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Deficiência intelectual (DI) é reconhecida como um transtorno do desenvolvimento neurológico, caracterizado por diminuição significativa em ambas funções cognitivas e adaptativas, originada antes dos 18 anos de idade. Estima-se que em torno de 1–3% das crianças ao redor do mundo são afetadas pela DI e que no Brasil acomete aproximadamente 0,8% da população. As variações no número de cópias (CNVs) são responsáveis por cerca de 15–20% dos casos de DI, que podem comprometer o funcionamento de diversos genes. O objetivo do estudo foi avaliar a ocorrência de CNVs, identificadas pelo CMA com filtro de tamanho < 100 kb, que abrigassem genes funcionalmente associados à DI em pacientes do SUS com diagnóstico clínico de DI. Durante o período de janeiro 2013 a dezembro de 2016, foi realizado o cariótipo por bandeamento GTG em 325 pacientes com indicação clínica de DI, chegando ao diagnóstico genético em 57,2% dos pacientes, demonstrando ser uma importante metodologia de triagem para os pacientes com DI. No entanto, 42,8% dos pacientes apresentaram o cariótipo sem alterações estruturais ou numéricas. Foi realizado nestes pacientes o CMA com filtro de tamanho ≥ 100 kb, onde foi possível elucidar o diagnóstico genético em 29,8% dos casos, apresentando um incremento na taxa de diagnóstico de 7,1%. Os casos que permaneceram sem elucidação, foram submetidos ao CMA com filtro de tamanho < 100 kb, no qual foram identificadas CNVs de perda em regiões que continham os genes CNTNAP2, FGF13, MID1, MID2, SHANK3, IL1RAPL1, DMD e PAK3. A redução do filtro de tamanho demonstrou um incremento na taxa de diagnóstico em 12%, ampliando o espectro de identificação de CNVs presentes em regiões que abrigam genes implicados à manifestação clínica da DI. A aplicação em conjunto das metodologias cariótipo por bandeamento GTG e CMA com filtro de tamanho ≥ 100 kb e posteriormente filtro de tamanho < 100 kb, permitiu um aumento no diagnóstico genético da DI e outras comorbidades, promovendo uma compreensão mais ampla dos aspectos genéticos relacionados à essas afecções e possibilitando um manejo adequado das famílias. Finalmente, o acesso ao aconselhamento genético proporciona uma melhor compreensão das causas genéticas da DI, as implicações familiares da contribuição genética e a chance de recorrência. Intellectual disability (ID) is characterized by significant impairment in both cognitive and adaptive functions, originating before the age of 18 years. In addition, it is a common phenotype sign in a cluster of heterogeneous syndromic or non-syndromic disorders, associated with some comorbidities such as autism and congenital malformations. In the worldwide, ID affects around 1–3% of the general population and in Brazil ID affects approximately 0.8% of the population. The Copy number variations account for about 15– 20% of children with unexplained ID, compromising the functioning of several genes, with more than 1,416 genes described as causative of this phenotype sign. The aim of the study was to evaluate the occurrence of CNVs, identified by CMA with the size filter of < 100 kb, harboring genes functionally associated with ID in patients from SUS with a clinical diagnosis of ID referred for the genetic diagnosis. During January 2013 to December 2016, GTG banding karyotype was performed in 325 patients with ID, achieve the genetic diagnostic in 57.2%, demonstrating to be an important screening approach for patients with DI. However, 42,8% of the patients showed the karyotype with no visible numerical or structural alterations. The CMA analysis with the size filter of ≥ 100 kb was performed in these patients, where it was possible to elucidate the genetic diagnose in 29.8% of the patients, demonstrating 7,1 % of the increment on the diagnostic. All the cases remained without a diagnosis were submitted to the CMA analysis with size filter of < 100 kb, where it was identified loss CNVs in regions harboring CNTNAP2, FGF13, MID1, MID2, SHANK3, IL1RAPL1, DMD, and PAK3 genes. The reduction of the size filter demonstrated an increase of 12% in the ratio of diagnosis, expanding the spectrum of CNVs identification in regions which harboring genes related to the clinical manifestation of ID. The application of both GTG banding and CMA with the size filter of ≥ 100 kb and later the size filter of < 100 kb allowed an increase in the genetic diagnosis of ID and comorbidities, giving a broad understanding of the genetic aspects related to these conditions and allowing the adequate management of families. Finally, the genetic counseling provides a better understanding of the genetic causes of ID, the familial implications of the genetic contribution and the chance of recurrence. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG

X-linked intellectual disability accounts for 10-12% of cases of cognitive impairment in males. Mutations in IL1RAPL1 are an emerging form of apparently non-syndromic X-linked intellectual disability. We report a 8-year-old intellectually disabled boy with speech delay, and unusual facial and digital anomalies who showed a novel and complex IL1RAPL1 rearrangement. It was defined by two intragenic non-contiguous duplications inherited from the unaffected mother. Chromosome X inactivation study on the mother's blood leukocytes, urinary sediment and buccal swab did not show a significant skewed inactivation. Comparison with previously described patients with IL1RAPL1 disruption was carried. Although data on craniofacial features were scanty in many papers, subtle facial dysmorphism with a thin upper lip seemed a quietly represented picture without any other genotype-phenotype correlations. Our study expands the molecular repertoire of IL1RAPL1 mutations in intellectual disability and points out the need of more accurate clinical descriptions to better define the related phenotype.

Advanced colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are highly aggressive and heterogeneous diseases which urgently require the development of more effective treatment strategies. Novel membrane proteins overexpressed in CRC and PDAC, could have the potential to represent attractive molecular targets for development of antibody-based targeted therapeutics. Candidate molecular targets were identified by Bioinformatic, transcriptomic analysis of publicly available colon cancer gene microarray datasets. Lists were generated of genes overexpressed in colon adenoma and adenocarcinoma vs. normal colon with predicted membrane localisation. Seven novel candidate targets (LY6G6F, IL1RAPL1, IL20RA, BACE2, NTM, LRRC8E and EPHX4) were selected for protein validation in normal and colon cancer tissue sections. Two targets, LY6G6F and IL1RAPL1, both previously unexploited in cancer were found to be minimally expressed in normal colon and show strong expression in CRC, warranting further investigation and functional characterisation. LY6G6F is a type 1 transmembrane protein, first identified as a novel platelet plasma membrane protein, linking to downstream signal transduction pathways upon platelet activation. However, a ligand for LY6G6F remains unknown. LY6G6F was found in this study to be significantly overexpressed in colon adenocarcinoma, PDAC and gastric adenocarcinoma, with minimal expression observed in normal proliferating cells and tissues. IHC analysis of a 57 PDAC patient cohort revealed that 78.9% showed strong LY6G6F immunoreactivity; using Kaplan-Meier survival analysis, a clear trend was observed between high LY6G6F expression and decreased survival in this cohort, however this failed to reach significance (p-value: 0.182). High LY6G6F mRNA expression is significantly associated with decreased survival in gastric cancer using KM plotter survival analysis (logrank-p: 4.3e-05). Transient protein knockdown of LY6G6F in the colon cancer cell line HCT116 and the PDAC cell line MIA PaCa-2 significantly inhibits proliferation, 2D colony formation, migration and invasion of these cells in vitro. These effects were found to be potentially mediated by a decrease in FAK activation and an increase in apoptosis. Thus, taken together these results implicate LY6G6F in the proliferation and survival of these cancers. IL1RAPL1 is a type 1 transmembrane protein highly expressed in brain neurons, with a reported role in synapse formation. Deletions and mutations in IL1RAPL1 have been associated with X-linked intellectual disability. IL1RAPL1 was found to be significantly overexpressed in all CRC subtypes vs. normal colon and showed limited expression in a range of other normal tissues and highly proliferating cells. Furthermore, IL1RAPL1 expression was associated with oesophageal squamous cell carcinoma (OSCC), with significant overexpression compared to normal oesophagus and also other oesophageal cancer subtypes. The potential functional role of IL1RAPL1 in the colon cancer cell phenotype could not be determined. LY6G6F and IL1RAPL1 both represent novel candidate proteins overexpressed in these cancer types with restricted expression in normal tissues and should be investigated further as potential molecular targets for antibody-based therapeutic targeting.

Made available in DSpace on 2022-04-29T07:26:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-01 Detailed molecular characterization of chromosomal rearrangements involving X-chromosome has been a key strategy in identifying X-linked intellectual disability-causing genes. We fine-mapped the breakpoints in four women with balanced X-autosome translocations and variable phenotypes, in order to investigate the corresponding genetic contribution to intellectual disability. We addressed the impact of the gene interruptions in transcription and discussed the consequences of their functional impairment in neurodevelopment. Three patients presented with cognitive impairment, reinforcing the association between the disrupted genes (TSPAN7-MRX58, KIAA2022-MRX98, and IL1RAPL1-MRX21/34) and intellectual disability. While gene expression analysis showed absence of TSPAN7 and KIAA2022 expression in the patients, the unexpected expression of IL1RAPL1 suggested a fusion transcript ZNF611-IL1RAPL1 under the control of the ZNF611 promoter, gene disrupted at the autosomal breakpoint. The X-chromosomal breakpoint definition in the fourth patient, a woman with normal intellectual abilities, revealed disruption of the ZDHHC15 gene (MRX91). The expression assays did not detect ZDHHC15 gene expression in the patient, thus questioning its involvement in intellectual disability. Revealing the disruption of an X-linked intellectual disability-related gene in patients with balanced X-autosome translocation is a useful tool for a better characterization of critical genes in neurodevelopment. Department of Morphology and Genetics Genetics Division Universidade Federal de São Paulo Jena University Hospital Friedrich Schiller University Institute of Human Genetics Department of Psychobiology Universidade Federal de São Paulo Departament of Genetics Instituto de Biocincias de Botucatu Universidade Estadual de São Paulo Departament of Genetics Instituto de Biocincias de Botucatu Universidade Estadual de São Paulo

Preserving the integrity of neuronal synapses is important for the development and maintenance of cognitive capacities. Mutations on a growing number of genes coding for synaptic proteins are associated with intellectual disability (ID), a neurodevelopmental disease characterized by deficits in adaptive and intellectual functions. The present work is dedicated to the study of one of those genes, IL1RAPL1, and the role of its encoding protein in synapse formation and function. IL1RAPL1 is a trans-membrane protein that is localized at excitatory synapses, where it interacts with the postsynaptic proteins PSD-95, RhoGAP2 and Mcf2l. Moreover, the extracellular domain of IL1RAPL1 interacts trans-synaptically with the presynaptic phosphatase PTPd. We studied the functional consequences of two novel mutations identified in ID patients affecting this IL1RAPL1 domain. Those mutations lead either to a decrease of the protein expression or of its interaction with PTPd, affecting in both cases the IL1RAPL1-mediated excitatory synapse formation. In the absence of IL1RAPL1, the number or function of excitatory synapses is perturbed, leading to an imbalance of excitatory and inhibitory synaptic transmissions in specific brain circuits. In particular, we showed that this imbalance in the lateral amygdala results in associative memory deficits in mice lacking Il1rapl1. Altogether, the results included in this work show that IL1RAPL1/PTPd interaction is essential for synapse formation and suggest that the cognitive deficits in ID patients with mutations on IL1RAPL1 result from the imbalance of the excitatory and inhibitory transmission. These observations open therapeutic perspectives aiming to reestablish this balance in the affected neuronal circuits., L’intégrité des synapses neuronales est primordiale pour le développement et le maintien des capacités cognitives. Des mutations dans des gènes codant pour des protéines synaptiques ont été trouvées chez des patients atteints de déficience intellectuelle (DI), qui est une maladie neurodéveloppementale ayant des conséquences sur les fonctions intellectuelles et adaptatives. Ce travail de thèse porte sur l’étude de l’un de ces gènes, IL1RAPL1, dont les mutations sont responsables d’une forme non-syndromique de DI liée au chromosome X, et sur le rôle de la protéine IL1RAPL1 dans la formation et le fonctionnement des synapses. IL1RAPL1 est une protéine trans-membranaire qui est localisée dans les synapses excitatrices où elle interagit avec les protéines post-synaptiques PSD-95, RhoGAP2 et Mcf2l. De plus, IL1RAPL1 interagit en trans- avec une protéine phosphatase présynaptique, PTPd, via son domaine extracellulaire. Nous avons étudié les conséquences fonctionnelles de deux nouvelles mutations qui affectent le domaine extracellulaire d’IL1RAPL1 chez des patients présentant une DI. Ces mutations conduisent soit à une diminution de l’expression de la protéine, soit à une réduction de l’interaction avec PTPd affectant ainsi la capacité d’IL1RAPL1 à induire la formation de synapses excitatrices. En absence d’IL1RAPL1, le nombre ou la fonction des synapses excitatrices est diminué, ce qui mène à un déséquilibre entre les transmissions synaptiques excitatrice et inhibitrice dans des régions spécifiques du cerveau. Dans le cas particulier de l’amygdale latérale, nous avons montré que ce déséquilibre conduit à des défauts de mémoire associative chez la souris déficiente en Il1rapl1. L’ensemble des résultats qui font partie de ce travail montre que l’interaction IL1RAPL1/PTPd est essentielle pour la formation des synapses et suggère que les déficits cognitifs des patients avec une mutation dans il1rapl1 proviennent du déséquilibre de la balance excitation/ inhibition. Ces observations ouvrent des perspectives thérapeutiques visant à rétablir cette balance dans les réseaux neuronaux affectés.

Intellectual disability (ID) comprises a highly heterogeneous collection of neurodevelopmentaldisorders that arise during childhood. They have an incidence of 1-3% in the population withimpairments in mental and adaptive functions. While the etiologies of IDs are thought to bevery heterogeneous, a significant proportion of ID has genetic origins. Mutations in single IDgenes lead to dysfunctions in proteins that fulfill highly different biological functions in thebrain. Interestingly, ID-related proteins are often found enriched at synapses, suggesting thatcognitive impairments defining ID could be related to alterations of synaptic function. The maingoal of our research team is to understand the role of ID-related proteins in synaptic functionand cognition using mouse models bearing gene mutations associated to ID in humans. Myresearch focused on the study of Il1rapl1, a gene coding for the Interleukin-receptor-accessoryprotein-like-1 protein. Micro-deletions or point mutations in this gene are directly linked to thedevelopment of ID and autism spectrum disorder in humans. In neurons, Il1rapl1 encodes atrans-membrane protein and several in vitro experiments point to its important role in thedifferentiation and formation/stabilization of excitatory synapses trough interactions withpresynaptic, trans-synaptic or postsynaptic partners. However, the consequences of Il1rapl1deficiency at more integrated levels remains poorly understood. The principal objective of mythesis is to explore the link between synaptic deficits and behavioral impairments in Il1rapl1-deficient mice. To achieve that, wild-type and mutant animals were first submitted to fearlearning tasks. I then used a combination of in vivo, ex vivo and in vitro functional essays tocharacterize synaptic functions in behaviorally relevant neuronal circuits. Ultimately, ourworking hypothesis were challenged in vivo by pharmacological and optogenetic approaches tonormalize behavioral deficits in Il1rapl1 KO mice. Altogether my work demonstrates thatInhibitory/Excitatory imbalances associated with the absence of Il1rapl1 impaired both thecapacity to form new memories as well as the expression of previously formed memories., Les désordres intellectuels (DI) comprennent une collection hétérogène de désordresneurodéveloppementaux qui émergent pendant l’enfance. Ils ont une incidence de 1 à 3% dansla population et sont associés avec des déficits dans les fonctions mentales et adaptives. Denombreuses mutations ont été identifiées dans des gènes codant pour des protéines quiremplissent des fonctions biologiques très diverses dans le cerveau. Parmi ces protéines,certaines sont enrichies à la synapse, supposant que les déficits cognitifs associés aux DIpourraient être reliés à des déficits synaptiques. L’objectif scientifique de notre équipe et decomprendre le rôle de certaines protéines dans la fonction synaptique et la cognition enutilisant des souris génétiquement modifiées portant des mutations dans le gènecorrespondant. Je me suis concentré sur Il1rapl1, un gène codant pour la protéine Interleukinreceptor-accessory-protein-like-1. Des mutations ou micro-délétions dans ce gène sont liés audéveloppement de DI chez l’homme. Dans les neurones, Il1rapl1 code pour une protéinetransmembranaire qui serait impliquée dans la formation et/ou la stabilisation de synapsesexcitatrices. Les conséquences de l’absence d’IL1RAPL1 à des niveaux plus intégrés restaientpeu étudiées lors du début de ma thèse. J’ai utilisé une souris déficiente pour IL1RAPL1 (KO) afinde comprendre le lien entre les déficits comportementaux et la fonction synaptique. Pour cela,j’ai soumis des souris KO à des taches comportementales de peur conditionnée. J’ai ensuiteutilisé une combinaison d’approches in vitro, ex vivo et in vivo afin de caractériser la fonctionsynaptique dans les circuits neuronaux dédiés : l’amygdale latérale et basolatérale. Desenregistrements electrophysiologiques ont montré une dérégulation de la balance entre latransmission inhibitrice et excitatrice (I/E) dans l’amygdale de souris Il1rapl1 KO, causant ainsides déficits dans la capacité d’acquérir et d’exprimer la mémoire de peur conditionnée. Lacorrection de ce déficit synaptique in vivo par pharmacologie ou par optogénétique a permis derestaurer le comportement chez les souris KO.

The genetic exploration of intellectual disability (ID) has been revolutionized with the improvement in sequencing technologies during last decade with characterization of mutational spectrum of genes involved in ID as well as to identify new genes associated with it. In this study we used high resolution (comparative genomic hybridization array) CGH-array and high throughput sequencing technique to find the genetic cause in patients with unexplained ID. Aicardi syndrome is a rare sporadic neurodevelopmental syndrome, characterized by classic triad of agenesis of corpus callosum, chorioretinal lacunes and infantile spasms. This syndrome is exclusively present in females with plausible hypothesis of X linked dominant mutation. We first tested DNA of 22 patients diagnosed with Aicardi syndrome by using a high resolution oligonucleotide CGH-array 1M specifically designed for X-chromosome without identifying any pathogenic CNV or deleterious rearrangements involved in the disease. High throughput sequencing for exome of X chromosome was carried out in one trio (patient-parents) and two patients with typical Aicardi syndrome diagnosis. Sequencing results detected 59 mutations in 51 genes. 13 mutations were inherited from mother, 8 inherited from father, 36 false positive and 2 were SNP?s. Second approach was based on High throughput sequencing for complete exome of five trios (patient-parents) DNA. We presented and commented different strategies for data analysis in search of a candidate gene. These results highlighted difficulties in terms of depth and alignment of sequencing reads which generated various false positive SNP?s and indels. In second cohort from reference centre of rare diseases CGH-array has identified two intragenic rearrangements of IL1RAPL1 gene: two unique duplications and one deletion. We analyze genotype-phenotype correlations with cases described in literature which emphasizes the clinical variability of expression in these patients. Two de novo intragenic deletions and a de novo intragenic duplication were detected in MBD5 gene in patients with ID. The de novo duplication of MBD5 resulted in an aberrant transcripts leading to a premature termination codon. A selected cohort of 78 patients were sequenced for MBD5 gene which revealed a de novo nonsense mutation in a male patient associated with a much more damaging phenotype. This study highlighted the advantages of pangenomic analysis by CGH-array and at the same time it identified the complexity and limitations in interpretation of results particularly for High throughput sequencing, L'exploration génétique de la déficience intellectuelle (DI) a été révolutionnée par l'amélioration des technologies de séquençage depuis ces dernières années avec la caractérisation du spectre mutationnel des gènes impliqués dans la DI, ainsi que l'identification de nouveaux gènes associés. Dans notre étude, nous avons utilisé la technique d'analyse sur microréseau d'ADN (Hybridation Génomique Comparative ou CGH-array) à haute résolution, puis celle du séquençage haut débit pour rechercher une altération à l'origine de la DI inexpliquée. Le syndrome d'Aicardi est une maladie neurodéveloppementale rare et sporadique, caractérisée par la triade: spasmes infantiles, agénésie du corps calleux, et lacunes choriorétiniennes. Ce syndrome est décrit exclusivement chez les filles avec l'hypothèse la plus probable d'une mutation dominante liée au chromosome X. Nous avons d'abord analysé les ADN de 22 patientes atteintes du syndrome d'Aicardi par CGH-array, à l'aide d'un microréseau d'oligonucléotides haute résolution (1M) spécifique du chromosome X, sans identifier de remaniements ou CNV pouvant être impliqués dans la maladie. Un premier séquençage haut débit de l'exome du chromosome X, a été effectué sur l'ADN d'un trio (patiente et parents) et deux autres patientes présentant des signes typiques du syndrome d'Aicardi. Les résultats ont révélé 59 mutations dans 51 gènes. Il s'agit de 13 variants hérités de la mère, 8 hérités du père, de 36 faux positifs, et 2 SNP. Un deuxième séquençage haut débit, sur l'exome complet, a ensuite été réalisé, à partir de l'ADN de cinq trios (patientes et parents). Nous présentons et commentons les différentes stratégies d'analyses utilisées à la recherche d'un gène candidat. Les résultats obtenus pour les SNP soulignent les difficultés rencontrées en terme de profondeur du séquençage générant de nombreux contrôles et les difficultés d'alignement des séquences ne rendant pas performant l'analyse des indels. Parallèlement, dans notre cohorte de patients du centre de référence maladies rares, la CGH-array a identifié des altérations intragéniques du gène IL1RAPL1 dont deux duplications originales et une délétion. Nous analysons les corrélations génotype - phénotype au regard des données de la littérature avec notamment la variabilité d'expression clinique. Deux délétions intragéniques et une duplication intragénique du gène MBD5, survenue de novo ont été aussi détectées chez des patients atteints de DI. Cette duplication conduit à des transcrits aberrants avec codon stop prématuré. Le gène MBD5 a été séquencé sur une cohorte de 78 patients phénotypiquement sélectionnés révélant une mutation nonsens de novo détecté chez un garçon associé avec un phénotype sévère. Nos travaux témoignent des avantages de ces stratégies d'analyse pangénomiques, dont l'analyse sur microréseau, mais souligne aussi la complexité, les limites en terme d'interprétation des résultats, tout particulièrement pour le séquençage de nouvelle génération

The genetic exploration of intellectual disability (ID) has been revolutionized with the improvement in sequencing technologies during last decade with characterization of mutational spectrum of genes involved in ID as well as to identify new genes associated with it. In this study we used high resolution (comparative genomic hybridization array) CGH-array and high throughput sequencing technique to find the genetic cause in patients with unexplained ID. Aicardi syndrome is a rare sporadic neurodevelopmental syndrome, characterized by classic triad of agenesis of corpus callosum, chorioretinal lacunes and infantile spasms. This syndrome is exclusively present in females with plausible hypothesis of X linked dominant mutation. We first tested DNA of 22 patients diagnosed with Aicardi syndrome by using a high resolution oligonucleotide CGH-array 1M specifically designed for X-chromosome without identifying any pathogenic CNV or deleterious rearrangements involved in the disease. High throughput sequencing for exome of X chromosome was carried out in one trio (patient-parents) and two patients with typical Aicardi syndrome diagnosis. Sequencing results detected 59 mutations in 51 genes. 13 mutations were inherited from mother, 8 inherited from father, 36 false positive and 2 were SNP?s. Second approach was based on High throughput sequencing for complete exome of five trios (patient-parents) DNA. We presented and commented different strategies for data analysis in search of a candidate gene. These results highlighted difficulties in terms of depth and alignment of sequencing reads which generated various false positive SNP?s and indels. In second cohort from reference centre of rare diseases CGH-array has identified two intragenic rearrangements of IL1RAPL1 gene: two unique duplications and one deletion. We analyze genotype-phenotype correlations with cases described in literature which emphasizes the clinical variability of expression in these patients. Two de novo intragenic deletions and a de novo intragenic duplication were detected in MBD5 gene in patients with ID. The de novo duplication of MBD5 resulted in an aberrant transcripts leading to a premature termination codon. A selected cohort of 78 patients were sequenced for MBD5 gene which revealed a de novo nonsense mutation in a male patient associated with a much more damaging phenotype. This study highlighted the advantages of pangenomic analysis by CGH-array and at the same time it identified the complexity and limitations in interpretation of results particularly for High throughput sequencing, L'exploration génétique de la déficience intellectuelle (DI) a été révolutionnée par l'amélioration des technologies de séquençage depuis ces dernières années avec la caractérisation du spectre mutationnel des gènes impliqués dans la DI, ainsi que l'identification de nouveaux gènes associés. Dans notre étude, nous avons utilisé la technique d'analyse sur microréseau d'ADN (Hybridation Génomique Comparative ou CGH-array) à haute résolution, puis celle du séquençage haut débit pour rechercher une altération à l'origine de la DI inexpliquée. Le syndrome d'Aicardi est une maladie neurodéveloppementale rare et sporadique, caractérisée par la triade: spasmes infantiles, agénésie du corps calleux, et lacunes choriorétiniennes. Ce syndrome est décrit exclusivement chez les filles avec l'hypothèse la plus probable d'une mutation dominante liée au chromosome X. Nous avons d'abord analysé les ADN de 22 patientes atteintes du syndrome d'Aicardi par CGH-array, à l'aide d'un microréseau d'oligonucléotides haute résolution (1M) spécifique du chromosome X, sans identifier de remaniements ou CNV pouvant être impliqués dans la maladie. Un premier séquençage haut débit de l'exome du chromosome X, a été effectué sur l'ADN d'un trio (patiente et parents) et deux autres patientes présentant des signes typiques du syndrome d'Aicardi. Les résultats ont révélé 59 mutations dans 51 gènes. Il s'agit de 13 variants hérités de la mère, 8 hérités du père, de 36 faux positifs, et 2 SNP. Un deuxième séquençage haut débit, sur l'exome complet, a ensuite été réalisé, à partir de l'ADN de cinq trios (patientes et parents). Nous présentons et commentons les différentes stratégies d'analyses utilisées à la recherche d'un gène candidat. Les résultats obtenus pour les SNP soulignent les difficultés rencontrées en terme de profondeur du séquençage générant de nombreux contrôles et les difficultés d'alignement des séquences ne rendant pas performant l'analyse des indels. Parallèlement, dans notre cohorte de patients du centre de référence maladies rares, la CGH-array a identifié des altérations intragéniques du gène IL1RAPL1 dont deux duplications originales et une délétion. Nous analysons les corrélations génotype - phénotype au regard des données de la littérature avec notamment la variabilité d'expression clinique. Deux délétions intragéniques et une duplication intragénique du gène MBD5, survenue de novo ont été aussi détectées chez des patients atteints de DI. Cette duplication conduit à des transcrits aberrants avec codon stop prématuré. Le gène MBD5 a été séquencé sur une cohorte de 78 patients phénotypiquement sélectionnés révélant une mutation nonsens de novo détecté chez un garçon associé avec un phénotype sévère. Nos travaux témoignent des avantages de ces stratégies d'analyse pangénomiques, dont l'analyse sur microréseau, mais souligne aussi la complexité, les limites en terme d'interprétation des résultats, tout particulièrement pour le séquençage de nouvelle génération

of the thesis Glutamate is a main excitatory neurotransmitter in the brain of mammals, which activates both ionotropic and metabotropic glutamate receptors. Ionotropic receptors are responsible for fast synaptic transmission leading to membrane depolarization and Ca2+ influx into the cell. On the other hand mGlu receptors play an important role in regulation of the transmission via heterotrimeric G-proteins and activation of various signaling pathways. Postsynaptically localized group I mGlu receptors (mGluR1, 5) together with ionotropic NMDA and AMPA receptors share common large receptor signaling complexes, or signalosome facilitating glutamate signal transductions. Individual mGluR1 splice variants are differently associated with signalosome including scaffold proteins like PSD-95 which organize postsynaptic density (PSD). Heterodimerization of different mGluR1 splice variants is a focal point of my thesis together with investigation of recently discovered protein IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) and its role in organization of postsynaptic signalosome. Using biochemical, immunocytochemical and functional assays we showed heterodimers of mGluR1a/1b were expressed on the plasma membrane and that heterodimers are fully functional in the recombinant system. Next we showed...