La déficience intellectuelle (DI) est définie comme un dysfonctionnement intellectuel général inférieur à la moyenne, qui s'accompagne de limitations significatives du fonctionnement adaptatif (DSM-V). Il s'agit d'un handicap fréquent qui concerne près de 3% de la population générale. L'identification de l'étiologie d'une DI est une question primordiale car elle permet d'optimiser la prise en charge des patients sans risque de passer à côté d'une cause curable, et d'évaluer le risque de récidive dans la famille afin d'offrir un conseil génétique pour les grossesses à venir. Malgré les récents progrès, l'étiologie de la maladie reste inconnue dans près de 40% des cas. Le démembrement des causes génétiques et la compréhension des bases physiopathologiques des DI constituent donc un grand défi scientifique et médical. Par ailleurs, l'identification des gènes impliqués dans les DI et le décryptage des processus cellulaires sous-jacents à ces phénotypes sont une approche de choix pour étudier le développement et la plasticité cérébrale chez l'homme d'une part et entrevoir le développement de nouvelles stratégies thérapeutiques d'autre part. Le travail de thèse présenté dans ce manuscrit s'inscrit dans cette thématique de recherche et a porté sur l'analyse, par la méthode de Whole Exome Sequencing (WES), de cinq familles indépendantes dans lesquelles ségrège une forme syndromique de DI. La première partie détaille les résultats obtenus pour l'analyse de trois familles consanguines dans lesquelles ségrège une DI autosomique récessive. La seconde partie présente l'étude de deux familles indépendantes dont les enfants atteints présentent une clinique très semblable. Au total, ce mémoire décrit l'identification de i) deux gènes précédemment associés à la DI (WDR62 et AP4M1), ii) deux gènes candidats (RAD54B et HERC2), potentiels modificateurs des symptômes observés, puis iii) la définition d'un nouveau mode d'hérédité, et enfin iv) la caractérisation de deux nouveaux gènes impliqués dans la DI (TTI2 et NONO) suivie des études fonctionnelles des efiets des mutations sur les cellules de patients et l'analyse d'un modèle murin Nonogt. / Intellectual deficiency (ID) is characterized by a broad range of deficits in higher brain functions that result in significant limitations in adaptive and cognitive capacities required for competence in daily living, communication, social interaction and integration, self-direction, and work (DSM-V). ID affects approximately 3% of the population. Identifying ID causes is essential to improve patients' care services with no risk to miss a curable cause, but also to provide genetic counselling to the family for future pregnancies. Little is known about the biological bases of these conditions. Indeed, despite recent advances in cytogenetic and molecular genetics, the cause of the mental handicap remains unexplained in 40% of the cases. Understanding the molecular bases of these disorders is therefore an important medical challenge for the next years. Also, ID genes identification and analysis of the cellular mechanisms underlying these conditions should provide significant insight into the molecular and cellular pathways involved in cognition and may lead to new therapeutic trials aiming at improving the daily living of these patients and their families. The PhD work presented here report on the analysis, using Whole Exome Sequencing (WES), of five different families presenting with syndromic ID. The first part develops results from the analysis of three consanguineous families with an autosomal recessive form of ID. The second part presents the study of 2 unrelated male ID patients who presented the same clinical features. Overall, this work allowed the identification of i) two genes previously associated with ID (WDR62 and AP4M1), ii) two candidate genes (RAD54B and HERC2), potential modifiers of the phenotype, then iii) the definition of a novel hereditary mode, and finally iv) the characterization of two new genes of ID (TTI2 and NONO) followed by the functional analysis of mutations effects in patients' cells and the Nonogt mouse model.

Les syndromes oro-facio-digitaux (OFD) sont caractérisés par la présence d'une atteinte orale, faciale et digitale et classés en 13 sous-types. Pendant longtemps seul le gène OFD1, responsable du type I et codant pour une protéine centrosomale et du corps basal, était principalement connu, faisant suspecter l’implication du cil primaire dans les syndromes OFD. Des mutations ont été rapportées plus récemment dans les gènes TMEM216, DDX59, SCLT1, TBC1D32 et TCTN3 chez un ou deux patients. Dans le but d’identifier de nouveaux gènes impliqués dans ces syndromes, nous avons réalisé une stratégie de séquençage haut débit d’exome chez 24 patients. Cette stratégie a permis d’identifier 5 nouveaux gènes (C2CD3, TMEM107, INTU, KIAA0753, IFT57), d'étendre aux syndromes OFD le spectre clinique de 3 gènes déjà connus dans d'autres ciliopathies (C5orf42, TMEM138, TMEM231) et de confirmer l'implication de 3 gènes déjà connus comme responsable de syndrome OFD (OFD1, DDX59, WDPCP). Les analyses fonctionnelles ont montré l’implication de la croissance centriolaire, de la zone de transition et du transport intraflagellaire, avec notamment la caractérisation de 3 complexes protéiques principaux : le complexe ternaire KIAA0753/OFD1/FOPNL, régulant la croissance centriolaire, le complexe MKS (TMEM107, TMEM231, TMEM216) constituant majeur de la zone de transition et le complexe CPLANE (INTU/FUZ/WDPCP), favorisant l’assemblage des protéines périphériques du complexe de transport intraflagellaire IFT-A. En conclusion, cette étude, la plus importante consacrée aux syndromes OFD, démontre la très grande hétérogénéité clinique et génétique de ces syndromes, de même que de nombreux allélismes avec d’autres ciliopathies. Elle étend à 15 le nombre de gènes causaux, rendant la classification clinique initiale totalement obsolète et permettant de considérer les syndromes OFD comme un nouveau sous-groupe de ciliopathies à part entière. / Oral-facial-digital syndromes (OFDS) are characterized by the association of oral, facial and digital anomalies. The different modes of inheritance and additional features lead to clinically delineate 13 subtypes. For a long time, only the OFD1 gene, responsible for OFDI subtype and coding for a centrosomal protein, has been known, suggesting the involvement of the primary cilium in OFDS. Mutations have recently been reported in the TMEM216, DDX59, SCLT1, TBC1D32 and TCTN3 genes in anecdotic cases. To identify new genes involved in OFDS, we performed whole-exome sequencing in 24 patients. In 14/24 cases, we identified 5 novel genes (C2CD3, TMEM107, INTU, KIAA0753, IFT57), enlarged the clinical spectrum of OFDS of 3 known genes responsible for other ciliopathies (C5orf42, TMEM138, TMEM231) and confirmed the involvement of 3 known genes in OFDS (OFD1, DDX59, WDPCP). Functional studies demonstrated the involvement of the centriolar growth, the transition zone and the intraflagellar transport, through the characterization of 3 major protein complexes: the KIAA0753/OFD1/FOPNL complex controlling the centriole elongation, the MKS module (TMEM107/TMEM231/TMEM216), an essential component of the transition zone, and the CPLANE complex (INTU/FUZ/WDPCP) enabling in the IFT-A assembly. We demonstrated the large clinical and genetic heterogeneity of OFDS, yielding the initial classification in 13 subtypes obsolete, extending the number of 15 causal genes, and confirming OFDS as a new full subgroup of ciliopathies.

L’histiocytose langerhansienne (HL) est la plus fréquente des histiocytoses, liée à l’accumulation de cellules pathologiques de phénotype langerhansien. La découverte de la mutation somatique BRAFV600E dans environ 50% des cas à ouvert un nouveau champ d’investigation pour tirer bénéfice de ce statut moléculaire pour la prise en charge des patients.Tout d’abord, nous avons montré l’efficacité des inhibiteurs de BRAF sans rapporter de résistance dans les formes actives d’HL, en particulier dans les formes multisystémiques avec atteinte des organes à risque (MS OR+) du nourrissons, confirmant le rôle driver de cette mutation dans l’HL. Toutefois, après l’arrêt du traitement administré durant 2 à 6 mois, de nombreuses récidives ont été constatées.Ensuite, nous avons montré que pour les enfants atteints d’HL, la mutation BRAFV600E était significativement associée aux formes MS OR+, retrouvée dans 87,8% de ces cas. Comparés aux patients non mutés BRAF, les patients avec la mutation BRAFV600E présentaient un taux de résistance plus élevé à la chimiothérapie de première ligne velbé - corticoïde (21,9% contre 3,3%), un taux plus élevé de réactivation à 5 ans (42,8% contre 28,1%) et un taux de séquelles supérieur (27,9% contre 12,6%).Par ailleurs, nous avons montré que, pour les HL BRAFV600E mutées, la quantification de BRAFV600E dans l’ADN libre circulant par PCR digitale en gouttelette était un biomarqueur pertinent pour les cas d’HL MS OR+ et les présentations résistantes au traitement de première ligne.Enfin, après un criblage de points chauds mutationnels d’une série d’échantillons tissulaires d’HL ayant permis de mettre en évidence un cas avec la mutation somatique PIK3CAE542K, 9 couples d’échantillons tumeur/constitutionnel ont été étudiés par séquençage d’exome. Cela nous a permis de mettre en évidence une mutation récurrente (n=2) de BRAF au niveau du site d’épissage 5’ (donneur) de l’intron 12. Selon l’analyse de l’ARN, cette mutation conduirait à l’insertion de 3 acides aminés (LLR) dans le domaine kinase de la protéine mutée, dont l’analyse fonctionnelle est en cours. / Langerhans cell histiocytosis (LCH) is the most common type of histiocytosis owing to accumulation of pathologic CD1a+ CD207+ histiocytes. The identification of BRAFV600E in more than half of patients with LCH has launched a new field of investigation to study potential patient’s management benefits and implications from this molecular status.First, in BRAFV600E mutated LCH, we reported the effectiveness of BRAF inhibitors. Efficacy with no resistance to vemurafenib was reported in all cases with active LCH disease, especially for multi-system LCH with risk organ (MS RO+) involvement, confirming the driver status of this mutation in LCH. However, after discontinuation of this treatment administered during 2-6 months, many recurrences were observed.Then, we showed that children with BRAFV600E mutated LCH manifested more severe disease, comprised 87.8% of patients with MS RO+ involvement. Compared to patients with wild-type BRAF, patients with BRAFV600E more commonly displayed resistance to combined vinblastine and corticosteroid therapy (21.9% vs. 3.3%), showed a higher 5-year reactivation rate (42.8% vs. 28.1%) and had more long-term permanent consequences (27.9% vs. 12.6%).Moreover, we showed that BRAFV600E quantification in circulating cell-free DNA by droplet digital PCR is a relevant biomarker to monitor response to therapy for MS RO+ LCH and RO- LCH children who failed to respond to first line chemotherapy.Finally, after the screening of LCH biopsy (n=86) for the BRAF, KRAS, NRAS and PI3KCA most common mutations, leading to highlight one case with the PIK3CAE542K somatic mutation, 9 paired tumor-normal samples from children with LCH were analyzed by whole exome sequencing. Data showed a new BRAF recurrent mutation (n=2) in the 5′ splice sites of the intron 12. According to RNA analysis, this mutation would lead to the insertion of 3 amino acids (LLR) in the smaller N-terminal lobe of the BRAF kinase domain. Functional analysis is ongoing.

Bc. Klára Horáčková DIPLOMA THESIS Mechanisms of immune dysregulation leading to inflammatory bowel disease Abstract Inflammatory bowel disease (IBD) is a complex disorder characterized by chronic inflammation of the gastrointestinal tract. Classical IBD is a multifactorial disease with adulthood or later-childhood onset. However, children with very early onset IBD (VEO-IBD, before 6 years of age) are a specific cohort, whose pathology can be caused by severe genetic defects in genes connected to immune homeostasis in the gut. We aimed to identify the causal genetic variants in 20 pediatric patients diagnosed with IBD (age of onset from 3 to 154 months) using whole exome sequencing (WES). We evaluated several bioinformatical approaches for WES data analysis. This included a comparison of two methods of variant identification using VarScan2 or GATK4-based tools. Furthermore, we compared 4 gene lists ("virtual panels") for variant filtering, one of which was compiled purposefully for this thesis. We identified and validated via segregation analysis 5 causal variants in 4 genes (DUOX2 compound heterozygote, FOXP3, NLRP3 and NOD2) accounting for 20 % of the cohort. NOD2 (p.A755V) variant has already been reported in IBD cases, while DUOX2 (p.R1216W + p.A1131T), FOXP3 (p.H400L) and NLRP3 (p.V200M) were newly...

Le système immunitaire est particulièrement dépendant des mécanismes de réparation de l’ADN, en effet le développement du système immunitaire adaptatif nécessite certains mécanismes de réparation de l’ADN, lors de la recombinaison V(D)J et lors de la commutation de classe des immunoglobulines. De plus, le système hématopoïétique est par sa nature très sensible aux lésions spontanées de l’ADN. Il existe chez l’homme de nombreux déficits immunitaires directement liés à un défaut de réparation de l’ADN. L’identification du gène responsable est importante pour un conseil génétique familial approprié et pour la prise en charge médicale. Nous avons accès aujourd’hui à de puissants outils de dépistage génétique grâce au séquençage à haut débit et la liste des gènes responsables d’un déficit immunitaire s’allonge de plus en plus en rapidement. La première partie de ce travail porte sur la mise au point d’un nouvel outil de dépistage rapide des déficits de la réparation de l’ADN, en particulier dans le cas de déficit immunitaires. Ce test est fondé sur l’observation d’un biais du répertoire du TCRdes lymphocytes T circulants lorsque les thymocytes ont une durée de vie diminuée, or un défaut de réparation de l’ADN entraîne une diminution de la survie thymocytaire. Nous avons mis au point deux techniques, par biologie moléculaire et par cytométrie en flux, pour détecter un éventuel biais du répertoire du TCRα et évaluer la pertinence de ce test dans les déficits immunitaires liés à un défaut de réparation de l’ADN. Un biais a notamment été détecté dans les cas de déficit en facteur du NHEJ et en ATM. Nous avons également établi en collaboration avec le service d’Immunologie Clinique de l’hôpital Saint-Louis une cohorte de patients atteints de déficit immunitaire commun variable (DICV) dont la présentation clinique est évocatrice d’un défaut de réparation de l’ADN. Une série de test fonctionnels de dépistages de déficit de la réparation de l’ADN ainsi que des analyses génétiques (CGH array, séquençage complet de l’exome) ont été fait chez ces patients afin d’identifier de nouveaux gènes impliqués dans les DICV. Parmi les 18 patients analysés, dans 5 cas on retrouve une sensibilité cellulaire accrue aux agents génotoxiques et chez 15 patients, un gène candidat a été identifié. Ces résultats sont encore préliminaires et la caractérisation génétique et fonctionnelle des mutations identifiées sera poursuivie par notre équipe. Pour finir, nous avons entrepris l’exploration génétique et fonctionnelle de deux mutations identifiées chez une jeune patiente atteinte de déficit immunitaire combiné (CID) associé à un syndrome lymphoprolifératif et une auto-immunité, et chez qui une hypersensibilité cellulaire à la Mitomycine C, agent pontant de l’ADN, a été détectée. La première mutation a été identifiée dans le gène ELKS qui code pour un facteur impliqué dans la réparation de l’ADN. La complémentation fonctionnelle de ce gène prouve l’implication de cette mutation dans l’hypersensibilité des cellules de la patiente à la MMC. Nous avons développé un modèle murin KO conditionnel de ce gène dans les cellules hématopoïétiques qui n’a pas montré de défaut de développement du système immunitaire. La deuxième mutation identifiée se situe dans le gène BACH2 codant pour un répresseur transcriptionnel très impliqué dans le développement du système immunitaire. Les souris KO pour ce gène ont un phénotype proche du déficit immunitaire décrit chez cette patiente. Les investigations de cette mutation sont en cours chez elle et chez les membres de sa famille également porteurs de la mutation. / The immune system is particularly dependent on DNA damage response (DDR) pathways. The development of the adaptive immune system requires certain DDR mechanisms, in particular during the V(D)J recombination and during class switch recombination (CSR), furthermore, the hematopoietic system is very sensitive to spontaneous DNA lesions. Therefore, there are many immune deficiencies in human directly related to a DDR deficiency. The identification of the responsible gene is important for appropriate genetic counseling. Today, we have access to powerful genetic screening tools, in particular next generation sequencing (NGS) and the list of genes responsible for immune deficiency is growing rapidly. The first part of this work focuses on the development of a new screening tool for DDR defects, in particular in the case of immune deficiency, and evaluation of clinical interest. This test is based on the observation of a bias of the TCRα repertoire in circulating T lymphocytes when thymocytes lifespan is diminished and we know that DDR defect causes decreased thymocyte survival. We have developed two techniques, by molecular biology and by flow cytometry, to detect a potential bias of the TCRα repetoire and assess the suitability of this test in some immunodeficiencies linked to a DDR defect. A significant bias was detected in the case of ATM and NHEJ factor deficiency. Furthermore, we have established a cohort of patients suffering from common variable immunodeficiency (CVID) with a clinical presentation highly suggestive of DDR defect, in collaboration with the Clinical Immunology Service of Hôpital Saint-Louis (Paris). Functional test for DDR defect and genetic analysis (CGHarray, whole exome sequencing) were performed in these patients to identify new genes involved in CVID. Among the 18 patients analyzed until now, five cases of cellular sensitivity to genotoxic agents have been detected and a candidate gene was identified in 15 of them. These results are still preliminary and our team will pursue genetic and functional characterization of the identified mutations. Finally, we undertook genetic and functional exploration of two mutations identified in a young patient with combined immunodeficiency (CID) associated with a lymphoproliferative disease and autoimmunity, and in whom a cellular hypersensitivity to mitomycin C, a DNA crosslinking agent, was detected. The first mutation was identified in the ELKS gene, which codes for a factor involved in DNA repair. Functional complementation of this gene demonstrates the involvement of this mutation in the hypersensitivity of patient’s cells to MMC. We have developed a conditional knockout mouse model of this gene in hematopoietic cells that did not show any defect in development of the immune system. The second mutation was identified in BACH2 gene encoding a transcriptional repressor involved in the development of the immune system. Knockout mice for this gene have a similar phenotype to the immune deficiency described in this patient. Investigations on this mutation are ongoing in the patient and among family members that also carry the mutation.

Le système immunitaire est particulièrement dépendant des mécanismes de réparation de l’ADN, en effet le développement du système immunitaire adaptatif nécessite certains mécanismes de réparation de l’ADN, lors de la recombinaison V(D)J et lors de la commutation de classe des immunoglobulines. De plus, le système hématopoïétique est par sa nature très sensible aux lésions spontanées de l’ADN. Il existe chez l’homme de nombreux déficits immunitaires directement liés à un défaut de réparation de l’ADN. L’identification du gène responsable est importante pour un conseil génétique familial approprié et pour la prise en charge médicale. Nous avons accès aujourd’hui à de puissants outils de dépistage génétique grâce au séquençage à haut débit et la liste des gènes responsables d’un déficit immunitaire s’allonge de plus en plus en rapidement. La première partie de ce travail porte sur la mise au point d’un nouvel outil de dépistage rapide des déficits de la réparation de l’ADN, en particulier dans le cas de déficit immunitaires. Ce test est fondé sur l’observation d’un biais du répertoire du TCRdes lymphocytes T circulants lorsque les thymocytes ont une durée de vie diminuée, or un défaut de réparation de l’ADN entraîne une diminution de la survie thymocytaire. Nous avons mis au point deux techniques, par biologie moléculaire et par cytométrie en flux, pour détecter un éventuel biais du répertoire du TCRα et évaluer la pertinence de ce test dans les déficits immunitaires liés à un défaut de réparation de l’ADN. Un biais a notamment été détecté dans les cas de déficit en facteur du NHEJ et en ATM. Nous avons également établi en collaboration avec le service d’Immunologie Clinique de l’hôpital Saint-Louis une cohorte de patients atteints de déficit immunitaire commun variable (DICV) dont la présentation clinique est évocatrice d’un défaut de réparation de l’ADN. Une série de test fonctionnels de dépistages de déficit de la réparation de l’ADN ainsi que des analyses génétiques (CGH array, séquençage complet de l’exome) ont été fait chez ces patients afin d’identifier de nouveaux gènes impliqués dans les DICV. Parmi les 18 patients analysés, dans 5 cas on retrouve une sensibilité cellulaire accrue aux agents génotoxiques et chez 15 patients, un gène candidat a été identifié. Ces résultats sont encore préliminaires et la caractérisation génétique et fonctionnelle des mutations identifiées sera poursuivie par notre équipe. Pour finir, nous avons entrepris l’exploration génétique et fonctionnelle de deux mutations identifiées chez une jeune patiente atteinte de déficit immunitaire combiné (CID) associé à un syndrome lymphoprolifératif et une auto-immunité, et chez qui une hypersensibilité cellulaire à la Mitomycine C, agent pontant de l’ADN, a été détectée. La première mutation a été identifiée dans le gène ELKS qui code pour un facteur impliqué dans la réparation de l’ADN. La complémentation fonctionnelle de ce gène prouve l’implication de cette mutation dans l’hypersensibilité des cellules de la patiente à la MMC. Nous avons développé un modèle murin KO conditionnel de ce gène dans les cellules hématopoïétiques qui n’a pas montré de défaut de développement du système immunitaire. La deuxième mutation identifiée se situe dans le gène BACH2 codant pour un répresseur transcriptionnel très impliqué dans le développement du système immunitaire. Les souris KO pour ce gène ont un phénotype proche du déficit immunitaire décrit chez cette patiente. Les investigations de cette mutation sont en cours chez elle et chez les membres de sa famille également porteurs de la mutation. / The immune system is particularly dependent on DNA damage response (DDR) pathways. The development of the adaptive immune system requires certain DDR mechanisms, in particular during the V(D)J recombination and during class switch recombination (CSR), furthermore, the hematopoietic system is very sensitive to spontaneous DNA lesions. Therefore, there are many immune deficiencies in human directly related to a DDR deficiency. The identification of the responsible gene is important for appropriate genetic counseling. Today, we have access to powerful genetic screening tools, in particular next generation sequencing (NGS) and the list of genes responsible for immune deficiency is growing rapidly. The first part of this work focuses on the development of a new screening tool for DDR defects, in particular in the case of immune deficiency, and evaluation of clinical interest. This test is based on the observation of a bias of the TCRα repertoire in circulating T lymphocytes when thymocytes lifespan is diminished and we know that DDR defect causes decreased thymocyte survival. We have developed two techniques, by molecular biology and by flow cytometry, to detect a potential bias of the TCRα repetoire and assess the suitability of this test in some immunodeficiencies linked to a DDR defect. A significant bias was detected in the case of ATM and NHEJ factor deficiency. Furthermore, we have established a cohort of patients suffering from common variable immunodeficiency (CVID) with a clinical presentation highly suggestive of DDR defect, in collaboration with the Clinical Immunology Service of Hôpital Saint-Louis (Paris). Functional test for DDR defect and genetic analysis (CGHarray, whole exome sequencing) were performed in these patients to identify new genes involved in CVID. Among the 18 patients analyzed until now, five cases of cellular sensitivity to genotoxic agents have been detected and a candidate gene was identified in 15 of them. These results are still preliminary and our team will pursue genetic and functional characterization of the identified mutations. Finally, we undertook genetic and functional exploration of two mutations identified in a young patient with combined immunodeficiency (CID) associated with a lymphoproliferative disease and autoimmunity, and in whom a cellular hypersensitivity to mitomycin C, a DNA crosslinking agent, was detected. The first mutation was identified in the ELKS gene, which codes for a factor involved in DNA repair. Functional complementation of this gene demonstrates the involvement of this mutation in the hypersensitivity of patient’s cells to MMC. We have developed a conditional knockout mouse model of this gene in hematopoietic cells that did not show any defect in development of the immune system. The second mutation was identified in BACH2 gene encoding a transcriptional repressor involved in the development of the immune system. Knockout mice for this gene have a similar phenotype to the immune deficiency described in this patient. Investigations on this mutation are ongoing in the patient and among family members that also carry the mutation.

Mutações nos genes envolvidos na embriogênese hipofisária já foram descritas relacionadas a quadros isolados de deficiência hormonal múltipla e/ou associado a fenótipos extra-pituitários. As mutações encontradas em humanos foram descritas em genes envolvidos na embriogênese hipofisária e cujos fenótipos foram gerados em animais a partir de nocaute, servindo de ponto de partida para sua busca em pacientes com fenótipo similar. Essa estratégia é conhecida como busca por gene candidato, e é feita pela técnica de sequenciamento tradicional Sanger. Na última década, com o avanço de novas tecnologias de sequenciamento, diversos genes foram associados ao hipopituitarismo, principalmente utilizando-se a metodologia de exoma. Contudo, ainda há uma grande parcela dessa população sem diagnóstico molecular, como evidenciado em um levantamento na literatura por Fang e colaboradores e cuja tendência foi observada no ambulatório de Endocrinologia do Desenvolvimento do Hospital das Clínicas, onde apenas 14% dos pacientes tiveram o seu diagnóstico molecular determinado. Com isso, as tecnologias de sequenciamento de última geração, passaram a ser uma ferramenta promissora para determinação molecular dos fenótipos dos pacientes. Logo, alguns pacientes em seguimento no ambulatório de endocrinologia do desenvolvimento tiveram o exoma sequenciado, e uma análise das métricas do sequenciamento evidenciou regiões de cobertura muito baixa, o que não permitiu a conclusão sobre a presença ou ausência de variantes nessas regiões. Entre essas regiões estão os genes SOX2 e SOX3, os quais possuem variantes conhecidas causadoras do fenótipo. Esse trabalho tem como objetivo analisar a cobertura dos genes envolvidos na embriogênese hipofisária assim como os relacionados ao hipopituitarismo congênito em quatro diferentes kits de preparação de biblioteca para exoma, a fim de identificar a melhor metodologia para um diagnóstico molecular dos pacientes alem determinar variantes especificas da população brasileira na região de interesse através de busca no site ABraOM. Foram analisados 76 genes em um total de 119 amostras separadas em três grupos, sendo o primeiro grupo de amostras HapMap, o segundo de um paciente com hipopituitarismo e sua mãe e o terceiro de amostras brasileiras aleatórias. Os kits utilizados foram NimbleGen (Roche), Nextera (Illumina), SureSelect e SureSelect+UTR (Agilent). Para isso, foram utilizados diversos programas de bioinformática, tendo entre eles o FASTQC, BWA, GATK, Annovar, Qualimap e bedTools. Análises da qualidade do sequenciamento, assim como a taxa de mapeamento e duplicação mostraram que as amostras utilizadas apresentavam qualidades adequadas e similares entre si para a análise. De acordo com os resultados obtidos em relação a cobertura, o kit da NimbleGen apresenta uma queda em sua cobertura dos genes de interesse em relação a sua capacidade de cobertura do exoma global, algo que pode ser devido à alta taxa de GC na região de interesse, uma vez que a capacidade do kit nessas regiões é deficiente em relação aos demais. Os genes com piores coberturas em todas as quatro tecnologias foram os genes HES5, que apesar de fazer parte da embriologia hipofisária, não possui variante relacionada ao fenótipo em humanos, e o SOX3 que, apesar de ter muita baixa cobertura na NimbleGen, é bem coberto na SureSelect. Isso corrobora com a análise de capacidade de cobertura em regiões com alta taxa de GC. Somado a isso observou-se que a população brasileira tem 885 variantes únicas e exclusivas. Concluímos, portanto, que o kit SureSelect, da Agilent, tem o melhor desempenho na região de interesse, assim como no exoma global, sendo o indicado para estudos em coortes de hipopituitarismo e a população brasileira possui variantes únicas inerentes a ela / Mutations in the genes involved in pituitary embryogenesis have been described related to isolated cases of multiple hormonal deficiency and/or associated with extra-pituitary phenotypes. Mutations found in humans were described in genes involved in pituitary embryogenesis by generating phenotypes knockout animals, serving as the starting point for their search in patients with similar phenotype. This strategy is known as gene candidate search and is performed by the traditional Sanger sequencing technique. In the last decade, with the advancement of new sequencing technologies, several genes have been associated with hypopituitarism, mainly using the exome methodology. However, there is still a large portion of this population without molecular diagnosis, as evidenced by a survey in the literature by Fang et al., This trend was also observed in the outpatient clinic of Developmental Endocrinology of Hospital das Clínicas, where only 14% of the patients had their molecular diagnosis. With this, high throughput sequencing technologies have become a promising tool for the molecular determination of patients\' phenotypes. Therefore, we sequenced the exome of some of our patients, and an analysis of the sequencing quality showed very low coverage regions, which harms the researcher\'s ability to reach a conclusion regarding presence or lack of variants in these regions. Among these regions are the SOX2 and SOX3 genes, which have many variants that are known to cause the phenotype. This work aims to analyze the coverage of the genes involved in pituitary embryogenesis as well as those related to congenital hypopituitarism in four different exome library preparation kits in order to identify the best methodology for a molecular diagnosis of the patients and to determine specific variants of the Brazilian population in the region of interest by searching the ABraOM website. A total of 76 genes were analyzed in a total of 119 samples in three groups: the first group of HapMap samples, the second of a patient with hypopituitarism and his mother, and the third group of random Brazilian samples. The kits used were NimbleGen (Roche), Nextera (Illumina), SureSelect and SureSelect + UTR (Agilent). For this, several bioinformatics programs were used, among them FASTQC, BWA, GATK, Annovar, Qualimap and bedTools. Sequencing quality analysis, as well as the mapping and duplication rate, showed that the samples used presented adequate and similar qualities for the comparison. According to the results obtained in relation to the coverage, the NimbleGen kit shows a drop in its coverage of the genes of interest in relation to its capacity to cover the global exome, something that may be due to the high GC rate in the region of interest, once the capacity of the kit in these regions is not as good as the others. The genes with the worst coverage in all four technologies were the HES5 gene, which despite being part of the pituitary embryology, have no phenotype-related variant in humans, and SOX3 which, despite having very low coverage in NimbleGen, is well covered on SureSelect. This corroborates the analysis of coverage capacity in regions with a high GC rate. In addition to this it was observed that the Brazilian population has 885 unique and exclusive variants. Therefore, we conclude that the Agilent\'s SureSelect kit has the best performance in the region of interest, as well as in the global exome, being recommended for studies in hypopituitarism cohorts, and that the Brazilian population has unique variants inherent to it

Le système immunitaire est particulièrement dépendant des mécanismes de réparation de l’ADN, en effet le développement du système immunitaire adaptatif nécessite certains mécanismes de réparation de l’ADN, lors de la recombinaison V(D)J et lors de la commutation de classe des immunoglobulines. De plus, le système hématopoïétique est par sa nature très sensible aux lésions spontanées de l’ADN. Il existe chez l’homme de nombreux déficits immunitaires directement liés à un défaut de réparation de l’ADN. L’identification du gène responsable est importante pour un conseil génétique familial approprié et pour la prise en charge médicale. Nous avons accès aujourd’hui à de puissants outils de dépistage génétique grâce au séquençage à haut débit et la liste des gènes responsables d’un déficit immunitaire s’allonge de plus en plus en rapidement. La première partie de ce travail porte sur la mise au point d’un nouvel outil de dépistage rapide des déficits de la réparation de l’ADN, en particulier dans le cas de déficit immunitaires. Ce test est fondé sur l’observation d’un biais du répertoire du TCRdes lymphocytes T circulants lorsque les thymocytes ont une durée de vie diminuée, or un défaut de réparation de l’ADN entraîne une diminution de la survie thymocytaire. Nous avons mis au point deux techniques, par biologie moléculaire et par cytométrie en flux, pour détecter un éventuel biais du répertoire du TCRα et évaluer la pertinence de ce test dans les déficits immunitaires liés à un défaut de réparation de l’ADN. Un biais a notamment été détecté dans les cas de déficit en facteur du NHEJ et en ATM. Nous avons également établi en collaboration avec le service d’Immunologie Clinique de l’hôpital Saint-Louis une cohorte de patients atteints de déficit immunitaire commun variable (DICV) dont la présentation clinique est évocatrice d’un défaut de réparation de l’ADN. Une série de test fonctionnels de dépistages de déficit de la réparation de l’ADN ainsi que des analyses génétiques (CGH array, séquençage complet de l’exome) ont été fait chez ces patients afin d’identifier de nouveaux gènes impliqués dans les DICV. Parmi les 18 patients analysés, dans 5 cas on retrouve une sensibilité cellulaire accrue aux agents génotoxiques et chez 15 patients, un gène candidat a été identifié. Ces résultats sont encore préliminaires et la caractérisation génétique et fonctionnelle des mutations identifiées sera poursuivie par notre équipe. Pour finir, nous avons entrepris l’exploration génétique et fonctionnelle de deux mutations identifiées chez une jeune patiente atteinte de déficit immunitaire combiné (CID) associé à un syndrome lymphoprolifératif et une auto-immunité, et chez qui une hypersensibilité cellulaire à la Mitomycine C, agent pontant de l’ADN, a été détectée. La première mutation a été identifiée dans le gène ELKS qui code pour un facteur impliqué dans la réparation de l’ADN. La complémentation fonctionnelle de ce gène prouve l’implication de cette mutation dans l’hypersensibilité des cellules de la patiente à la MMC. Nous avons développé un modèle murin KO conditionnel de ce gène dans les cellules hématopoïétiques qui n’a pas montré de défaut de développement du système immunitaire. La deuxième mutation identifiée se situe dans le gène BACH2 codant pour un répresseur transcriptionnel très impliqué dans le développement du système immunitaire. Les souris KO pour ce gène ont un phénotype proche du déficit immunitaire décrit chez cette patiente. Les investigations de cette mutation sont en cours chez elle et chez les membres de sa famille également porteurs de la mutation. / The immune system is particularly dependent on DNA damage response (DDR) pathways. The development of the adaptive immune system requires certain DDR mechanisms, in particular during the V(D)J recombination and during class switch recombination (CSR), furthermore, the hematopoietic system is very sensitive to spontaneous DNA lesions. Therefore, there are many immune deficiencies in human directly related to a DDR deficiency. The identification of the responsible gene is important for appropriate genetic counseling. Today, we have access to powerful genetic screening tools, in particular next generation sequencing (NGS) and the list of genes responsible for immune deficiency is growing rapidly. The first part of this work focuses on the development of a new screening tool for DDR defects, in particular in the case of immune deficiency, and evaluation of clinical interest. This test is based on the observation of a bias of the TCRα repertoire in circulating T lymphocytes when thymocytes lifespan is diminished and we know that DDR defect causes decreased thymocyte survival. We have developed two techniques, by molecular biology and by flow cytometry, to detect a potential bias of the TCRα repetoire and assess the suitability of this test in some immunodeficiencies linked to a DDR defect. A significant bias was detected in the case of ATM and NHEJ factor deficiency. Furthermore, we have established a cohort of patients suffering from common variable immunodeficiency (CVID) with a clinical presentation highly suggestive of DDR defect, in collaboration with the Clinical Immunology Service of Hôpital Saint-Louis (Paris). Functional test for DDR defect and genetic analysis (CGHarray, whole exome sequencing) were performed in these patients to identify new genes involved in CVID. Among the 18 patients analyzed until now, five cases of cellular sensitivity to genotoxic agents have been detected and a candidate gene was identified in 15 of them. These results are still preliminary and our team will pursue genetic and functional characterization of the identified mutations. Finally, we undertook genetic and functional exploration of two mutations identified in a young patient with combined immunodeficiency (CID) associated with a lymphoproliferative disease and autoimmunity, and in whom a cellular hypersensitivity to mitomycin C, a DNA crosslinking agent, was detected. The first mutation was identified in the ELKS gene, which codes for a factor involved in DNA repair. Functional complementation of this gene demonstrates the involvement of this mutation in the hypersensitivity of patient’s cells to MMC. We have developed a conditional knockout mouse model of this gene in hematopoietic cells that did not show any defect in development of the immune system. The second mutation was identified in BACH2 gene encoding a transcriptional repressor involved in the development of the immune system. Knockout mice for this gene have a similar phenotype to the immune deficiency described in this patient. Investigations on this mutation are ongoing in the patient and among family members that also carry the mutation.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-04-20T13:30:09Z No. of bitstreams: 1 FelipeAlbuquerqueMarquesTese2015.pdf: 5827285 bytes, checksum: 3dfbabb913ebff137b6f41b2cd1ba5fc (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-04-20T13:30:28Z (GMT) No. of bitstreams: 1 FelipeAlbuquerqueMarquesTese2015.pdf: 5827285 bytes, checksum: 3dfbabb913ebff137b6f41b2cd1ba5fc (MD5) / Made available in DSpace on 2017-04-20T13:30:28Z (GMT). No. of bitstreams: 1 FelipeAlbuquerqueMarquesTese2015.pdf: 5827285 bytes, checksum: 3dfbabb913ebff137b6f41b2cd1ba5fc (MD5) Previous issue date: 2015-09-25 / Genetic diseases of the skeleton affect the genesis of skeletal system. They are caused by mutations in genes which act on the cartilage and/or growth plate. The current classification of skeletal anomalies describes more than 456 distinct phenotypes organized into 40 groups. Of this total, 360 phenotypes are associated with defects in 336 genes (thus, 90 diseases remain to have their cause elucidated). The development of high resolution techniques for genomic analysis has enabled more genetic diseases, including skeletal phenotypes, to have molecular basis clarified. This research aimed to identify genes or regions in the human genome associated with genetic diseases of the skeleton. To this end, a pipeline was developed involving experiments and data analysis. Investigation of single nucletoide variation (SNV) was carried out using whole exome sequencing (WES) and submicroscopic structural variations were analyzed by Chromossomal Microarray Analysis (CMA). Moreover, Sanger sequencing, Fluorescence in situ Hybridization, in vitro functional tests (cell culture, qRT-PCR, Western Blot, Immunofluorescence and transcriptome), Immunohistochemistry and histochemistry were employed. 14 patients with rare diseases of skeleton (Craniosynostosis, s. FATCO sindrome, Catel-Manzke-like sindrome, Nager Syndrome and Rodriguez Syndrome) were selected. Craniosynostosis: of three patients, two had their molecular diagnoses elucidated, one with mutation in FGFR3 and the other with a translocation involving chromosomes 17q and 20q. s. FATCO: it wasn't possible to identify the causative mutation for this disease. Catel-Manzke-like Syndrome: initially this patient was diagnosed as Catel-Manzke Syndrome, and there was found a mutation in EXT2. Thus, this patient was reclassified as a new syndrome recently reported as seizures-scoliosis-macrocephaly. Nager and Rodriguez Syndrome: two of four have been diagnosed with mutation in SF3B4. For these patients, the results of qRT-PCR, Western Blot and Immunofluorescence together suggested that the phenotype is caused by SF3B4 haploinsuficiency. Immunohistochemistry and Histochemistry showed the expression of SF3B4 in cartilage tissue and the disorganization of hypertrophic cells in growth plate, respectively. The transcriptome result from cartilage tissue of one patient with SF3B4 mutation showed 12 underexpressed genes involved in skeletogeneses. The combination of techniques like classical cytogenetics and molecular cytogenetics as well as sequencing and in vitro assays were effective to achieve a diagnosis. Although there was an investigative core common to all diseases, investigations were customized to each case, seeking greater efficiency in the detection of the molecular basis and cost optimization of molecular research. / As doen??as gen??ticas do esqueleto s??o anomalias que envolvem a g??nese do sistema esquel??tico, causadas por altera????es em genes que atuam principalmente na cartilagem e/ou no n??cleo de crescimento. Na classifica????o atual h?? 456 doen??as do esqueleto categorizadas em 40 grupos. Destes, 360 patologias esquel??ticas est??o associadas a defeitos em 336 genes (portanto, existem 90 displasias esquel??ticas sem causa definida). O surgimento de t??cnicas de alta resolu????o de an??lise gen??mica tem permitido que cada vez mais doen??as gen??ticas, incluindo as doen??as gen??ticas do esqueleto, possam ter suas bases moleculares elucidadas. Esta pesquisa teve como objetivo a identifica????o e caracteriza????o de genes e regi??es do genoma humano associado a doen??as gen??ticas raras do esqueleto. Para isso, implantou-se um pipeline envolvendo experimentos e an??lise de dados. Esta tese investigou varia????es de nucleot??deo ??nico (SNVs) pelo uso Whole Exome Sequencing (WES) e varia????es estruturais submicrosc??picas, pelo uso Chromossomal Microarray Analysis (CMA). Al??m disso, fez-se uso de Sequenciamento de Sanger, Fluorescence in situ Hybridization, testes funcionais in vitro (cultura celular, qRT-PCR, Western Blot, Imunofluoresc??ncia e Transcritoma), Imunohistoqu??ca e histoqu??mica. Foram selecionados 14 pacientes com doen??as raras do esqueleto (Craniossinostose, s??ndrome FATCO, s??ndrome Catel-Manzke-like, s??ndrome de Nager e s??ndrome Rodriguez). Craniossinostose: dos quatro pacientes, dois foram diagnosticados com muta????o em FGFR3 e outro com uma transloca????o envolvendo os cromossomos 17q e 20q. S??ndrome de FATCO: n??o foi poss??vel identificar as bases moleculares da doen??a. ???Catel-Manzke-Like???: inicialmente diagnosticado com Sindrome de Catel-Manzke, o paciente teve uma muta????o detectada em EXT2, sendo reclassificado como uma nova s??ndrome (S??ndrome Convuls??o-Escoliose-Microcefalia). S??ndromes de Nager e Rodriguez: foram diagnosticado muta????es no gene SF3B4 em dois dos quatro pacientes. Nestes mesmos pacientes foram realizados qTR-PCR, Western Blot e Imunofluoresc??ncia que juntos sugeriram que estas patologias sejam causadas pela haploinsufici??ncia do SF3B4. A imunohistoqu??mica e histoqu??mica mostraram a express??o de SF3B4 na placa de crescimento e desorganiza????o de condr??citos hipertr??ficos, respectivamente. O transcritoma de um dos pacientes, com muta????o em SF3B4, evidenciou 12 genes ligados a esqueletog??nese com express??o diminu??da no tecido cartilaginoso. A combina????o de t??cnicas seja citogen??tica cl??ssica, citogen??tica molecular, sequenciamento, bem como an??lises in vitro se mostraram eficientes para se alcan??ar o diagn??stico. Embora houvesse um cerne investigativo em comum a todas as doen??as, as investiga????es foram personalizadas para cada caso, visando maior efici??ncia na detec????o das bases moleculares e otimiza????o dos custos da investiga????o molecular.

A imunodeficiência combinada grave (SCID) é uma doença caracterizada por profunda deficiência de células T, que afeta as imunidades celular e humoral e gera anormalidades graves no desenvolvimento e funções do sistema imune. Recém-nascidos com SCID apresentam a doença nos primeiros meses de vida e tem grande susceptibilidade a infecções. Sem tratamento, essas condições são invariavelmente fatais, porém se reconhecidas precocemente, há a possibilidade da realização do transplante de células-tronco hematopoiéticas, o tratamento curativo, o que torna a SCID uma emergência pediátrica. A investigação do defeito genético é uma prerrogativa para o condicionamento adequado do transplante, a terapia gênica, o aconselhamento genético e o diagnóstico pré-natal. No Brasil, o conhecimento sobre SCID é incipiente e não existem dados moleculares sobre pacientes com a doença. Sendo assim, este estudo teve por objetivo investigar defeitos genético-moleculares de pacientes brasileiros com SCID. Foram incluídos 13 pacientes, todos com início precoce dos sintomas e manifestações clínicas esperadas em SCID (principalmente infecções respiratórias, de pele, diarreia crônica e atraso de crescimento). Os patógenos isolados foram vírus, bactérias e fungos oportunistas comumente encontrados em pacientes SCID. A partir da quantificação de TRECS e KRECs e imunofenotipagem de linfócitos, foi montado o perfil imunológico de cada paciente, que guiou o sequenciamento direto de Sangerdos genes mais frequentemente mutados em cada imunofenótipo de SCID. Mutações em 3 pacientes foram identificadas por Sanger e, posteriormente, 8 pacientes cujas mutações não foram encontradas no Sanger foram encaminhados para o sequenciamento completo de exoma, que resultou na identificação do gene afetado em 62,5% dos casos. Ao todo, foram identificadas mutações patogênicas em 8 dos 13 pacientes. Os resultados revelaram 6 alterações em 5 genes de SCID clássica (IL7R, RAG2, DCLRE1C, JAK3, IL2RG), 1 mutação no gene CD3G e 2 alterações em CECR1. Das 9 mutações encontradas, 5 não possuíam registro na literatura. O estudo genético de SCID em nosso país é problemático, principalmente porque ainda hoje, a esmagadora maioria dos pacientes não é diagnosticada. A implementação da quantificação de TRECs e KRECs como triagem neonatal para linfopenias graves é uma ferramenta fundamental para que os pacientes SCID possam ser identificados, investigados e tratados adequadamente. / Primary immunodeficiencies are a heterogeneous group of genetic diseases that lead to increased susceptibility to infections and affect mostly children. Severe Combined Immunodeficiency (SCID) is the most severe of all these diseases and is characterized by profound T cell deficiency, which affects cellular and humoral immunities and leads to severe abnormalities in the development and function of the immune system. Newborns with SCID present the disease in the first months of life and are highly susceptible to infections. Without treatment, these conditions are invariably fatal, but if recognized early, there is the possibility of hematopoietic stem cell transplantation, the curative treatment, which makes SCID a pediatric emergency. Identifying the genetic defect of SCID patients is a prerequisite for proper transplant conditioning, gene therapy, genetic counseling and prenatal diagnosis. Knowledge about SCID is still incipient in Brazil, and there are virtually no molecular data on patients with the disease. Therefore, this study aimed to investigate genetic-molecular defects of Brazilian patients with SCID. Thirteen patients were recruited, all with early onset of symptoms and clinical manifestations expected of classic SCIDs (mainly respiratory and skin infections, chronic diarrhea and failure to thrive). The pathogens isolated were opportunistic viruses, bacteria and fungi often reported in SCID patients. The immunological profile from each patient was defined by the quantification of TRECS and KRECs and lymphocyte immunophenotyping, which was meant to guide direct sequencing by Sanger of the most frequently mutated genes of each SCID immunophenotype. Mutations in 3 patients were identified by Sanger and, subsequently, 8 patients whose mutations were not identified by Sanger were referred for whole exome sequencing, which resulted in the identification of the affected gene in 62,5% of cases. Pathogenic mutations were identified in 8 of the 13 patients. The results revealed 6 mutations in 5 genes associated to classical SCID genes (IL7R, RAG2, DCLRE1C, JAK3, IL2RG), 1 mutation in the CD3G gene, and 2 mutations in CECR1. Five of the 9 mutations found had no record in the literature. SCID genetic investigation in our country is troublesome, mainly because even nowadays, the vast majority of patients are not diagnosed properly. Newborn screening for SCID and other severe lymphopenias by the quantification of TRECs and KRECs is key for the identification, investigation and proper treatment of SCID patients.