: First; the identification of a change that modulates the function of the CACNA1A channel. This study contributes to explain the genetic contribution in the clinical heterogeneity of one family and to know more about the molecular mechanism of the P/Q calcium channel. Second; a report of a patient that presents an acute stratial necrosis that had clinical relevance because of the early onset of the neurological symptoms previous to the hemiplegic attacks. Third; a mutational screening of ATP1A2 and CACNA1A genes in 19 patients with FHM. 5 previously described mutations and two new mutations were found. Functional studies were carried out for the newly mutations.

La migranya és un trastorn neurològic molt prevalent a la població caracteritzat per episodis de cefalea recurrents i incapacitants. És una malaltia amb herència complexa, causada per múltiples gens que actuen en combinació amb factors ambientals.Aquest treball té per objectiu aprofundir en el coneixement dels factors genètics implicats en l'etiologia de les dues formes més comunes de migranya: la migranya sense aura (MO) i la migranya amb aura (MA).S'ha avaluat la participació en la susceptibilitat a la migranya comuna o a algun dels subtipus clínics principals de 45 gens candidats mitjançant estudis d'associació de tipus cas-control de 255 variants polimòrfiques, seleccionades principalment en base a criteris de cobertura gènica. La selecció de gens s'ha centrat en sistemes de gens candidats que codifiquen proteïnes funcionalmente relacionades.En primer lloc, l'estudi de polimorfismes sinònims del gen que codifica el receptor d'estrògens ESR1 i d'una inserció Alu del gen del receptor de progesterona PGR no recolzen la participació d'aquestes variants en la migranya, en oposició a estudis previs realitzats en altres poblacions. A continuació s'han estudiat 19 gens del sistema de neurotransmissió serotoninèrgica en una població àmplia i s'han identificat haplotips de risc en els gens del receptor 2B de serotonina (HTR2B) i de la monoamina oxidasa A MAOA) en el grup de MO, i en el gen de la decarboxilasa d'aminoàcids aromàtics (ADDC o DDC) en el grup de MA, resultats que es mantenen significatius en aplicar correccions per comparacions múltiples. La AADC i la MAOA participen en la síntesi i la degradació, respectivament, de serotonina i dopamina.En la tercera part del treball s'han analitzat SNPs de 9 gens relacionats amb el sistema dopaminèrgic seguint un disseny en dues etapes. S'han identificat associacions nominals en cinc gens, dues de les quals es mantenen significatives després d'aplicar correccions per múltiples comparacions. Malauradament, cap d'aquestes associacions s'ha replicat en una segona mostra.La darrera part del treball s'ha centrat en l'estudi de nous gens candidats pels que no s'havia avaluat encara la seva possible relació amb el fenotip migranós. L'anàlisi del gen de la sintaxina 1A (STX1A) a la migranya ha mostrat en un primer estudi una associació nominal que es manté en doblar els controls. Els resultats obtinguts en ampliar el número de casos i de controls en un segon estudi indiquen que es podria tractar d'un fals positiu. A continuació s'ha estudiat la participació en la migranya de 15 nous gens candidats que codifiquen proteïnes implicades en la regulació de la neurosecreció. S'ha identificat un haplotip de susceptibilitat a MO en el gen de la complexina 2 (CPLX2), que codifica una proteïna reguladora dels passos finals de la fusió de membranes per l'alliberament de neurotransmissors. D'altra banda, s'ha identificat una associació entre un SNP del gen NSF, que codifica la proteïna responsable de la reactivació dels elements de fusió a la membrana presinàptica, i el grup de MA. Aquestes associacions estan pendents de rèplica, però podrien aportar una nova evidència a favor de la hipòtesi d'una disfunción sinàptica en alguns trastorns neurològics paroxístics.La identificació d'associacions específiques de MO o MA suggereix que aquests trastorns tenen, com a mínim en part, factors de susceptibilitat específics. D'altra banda s'han identificat alguns polimorfismes associats a ambdós grups, tot recolzant també l'existència de factors etiològics comuns., AUTHOR: Roser Corominas CastiñeiraTITLE: Evaluation of the genetic contribution to common forms of migraineTEXT:Migraine is a highly prevalent neurological disorder with a complex inheritance pattern. Many genes in interaction with environmental factors participate in the risk to develop the disease. However, the precise mechanisms underlying the pathogenesis of migraine are not completely understood.The aim of our project was to get more insight into the genetic factors involved in the etiology of the common forms of migraine, migraine without aura (MO) or migraine with aura (MA), investigating the role of 45 candidate genes through case-control association studies of 255 genetic polymorphic variants selected according to genetic coverage parameters.Our study in estrogen receptor and progesterone receptor genes does not support a major contribution to the pathogenesis of migraine.We also evaluated the possible contribution of 19 serotonin-related genes to the disorder. Multiple-marker analysis revealed risk haplotypes in the HTR2B and MAOA genes conferring susceptibility to MO, and an haplotype in DDC specific to MA.The contribution to migraine susceptibility of eight additional genes involved in dopaminergic neurotransmission was evaluated through a two-stage case-control association study. The positive results obtained in the first population were not replicated in the second independent cohort.Finally, we investigated the involvement of genes encoding proteins involved in the regulation of synaptic exocytosis of neurotransmitters. In an initial study, we assessed the contribution of STX1A to migraine and identified a significant association with migraine. However, these results were not maintained when the population was enlarged. The study of 15 genes, including STX1A, in a larger sample reveals several nominal associations, but only two SNPs in CPLX2 for MO and one SNP in NSF for MA remained significant after corrections for multiple testing. Further studies are required to confirm the participation of these genes in migraine susceptibility.

La migranya és un mal de cap primari, episòdic crònic i incapacitant molt comú a la població. Ha estat identificada per la Organització Mundial de la Salut (OMS) entre les principals causes d'incapacitat. La migranya té una etiologia complexa, amb contribució de múltiples gens d'efecte menor i factors ambientals que conjuntament determinen una certa predisposició a la patologia. No obstant, hi ha formes rares de migranya en què la causa és monogènica, amb un únic gen d'efecte major que determina l'aparició del fenotip.L'objectiu principal d'aquest treball ha estat aprofundir en l'estudi de la base genètica de les formes monogèniques de migranya i més específicament, identificar nous loci responsables del fenotip migranyós i nous al.lels mutats en gens ja reconeguts com a responsables d'aquest trastorn genèticament heterogeni.D'una banda, s'ha mapat el gen responsable de la migranya, de forma independent, en dues famílies catalanes extenses no emparentades, amb una herència dominant, a la regió cromosòmica 14q32. Tanmateix, la identificació del gen causant de la malaltia en aquestes famílies resta encara per resoldre. D'altra banda, s'han identificat 9 mutacions en el gen CACNA1A, totes elles de canvi de sentit, en individus espanyols amb diferents variants de migranya (HM, MA i CPS) i atàxia episòdica de tipus 2 (EA2). Tres de les mutacions són responsables del fenotip FHM, una de les quals es va identificar en un individu que manifestava símptomes de BPT i que va evolucionar a BPV i finalment a FHM. La quarta mutació es va trobar en un pacient que des dels 7 mesos de vida manifestava episodis d'hipotèrmia amb símptomes acompanyants i que podria correspondre a una variant, encara no ben caracteritzada clínicament, de CPS. S'han detectat dos canvis més en pacients amb manifestacions d'EA2. El setè canvi es va detectar en un pacient afectat d'EA2 i FHM, i el vuitè en un pacient amb EA2 i MA. La novena mutació s'ha identificat en dues pacients amb MA, mare i filla, que pertanyen a una família en què segrega FHM. En aquest darrer cas s'han pogut fer, en col·laboracio, estudis funcionals de la proteïna mutada, que revelen que determinades variacions en el gen CACNA1A poden tenir un paper modificador i no causant, com fins ara s'havia pensat, del fenotip migranyós.L'estudi de les formes monogèniques de migranya presenta diverses complicacions com l'heterogeneïtat genètica, la variabilitat en l'expressió clínica, la penetració incompleta, les fenocòpies i les genocòpies i l'efecte dels factors ambientals, el seu caràcter episòdic i la manca de marcadors biològics, la variabilitat de la simptomatologia en funció de l'edat de l'individu, la dificultat d'agrupar pacients en categories fenotípicament homogènies, la difícil obtenció del teixit d'interès -sistema nerviós central- i la dificultat d'interpretar i reproduir una simptomatologia basada en el testimoni del pacient, en animals d'experimentació. Els estudis de lligament que es presenten en aquest treball proporcionen evidències significatives de l'existència d'un nou locus comú a FHM i MA en dues famílies catalanes. La identificació d'un nou locus de migranya i els resultats de l'anàlisi mutacional realitzada en el gen CACNA1A augmenten la reconeguda heterogeneïtat genètica no al·lèlica i al·lèlica de les formes monogèniques i rares de migranya. Identificar el nou gen de migranya que hem localitzat al cromosoma 14 i estudiar la seva rellevància entre els pacients migranyosos a nivell espanyol i mundial contribuirà de ben segur a una millor comprensió dels mecanismes moleculars de la migranya i també a la identificació de possibles dianes terapèutiques en benefici dels pacients que la pateixen., Migraine is a chronic, episodic and disabling prymary headache. The World Health Organization (WHO) rated migraine among the most disabling chronic disorders. Migraine has a complex etiology involving multiple susceptibility genes and environmental factors. However, there are rare forms of migraine in which the cause is monogenic.The main goal of this work has been to study the genetic basis on the monogenic forms of migraine and more specifically, to identify new loci responsible for the migraine phenotype and new mutated alleles in genes responsibles for this genetically heterogeneous disorder.A gene responsible for migraine has been mapped in two extensive unrelated families at chr14q32. However, the identification of the causative gene rest to be identified. On the other hand, nine missense mutations have been identified in the CACNA1A gene in Spanish patients with hemiplegic migraine (HM), migraine with aura (MA), chilhood periodic syndromes (CPS) and episodic ataxia type 2 (EA2). Three of the mutations are responsible for the FHM phenotype, one of them was identified in an individual who manifested symptoms of childhood benign paroxysmal torticolis that evolved to benign paroxysmal vertigo and finally to FHM. The fourth mutation found in a patient that manifested episodes of hypothermia with accompanying symptoms, could correspond to a CPS variant. Two more changes have been detected in patients with EA2. The seventh change was detected in an affected patient of EA2 and FHM, and the eighth in a patient with EA2 and MA. The ninth mutation has been identified in two patients with MA belonging to a HM family. Functional studies of p.Ala454Thr CACNA1A protein revealed that some variants can have a not causative but modifier role.The linkage studies performed in this work provide significant evidence of the existence of a new common locus to FHM and MA. Its identification and the results of the mutational analysis carried out in CACNA1A gene increase the recognized genetic heterogeneity in monogenic forms of migraine. Identifying the new migraine gene will contribute to better understand the molecular mechanisms of migraine and the identication of new therapeutic targets.