Dissertations / Theses on the topic 'No mutation identified'

Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder characterized by development of hamartomas, intellectual disability, seizures and autism. TSC is caused by inactivating mutations in either the TSC1 or the TSC2 genes. A pathogenic variant is not identified in up to 10% of the patients with a clinical diagnosis of TSC despite full molecular assessment. These individuals are referred to as NMI (No Mutation Identified), and it is not clear if they should be monitored and/or treated in similar fashion to those with known etiology of TSC. To identify the genetic cause of TSC in these patients, we selected ten individuals with a definite clinical diagnosis of TSC and NMI and performed a pilot study. Three different technologies were used and their results were compared: chromosomal microarray, trio whole genome sequencing, and targeted deep-coverage next generation sequencing of TSC1 and TSC2. We identified mosaic variants in TSC1/TSC2 in six patients. No variants in other genes were detected in the remaining individuals. Based on the results of the pilot study and on recent literature, we then performed targeted deep-coverage TSC1/TSC2 sequencing on 200 affected individuals using peripheral blood DNA and saliva or other tissue samples where available. We identified 24 patients with mosaic pathogenic variants in TSC1 (n=2) or TSC2 (n=22), defining a rate of mosaicism of 12%. Mosaic variant allele frequency (VAF) ranged from 2% to 32% in blood and from 2% to 35% in saliva. Most affected individuals had low-level mosaicism (VAF ≤10%). We performed an extensive analysis of the phenotype, and show that individuals with a mosaic variant in TSC1/TSC2 often display normal cognitive functioning, although other TSC-associated neuropsychiatric disorders (TAND) are seen in 62% of the patients. Cortical tubers are invariably present and seizures are diagnosed in 54% of the cohort, but infantile spasms are rare. The number of cutaneous manifestations in these patients is often insufficient to meet diagnostic criteria, except for facial angiofibromas. We observed a high frequency of pulmonary and renal manifestations in our mosaic cohort, which are as severe as those seen in the general TSC population. None of the patients who have reproduced transmitted the variant to their offspring. In conclusion, our study shows that genome sequencing fails to identify rare variants in new genes related to TSC, and a third gene is therefore unlikely to exist. We demonstrated that at least one out of 10 patients with a clinical diagnosis of TSC carries a mosaic pathogenic variant in TSC1 or TSC2. We also showed that individuals with mosaic variants have a distinctive phenotypic severity, with important implications for surveillance.

L'annotation fonctionnelle de mutations somatiques est un point focal des études de génomique du cancer. Jusque récemment, la recherche s'est concentré sur des mutations dans la fraction codante du génome, pour lesquelles de puissants outils bioinformatiques ont été développés afin de distinguer des mutations délétères des mutations neutres. On identifie un nombre croissant de variants associés à des maladies dans le génome non-codant. L'interprétation des mutations non-codantes dans le cancer est donc devenue une tâche urgente. Des projets de grande envergure tels que ENCODE ont rendu possible l'interprétation fonctionnelle de variants dans les cancers. Plusieurs programmes ont été produits sur la base de ces informations fonctionnelles. Ces outilssont encore limités, notamment, une bas précision de la prédiction, le manque d'information de la mutation de cancer et biais de constatation importante. Dans le chapitre 2 de cette thèse, pour interpréter fonctionnellement les mutations non-codantes dans les cancers, nous avons développé deux modèles de forêts aléatoires indépendants, appelées SNP et SOM. Compte tenu de la combinaison de caractéristiques fonctionnelles à une position donnée du génome, le modèle SNP prédit la fraction de SNP rares (une mesure de la sélection négative), et le modèle SOM prédit la densité de mutations somatiques attendue à cette position. Nous avons appliqué nos deux modèles pour évaluer des clinvariant and HGMD variants asociés à des maladies, et un ensemble de SNP-contrôle aléatoires. Les résultats ont montré que les variants associés à des maladies ont des scores plus élevés que les SNP-contrôle avec le modèle SNP et inférieures avec le modèle SOM, confortant notre hypothèse selon laquelle la sélection négative, telle que mesurée par fraction de SNP rares et de densité de mutation somatiques, nous informe sur l'impact fonctionnel des mutations tumorales dans le génome non-codant. Jusqu'à présent, les chercheurs ont surtout considéré les gènes protéiques comme critiques dans l'initiation et la progression des cancers. Toutefois, des preuves récentes ont montré que les ARN non-codants, en particulier les lncRNAs, sont activement impliqués dans divers processus de cancer. Un chapitre de cette thèse est consacré à cette classe de transcripts non codants. Comme pour les gènes codants, il pourrait exister un grand nombre de lncRNAs driver de cancer. Le développement d'outils bioinformatiques pour identifier et hiérarchiser les lncRNA et autres ARN non-codants est devenu un important objet de recherche en oncologie.La dernière partie de cette thèse est consacrée à la mise en œuvre de méthodes pour découvrir des éléments non-codants potentiellement driver de cancer. Nous avons d'abord appliqué trois outils tierces, CADD, funSeq2, GWAVA, ainsi que nos modèles SNP et SOM, pour évaluer l'impact des mutations non-codantes dans tout le génome. Pour chaque locus, nous calculons la moyenne des scores de tous les variants observés à l'aide de l'un des modèles, et nous prenons au hasard le même nombre de variants et calculons leur score moyen 1 million de fois pour former une distribution nulle et obtenir une P-valeur pour ce locus. Pour valider notre hypothèse et notre modèle de permutation, nous avons testé ce système sur 452 gènes codants et 61 lncRNA liés au cancer, en utilisant des données de mutation somatique de cancer du foie, cancer du poumon, CLL et mélanome. Nous avons constaté que les lncRNAs et gènes codants associés au cancer avaient des valeurs-P significativement plus faibles que l'ensemble de lncRNAs et gènes codant. Appliquer ce test de permutation à des lncRNAs avec cinq systèmes de notation différents nous a permis de prioriser les centaines de candidats potentiellement liés au cancer.Ces candidats peuvent maintenant être soumis à validation expérimentale<br>Functional annotation of somatic mutations have been a consistent hotspot of cancer genomics studies. In the past, researchers preferentially focused on mutations in the coding fraction of the genome, for which ample bioinformatics tools were developed to distinguish cancer-driver mutations from neutral ones. In recent years, as an increasing number of variants were being identified as disease-associated in the non-coding genome, interpreting non-coding cancer mutations has become an urgent task. The completion of large scale projects such as ENCODE, has made functional interpretation of cancer variants achievable, and several programs were produced based on this functional information. However, there still exists some limitations as to these prediction tools, such as low prediction accuracy, lack of cancer mutation information and significant ascertainment bias. In chapter 2 of this thesis, in order to functionally interpret non-coding mutations in cancer, we developed two independent random forest models, referred to as SNP and SOM. Given a combination of features at a given genome positions, the SNP model predicts the expected fraction of rare SNPs (a measure of negative selection), and the SOM model predicts the expected mutation density at this position. We applied our two models to score these non-coding disease-associated clinvariant and HGMD variants and a set of random control SNPs. Results showed that disease-associated variants were scored higher than control SNPs with the SNP model and lower than control SNPs with the SOM model, supporting our hypothesis that purifying selection as measured by fraction of rare SNPs and mutation density is informative for the evaluation of the functional impact of cancer mutations in the non-coding genome. In the past, researchers have preferentially considered protein-coding genes as critical to the initiation and progression of cancers. However, recent evidences have shown that ncRNAs, in particular lncRNAs, are actively implicated in various cancer processes. A chapter of this thesis is devoted to this class of non-coding transcripts. Similar to protein coding genes, there might be a large number of lncRNAs with cancer-driving functions. The development of bioinformatics tools to prioritize them has become a new focus of research for computational oncologists.The last part of this thesis is devoted to the implementation of methods for discovering potential cancer-driving non-coding elements in lncRNA and protein-coding genes. We applied three scoring tools, CADD, funSeq2, GWAVA, together with our SNP and SOM scoring systems to prioritize cancer-associated elements using a permutation-based algorithm. For each locus, we compute the average score of all observed variants using one of the models, and we randomly take the same number of variants and compute their average score 1 million times to form a null distribution and obtain a P value for this locus. To validate our hypothesis and permutation model, we tested this system on 61 cancer-related lncRNA and 452 cancer genes using somatic mutation data from liver cancer, lung cancer, CLL and melanoma. We observed that both cancer lncRNAs and protein-coding genes had significantly lower average P values than total lncRNAs and protein-coding genes in all cases. Applying the permutation test to lncRNAs with five different scoring systems enabled us to prioritize hundreds to thousands of cancer-related lncRNA candidates. These candidates can be used for future experimental validation

Mutations of the AR are implicated as one of the key components in the development of prostate cancer and its poor prognosis to treatment. As prostate cancer is the second leading cause of cancer related death in Caucasian men much interest is currently focused on understanding it mechanisms of development. At present around fifty AR prostate cancer mutations have been reported in the androgen receptor mutation database, most of which have been either incompletely or poorly characterised. This study aims to clarify some of the molecular consequences of specific prostate cancer mutations that have been mapped to the receptor ligand-binding domain (LBD). It was observed that certain mutations such as the T877A and H874Y mutations were altered in ligand responsiveness to non-androgen ligands as has been previously reported, and that the Q798E mutation was also activated to some extent with progesterone. Other mutations such as D879G and V757A appeared to have a reduced transactivation response when stimulated by androgens in cell culture experiments. Gross experimental analysis of the mutant protein structures revealed that there were no major changes in the conformation of any of the proteins compared to wildtype. While modelling analysis of the ligand binding domain DHT bound crystal structure using the Swissprot Deepview programme revealed that several of the mutations resulted in more minor structural alterations such as altered H bonding or steric clashes. Finally several mutant proteins were revealed to have an altered interaction with p160 co-activator proteins in vitro. Most significant of these were the H874Y and the K720E AR mutants, which were also shown to be enhanced to a greater extent by the presence of certain co-activators in in vivo cell culture experiments.

The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting (HRM) analysis assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA genes for variants. DNA from 96 unaffected reference individuals, 72 patients with complementation confirmed cblA, and 181 patients with elevated isolated methylmalonic acid, who could not be diagnosed using complementation analysis, were scanned by HRM. Suspected variants were confirmed using Sanger sequencing. In the cblA cohort, HRM correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (C.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants, listed above, affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D).<br>Le produit génique du MMAA est nécessaire pour le métabolisme de la cobalamine intracellulaire (Cbl). Des mutations dans ce gène conduisent à la classe de maladies cblA, caractérisé par l'acidurie méthylmalonique isolée. Nous avons été concernés que les méthodes de diagnostic de cellules somatiques peuvent manquer les patients atteints phénotypes cellulaires moins sévère. Une teste de fusion à haute résolution a été développé pour balayer rapidement les exons codantes et les régions introniques adjacentes du gène MMAA pour des variantes. Nous avons testé l'ADN à partir de 96 personnes de référence qui ne sont pas touchés, 72 patients atteints de cblA confirmé par complémentation et 181 patients présentant une élévation de l'acide méthylmalonique isolée, qui ne pouvaient pas être diagnostiquée à l'aide d'analyse de complémentation. Les variantes suspectes ont été confirmées à l'aide de séquençage Sanger. Dans la cohorte cblA, l'analyse de fusion à haute résolution a correctement identifié toutes les mutations connues antérieurement, ainsi que 22 autres variantes, dont 10 n'avaient pas été signalés précédemment. Nouveaux variantes inclus une duplication (C.551dupG, p.C187LfsX3), une délétion (c.387delC, p.Y129YfsX13), une mutation du site d'épissage (c.440-2A> G, site d'épissage), 4 mutations faux-sens (c. 748G> A, p.E520K; c.820G> A, p.G274S; c.627G> T, p.R209S; c.826A> G, p.K276E), et 3 mutations non-sens (c.960G> A, p.W320X; c.1075C> T, p.E359X; c.1084C> T, p.Q362X). Toutes les variantes faux-sens nouveaux, énumérés ci-dessus, affectent des résidus hautement conservés et sont prévus pour être endommageant. L'analyse de MMAA dans les 181 échantillons non diagnostiqués a révélé un seul changement faux-sens hétérozygote (c.821G> A, p.G274D).

Pathogenic variants in the MMAB gene (OMIM 607958) are responsible for the cblB class of cobalamin-responsive methylmalonic aciduria (MMA) (OMIM 251110). MMAB encodes cobalamin adenosyltransferase, a mitochondrial enzyme responsible for the formation of adenosylcobalamin (AdoCbl). AdoCbl subsequently functions as a cofactor for methylmalonyl-CoA mutase (MCM) during the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Somatic cells studies have been used to evaluate patient samples for cobalamin related disorders. Due to high basal levels of propionate incorporation, some patients with mild MMA biochemical phenotypes cannot be diagnosed by complementation analysis. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions for variants in the MMAB gene.Three cohorts of samples were scanned by HRMA: an unaffected reference population, 42 samples assigned to the cblB by complementation analysis and 181 patients with unresolved isolated MMA. HRMA correctly identified all of the previously reported mutations in the cblB cohort as well as seven additional variants including a novel nonsense variant (c.12C>A, p.C4X). Scanning of the unresolved MMA cohort identified six samples containing MMAB variants. Two samples, WG3948 and WG4034, contained compound heterozygous variants. They shared a c.572 G>A (p.R191Q) mutation. WG3948, the index case for this study, was found to have c.398 C>T (p.S133F) as the second mutation, and WG4034, the second patient, contained a novel variant c.394 C>T (p.C132R). Samples from four other affected patients contained a single variant. The c.572 G>A (p. R191Q) was found in both WG3546 and WG4090. WG3759 contained a c.521C>T ( p.S174L) substitution, and WG4029 contained a novel c.185 C>T (p.T62M) substitution. The identification of two patients with compound heterozygous variants in the MMAB gene suggests the existence of an infrequent but distinct atypical cblB phenotype. This subclass is characterized by levels of propionate incorporation and of AdoCbl synthesis within reference ranges, preventing diagnosis by somatic cell studies.<br>Des variantes pathogéniques dans le gène MMAB (OMIM 607958) sont responsables de la classe cblB d'acidurie méthylmalonique (AMM) respondant à la cobalamine (OMIM 251110). MMAB encode cobalamine adénosyltranférase, une enzyme mitochondriale responsable de la formation de l'adénosylcobalamine (AdoCbl). AdoCbl fonctionne par la suite en tant que cofacteur pour méthylmalonyl-CoA mutase (MCM) durant l'isomérisation de L-méthylmalonyl-CoA vers succinyl-CoA. Des analyses sur des cellules somatiques ont été utilisées pour évaluer des échantillons de patients pour des troubles reliés à la cobalamine. En raison de niveaux de base élevés d'incorporation de propionate, certains patients présentant des phénotypes biochimiques bénins d'AMM ne peuvent être diagnostiqués par analyse de complémentation. Une analyse de fusion à haute résolution (AFHR) a été développée pour balayer rapidement les exons codants et les régions introniques avoisinnantes pour des variantes dans le gène MMAB.Trois cohortes d'échantillons ont été balayées par AFHR : une population de référence non-affectée, 42 échantillons assignés au groupe cblB par analyse de complémentation et 181 patients avec une AMM isolée sans diagnostique. L'AFHR a correctement identifié toutes les mutations précédemment rapportées dans la cohorte cblB ainsi que sept variantes additionelles, incluant une nouvelle variante non-sens (c.12C>A, p.C4X). Le balayage de la cohorte avec de l'AMM isolée a identifié six échantillons contenant des variantes dans MMAB. Deux échantillons, WG3948 et WG4034, étaient des porteurs de variantes hétérozygotes composés. Ils partageaient la mutation c.572G>A (p.R191Q). WG3948, le cas index pour cette étude, était porteur du c.398C>T (p.S133F) pour la deuxième mutation et WG4034, le deuxième patient, contenait une nouvel variante c.394C>T (p.C132R). Les échantillons provenant de quatre autres patients atteints contenait une seule variante. Le c.572G>A (p.R191Q) a été trouvé dans WG3546 et WG4090. WG3759 contenait une substitution c.52C>T (p.S174L), et WG4029 contenait une nouvelle substitution c.185C>T (p.T62M).L'identification de deux patients avec des variantes hétérozygotes composées dans le gène MMAB suggère l'existence d'un phénotype rare mais distinct de cblB. Cette sous-classe est charactérisée par des niveaux d'incorporation de propionate et de synthèse d'AdoCbl dans les valeurs normales, empêchant le diagnostique par analyse des cellules somatiques.

Background The automated retrieval and integration of information about protein point mutations in combination with structure, domain and interaction data from literature and databases promises to be a valuable approach to study structure-function relationships in biomedical data sets. Results We developed a rule- and regular expression-based protein point mutation retrieval pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval task on a benchmark dataset. In order to link mutations to their proteins, we utilize a named entity recognition algorithm for the identification of gene names co-occurring in the abstract, and establish links based on sequence checks. Vice versa, we could show that gene recognition improved from 77% to 91% F-measure when considering mutation information given in the text. To demonstrate practical relevance, we utilize mutation information from text to evaluate a novel solvation energy based model for the prediction of stabilizing regions in membrane proteins. For five G protein-coupled receptors we identified 35 relevant single mutations and associated phenotypes, of which none had been annotated in the UniProt or PDB database. In 71% reported phenotypes were in compliance with the model predictions, supporting a relation between mutations and stability issues in membrane proteins. Conclusion We present a reliable approach for the retrieval of protein mutations from PubMed abstracts for any set of genes or proteins of interest. We further demonstrate how amino acid substitution information from text can be utilized for protein structure stability studies on the basis of a novel energy model.

Background The automated retrieval and integration of information about protein point mutations in combination with structure, domain and interaction data from literature and databases promises to be a valuable approach to study structure-function relationships in biomedical data sets. Results We developed a rule- and regular expression-based protein point mutation retrieval pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval task on a benchmark dataset. In order to link mutations to their proteins, we utilize a named entity recognition algorithm for the identification of gene names co-occurring in the abstract, and establish links based on sequence checks. Vice versa, we could show that gene recognition improved from 77% to 91% F-measure when considering mutation information given in the text. To demonstrate practical relevance, we utilize mutation information from text to evaluate a novel solvation energy based model for the prediction of stabilizing regions in membrane proteins. For five G protein-coupled receptors we identified 35 relevant single mutations and associated phenotypes, of which none had been annotated in the UniProt or PDB database. In 71% reported phenotypes were in compliance with the model predictions, supporting a relation between mutations and stability issues in membrane proteins. Conclusion We present a reliable approach for the retrieval of protein mutations from PubMed abstracts for any set of genes or proteins of interest. We further demonstrate how amino acid substitution information from text can be utilized for protein structure stability studies on the basis of a novel energy model.

Mutations in the BRCA1 tumour suppressor gene are associated with significantly elevated risks of develop breast or ovarian carcinomas, up to 80% or 40% respectively by age 70. The anti-tumourigenic activity of the wild-type protein product is the result of participation in a number of distinct but overlapping protective pathways; DNA damage repair, transcription & splicing, cell cycle regulation, ubiquitination & regulation of apoptosis. Through modulating these cellular mechanisms, BRCA1 is able to participate in the preservation of genomic integrity. Consequently, cells lacking functional BRCA1 exhibit traits associated with genetic instability; gross chromosomal translocations, often involving multiple, non-homologous chromosomes; as well as deletions and/or amplifications of genetic material. Furthermore, BRCA1 deficient cells lack fully functional DNA repair processes and are sensitive to genotoxic agents. It has been demonstrated that BRCA1 acts principally as a scaffold protein, and mediates the formation of a number of complexes in the cell with unique functions. The BRCA1-associated factors in these complexes participate in the pathways described above, and ultimately contribute to BRCA1-mediated tumour suppression. Accordingly, it is reasonable to suggest that a significant proportion of BRCA1 function is a product of the factors with which it interacts. Protein interaction sites have been described across the entire length of the BRCA1 protein sequence, and some specific regions have been broadly associated with particular functions or pathways, for instance the C-terminus is associated with DNA damage response, whilst the N-terminus is required for ubiquitination. However, there is limited information as yet associating specific domains of the BRCA1 protein with preventing tumour formation or disease progression. In this study, we have attempted to assess the contribution of specific domains to BRCA1 function and tumour suppression through directly analysing the effect of pathogenic mutations on BRCA1 function and how phosphorylation of BRCA1 contributes to complex formation.

Les ciliopathies sont un groupe de maladies génétiques multi-systémiques liées à un dysfonctionnement du cil primaire, une structure sensorielle présente à la surface des cellules qui régule des voies de signalisation clés au cours du développement et de l'homéostasie tissulaire. Afin d'identifier de nouveaux gènes responsables de ciliopathies développementales sévères, ~ 500 patients / fétus ont été analysés par une approche de séquençage à haut débit de l'exome ciblant &gt; 1 200 gènes ciliaires ("ciliome"). Nous avons identifié huit nouvelles mutations dans le gène NEK8/NPHP9 chez cinq familles dont les syndromes se chevauchent. NEK8/NPHP9 code une protéine kinase de la famille des NIMA qui se localise au niveau du compartiment Inversine du cil primaire et agit comme un régulateur de la signalisation Hippo, une voie essentielle contrôlant la taille des organes. Nous montrons pour la première fois que les mutations du gène NEK8 sont associées à une agénésie rénale et une hypodysplasie. De plus, notre travail met en évidence une corrélation génotype/phénotype: les mutations "perte de fonction" de NEK8 conduisant à reins élargies et kystiques, des kystes pancréatiques et hépatique, alors que les mutations faux-sens de NEK8 causent une hypodysplasie/agénésie rénale associée à une cardiopathie et une paucité des canaux biliaires. La première partie de mon projet de thèse porte sur l'étude de l'impact des mutations faux-sens de NEK8 sur divers processus cellulaires et des voies de signalisation dépendantes de NEK8. Nous avons démontré un effet "gain de fonction" des mutations faux-sens de NEK8 puisqu'elles affectent la ciliogenèse et la composition du compartiment Inversine (localisation ciliaire de ANKS6). De plus, ces mutations altèrent la localisation nucléaire de YAP, le principal acteur de la voie Hippo, ainsi que l'expression des gènes cibles de YAP dans les fibroblastes de patients et dans la lignée cellulaire rénale (mIMCD3) invalidée pour NEK8. De même, nous avons montré une accumulation anormale de YAP nucléaire dans les reins polykystiques de la souris Jck, porteuse d'une mutation faux-sens de Nek8. Un déséquilibre de la voie Hippo serait donc à l'origine des défauts de morphogenèses épithéliales. En effet, les cellules mIMCD3 invalidées pour NEK8 forment en culture 3D des structures anormales et/ou des sphères élargies qui s'accompagnent d'une persistance du marquage nucléaire de YAP et Ki-67 et forment de grandes sphères par rapport aux cellules contrôles. Des défauts plus sévères ont été observés pour les cellules ré-exprimant les différents mutants de NEK8, confirmant la pathogénicité de ces mutations et leur effet "gain de fonction". Enfin, le traitement par la Vertéporfine, un inhibiteur spécifique de l'activité transcriptionnelle de YAP, améliore non seulement le phénotype des fibroblastes de patients et des cellules rénales invalidées pour NEK8 en culture 3D, mais également in vivo les anomalies observées chez les embryons de poisson zèbre dues à la surexpression de la forme NEK8 humaine, confirmant ainsi l'implication d'une dérégulation de YAP dans les mécanismes physiopathologiques. Par ailleurs, nous avons observé que les mutants de NEK8 s'accumulent de manière anormale au niveau de l'appareil de Golgi dans les fibroblastes de patients, et que cet appareil de Golgi apparait dispersé. Nos résultats montrent que le recrutement de NEK8 au Golgi est sensible à la Brefeldine A et dépendrait donc de ARF1, une petite GTPase impliquée dans le trafic de protéines entre les compartiments du Golgi et du réticulum endoplasmique. Nous avons démontré que NEK8 interagit et co-localise préférentiellement avec la forme d'ARF1 liée au GDP, suggérant pour NEK8 une possible fonction de facteur d'échange d'ARF1 à des sites spécifiques (appareil de Golgi, membranes, cil) afin de promouvoir le trafic vésiculaire de protéines telles que les protéine ciliaires. (...)<br>Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, ~500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of &gt; 1200 ciliary genes. We have identified eight novel mutations in NEK8/NPHP9 in five independent families with severe overlapping syndromic disorders. NEK8/NPHP9 encodes a NIMA-related kinase that localizes at the inversin compartment of the primary cilium and acts as a regulator of Hippo signaling, a pathway that is essential for control of organ size during development. We show for the first time that NEK8 mutations are associated with renal agenesis and hypodysplasia, and our work highlights a genotype/phenotype correlation with NEK8 loss-of-function mutations leading to enlarged cystic kidney, pancreas and liver, whereas NEK8 gain-of-function (missense) mutations cause renal hypodysplasia, cardiopathy and paucity of bile ducts. The first part of my thesis project focuses on the study of the impact of these NEK8 missense mutations on various cellular processes and NEK8-dependent signaling pathways. We demonstrate that NEK8 missense mutations impair the Inversin (INVS) compartment composition and ciliogenesis, and also alter the nuclear localization of the main Hippo signalling effector, YAP, as well as expression of its target genes in patient fibroblasts and renal cell lines. We also demonstrated that this Hippo pathway imbalance causes epithelial morphogenesis defects in 3D matrigel culture. Indeed, mIMCD3 cells depleted for NEK8 showed persistent YAP and Ki-67 staining and formed bigger spheres compared to control cells. Abnormal sphere volume was also observed in cells re-expressing NEK8-GFP mutations, suggesting their pathogenicity. We confirm these data in vivo in Jck mice, a model of polycystic kidney disease bearing a Nek8 missense mutation. Finally, treatment with Verteporfin, a specific inhibitor of YAP transcriptional activity, improves the mutant phenotype of both cellular models and zebrafish embryos overexpressing human NEK8, further supporting the involvement of YAP dysregulation in the pathogenic cellular mechanisms. Surprisingly, in patient fibroblasts, we showed that mutated NEK8 accumulates at the Golgi that appeared dispersed. NEK8 recruitment at the Golgi apparatus is dependent on ARF1 (Brefeldin A sensitive), a small GTPase involved in protein trafficking between Golgi compartments and ER. We notably demonstrated that NEK8 mostly interacts and localizes with the dominant negative form of ARF1 (T31N), suggesting that NEK8 could act as an activator (GEF) of ARF1 to promote vesicular trafficking of ciliary proteins. The second part of my project focuses on a new candidate gene for which a missense homozygous mutation has been identified in 3 individuals presenting a late onset NPH with hepatic fibrosis. This gene encodes ANKS3, an evolutionarily conserved protein whose function is still poorly characterized. Interestingly, ANKS3 has been reported to be a partner of NEK8, even though we showed it does not localize at the INVS compartment with NEK8 but is rather present at the base of cilia in fibroblasts. We showed that the missense mutation does not affect ANKS3 localization but leads to longer cilia and abnormal accumulation of NEK8 at the cilium base in patient fibroblasts and kidney tubules. Altogether, my work focused on NEK8 and its partners, ANKS6 and ANKS3, each of whose related gene is mutated in patients presenting a broad clinical spectrum of phenotypes. (...)

Thesis (Ph. D.)--University of California, San Diego, 2009.<br>Title from first page of PDF file (viewed June 16, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 142-172).

Background: In the management of metastatic colorectal cancer (mCRC), KRAS, NRAS and BRAF mutational status individualizes therapeutic options and identify a cohort of patients (pts) with an aggressive clinical course. We hypothesized that relapsed and refractory mCRC pts develop unique mutational signatures that may guide therapy, predict for a response and highlight key signaling pathways important for clinical decision making. Methods: Relapsed and refractory mCRC pts (N=32) were molecularly profiled utilizing commercially available next generation sequencing (NGS) platforms. Web-based bioinformatics tools (Reactome/Enrichr) were utilized to elucidate mutational profile linked pathways-networks that have the potential to guide therapy. Results: Pts had progressed on fluoropyrimidines, oxaliplatin, irinotecan, bevacizumab, cetuximab and/or panitumumab. Most common histology was adenocarcinoma (colon N=29; rectal N=3). Of the mutations TP53 was the most common, followed by APC, KRAS, PIK3CA, BRAF, SMAD4, SPTA1, FAT1, PDGFRA, ATM, ROS1, ALK, CDKN2A, FBXW7, TGFBR2, NOTCH1 and HER3. Pts had on average had >= 5 unique mutations. The most frequent activated signaling pathways were: HER2, fibroblast growth factor receptor (FGFR), p38 through BRAF-MEK cascade via RIT and RIN, ARMS-mediated activation of MAPK cascade, and VEGFR2. Conclusions: Dominant driver oncogene mutations do not always equate to oncogenic dependence, hence understanding pathogenic ` interactome(s)' in individual pts is key to both clinically relevant targets and in choosing the next best therapy. Mutational signatures derived from corresponding ` pathway-networks' represent a meaningful tool to (I) evaluate functional investigation in the laboratory; (II) predict response to drug therapy; and (III) guide rational drug combinations in relapsed and refractory mCRC pts.

La mucoviscidose, la maladie génétique létale la plus fréquente dans la population Caucasienne, se caractérise par une expression phénotypique variée allant d'une forme classique et complète de la mucoviscidose (CF, Cystic Fibrosis) à des formes monosymptomatiques comme l'agénésie bilatérale des canaux déférents (ABCD). Ces désordres résultent de mutations sur le gène Cystic Fibrosis Transmembrane conductance Regulator (CFTR). A ce jour, plus de 1600 alérations moléculaires ont été identifiées avec une grande majorité de mutations privées, rendant difficile la définition de leur pathogénécité d'autant plus si ces variants se situent au niveau de séquences non traduites. Seuls quelques travaux ont évalué l'effet délétère d'altérations moléculaires présentes dans ces régions peu explorées, permettant de caractériser de nouveaux éléments cis- et trans-régulateurs sur l'ensemble de ce gène. Dans le but d'élucider l'impact de certains variants rares situés dans des régions non codantes identifiés chez des patients CF ou atteints d'ABCD, nous avons caractérisé leur rôle sur l'expression du gène CFTR par une combinaisond'approches de biologie moléculaire et cellulaire. Nos résultats indiquent que des variations de séquence (-94&gt;T et -33G&gt;A) situées dans le promoteur minimal du gène CFTR sont capables de diminuer la transcription de ce gène, en pertubant la fixation de facteurs de transcription ubiquitaires (Specificity protein : Sp l et Upstream Stimulatory Factor : USF) et tissu-spécifiques (protéines Forkhead Box : FOX). Un mécanisme de régulation complexe impliquant trois facteurs dont un élément régulateur tissu-spécifique C/EBP (CAAT/enhancer binding protein) a également été mis en évidence. Par ailleurs, nous avons démontré que la diminution du nombre de répétitions d'un motif (TAAA)n présent dans l'intron 9 du gène CFTR, altère l'expression de ce gène. La caractérisation de l'effet pathogène des variants naturels identifiés dans les régions non codantes du gène CFTR ainsi que l'identification des éléments trans-régulateurs devraient permettre d'envisager de nouvelles cibles pouvant moduler plus spécifiquement l'expression du gène CFTR<br>Cystic fibrosis (CF), the most frequent lethal genetic disease in Caucasian population, is characterized by a great variability in disease presentation from a classical CF to monosymptomatic forms such as congenital bilateral absence of the vas deferens (CBAVD). More than 1,600 molecular alterations have hitherto been identified on the CFTR (Cystic Fibrosis Transmembrane conductance regulator) gene. Because the large majority of them are rare mutations, the establishing of their pathogenecity is problematic. Evaluating the effect of molecular alterations in untranslated CFTR region (UTR) is poorly documented. Some studies allowed characterizing news cis- and trans regulatory elements on the CFTR gene. To elucidate the impact of rare variants located in on-coding regions identified in either CF or CBAVD patients, we investigated their role on the CFTR gene expression by using molecular and cellular biology approaches. Our results indicate that sequence variations (-94&gt;T et -33G&gt;A) identified in the minimal promoter, repress the CFTR transcriptional activity, perturbing the binding of ubiquitous (Specificity protein : Spl et Uspstream Stimulatory Factore : USF) and tissue-specific (Forkhead Box proteins:FOX) transcription factors. A complex regulatory mechanism involving threr proteins including the tissue-specific C/EBP (CAAT/enhancer binding proteins) factor has been showed. In addition, we demonstrated that the decrease in the number of (TAAA)n motif repeat located in the CFTR into 9, alters the expression of this gene. Identification of trans-regulatory elements through the charactérization of the deleterious effect of naturally occuring variants within CFTR UTR parts may shed light on molecular mechanism controlling tissue-specific expression of the CFRT gene

Infection with human cytomegalovirus (HCMV), usually asymptomatic in healthy individuals, can cause severe or fatal disease in infants and immunocompromised patients. The generation of a potent protective vaccine is necessary to protect vulnerable people. Because of host restriction, murine cytomegalovirus (MCMV) is used as a model for HCMV. Previously, we have generated a temperature-sensitive mutant, tsm5, which failed to replicate in mice yet protected them against virus challenge. Several mutations have been identified in this mutant by Comparative Genome Sequencing (GCS) (Roche NimbleGen); 10 synonymous and 15 non-synonymous single nucleotide polymorphisms (SNPs). Among these are m139 (Y565X) and m141 (V195M), shown to be essential for replication in macrophages but not in fibroblasts, m143 (M232I), shown to play an important role in the inhibition of the PKR-mediated host antiviral response, M27 (A658S ), involved in interference with interferon- signalling, and M36 (V54I), an anti-apoptotic protein. In the present study, the above mentioned mutations were introduced individually into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. An in vitro phenotypical analysis revealed that only the double (Mt[M27\(^A\)\(^6\)\(^5\)\(^8\)\(^S\)M36\(^V\)\(^5\)\(^4\)\(^I\)]) and the m139 (Mt[m139\(^Y\)\(^5\)\(^6\)\(^5\)\(^X\)]) mutants showed a temperature sensitive phenotype in MEF and/or Raw 264.7 macrophages cells

Le facteur de transcription PU.1/SPI1 de la famille ETS est un régulateur majeur de l'hématopoïèse, et de la différenciation des cellules souches, myéloïdes et lymphoïdes. Précédemment identifié comme un suppresseur de tumeur dans les malignités myéloïdes humaines, nous avons identifié une mutation somatique récurrente faux sens (Q226E) du gène PU.1 dans la macroglobulinémie de Waldenström, un syndrome lymphoprolifératif à cellules B. La mutation affecte la liaison à l'ADN de la protéine et permet au mutant de se lier et d'activer plus fréquemment les régions promotrices par rapport à la protéine de type sauvage. La liaison du mutant aux promoteurs active la transcription de gènes généralement activés par d'autres facteurs ETS, comme Ets1, entraînant une prolifération accrue dans les modèles de lignées cellulaires et les lymphocytes B primaires de souris. Pour analyser les propriétés du mutant dans des conditions physiologiques, nous avons développé un modèle de souris portant un allèle conditionnel Pu.1 Q226E Knock-In. L'utilisation d'un transgène CD19-Cre induit l'expression de la protéine mutée dans la lignée lymphoïde B. L'analyse du développement précoce des cellules B montre une augmentation de la réponse des cellules pré-B à l'IL-7, entraînant l'amplification de cette population in vivo. La protéine mutante stimule la prolifération cellulaire des lymphocytes B et augmente la différentiation des lymphocytes B en plasmocytes (CD138+). Plus précisément, ce mutant de Pu.1 semble augmenter précocement l'expression des facteurs de transcription spécifiques des plasmocytes, comme Blimp1 et Xbp1, et augmente l'activation de la voie UPR, permettant une différenciation plus importante des cellules B matures en plasmocytes. Ces résultats décrivent un mécanisme de subversion oncogénique de la fonction d’un facteur de transcription suite à la modification subtile de la spécificité de liaison à l'ADN de la protéine, qui affecte la prolifération et la différenciation<br>The ETS transcription factor PU.1/SPI1 is a major regulator of hematopoiesis. It is implicated in HSC, myeloid but also lymphoid biology and has been described as a tumor suppressor in human myeloid malignancies. We identified a PU.1 activating missense mutation in Waldenström macroglobulinemia (Q226E), a B-cell lympho-proliferative neoplasm, highlighting new oncogenic features for this transcription factor. This mutation affects the DNA-binding affinity of the protein and allows the mutant to more frequently bind to promoter regions with respect to wild-type protein, resulting in transcriptional activation of gene sets typically regulated by other ETS factors, such as ETS1, resulting in enhanced proliferation in model cell lines and murine primary B-cells. To analyze the properties of the mutant protein in physiological conditions, I developed mouse model carrying a Pu.1 Q226E Knock-In conditional allele. The use of a CD19-Cre transgene triggers the expression of the mutated protein in the B lymphoid lineage. Analysis of the early B cells development shows an increase response of pre-B cells to IL-7, associated to the accumulation of this population in vivo. I confirmed that the mutant protein stimulates B cell proliferation and showed that it also increases the differentiation of B cells into plasma cells (CD138+). Specifically, Pu.1 mutant induce an early increase of the expression of plasma-specific transcription factors, such as Blimp1 or Xbp1, and increases activation of the UPR pathway, which likely increases differentiation of mature B cells into plasma cells. These results describe a mechanism of oncogenic subversion of the function of a transcription factor as a result of the subtle modification of the DNA binding specificity of the protein, which affects proliferation and differentiation

L'hormone de croissance (gh) exerce de multiples effets sur la croissance de l'organisme. La gh se lie a deux molecules de recepteur membranaire, le ghr, dont la dimerisation induit l'activation de la tyrosine kinase cytoplasmique jak2, initiant ainsi les voies de signalisation de la gh. Nous avons etudie deux aspects de cette signalisation, l'un par l'etude de nouveaux defauts de gene du ghr (syndrome de laron), l'autre par analyse de sequence de jak2. Le syndrome de laron est une maladie provoquee par une resistance totale a la gh due a un defaut du ghr. Trois nouvelles mutations du gene du ghr ont ete identifiees chez des patients atteints de ce syndrome : i153t, q154p et v155g. Nous les avons etudiees in vitro et avons revele deux anomalies, dont la combinaison peut expliquer la pathologie in vivo : defaut d'affinite de liaison du ghr pour la gh et defaut d'adressage du ghr a la membrane plasmique (les recepteurs mutes restent dans le reticulum endoplasmique). Par analyse de sequence de jak2, nous avons caracterise le domaine d'interaction avec le ghr, un domaine jef, dont nous avons montre que les cent premiers residus se repliaient selon la topologie du domaine ra, domaine de liaison a la gtpase ras. Ces travaux permettront de modeliser jak2 par homologie. Nous avons enfin etudie la modelisation des boucles des proteines. Ces boucles jouent un role important dans la fonction, la stabilite ou le repliement des proteines mais leur grande variabilite les rend difficiles a modeliser. Afin de comprendre leurs caracteristiques structurales, nous avons constitue une banque de boucles. Apres les avoir regroupees en familles, nous avons montre une relation sequence/structure dans les boucles que nous avons utilise pour developper un algorithme de modelisation. Par analyse statistique, nous fournissons pour chaque resultat un facteur de fiabilite qui est correle a la qualite de la prediction, proposant ainsi un outil pratique de modelisation.

He, Jun.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.<br>Includes bibliographical references (leaves 136-156).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) is in the family of structural renal tract birth defects. CAKUT is the major cause of chronic kidney disease and renal failure in children and adults <40 years of age. ROBO2 is a receptor for the SLIT2 ligand. ROBO2/SLIT2 signaling has been shown to play important roles in neuronal migration and in early renal tract development. Our laboratory has recently identified ROBO2 as a novel CAKUT-causing gene. So far, total 26 ROBO2 mutations have been identified in patients with CAKUT. However, most of these mutations are missense amino acid substitutions and their functional significances are unclear, although they are predicted to be disease-causing by several bioinformatics prediction software. OBJECTIVE: To clarify uncertainties and confusions in the CAKUT field regarding the causality for ROBO2 missense mutations, we performed functional analysis of a ROBO2 missense mutation p.G114W (c.340G>T) that was identified in a CAKUT family. This p.G114W ROBO2 mutation is located in the first Ig domain of the ROBO2 extracellular region. The ROBO2 first Ig domain is the binding site for ligand SLIT2 that is required for ROBO2-SLIT2 signaling chemorepulsive activity in neuronal migration. We hypothesize that this p.G114W ROBO2 mutation would disrupt the SLIT2-ROBO2 binding and compromise its chemorepulsive activity in a sensitive functional neuronal migration assay. METHODS: Site directed mutagenesis was used to introduce c.340G>T point mutation into a ROBO2 cDNA fusion construct that contains the first Ig domain. Point mutation was verified using Sanger sequencing. Mutant ROBO2 cDNA and wildtype control constructs were purified using Qiagen Midiprep kit and transfected in HEK cells via calcium phosphate co-precipitation. The conditioned medium (CM) containing ROBO2 fusion proteins were analyzed by Western Blot. Neuronal migration assays were performed using postnatal anterior subventricular zone (SVZa) tissue explants that were isolated from postnatal day 1 to 5 (P1-5) Sprague-Dawley rat brain. RESULTS: By Sanger sequencing, we verified the c.340G>T point mutation in the ROBO2 cDNA fusion construct. By GFP fluorescence and Western blot analysis, we found abundant expressions of ROBO2 fusion protein in the conditioned medium of transfected HEK cells. In SVZa neuronal migration assays, we found that, when compared to the wild type fusion protein, the mutant ROBO2 fusion protein with the p.G114W amino acid substitution lost its function to block SLIT2-medicated inhibition of neuronal migration at both 50% conditioned medium and 100% conditioned medium concentrations. CONCLUSION: Our results show that ROBO2 p.G114W is a loss-of-function mutation disrupting normal SLIT2-ROBO2 chemorepulsive activity on SVZa neuronal migration, suggesting that the presence of this missense mutation compromises SLIT2-ROBO2 signaling and contributes to the development of CAKUT.<br>2020-07-03T00:00:00Z

Dissertação de Mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.<br>Mitosis is the phase of the cell cycle in which the cell divides into two genetically identical daughter cells. The spindle assembly checkpoint (SAC), also known as mitotic checkpoint, safeguards the transition from metaphase to anaphase and is responsible for correct chromosome segregation by delaying the onset of anaphase until the chromosomes are properly attached to the mitotic spindle. The SAC is comprised of the mitotic checkpoint complex (MCC), which is formed by Mad2, BubR1, Bub3 and Cdc20. Apart from the MCC many more proteins are essential for the proper function of the SAC, including the kinases Aurora B and Mps1. Mps1 is an essential threonine/tyrosine kinase that not only controls the recruitment of SAC proteins to unattached kinetochores starting the SAC signal but also monitors the formation of correct kinetochore-microtubule attachments. Depletion of Mps1 causes shortening of the time cells spend in mitosis due to the premature satisfaction of the SAC. This results in errors in chromosome segregation, which is associated with aneuploidy and chromosomal instability (CIN). Although aneuploidy and CIN have often been associated with tumorigenesis and tumor progression, cells can only cope with a certain level of chromosome imbalances. Overexpression of Mps1 has already been described as a protective mechanism against aneuploidy in several cancer types. These observations led to the development of small molecule inhibitors against cell cycle kinases, such as Mps1, which explore the concept that increased missegregation rates are detrimental for cell survival. Although described to exhibit promising results in cell culture and xenograft models, resistance to some of the developed inhibitors, such as Aurora B and PLK1, has already been reported. On the other hand, resistance to Mps1 inhibitors has not been described so far and is a very relevant point to be addressed. After characterization of four mutations present in the kinase domain of Mps1 and identified in tumor samples, we show that certain mutations can confer tumors resistance to specific Mps1 inhibitors. Besides this, two more mutations also identified in tumor samples are studied to better understand Mps1 contribution to tumorigenesis. However, characterization of these two new Mps1 mutations did not confer new insights on the role of this kinase in the process of tumorigenesis.<br>A mitose é a fase do ciclo celular na qual a célula divide-se em duas células-filhas idênticas. O ponto de controlo mitótico controla a transição de metáfase para anáfase e é responsável pela correta segregação de cromossomas ao atrasar a entrada em anáfase até garantir que todos os cromossomas estão corretamente ligados ao fuso mitótico. O ponto de controlo mitótico é composto por um complexo formado pelas proteínas Mad2, BubR1, Bub3 e Cdc20. Além deste complexo, muitas outras proteínas são essenciais para o seu correto funcionamento, incluindo a Aurora B e a Mps1. A Mps1 é uma quinase treonina/tirosina essencial que não só controla o recrutamento de proteínas do ponto de controlo mitótico para os cinetócoros livres dando início ao sinal ativador deste mas também monitoriza a formação das ligações corretas entre os cinetócoros e os microtúbulos. Depleção da Mps1 leva ao encurtamento do tempo que a célula passa em mitose devido à satisfação prematura do ponto de controlo mitótico. Isto resulta em erros de segregação dos cromossomas, o que está associado a aneuploidia e instabilidade cromossómica (IC). Apesar da aneuploidia e a IC estarem associados a tumorigénese e progressão tumoral, as células conseguem apenas lidar com certos níveis de desequilíbrios cromossómicos. Sobrexpressão da Mps1 já foi descrita como sendo um mecanismo protetor para a aneuploidia em vários tipos de tumores. Estas observações levaram ao desenvolvimento de inibidores contra várias quinases do ciclo celular, entre as quais a Mps1, que pretendem explorar o conceito de que elevados níveis de erros de segregação são prejudiciais para a sobrevivência das células. Apesar de terem mostrado resultados promissores em cultura de células e modelos xenográficos, resistência contra alguns dos inibidores, como contra a Aurora B e PLK1, já foi reportada. No entanto, resistência a inibidores da Mps1 não foi ainda descrita e parece um ponto bastante relevante que deve ser endereçado. Após caracterização de quatro mutações no domínio quinase da Mps1 identificadas em amostras tumorais, é aqui mostrado que certas mutações conferem aos tumores resistência a inibidores da Mps1. Duas outras mutações, também identificadas em amostras tumorais, são estudadas para melhor compreender a contribuição da Mps1 para a tumorigénese. No entanto, caracterização destas duas mutações não contribuiu para o melhor conhecimento do papel desta quinase neste processo.

<p>Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations. </p><p>We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization. </p><p>Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.</p><p>Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association. </p><p>Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases. </p><p>Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.</p><br>Dissertation

The discovery of the Arabidopsis ttg1 mutant almost three decades ago provided a unique opportunity for the study of how several cell fates and organ identity pathways are co-regulated. Besides showing a lack of flavonoid based pigments, the pleiotropic ttg1 mutant is also deficient for the development of several epidermal characters including plant hair cells (trichomes), the non-hair cells of the root and the mucilage-secreting cells of the seed coat epidermis. Ectopic expression of the maize R bHLH transcriptional regulator of the flavonoid pigment pathway could completely suppress all the ttg1 mutant phenotypes, providing the first clue to the nature of the control mechanisms governing TTG1-dependent traits. Because it was established that a bHLH and a Myb protein are required for the regulation of anthocyanin pigment production in several plant species and an Arabidopsis Myb gene was necessary for trichome initiation, the existence of bHLH and Myb proteins that would regulate all the TTG1-dependent developmental pathways was hypothesized. This study works towards the elucidation of the transcriptional control mechanisms that regulate the TTG1-dependent developmental pathways. The identification and characterization of a key regulator, EGL3, uncovered the redundant nature of bHLH proteins operating under the TTG1 regulatory umbrella. As a result, bHLH regulators were assigned to all TTG1-dependent epidermal cell fate pathways and new roles for previously identified bHLH proteins were revealed. Roles suggested in the literature for Arabidopsis Myb factors suspected of regulating the flavonoid pigment pathway were at odds with findings from other plant models. Analysis of Myb loss-of-function RNAi lines and TTG1:GR and GL3:GR fusion lines presented here provides a clarified understanding of the regulation of anthocyanin biosynthesis by the Myb/bHLH/WDrepeat complex in Arabidopsis. Missing from the combinatorial complex model is the Myb component controlling the differentiation of the seed coat epidermis. Work presented here characterizes Myb5 as the primary Myb regulator of this differentiation pathway and defines a new role for TT2 as partially redundant with Myb5 for testa epidermis development. Myb5 also plays a minor role in trichome development and PA biosynthesis. Thus pleiotropy among the TTG1-dependent Myb regulators previously unobserved is first noted here. A more complete Myb/bHLH combinatorial transcription factor network model for the regulation of the TTG1-dependent pathways is proposed based on the results of work presented in this dissertation.<br>text

Plant defense responses vary depending on the pathogen and intensity of attack. These responses are mediated through two levels of defense, with the first level being pathogen-triggered immunity (PTI) that is triggered by host recognition of microbe-associated molecular patterns (MAMPs). Successful pathogens are able to evade PTI by secreting effector molecules into host cells. These effectors are designed to suppress host defenses. In turn, effectors are inhibited by the second level of plant defense called effector-triggered immunity (ETI). In ETI, intracellular resistance proteins recognize and block effector dampening of host defenses. ETI results in gene expression changes that can lead to localized cell death known as the hypersensitive response (HR) as well as a plant-wide systemic acquired resistance. The Arabidopsis thaliana mutant alf3-1 (aberrant lateral root formation 3-1) was characterized as the first and only case of HR in the root system. The alf3-1 mutant’s primary and lateral roots die unless they are grown in auxin-supplemented medium or at elevated ambient temperature. This thesis describes further characterization of the mutant phenotype and identifies a candidate gene for ALF3. Consistent with an autoimmune response, we found that the alf3-1 mutant has increased production of phenylalanine- and tryptophan-derived defense compounds, as well as increased production of salicylic acid (SA), a plant hormone that mediates innate immunity. Based on gene expression profiling, we found that many immune and defense response genes were expressed highly in alf3-1 compared to wild type (WT). These genes include the SA-responsive PR1 and PBS3 as well as several WRKY transcription factors, a gene family implicated in plant defense. Importantly, we found that the vast majority of defense-related phenotypes dysregulated in alf3-1 returned to WT levels when the mutant was grown at elevated temperatures or in medium supplemented with auxin, conditions that suppress innate immunity. To determine the identity of ALF3, we used whole genome re-sequencing to identify a candidate gene that encodes an uncharacterized TIR domain protein. Because characterized plant TIR domain proteins have been shown to function in plant innate immunity, we hypothesize that alf3-1 is a gain-of-function mutation that causes an autoimmune phenotype in roots.<br>2020-07-12T00:00:00Z