Abstract Novel genetic aetiologies for epileptic encephalopathies and movement disorders have been discovered by using next-generation sequencing methods. The phenotypic and genotypic variability in these conditions is very wide. The aim of this study was to discover novel genetic causes and phenotypes of childhood-onset drug-resistant epilepsy and epileptic or developmental encephalopathies that occur separately or together with movement disorders, and familial movement disorders. Furthermore, the use of whole-exome sequencing (WES) as a diagnostic tool in clinical practice was evaluated. Altogether, 12 children with undefined aetiology, who fulfilled the inclusion criteria, were included in the study. GABRG2 gene was identified as a genetic cause of epileptic encephalopathies. Novel GABRG2-associated phenotypes included progressive neurodegeneration, epilepsy in infancy with migrating focal seizures, and autism spectrum disorder. New pathogenic variants, GABRG2 p.P282T and p.S306F, were discovered. The pathogenic NACC1 variant caused focal epilepsy, developmental disability, bilateral cataracts, and dysautonomia. The novel phenotype associated with the NACC1 p.R298W variant included hyperkinetic movement disorder. SAMD9L was found to be the genetic cause for the familial movement disorder. The phenotype associated with the novel SAMD9L p.I891T variant was very variable. Neuroradiological findings included cerebellar atrophy and periventricular white matter changes. After publication of these results, SAMD9L was reported to be one of the most common genetic aetiologies of childhood bone marrow failure and myelodysplastic syndrome. The pathogenic homozygous MTR variant was found to cause early-onset epileptic encephalopathy that occurred together with movement disorder and haematological disturbances. Drug resistant seizures responded to cofactor and vitamin treatments. Whole-exome sequencing for 10 patients with drug-resistant epilepsy or epileptic or developmental encephalopathy provided a genetic diagnosis for two patients (20%). This study confirmed that, for epileptic encephalopathies and movement disorders in which the genetic causes and phenotypes are heterogeneous and sometimes treatable, WES is a useful tool for diagnostics and in the search for novel aetiologies, which might turn out to be more common than expected. / Tiivistelmä Uusien sekvensointimenetelmien käyttöönotto on mahdollistanut epileptisten enkefalopatioiden ja liikehäiriöiden uusien geneettisten syiden löytymisen. Näissä sairausryhmissä geenien ja ilmiasujen vaihtelevuus on suurta. Tutkimuksen tarkoituksena oli löytää uusia geneettisiä syitä ja ilmiasuja lapsuusiällä alkavissa vaikeahoitoisissa epilepsioissa ja epileptisissä tai kehityksellisissä joko itsenäisesti tai yhdessä liikehäiriön kanssa esiintyvissä enkefalopatioissa sekä perheittäin esiintyvissä liikehäiriöissä. Lisäksi selvitettiin eksomisekvensoinnin käyttökelpoisuutta kliinisessä diagnostiikassa näiden potilasryhmien kohdalla. Tutkimukseen osallistui yhteensä 12 sisäänottokriteerit täyttävää lasta, joiden sairauden syy oli jäänyt tuntemattomaksi. GABRG2-geenin mutaatiot aiheuttivat epileptisiä enkefalopatioita, joiden uutena ilmiasuna oli etenevä taudinkuva, johon liittyivät aivojen rappeutuminen, migroiva imeväisiän paikallisalkuinen epilepsia sekä autismikirjon häiriö. Tutkimuksessa löydettiin uusia GABRG2-mutaatioita: p.P282T ja p.S306F. NACC1-geenin mutaatio aiheutti epilepsian, kehitysvammaisuuden, molemminpuolisen kaihin ja autonomisen hermoston toiminnan häiriön. Hyperkineettinen liikehäiriö oli uusi NACC1 p.R298W -mutaatioon liittyvä ilmiasu. SAMD9L-geenin mutaatio aiheutti perheessä esiintyvän liikehäiriön. Neurologinen ja hematologinen ilmiasu olivat hyvin vaihtelevia. Aivojen kuvantamislöydöksiin sisältyi pikkuaivojen rappeutumista ja valkoisen aivoaineen muutoksia aivokammioiden ympärillä. Näiden tutkimustulosten julkaisemisen jälkeen SAMD9L-geenin mutaatioiden on todettu olevan yksi yleisimmistä perinnöllisistä luuytimen vajaatoiminnan ja myelodysplasian syistä. Homotsygoottinen MTR-geenin mutaatio aiheutti varhain alkaneen epileptisen enkefalopatian, liikehäiriön ja hematologisen häiriön. Kofaktori- ja vitamiini hoidot vähensivät epileptisiä kohtauksia, joihin tavanomainen lääkitys ei tehonnut. Geneettiset syyt ja ilmiasut ovat epileptisissä enkefalopatioissa ja liikehäiriöissä hyvin vaihtelevia, ja osaan on olemassa spesifi hoito. Eksomisekvensointi on käyttökelpoinen diagnostiikan ja uusien geneettisten syiden etsimisen apuna. Tässä tutkimuksessa eksomisekvensoinnin avulla kymmenestä potilaasta kahdelle (20%) saatiin varmistettua geneettinen diagnoosi.

Autism spectrum disorder (ASD) is a neurodevelopment disorder characterized by impairment in communication skills, behavior and social interactions that affects around 1-2% worldwide. To date the etiology of ASD has not yet been fully understood, but in the last 18 years many advances have been made to understand the genetic component related to the development of the clinical phenotype. With the advent of genomic scan analysis such as chromosome analysis by microarray and whole exome sequencing (WHE) many advances have been made to understand the pathophysiology of the disease. About 10-15% of the cases can be explained by large losses or gains (deletions or duplications greater than 1000 base pairs) of the genetic material, which generally involve the disruption of one or more genes. Next generation sequencing methodologies were fundamental in the description of point mutations and small insertions and deletions associated with ASD. The WES has allowed many discoveries to be made about new candidate genes and mechanisms for the development of the disease. It is now claimed that de novo (non-inherited) and likely gene disruptive mutations, such as loss-of-function and non-synonymous changes with high prediction of damage by computational tools, in genes related to neurodevelopment are a major contributor to the disease mechanism. However, these mutations, in addition to not explaining the majority of cases, are rarely recurrent in the population, which makes it difficult to establish a definitive molecular diagnosis for most patients. WES is already a practice in clinical genetics laboratories and demonstrates high effectiveness for diseases that follow a Mendelian pattern of inheritance, and have an established genetic cause. In clinical practice WES is requested for cases of ASD, despite having different modes of inheritance and having more than 1,000 genes associated with the disease. Due to these characteristics the analysis of WES for ASD is a major challenge for the clinical laboratory. This study proposes the construction of a computerized WES analysis routine that can test different candidate genes for their sensitivity and specificity for the detection of affected individuals. The proposed approach consists in the counting of variants separated by their possible protein damage and population frequency for each individual from affected and control groups, this study analyzed 168 WES, being 49 with ASD and 119 controls. After counting formulation, these values are subjected to a sequence of statistical tests, seeking a significant difference in the amount of mutations of all the variants alone, loss-of-function or damaging missense mutations, and the application of models of multivariate analysis such as: logistic regression, decision tree, neural network, vector support machine and principal component analysis for the elaboration of more complex models for disease development. A total of 21 lists of genes were tested, of which 19 presented at least one significant result, and the analysis of variants alone was the one that obtained the largest number of significant events. From apparently protective variants (higher number in the control group), such as the missense variants in RAS/MAPK pathway as variants of stopgain with population frequency above 0.05 in chromatin genes in greater number in individuals with ASD. None of the multivariate analysis models had significant discrimination results between the two groups. Due to the small sample size, the results of this study should be interpreted with limitations, and it is necessary to replicate these scenarios in other databases. However, these findings suggest that different types and frequencies of variants may have distinct contributions to disease development depending on the genes analyzed, rather than complex relationships between variants of the same gene list / O transtorno do espectro autista (TEA) é um distúrbio do neurodesenvolvimento caracterizado por uma incapacidade de comunicação comportamento e interações sociais que afeta em torno de 1-2% da população mundial. Até o momento a etiologia do TEA ainda não é totalmente compreendida, mas nos últimos 18 anos muitos avanços foram feitos para entender o componente genético relacionado ao desenvolvimento do quadro clínico. Com o advento das análises de varredura genômica como a análise cromossômica por microarray e o sequenciamento completo de exoma (SCE) muitos avanços foram feitos para a compreensão da fisiopatologia da doença. Em torno de 10-15% dos casos podem ser explicados por grandes perdas ou ganhos (deleções ou duplicações superiores a 1000 pares de bases) do material genético, que geralmente envolvem a disrupção de um ou mais genes. As metodologias de sequenciamento de nova geração foram fundamentais para a descrição das mutações de ponto e pequenas inserções e deleções associadas ao TEA. O SCE permitiu que muitas descobertas fossem feitas sobre novos genes candidatos e mecanismos para o desenvolvimento da doença. Atualmente afirma-se que as alterações de novo (não herdadas) e de maior probabilidade de ruptura gênica, como as mutações de perda-de-função e as alterações não-sinônimas com alta predição de dano por ferramentas computacionais, em genes de susceptibilidade a doenças do neurodesenvolvimento sejam um grande contribuidor para o mecanismo da doença. Entretanto essas mutações, além de não explicar a totalidade dos casos raramente são recorrentes na população, o que dificulta o estabelecimento de um diagnóstico molecular definitivo para a maioria dos pacientes. O SCE já é uma prática nos laboratórios clínicos de genética e demonstra uma alta efetividade para as doenças que seguem um padrão de herança mendeliano, e têm uma causa genética estabelecida. Na prática clínica o SCE é solicitado para os casos de TEA, apesar de ter diferentes modos de herança e terem mais de 1,000 genes associados à doença. Devido a estas características o SCE para os casos de TEA são um grande desafio para o laboratório clínico. Este estudo propõem a construção de uma rotina computacional de análise do SCE que possa testar diferentes genes candidatos quanto à sua sensibilidade e especificidade para a detecção dos indivíduos afetados. A abordagem proposta é a contagem de variantes separadas por seu possível dano à proteína e frequência populacional para cada indivíduo de grupos afetado e controle em 168 indivíduos com SCE, sendo 49 com TEA e 119 controles. Após a formulação da contagem esses valores são submetidos a uma sequência de testes estatísticos, buscando diferença significativa em quantidade de mutações de todas as variantes isoladamente, das mutações de perda-de-função, ou não-sinônimas danosas como um conjunto e a aplicação de modelos de análise multivariada como: regressão logística, árvore de decisão, rede neural, máquinas de suporte de vetor e análise de componente principal para a elaboração de modelos mais complexos para o desenvolvimento na doença. Ao todo foram testadas 21 listas de genes, destas, 19 apresentaram ao menos um resultado significativo, sendo a análise de variantes isoladamente a que obteve maior número de eventos significativos. Desde variantes aparentemente protetoras (maior número no grupo controle), como as variantes não-sinônimas em via de RAS/MAPK quanto variantes de perda de códon de parada com frequência populacional acima de 0.05 em genes de cromatina em maior número nos indivíduos com TEA. Nenhum dos modelos de análise multivariada obteve resultados significativos na discriminação entre os dois grupos. Devido ao pequeno número amostral os resultados deste estudo devem ser interpretados com limitações, sendo necessária a replicação deste cenário em outros bancos de dados. Entretanto, estes achados sugerem que diferentes tipos e frequências de variantes podem ter contribuições distintas para o desenvolvimento da doença a depender dos genes analisados, mais de que relações complexas entre as variantes de uma mesma lista de genes

As fissuras orofaciais, ou fissuras labiopalatinas, são malformações prevalentes na população mundial, presente em cerca de um a cada 700 nascimentos. Dentro das fissuras orofaciais, um grupo etiologicamente distinto é composto pelas fissuras de lábio com ou sem fissura de palato, que, em 70% dos casos, não estão associadas a nenhuma comorbidade (fissuras de lábio com ou sem palato não sindrômicas, FL/P NS). A etiologia das FL/P NS é complexa, e em muitos casos apresenta herança multifatorial. A contribuição genética para as FL/P NS, embora sabidamente relevante, ainda é pouco conhecida. Ainda, os loci de suscetibilidade consistentemente associados às FL/P NS, não conferem um risco que explique a herdabilidade total da doença. O objetivo do presente trabalho foi investigar, por meio de diferentes estratégias, variantes de risco às FL/P NS. Utilizando sequenciamento de exoma em casos familiais, verificamos que o gene codificante da caderina epitelial, CDH1, contribui importantemente com variantes raras de efeito moderado a alto na etiologia das FL/Ps. Além disso, propusemos que também podem ter relevância etiológica genes envolvidos na via de polaridade planar celular, transição epitélio-mesênquima, adesão celular, regulação de ciclo celular ou de interação com microtúbulos. Por meio de um estudo de associação com correção para estratificação populacional, caracterizamos o intervalo de associação da região 8q24, o principal locus de suscetibilidade às FL/P, e identificamos associação significativa também para a região 20q12. Por fim, combinando o estudo de associação com mapeamento de eQTLs, encontramos pela primeira vez a associação entre marcadores na região 2p13, que regulam MRPL53, em FL/P NS. Em conclusão, este trabalho contribui para o melhor entendimento da relevância de variantes raras, de efeito moderado a alto, e comuns, de efeito pequeno, na etiologia das FL/P NS / Orofacial clefts (or cleft lip/palate) are congenital malformations with high prevalence in population (∼1:700 births). Among the orofacial cleft types, an etiologically distinct group is composed by cleft lip with or without cleft palate, which, in 70% of cases, is not accompanied by other malformations (nonsyndromic cleft lip with or without cleft palate, NSCL/P). NSCL/P presents complex etiology, often with multifactorial inheritance. Although important, the genetic contribution to NSCL/P is still poorly comprehended, and the susceptibility loci that have been associated with NSCL/P do not explain the totality of the disease\'s heritability. In light of this, our aim was to investigate risk variants for NSCL/P by means of different strategies. With exome sequencing for NSCL/P familial cases, we report that the epithelial cadherin-encoding gene contributes with rare, moderate-to-high risk variants to NSCL/P etiology. In addition, we suggest an etiological contribution of genes laying in planar cell polarity pathway, or involved with epithelial-mesenchymal transition, cell adhesion, cell cycle regulation, and interaction with microtubules. Using structured association approach, we narrowed the associated interval of 8q24 region in a Brazilian population, and also validated the association for 20q12. Finally, by combining association analysis with eQTL mapping, we found association of regulatory variants of MRPL53, in 2p13, with NSCL/P. In conclusion, this study contributes with a deeper comprehension of the etiological role of rare and common variants for NSCL/P

L’infertilité est considérée actuellement par l’organisation mondiale de la santé (OMS) comme une préoccupation majeure de santé affectant plus de 50 millions de couples dans le monde. Dans les pays occidentaux, la majorité des couples infertiles ont recours aux techniques d’assistance médicale à la procréation (AMP) pour obtenir une grossesse. Malgré le succès de ces techniques, près de la moitié des couples qui ont recours à l’AMP sortent du parcours de soin sans enfant. Une partie de ces échecs est expliquée par l’altération de la gamétogenèse. Chez l’homme, la spermatogenèse fait interagir des centaines de gènes spécifiquement exprimés dans le testicule. L’abondance de ces gènes suggère que les troubles de la spermatogenèse présentent une forte composante génétique. Récemment, les avancées techniques ont favorisé l’identification de gènes responsables de ces anomalies mais la grande majorité des cas d’infertilité masculine reste classée comme idiopathique. L’objectif de la thèse est d’identifier de nouvelles causes génétiques responsables d’infertilité masculine et d’élucider les mécanismes physiopathologiques associés à ces anomalies.Au cours de ma thèse j’ai participé avec l’équipe GETI (génétique, épigénétique et thérapies de l’infertilité) à l’exploration génétique et moléculaire de deux phénotypes distincts d’anomalies spermatiques liés à des défauts post-méiotiques de la spermatogenèse : une forme rare d’azoospermie non obstructive (ANO) et le phénotype d’anomalies morphologiques multiples du flagelle spermatique (AMMF). Enfin j’ai joué un rôle important dans la création et l’analyse de modèles murins pour caractériser la pathogénie de ces anomalies.L’analyse génétique de deux frères infertiles nés de parents consanguins et présentant une ANO idiopathique associée à un arrêt post-méiotique de la spermatogenèse nous a permis d’identifier un variant homozygote délétère dans le gène SPINK2 qui code pour un inhibiteur de sérine-protéases. L’étude des souris KO pour ce gène nous a permis d’observer que les souris mâles adultes sont infertiles et miment parfaitement les phénotypes spermatique et testiculaire observés chez nos patients. Nous avons montré que la protéine codée par ce gène est exprimée dans l’acrosome à partir du stade de spermatide ronde. En l’absence de Spink2, l’activité protéolytique non-neutralisée des protéases cibles qui transitent par le Golgi cause sa fragmentation et bloque la spermiogénèse au stade de spermatide ronde. Nous avons également pu observer que les spermatozoïdes provenant de patients et de souris hétérozygotes présentent un taux élevé d’anomalies morphologiques et une baisse de la mobilité progressive conduisant à une hypofertilité à expressivité variable. Ces résultats montrent pour la première fois que l’oligo-tératozoospermie et l’azoospermie peuvent constituer un continuum pathologique dû à une même pathogénie.Nous avons également réalisé le séquençage exomique complet d’une cohorte de 78 individus AMMF non apparentés et avons identifié chez 49 sujets des mutations bi-alléliques délétères dans 11 gènes candidats dont DNAH1, CFAP43, CFAP44, WDR66 et FSIP2, soit un rendement diagnostique de 63%. Ces résultats confirment l’hétérogénéité génétique du phénotype MMAF et l’efficacité diagnostique du séquençage haut débit dans son exploration. Nous avons également validé l’implication de certains gènes candidats (n=4) dans ce phénotype chez le modèle murin knock-out créé par la nouvelle technologie d’édition du génome, CRISPR/Cas9.Dans son ensemble, ce travail montre l’intérêt et l’efficacité de la combinaison du séquençage exomique et de la technique de CRISPR/Cas9 pour étudier les troubles de la spermatogenèse et l’infertilité masculine. / Infertility is currently considered by the World Health Organization (WHO) as a major health concern affecting more than 50 million couples worldwide. In western countries, the majority of infertile couples seek assisted reproductive technologies (ART) to achieve a pregnancy. Despite the success of these techniques, almost half of these couples fail to obtain a child. Part of these failures are explained by the alteration of gametogenesis. In humans, spermatogenesis involves hundreds of genes specifically expressed in the testis. The abundance of these genes suggests that spermatogenic defects are associated with a strong genetic component. Recently, technical advances have led to the identification of numerous causative genes, but the vast majority of male infertility cases remain idiopathic. The aim of the present thesis is to identify new genetic causes responsible for male infertility and to elucidate the physiopathological mechanisms associated with these anomalies.During my thesis, I participated with the team GETI (genetics, epigenetics and therapies of infertility) in the genetic exploration of two phenotypes of male infertility related to post-meiotic defects of spermatogenesis: a rare form of non-obstructive azoospermia and the phenotype of multiple morphological abnormalities of the sperm flagella (MMAF). I have also played a key role in creation and analysis of transgenic mice to better characterize the pathogeny of the identified genetic causes in Human.Genetic analyses performed on two infertile brothers born form consanguineous parents and presenting an-idiopathic non-obstructive azoospermia associated with a post-meiotic arrest of spermatogenesis allowed us to identify a homozygous variant in the SPINK2 gene that encodes a serine-protease inhibitor. Phenotypic analysis of Spink2-/- adult male mice showed that they are infertile and perfectly mimic the sperm and testicular phenotypes observed in our patients. We showed that Spink2 protein is expressed from the round spermatid stage and localized in the acrosome, a lysosomal-like vesicle rich in proteases that play a key role during fertilization. When Spink2 is absent, the deregulated proteolytic activity of the targeted proteases such as acrosin leads to the fragmentation of the Golgi apparatus and arrest of spermiogenesis at the round spermatid stage. We also showed that sperm from heterozygous human and mice present a high level of morphological abnormalities and a decrease of progressive motility leading to a variable subfertility. These results showed for the first time that oligo-teratozoospermia and azoospermia could present a pathological continuum due to the same pathogeny.We also performed exome sequencing in a cohort of 78 non related MMAF subjects and identified in 49 cases deleterious bi-allelic mutations in a total of 11 candidate genes including DNAH1, CFAP43, CFAP44, WDR66 and FSIP2 giving a genetic diagnosis yield of 63%. These results confirm the genetic heterogeneity of MMAF and the efficiency of high throughput sequencing in genetic exploration of this phenotype. We also demonstrated the pathogenic implication of certain candidate genes (n=4) using knock-out mice created by the new technology of genome editing, CRISPR/Cas9.Overall, this work demonstrates the interest and effectiveness of combining exome sequencing and CRISPR/Cas9 system to study spermatogenesis disorders and male infertility.

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes. The aim of the study was to describe genetic derangements in endocrine tumors. Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors. Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of  benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine. Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior. Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis. Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.

Ces dix dernières années, l’investigation des maladies génétiques a été bouleversée par l’émergence des techniques de séquençage haut-débit. Celles-ci permettent désormais de ne plus séquencer les gènes un par un, mais d’avoir accès à l’intégralité de la séquence génomique ou transcriptomique d’un individu. La difficulté devient alors d’identifier les variants causaux parmi une multitude d’artefacts techniques et de variants bénins, pour ensuite comprendre la physiopathologie des gènes identifiés.L’application du séquençage haut débit est particulièrement prometteuse dans le champ de la génétique de l’infertilité masculine car il s’agit d’une pathologie dont l’étiologie est souvent génétique, qui est génétiquement très hétérogène et pour laquelle peu de gènes ont été identifiés. Mon travail de thèse est donc centré sur la l’infertilité et comporte deux parties majeures : l’analyse des données issues du séquençage haut débit d’homme infertiles et de modèles animaux et la caractérisation moléculaire d’un phénotype spécifique d’infertilité, laglobozoospermie.Le nombre de variants identifiés dans le cadre d’un séquençage exomique pouvant s’élever à plusieurs dizaines de milliers, l’utilisation d’un outil informatique performant est indispensable. Pour arriver à une liste de variants suffisamment restreinte pour pouvoir être interprétée, plusieurs traitements sont nécessaires. Ainsi, j’ai développé un pipeline d’analyse de données issues de séquençage haut-débit effectuant de manière successive l’intégralité des étapes de l’analyse bio-informatique, c’est-à-dire l’alignement des reads sur un génome de référence, l’appel des génotypes, l’annotation des variants obtenus ainsi que le filtrage de ceux considérés comme non pertinents dans le contexte de l’analyse. L’ensemble de ces étapes étant interdépendantes,les réaliser au sein du même pipeline permet de mieux les calibrer pour ainsi réduire le nombre d’erreurs générées. Ce pipeline a été utilisé dans cinq études au sein du laboratoire, et a permis l’identification de variants impactant des gènes candidats prometteurs pouvant expliquer le phénotype d’infertilité des patients.L’ensemble des variants retenus ont ensuite pu être validés expérimentalement.J’ai également pris part aux investigations génétiques et moléculaires permettant la caractérisation du gène DPY19L2, identifié au laboratoire et dont la délétion homozygote entraine une globozoospermie, caractériséepar la présence dans l’éjaculât de spermatozoïdes à tête ronde dépourvus d’acrosome. Pour cela, j’ai contribué à caractériser les mécanismes responsables de cette délétion récurrente, puis, en utilisant le modèle murin Dpy19l2 knock out (KO) mimant le phénotype humain, j’ai réalisé une étude comparative des transcriptomes testiculaires de souris sauvages et de souris KO Dpy19l2-/-. Cette étude a ainsi permis de mettre en évidence la dérégulation de 76 gènes chez la souris KO. Parmi ceux-ci, 23 sont impliqués dans la liaison d’acides nucléiques et de protéines, pouvant ainsi expliquer les défauts d’ancrage de l’acrosome au noyau chez les spermatozoïdes globozoocéphales.Mon travail a donc permis de mieux comprendre la globozoospermie et de développer un pipeline d’analyse bioinformatique qui a déjà permis l’identification de plus de 15 gènes de la gamétogenèse humaine impliqués dans différents phénotypes d’infertilité. / In the last decade, the investigations of genetic diseases have been revolutionized by the rise of high throughput sequencing (HTS). Thanks to these new techniques it is now possible to analyze the totality of the coding sequences of an individual (exome sequencing) or even the sequences of his entire genome or transcriptome.The understanding of a pathology and of the genes associated with it now depends on our ability to identify causal variants within a plethora of technical artifact and benign variants.HTS is expected to be particularly useful in the field infertility as this pathology is expected to be highly genetically heterogeneous and only a few genes have so far been associated with it. My thesis focuses on male infertility and is divided into two main parts: HTS data analysis of infertile men and the molecular characterization of a specific phenotype, globozoospermia.Several thousands of distinct variants can be identified in a single exome, thereby using effective informatics is essential in order to obtain a short and actionable list of variants. It is for this purpose that I developed a HTS data analysis pipeline performing successively all bioinformatics analysis steps: 1) reads mapping along a reference genome, 2) genotype calling, 3) variant annotation and 4) the filtering of the variants considered as non-relevant for the analysis. Performing all these independent steps within a single pipeline is a good way to calibrate them and therefore to reduce the number of erroneous calls. This pipeline has been used in five studies and allowed the identification of variants impacting candidate genes that may explain the patients’ infertility phenotype. All these variants have been experimentally validated using Sanger sequencing.I also took part in the genetic and molecular investigations which permitted to demonstrate that the absence of the DPY192 gene induces male infertility due to globozoospermia, the presence in the ejaculate of only round-headed and acrosomeless spermatozoa. Most patients with globozoospermia have a homozygous deletion of the whole gene. I contributed to the characterization of the mechanisms responsible for this recurrent deletion, then, using Dpy19l2 knockout (KO) mice, I realized the comparative study of testicular transcriptome of wild type and Dpy19l2 -/- KO mice. This study highlighted a dysregulation of 76 genes in KO mice. Among them, 23 are involved in nucleic acid and protein binding, which may explain acrosome anchoring defaults observed in the sperm of globozoospermic patients.My work allowed a better understanding of globozoospermia and the development of a HTS data analysis pipeline. The latter allowed the identification of more than 15 human gametogenesis genes involved in different infertility phenotypes.

As fissuras orofaciais, ou fissuras labiopalatinas, são malformações prevalentes na população mundial, presente em cerca de um a cada 700 nascimentos. Dentro das fissuras orofaciais, um grupo etiologicamente distinto é composto pelas fissuras de lábio com ou sem fissura de palato, que, em 70% dos casos, não estão associadas a nenhuma comorbidade (fissuras de lábio com ou sem palato não sindrômicas, FL/P NS). A etiologia das FL/P NS é complexa, e em muitos casos apresenta herança multifatorial. A contribuição genética para as FL/P NS, embora sabidamente relevante, ainda é pouco conhecida. Ainda, os loci de suscetibilidade consistentemente associados às FL/P NS, não conferem um risco que explique a herdabilidade total da doença. O objetivo do presente trabalho foi investigar, por meio de diferentes estratégias, variantes de risco às FL/P NS. Utilizando sequenciamento de exoma em casos familiais, verificamos que o gene codificante da caderina epitelial, CDH1, contribui importantemente com variantes raras de efeito moderado a alto na etiologia das FL/Ps. Além disso, propusemos que também podem ter relevância etiológica genes envolvidos na via de polaridade planar celular, transição epitélio-mesênquima, adesão celular, regulação de ciclo celular ou de interação com microtúbulos. Por meio de um estudo de associação com correção para estratificação populacional, caracterizamos o intervalo de associação da região 8q24, o principal locus de suscetibilidade às FL/P, e identificamos associação significativa também para a região 20q12. Por fim, combinando o estudo de associação com mapeamento de eQTLs, encontramos pela primeira vez a associação entre marcadores na região 2p13, que regulam MRPL53, em FL/P NS. Em conclusão, este trabalho contribui para o melhor entendimento da relevância de variantes raras, de efeito moderado a alto, e comuns, de efeito pequeno, na etiologia das FL/P NS / Orofacial clefts (or cleft lip/palate) are congenital malformations with high prevalence in population (∼1:700 births). Among the orofacial cleft types, an etiologically distinct group is composed by cleft lip with or without cleft palate, which, in 70% of cases, is not accompanied by other malformations (nonsyndromic cleft lip with or without cleft palate, NSCL/P). NSCL/P presents complex etiology, often with multifactorial inheritance. Although important, the genetic contribution to NSCL/P is still poorly comprehended, and the susceptibility loci that have been associated with NSCL/P do not explain the totality of the disease\'s heritability. In light of this, our aim was to investigate risk variants for NSCL/P by means of different strategies. With exome sequencing for NSCL/P familial cases, we report that the epithelial cadherin-encoding gene contributes with rare, moderate-to-high risk variants to NSCL/P etiology. In addition, we suggest an etiological contribution of genes laying in planar cell polarity pathway, or involved with epithelial-mesenchymal transition, cell adhesion, cell cycle regulation, and interaction with microtubules. Using structured association approach, we narrowed the associated interval of 8q24 region in a Brazilian population, and also validated the association for 20q12. Finally, by combining association analysis with eQTL mapping, we found association of regulatory variants of MRPL53, in 2p13, with NSCL/P. In conclusion, this study contributes with a deeper comprehension of the etiological role of rare and common variants for NSCL/P

Parmi les couples avec un projet parental, le facteur masculin d’infertilité est responsable d’environ 20%. Malgré de longues années d’activités d’assistance médicale à la procréation, un nombre important de cas reste idiopathiques. Considérant le nombre élevé des gènes potentiellement impliqués dans la gamétogenèse, il est fort probable que la majorité des formes ‘idiopathiques’ sont d’origine génétique. Dans l'étude présente, nous avons d’identifier deux nouveaux gènes impliqués dans une infertilité masculine. Nos données suggèrent que la mutation dans TEX15 puisse corréler avec une diminution du nombre de spermatozoïdes au fil du temps. Un test diagnostique identifiant la mutation chez un patient pourrait fournir une indication d’organiser au plus tôt une cryopréservation du sperme. On a aussi identifié MAGEB4 liées à l’X comme un nouveau gène impliqué dans une infertilité masculine héritée. Cette étude fournit le premier indice sur la fonction physiologique d'une protéine MAGE. / Among couples with a desire for a child, male factor is responsible approximately 20%. Despite long years of assisted reproductive activities, a significant number of cases remain idiopathic. Considering the high predicted number of genes involved in male gametogenesis, it is likely that most ‘idiopathic’ forms may have a genetic origin. In the present study, we have defined two new genes implicated in male infertility. Our data suggested that a nonsense mutation in TEX15 correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age. We also identified MAGEB4 as a new X-linked gene involved in an inherited male infertility. This study provides the first clue on the physiological function of a MAGE protein.

Au cours de la dernière décennie, la recherche biomédicale et la pratique médicale ont été révolutionné par l'ère post-génomique et l'émergence des « Big Data » en biologie. Il existe toutefois, le cas particulier des maladies rares caractérisées par la rareté, allant de l’effectif des patients jusqu'aux connaissances sur le domaine. Néanmoins, les maladies rares représentent un réel intérêt, car les connaissances fondamentales accumulées en temps que modèle d'études et les solutions thérapeutique qui en découlent peuvent également bénéficier à des maladies plus communes. Cette thèse porte sur le développement de nouvelles solutions bioinformatiques, intégrant des données Big Data et des approches guidées par la connaissance pour améliorer l'étude des maladies rares. En particulier, mon travail a permis (i) la création de PubAthena, un outil de criblage de la littérature pour la recommandation de nouvelles publications pertinentes, (ii) le développement d'un outil pour l'analyse de données exomique, VarScrut, qui combine des connaissance multiniveaux pour améliorer le taux de résolution. / Over the last decade, biomedical research and medical practice have been revolutionized by the post-genomic era and the emergence of Big Data in biology. The field of rare diseases, are characterized by scarcity from the patient to the domain knowledge. Nevertheless, rare diseases represent a real interest as the fundamental knowledge accumulated as well as the developed therapeutic solutions can also benefit to common underlying disorders. This thesis focuses on the development of new bioinformatics solutions, integrating Big Data and Big Data associated approaches to improve the study of rare diseases. In particular, my work resulted in (i) the creation of PubAthena, a tool for the recommendation of relevant literature updates, (ii) the development of a tool for the analysis of exome datasets, VarScrut, which combines multi-level knowledge to improve the resolution rate.

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.