High-throughput genomics methodologies provide us the methods and solutions to the research fields of genomes, transcriptomes and proteomics. In my study, we utilized different high-throughput genomics methodologies to explore the exomes of human patients as well as transcriptomes of Drosophila melanogaster and Daphnia pulex. Exome sequencing, an efficient strategy to sequence the coding regions of the genome, helped us to identify potential disease causing mutations in two human diseases (Renal Agenesis and Cleft lip with or without palate). Although exome sequencing is mostly used to identify mutations underling Mendelian disorders, we chose informative pedigrees that were transmitted in Mendelian fashion for our study. Identification of new genes will be expected to significantly impact our understanding of human development and human diseases. RNA-Seq, a high-throughput sequencing technology to sequence cDNA in a sample, was utilized to explore the transcriptome of the microcrustacean Daphnia pulex. Daphnia pulex is a keystone species of freshwater ecosystems and are routinely used to determine the toxicity of aqueous solutions and to gauge the quality of inland water. Treatment of Daphnia pulex with different heavy metals followed by RNA-Seq analysis revealed us specific gene expression patterns that would provide new insights into their biological and toxicological responses to these environmental contaminants. Also these studies will help us to discover new genes that are not expressed during normal development. We also developed a new technology that utilizes microarray-based exome capture prior to RNA-Seq to faithfully annotate tissue specific transcriptomes in Drosophila melanogaster. We showed that this methodology was efficient for cataloging tissue-specific transcriptomes in which specific classes of genes or transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation.

Primary hyperparathyroidism (pHPT) is a pathology associated with one or multiple hyperfunctioning parathyroid glands.  The disease prevalence occurs in roughly 1-2% of the population primarily post-menopausal women.  The molecular pathology of the disease is poorly understood.  Elevated serum calcium levels in the setting of an inappropriately elevated parathyroid hormone level are indicative of the disease process.  The ultimate treatment of the disease is to remove the hyperfunctioning gland. The aim of this thesis was to examine potential genetic and epigenetic aberrations that are potentially disease causing. The methylation signature of normal and pathological parathyroid tissue has yet to be investigated.  DNA was bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident.  DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 known to be important in the development of parathyroid tumors were associated with reduced gene expression in both benign and malignant parathyroid tumors. Familial primary hyperparathyroidism (FPHPT) may occur due to an underlying germ-line mutation in the MEN1, CASR, or HRPT2/CDC73 genes.  Eighty-six young (≤45 years of age) patients with clinically non-syndromic PHPT underwent genetic analysis.  Eight of 86 (9.3%) young patients with clinically non-familial PHPT displayed deleterious germ-line mutations in the susceptibility genes (4 MEN1, 3 CASR, and 1 HRPT2/ CDC73). Accumulation of non-phosphorylated active β -catenin has been reported to commonly occur in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT).  We assessed possible β-catenin stabilizing mutations in a large series of parathyroid adenomas. A total of one hundred and eighty sporadic parathyroid adenomas were examined for mutations in exon 3 of the CTNNB1gene. The mutation S33C (TCT >TGT) was detected by direct-DNA sequencing of PCR fragments in 1 out of 180 sporadic parathyroid adenomas (0.68 %). Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing.  Four of eight tumors displayed a frame shift deletion or nonsense mutations within the MEN1 gene, which was accompanied by loss of heterozygosity of the remaining wild-type allele.  One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%).

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Schizophrenia is a debilitating disorder that occurs the world over. Although antipsychotics are largely effective in treating the positive symptoms of schizophrenia, the outcomes are non-optimal in many patients. As antipsychotic treatment response has been shown to be heritable, it is expected that the implementation of antipsychotic pharmacogenomics should aid in the optimization of antipsychotic treatments, however to date clinically applicable results are limited. Therefore this study utilized exome sequencing in a cohort of well characterized first episode schizophrenia patients to identify the genetic factors contributing to antipsychotic treatment response. The utility of exome sequencing for antipsychotic pharmacogenomic applications in the African context was assessed through examination of the literature and publically available data. Thereafter, a cohort of 104 well characterized South African first episode schizophrenia patients who were treated with flupenthixol decanoate for twelve months was collected. From this cohort, subsets of patients on extreme ends of the treatment response spectrum were identified for exome sequencing. Thereafter a bioinformatics pipeline was used to call and annotate variants. These variants and those that have previously been associated with antipsychotic response, along with a panel of ancestry informative markers, were prioritized for genotyping in the entire cohort of patients. After genotyping of the 393 variants, statistical analyses were performed to identify associations with treatment response outcomes. Examination of the literature revealed a need for exome sequencing in Africa. However, critical analyses of next generation sequencing data demonstrated that complex regions of the genome may not be well suited to these technologies. Thus, it may be necessary to combine exome sequencing with knowledge obtained from past research, as was done in this study to identify the genetic factors contributing to antipsychotic treatment response. Using this strategy, the current study highlighted the potential role that rare variants play in antipsychotic treatment response and additionally detected 11 variants that were significantly associated with antipsychotic treatment response outcomes (P=2.19x10-5). Nine of these variants were predicted to alter the function of the genes in which they occurred; of which eight were novel with regards to antipsychotic treatment response. The remaining two variants have been associated with antipsychotic treatment outcomes in previous GWAS. Examination of the function of the genes in which the variants occurred revealed that the variants associated with (i) positive symptom improvement were involved in the folate metabolism pathway and (ii) negative and general pathological symptoms improvement had potential links to neuronal development and migration. To our knowledge this study is the first to utilize exome sequencing for antipsychotic pharmacogenomic purposes. The ability of this study to identify significant associations, even after correction for multiple testing, has highlighted the importance of combining genomic technologies with well characterized cohorts. The results generated from this study have served both to replicate results from previous antipsychotic pharmacogenetic studies and to identify novel genes and pathways involved in antipsychotic response. These results should aid in improving our understanding of the biological underpinnings of antipsychotic treatment response and may ultimately aid in the optimization of these treatments. / AFRIKAANSE OPSOMMING: Skisofrenie is ‘n siekte wat wêreldwyd voorkom en lei tot erge funksionele inkorting. Alhoewel antipsigotiese medikasie redelik effektief is in die behandeling van die positiewe simptome van skisofrenie, is die funksionele uitkomste in baie pasiënte nie optimaal nie. Die reaksie op antipsigotiese behandeling blyk oorerflik te wees. Die verwagting is dus dat die implementering van antipsigotiese farmakogenomika met die optimalisering van antipsigotiese behandeling sal help. Tot dusver het die resultate van farmakogenomika studies egter beperkte kliniese toepassings opgelewer. Hierdie studie het dus eksoomvolgordebepaling in 'n groep van goed-karakteriseerde eerste-episode skisofrenie pasiënte gebruik om die genetiese faktore wat bydra tot die antipsigotiese behandelings-reaksies te identifiseer. Die gebruik van eksoom-volgordebepaling vir antipsigotiese farmakogenomika in die Afrikakonteks is deur die ondersoek van literatuur en openbaar-beskikbare data geëvalueer. Daarna is 'n groep van 104 goed-gekarakteriseerde Suid-Afrikaanse eerste-episode skisofrenie pasiënte, wat met flupenthixol dekanoaat vir twaalf maande behandel is, versamel. Uit hierdie groep is subgroepe van pasiënte op die teenoorgestelde eindpunte van die behandelings-reaksiespektrum vir eksoom-volgordebepaling geïdentifiseer. Hierna is 'n bioinformatika pyplyn gebruik om variante te identifiseer en te annoteer. Hierdie variante, asook variante wat voorheen met antipsigotiese reaksie geassosieer is, is saam met 'n paneel van afkoms-informatiewe merkers vir genotipering in die hele groep pasiënte geprioritiseer vir genotipering. Na genotipering van die 393 variante, is statistiese analises uitgevoer om assosiasies met behandelings-reaksie uitkomste te identifiseer. ‘n Ondersoek van die literatuur het getoon dat daar 'n behoefte vir eksoomvolgordebepaling in Afrika is. ‘n Kritiese analise van volgende-generasie volgordebepalings data het egter getoon dat komplekse dele van die genoom nie geskik is vir die gebruik van hierdie tegnologie nie. Om die genetiese faktore wat bydra tot suksesvolle antipsigotiese behandeling te identifiseer, mag dit nodig wees om eksoom-volgordebepaling te kombineer met bevindings verkry uit vorige navorsing, soos gedoen in hierdie studie. In die huidige studie het die gebruik van hierdie strategie die potensiële rol van skaars variante in antipsigotiese behandelings-reaksies beklemtoon en ‘n bykomende 11 variante is geïdentifiseer wat beduidend met antipsigotiese behandelingsrespons geassosieer is (P=2.19x10-5). Daar is voorspel dat nege van hierdie variante die funksie van die gene waarin hulle voorkom sal verander en agt van hierdie variante is vir die eerste keer met antipsigotiese behandelingsrespons geassosieer. Die oorblywende twee variante is met antipsigotiese behandelingsrespons in vorige GWAS geassosieer. ‘n Ondersoek na die funksie van die gene waarin die variasies voorgekom het, toon dat die variante wat geassosieer is met (i) verbetering van positiewe simptome ‘n rol speel in folaatmetabolisme, terwyl variante wat geassosieer is met (ii) die verbetering in negatiewe en algemene patologiese simptome potensiële skakels met neuron ontwikkeling en migrasie het. Na ons wete is hierdie die eerste studie wat eksoom-volgordebepaling vir antipsigotiese farmakogenomika doeleindes gebruik. Die vermoë van hierdie studie om beduidende assosiasies te identifiseer, selfs na korreksie vir veelvoudige toetse, onderstreep die belangrikheid van die kombinering van genomiese tegnologie met goed-gekarakteriseerde pasiënte. Die bevindinge van hierdie studie het nie net die resultate van vorige antipsigotiese farmakogenetiese studies bevestig nie, maar ook nuwe gene en variante wat betrokke is in antipsigotiese reaksie geïdentifiseer. Hierdie resultate sal hopelik ons begrip van die onderliggende biologiese faktore wat antipsigotiese behandelingsrespons beïnvloed verbeter en uiteindelik ook met die optimalisering van behandeling help.

Genus Danio includes zebrafish (Danio rerio) and several other phenotypically diverse species. To understand the history of these species and how they acquired the genetic differences underlying their diverse phenotypes, I performed two phylogenomic studies using Restriction-Site Associated DNA Sequencing and DNA hybridization-based exome enrichment. The results of these studies highlight important methodological considerations applicable to future experiments across taxa. Furthermore, these studies provide detailed understanding of the relationships within Danio including extensive introgression between lineages. The extent of introgression varies across the genome with regions of high recombination at the ends of chromosomes having the most evidence for introgression. Together, this work gives vital insight into the history of a model organism and the evolutionary processes that give rise to phenotypic diversity.

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases, the genetic basis is unknown. Via whole-exome sequencing and linkage analysis, we determined the most likely causative mutations in four families. While the mutations are in three different genes – FBLIM1, PLCG2 and PIP; all three genes are involved in Fcγ signaling and osteoclast activation. In a large cohort of 61 individuals with CRMO, we performed gene and pathway based association analysis using the 1000 genomes participants of European ancestry as controls. One gene from the family-based analyses, ANO6, was significantly enriched for rare variants in our cohort of cases. ANO6 is involved in P2RX7- mediated inflammasome activation and in the regulation of bone mineralization. While no pathways were enriched for rare variants in the CRMO cohort after genome-wide correction, four pathways were significantly enriched for rare variants in the control samples, indicating a protective effect of the variants. The second most significant pathway, activation of chaperone genes by XBP1s, is relevant to CRMO pathogenesis as XBP1s is a transcription factor that attenuates ER stress, and regulates the expression of genes involved in RANKL signaling and bone remodeling. An association analysis using a larger set of cases followed by functional validation of candidate genes is necessary to confidently declare the mutations isolated in the work presented here to be pathogenic. Our preliminary findings suggest that mutations in genes involved in both the inflammatory response and bone remodeling underlie the pathogenesis of CRMO.

Familial Pancreatic Cancer (FPC) is an autosomal dominant condition with greater then 80% of genetic causes unknown. We hypothesize that an integrative approach employing germline exome sequencing and somatic microarray analysis of FFPE DNA will identify novel tumour suppressor genes (TSGs). 55 FPC and 21 sporadic PDAC tumours were analyzed on the Affymetrix Oncoscan FFPE Express 2.0 SNP microarray and compared to data from 33 germline FPC cases analyzed on the Illumina GAIIx Analyzer. We have demonstrated that FPC is genetically heterogeneous with recurrent loss at CDKN2A/p16, TP53 and SMAD4. Analysis of 2 sisters has shown a shared loss region involving DCLK3 and SERPINF1. By an integrative approach, we have identified ATPAF1-AS1 and MAP3K6 as potential TSGs. Germline variants of these genes have been confirmed by Sanger sequencing and somatic fluorescence in-situ hybridization. Future functional studies will better characterize the importance of these regions and novel putative TSGs in FPC.

Familial Pancreatic Cancer (FPC) is an autosomal dominant condition with greater then 80% of genetic causes unknown. We hypothesize that an integrative approach employing germline exome sequencing and somatic microarray analysis of FFPE DNA will identify novel tumour suppressor genes (TSGs). 55 FPC and 21 sporadic PDAC tumours were analyzed on the Affymetrix Oncoscan FFPE Express 2.0 SNP microarray and compared to data from 33 germline FPC cases analyzed on the Illumina GAIIx Analyzer. We have demonstrated that FPC is genetically heterogeneous with recurrent loss at CDKN2A/p16, TP53 and SMAD4. Analysis of 2 sisters has shown a shared loss region involving DCLK3 and SERPINF1. By an integrative approach, we have identified ATPAF1-AS1 and MAP3K6 as potential TSGs. Germline variants of these genes have been confirmed by Sanger sequencing and somatic fluorescence in-situ hybridization. Future functional studies will better characterize the importance of these regions and novel putative TSGs in FPC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21254号 / 医博第4372号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 亮, 教授 Shohab YOUSSEFIAN, 教授 小杉 眞司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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La dystonie est une pathologie du contrôle du mouvement caractérisée par des contractions musculaires involontaires. Les causes génétiques de cette pathologie sont multiples. J’ai créé des bases de données locus spécifiques colligeant l’ensemble des diversités alléliques disponible pour 16 gènes de dystonie. L’objectif de ce travail est d’aider au diagnostic de cette pathologie et, à plus long terme et lorsque les données le permettent, d’établir des corrélations génotypes-phénotypes. Cela a été le cas pour le gène THAP1 (définissant la forme DYT6) pour lequel nous avons décrits plusieurs corrélations. J'ai recherché la mutation causale dans plusieurs familles par séquençage d'exome. Cela a permis d’identifier une famille porteuse d’une mutation prédite pathogène dans le gène ANO3 (DYT23). Une autre famille est porteuse d’une mutation dans un site d’épissage du gène ATP1A3 (DYT12) entrainant la rétention totale de l'intron 17. Pour une autres famille, un gène candidat a été identifié : ADD2 qui code l'adducine beta. Plusieurs résultats expérimentaux ont été obtenus. Tout d’abord j'ai observé des différences au niveau du cytosquelette d’actine. En effet la surexpression de la protéine sauvage provoque un comportement anormal de l’actine au niveau des fibres de stress. Par ailleurs des études de d’apprentissage par association dans un modèle C. elegans KO ADD2 ont montré un défaut de mémorisation à long-terme. Mes travaux de thèse ont permis d'approfondir les connaissances quant à la contribution de chaque gène déjà connu dans les dystonies, ainsi que d'élargir l'hétérogénéité génétique caractéristique de cette pathologie par l'identification d'un nouveau gène candidat. / Dystonia is a movement control disorder characterized by involuntary muscle contractions. The genetic causes of this disease are multiple. I have created databases " loci-specific " collecting all allelic diversity available in the literature for 16 dystonia genes. The goal of this work is to to assist in the diagnosis of this disease and in the longer term, when there are sufficient data, to establish genotype-phenotype correlations. This was the case for the THAP1 gene (responsible for DYT6 dystonia) for which we have described several correlations.I searched for the disease gene in several families using exome sequencing. I identified a pathogenic mutation in the predicted gene ANO3 (DYT23) carried by one family. Another family carries a mutation in a splice site of ATP1A3 (DYT12) resulting in the total retention of intron 17. In another family a candidate gene was identified: ADD2 gene, coding beta adducin. Several functional results were obtained. First, overexpression of wild type and mutated ADD2 enabled to view differences in the actin cytoskeleton. Indeed the overexpression of the wild type protein causes abnormal behavior of actin at the level of stress fibers and at the plasma membrane. Besides, learning by association studies in a Caenorhabditis elegans model KO for ADD2 gene have shown a long-term default memory compared to the wild type. This confirms the involvement of the protein in neuronal plasticity. My thesis work led to further knowledge about the contribution of each gene already known in dystonia , as well as broaden the genetic heterogeneity characteristic of this disease by identifying a new candidate gene.