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L’holoprosencéphalie (HPE) est la malformation congénitale cérébrale la plus fréquente chez l’Homme. Elle est caractérisée par une non-séparation plus ou moins importante des hémisphères cérébraux. Lorsqu’elle ne provient pas de défauts chromosomiques, une origine génétique est suspectée. Quatorze gènes sont impliqués, appartenant majoritairement aux voies de signalisation SHH, NODAL, FGF et NOTCH. Au total, les mutations retrouvées n’expliquent qu’environ un tiers des cas d’HPE non-chromosomique, et le mode de transmission n’est pas clairement établi. Afin d’améliorer le rendement diagnostique et le conseil génétique auprès des familles concernées, l’équipe GPLD (IGDR) cherche à identifier de nouveaux gènes impliqués. Dans ce but, le séquençage exomique de familles a été initié depuis 2013. Dans une famille consanguine, une mutation homozygote délétère dans le gène STIL a été identifiée. La protéine STIL est impliquée dans l’assemblage du cil primaire par lequel transite le signal SHH, dont la dysfonction est la première cause d’HPE. Dans une autre famille consanguine, une mutation homozygote candidate est présente dans FAT1, protocadhérine impliquée dans le développement cérébral et responsable d’HPE chez les modèles animaux. D’autres familles, non-consanguines, ont été analysées en trio. Les enfants de toutes ces familles portent une mutation dans un gène de l’HPE, héritée d’un de leur parent asymptomatique. Des mutations additionnelles ont été recherchées, en supposant une origine multigénique de l’HPE chez ces enfants. Un digénisme SHH/DISP1 est présent dans l’une d’elles, et des associations de mutations candidates ont été mises en évidence dans les autres familles, dont une impliquant également FAT1. En conclusion, ces travaux apportent de nouveaux éléments pour la compréhension des bases génétiques de l’HPE et notamment de nouveaux arguments en faveur d’une part importante de multigénisme. L’investigation de ces bases génétiques complexes nécessite le développement de nouvelles méthodes d’analyses, qui pourront être utiles à d’autres pathologies du développement pour lesquelles une origine multigénique est suspectée. / Holoprosencephaly (HPE) is the most common developmental disorder affecting the brain in humans. HPE is characterised by a lack of interhemispheric separation, on a varying scale of severity. When HPE is not due to chromosomal aberrations, a genetic origin is suspected. Alterations of fourteen genes have been implicated in HPE, mainly involved in SHH, NODAL, FGF and NOTCH signalling pathways, with an unclear mode of inheritance. In order to increase the molecular diagnosis yield and to improve genetic counselling, the goal of the GPLD team (IGDR) is to identify new genes. In one inbred family, a deleterious homozygous mutation in STIL gene has been identified. The STIL protein is involved in primary cilia assembly, through which SHH signalling transits. In another inbred family, a homozygous candidate mutation was located in FAT1, a protocadherin involved in brain development that causes HPE-like phenotypes in animal models. For other non-consanguineous families, exome sequencing data were analysed in trios. All children of these families have a previously identified mutation in a HPE gene that is transmitted from a healthy parent. The approach consisted in searching for additional genetic events, under the hypothesis of a multigenic inheritance. Thus, a digenic inheritance of mutations in SHH and DISP1 has been identified in one family. Further associations of candidate mutations have been identified in others, one also involving FAT1. In conclusion, this work provides new elements accounting for the understanding of HPE genetic bases and particularly new arguments in favour of a multigenic inheritance. The study of these complex genetics bases requires the development of new analytical methods that could be of use in relation to other developmental disorders in which a multigenic inheritance is suspected.

High-throughput sequencing technologies have changed the way we identify, study and understand the role of rare variation in Mendelian diseases. Sequencing in complex diseases have proven to be more challenging to interpret, but methods and approaches are being developed to aid in our understanding of variation in these diseases.

Orientador: Iscia Teresinha Lopes Cendes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T18:55:52Z (GMT). No. of bitstreams: 1 Borges_MuriloGuimaraes_M.pdf: 8164302 bytes, checksum: cc367cbf534276995150e7db644f6e94 (MD5) Previous issue date: 2015 / Resumo: Os avanços técnicos em sequenciamento alcançados em menos de uma década, atrelados ao desenvolvimento e barateamento do sequenciamento de alto desempenho, oferecem-nos a possibilidade de aplicação dessas tecnologias na medicina genômica. Nesse contexto, surge o sequenciamento do exoma humano, constituído das regiões codificantes do genoma, menor que 2% de sua totalidade. O sequenciamento do exoma (WES) se estabelece hoje como uma ferramenta custo-efetiva com a finalidade de identificar variantes de sequência relacionadas a várias doenças humanas. A análise através da bioinformática é essencial para lidar com o alto volume de dados gerados e realizar a ligação entre o experimento biológico e os dados obtidos. Objetivo: Aplicar e avaliar protocolos e aplicações disponíveis na análise dos dados gerados pelo sequenciamento de exomas humanos, bem como aplicar e aperfeiçoar protocolos e aplicações disponíveis para predizer variantes como potencialmente patológicas a partir de dados gerados pelo sequenciamento de exomas humanos. Materiais e métodos: Foram utilizadas as seguintes ferramentas: FastQC, Rqc, BWA, Picard, GATK e VEP. Estas foram então aplicadas às sequências do exoma humano possibilitando a identificação de variações nos perfis de qualidade das sequências, realinhamento local ao redor de inserções e deleções, recalibração da qualidade e posterior chamada das variantes potencialmente envolvidas nos fenótipos em estudo. No intuito de avaliar se a cobertura no exoma sofre variações mediante diferenças técnicas e étnicas, selecionamos amostras do Projeto 1000 Genomas. Resultados: A aplicação de nosso protocolo em 27 amostras WES resultou em gráficos de controle de qualidade pré e pós-alinhamento, que nos permitiram avaliar de modo global os perfis de qualidade destas sequências; realinhamento ao redor de inserções e deleções que ocorreu em mais de 15% da definição do exoma, realinhando mais de 79% das sequências; recalibração da qualidade que nos permitiu minimizar sua variação por ciclo da reação. Das sequências empregadas, 72% foram pareadas ao genoma, contudo 46% se estendem para fora da definição do exoma, com uma cobertura média de 59x para o exoma estendido e 66x para o exoma restrito. Temos que a cobertura para WES possui uma tendência a variar de acordo com a metodologia de captura empregada e ao grupo étnico de onde as amostras foram obtidas. Conclusão: A aplicação de um workflow para interrogação de variantes que considera a qualidade das sequências fornecidas pelo sequenciador, o alinhamento contra o genoma, realinhamento ao redor de regiões sabidamente conhecidas como portadoras de variações, recalibração da qualidade e anotação permitiu identificar variantes de sequência. Além disso, através da cobertura obtida pelo sequenciamento do exoma foi possível perceber diferenças técnicas e populacionais, refletindo que a complexidade do genoma pode interferir na reação de captura das sequências, influenciando na efetividade da técnica empregada / Abstract: The technical advances in sequencing made in less than a decade associated with the development and low costs of high throughput sequencing techniques allow their application in genomic medicine. Therefore, Whole Exome Sequencing (WES), which corresponds to less than 2% of the entire genome, emerges as a cost-effective tool that aims to identify variants related to human diseases. Bioinformatics is fundamental to process the big volume of data and link the obtained results with the biology. Objective: We aim to apply and evaluate protocols and applications designed for WES data analysis on human subjects. We also intend to apply and enhance protocols and applications designed to predict variants as potentially pathological from WES data. Materials and Methods: We used the following tools: FastQC, Rqc, BWA, Picard, GATK e VEP. We applied them to exome data, determining variation in quality profiles, local realignment, quality recalibration and variant calls. We also evaluated whether or not technical and population differences affect the depth profiles of samples from the 1000 Genomes Project. Results: We applied our protocol on 27 samples, resulting in pre and post-alignment quality control charts. Local realignment took place at more than 15% of the exome definition, extending to more than 79% of sequences. Quality recalibration minimized per cycle variation. In total, 72% of the sequences were paired against the genome, nevertheless 46% extended off-target. The mean coverage was 59X for the exome. We also detected that depth tends to vary based on technical and population differences between samples. Conclusion: We applied a variant-calling workflow that accounts for sequence quality, the alignment against the genome, local realignment, quality recalibration and annotation. In addition, we concluded that depth depends on technical and population differences, showing that genomic complexity may interfere with the capturing phase, affecting downstream analyses / Mestrado / Fisiopatologia Médica / Mestre em Ciências

[ES] Durante las últimas décadas, se han realizado importantes avances en el estudio de las causas genéticas de enfermedades raras y comunes, donde un gran número de variantes han sido identificadas y asociadas a múltiples enfermedades. Con las tecnologías de secuenciación de nueva generación, hoy en día somos capaces de investigar, con un alto rendimiento, la contribución de variantes de alta y baja frecuencia a distintos tipos de enfermedades, permitiéndonos así estudiar su importancia en el desarrollo de las mismas. En ésta tesis se ha utilizado la secuenciación del exoma como tecnología para el estudio de variantes raras en una enfermedad compleja, la alergia gastrointestinal inducida por múltiples alimentos. Para ello, se realizó la secuenciación del exoma completo de una cohorte de 31 individuos (ocho afectados y 23 no afectados) provenientes de siete familias diferentes. Se desarrolló un flujo de trabajo para procesar los datos generados a partir de diferentes librerías e instrumentos de secuenciación, así como un control de calidad exhaustivo con el fin de maximizar el número de variantes de alta calidad. Diferentes tipos de mutaciones fueron investigadas, incluyendo polimorfismos de nucleótido único, inserciones/deleciones, variantes del número de copia y haplotipos HLA, y se realizaron diferentes métodos de filtrado para su interpretación. Finalmente, se encontraron una serie de mutaciones que podrían estar asociadas con la enfermedad y se describe su posible papel en la patogénesis de las alergias gastrointestinales. Los resultados de esta tesis suponen importantes avances en el estudio de la compleja arquitectura genética de las alergias gastrointestinales y abren las puertas a futuras líneas de investigación, que serán necesarias para entender completamente las bases genéticas de esta enfermedad. / [CAT] Durant les últimes dècades, s'han realitzat importants avanços en l'estudi de les causes genètiques de malalties rares i comunes, on un gran nombre de variants han sigut identificades i associades a múltiples malalties. Amb les tecnologies de seqüenciació de nova generació, avui en dia som capaços d'investigar, amb un alt rendiment, la contribució de variants d'alta i baixa freqüència a diferents tipus de malalties, permetent-nos així estudiar la seva importància en el desenvolupament de les mateixes. En aquesta tesis s'ha utilitzat la seqüenciació del exoma com a tecnologia per a l'estudi de variants rares en una malaltia complexa, l'al·lèrgia gastrointestinal induïda per múltiples aliments. Per això, es va realitzar la seqüenciació del exoma complet d'una cohort de 31 individus (vuit afectats i 23 no afectats) provinents de set famílies diferents. Es va desenvolupar un flux de treball per a processar les dades generades a partir de diferents llibreries e instruments de seqüenciació, així com un control de qualitat exhaustiu amb la fi de maximitzar el nombre de variants d'alta qualitat. Diferents tipus de mutacions foren investigades, incloïent polimorfismes de nucleòtid únic, insercions/delecions, variants del nombre de còpia i haplotips HLA, i es realitzaren diferent mètodes de filtrat per a la seva interpretació. Finalment, es trobaren una sèrie de mutacions que podrien estar associades amb la malaltia i es descriu el seu possible paper en la patogènesis de les al·lèrgies gastrointestinals. Els resultats d'aquesta tesis suposen importants avanços en l'estudi de la complexa arquitectura genètica de les al·lèrgies gastrointestinals i obrin les portes a futures línies d'investigació, que seran necessàries per entendre completament les bases genètiques d'aquesta malaltia. / [EN] The study of genetics has been making significant progress towards understanding the causes of rare and common disease during the past decades. Across a wide range of disorders, there have been hundreds of associated loci identified and associated with multiple disorders. Now, with the advent of next-generation sequencing technologies, we are able to interrogate the contribution of high and low frequency variation to disease in a high throughput manner. This provides an opportunity to investigate the role of rare variation in complex disease risk, potentially offering insights into disease pathogenesis and biological mechanisms. In this thesis, it has been assessed the use of whole-exome sequencing technology to investigate the role of rare variation in a complex disease, gastrointestinal food allergy induced by multiple food proteins. For that, a cohort of 31 individuals (eight affected and 23 non-affected) from seven different families was whole exome sequenced. Data obtained from multiple sequencing systems and libraries were analysed, and a workflow was developed, focusing on a comprehensive quality control to maximise the number of real positive calls. Different types of genome variations were investigated, including single nucleotide variants, insertions/deletions, copy number variants and HLA haplotypes. By approaching different methods of variant filtering, a set of rare variants that could be associated with the disease was identified. The possible role of these candidate variants in the pathogenesis of gastrointestinal food allergies was also discussed. These results reveal important insights into the genetic architecture of gastrointestinal food allergies and lead to additional lines of investigation that will be required in order to fully understand the genetic basis of this disease. / Sanchis Juan, A. (2019). Exome Sequencing in Gastrointestinal Food Allergy Induced by Multiple Food Protein [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/134361 / TESIS

Le but de ce travail de thèse a été d’identifier par séquençage d’exomes, de nouveaux gènes de prédisposition dans 2 pathologies rares dont l’étiologie est peu connue. Le 1er projet a porté sur une famille à 3 cas de sarcomes, sans mutation du gène TP53. Nous avons identifié une mutation pathogénique du gène CDKN2A, dans les 3 cas. Le gène CDKN2A étant le gène majeur de prédisposition au mélanome, nous avons recherché parmi 3 collections différentes des cas de sarcomes associés à une mutation du gène CDKN2A. Nous avons identifié 8 cas de sarcomes indépendants, porteurs d’une mutation du gène CDKN2A et montré une perte d’hétérozygotie au niveau du site de la mutation constitutionnelle CDKN2A dans 5/7 cas, montrant ainsi une perte de fonction complète. Les sarcomes étant rares chez les porteurs de mutations CDKN2A, nous avons recherché des variants rares modificateurs potentiels, par séquençages d’exomes constitutionnels des 8 cas index, et identifié 3 variants du gène PDGFRA. Des études de modélisation ont montré que 2 de ces variants, pourraient avoir un impact sur la structure du domaine extracellulaire de la protéine PDGFRA. Nous avons ainsi démontré que le gène CDKN2A peut prédisposer aux sarcomes et identifié PDGFRA comme gène modificateur potentiel.Dans le second projet nous avons travaillé sur les mélanomes de l’enfant, d’apparence sporadique. Notre hypothèse de travail était que ces mélanomes surviennent suite à un accident génétique de novo. Nous avons analysé des exomes constitutionnels de 41 trios (enfant atteint et ses 2 parents sains). Nos analyses bio-informatiques ont montré l’existence de mutations de novo post-zygotiques de gènes impliqués dans le développement de la crête neurale ou le cancer, dans 5 cas. Les analyses plus complètes des exomes tumoraux sont en cours, ainsi que des études fonctionnelles des gènes et de leurs mutations, dans un modèle d’embryon de poulet. Ce travail permettra d’accroitre la compréhension des mécanismes moléculaires et cellulaires dérégulés dans ces maladies, ouvrant ainsi, de nouvelles perspectives thérapeutiques. / The aim of this thesis was to identify by sequencing of exomes new predisposing genes in 2 rare pathologies whose etiology is not well known.The first project involved a family with 3 cases of sarcomas, without mutation of TP53 gene. We identified a pathogenic mutation of the CDKN2A gene, in the 3 cases. Since CDKN2A gene is the major melanoma predisposing gene, we have searched in 3 different collections, sarcoma cases link to CDKN2A mutations. In total, we have identified 8 independent sarcomas cases, carrying a germline mutation of the CDKN2A gene and showed a loss of heterozygosity at the site of the constitutional mutation of CDKN2A in 5/7 cases, thus proving a complete loss of function. Since sarcomas are rare in carriers of CDKN2A mutations, we looked for potential modifying rare variants, by sequencing constitutional exomes of the 8 index cases, and identified 3 variants of the PDGFRA gene. Modeling studies have shown that 2 of these variants could have an impact on the structure of the extracellular domain of the PDGFRA protein. We have thus demonstrated that the CDKN2A gene can predispose to sarcomas and identified PDGFRA as a potential modifier gene. In the second project, we worked on childhood melanomas, of sporadic appearance. Our working hypothesis was that these melanomas occur as a result of a de novo genetic accident. We analyzed constitutional exomes of 41 trios (affected child and his 2 healthy parents). Our bioinformatics analyzes identified the existence of de novo post-zygotic mutations of genes involved in neural crest development or cancer, in 5 cases. More complete analyzes of tumor exomes are underway, as well as functional studies of genes and their mutations, in a chick embryo model.This work improves the understanding of molecular and cellular mechanisms that are deregulated in these diseases, thus opening up new therapeutic perspectives.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18156号 / 医博第3876号 / 新制||医||1003(附属図書館) / 31014 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 武藤 学, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Motivation: The advent of clinical exome sequencing will require new tools to handlecoverage data and making it relevant to clinicians. That means genes over targets, smartsoftware over BED-files, and full stack, automated solutions from BAM-files to genetic testreport. Fresh ideas can also provide new insights into the factors that cause certain regionsof the exome to receive poor coverage.Results: A novel coverage analysis tool for analyzing clinical exome sequencing data has beendeveloped. Named Chanjo, it’s capable of converting between different elements such astargets and exons, supports custom annotations, and provides powerful statistics andplotting options. A coverage investigation using Chanjo linked both extreme GC content andlow sequence complexity to poor coverage. High bait density was shown to increasereliability of exome capture but not improve coverage of regions that had already proventricky. To improve coverage of especially very G+C rich regions, developing new ways toamplify rather than enrich DNA will likely make the biggest difference.

Early-onset myopathies are genetically heterogeneous mendelian diseases. We have performed solo or trio whole exome sequencing (WES) in a cohort of 24 individuals with genetically undiagnosed early-onset myopathies from 21 families. We have identified single nucleotide variant (SNV) or copy number variants (CNV) in known genes in 52% of the patients. We contributed to the expansion of the phenotypic spectrum for two genes (TTN, TRIP4). In particular, through international collaborative efforts, we described the severe end of the phenotypic spectrum of congenital titinopathy. Fifteen cases, with biallelic TTN truncating or canonical splice site mutations, presented with severe in utero features and/or severe weakness and respiratory compromise at birth, leading to termination of pregnancy, neonatal death or survival with long term respiratory support. The presence of multiple congenital limb contractures, congenital long bone fracture and/or complete absence of muscle groups can be diagnostic clues. All severely affected patients that survived until birth had at least one mutation predicted to result in the production of at least some near-normal-length titin, a finding that seems to be essential for human in utero survival.WES also allowed us to identify three novel candidate genes (JAG2, R. U.). Through Matchmaker Exchange, we assembled a cohort of 23 individuals with undiagnosed muscular dystrophy from 13 unrelated families with rare homozygous or compound heterozygous variants in JAG2. Individuals with JAG2 variants had a wide spectrum of phenotypes and disease severity but the muscle MRI findings appear to be the unifying feature that helps to identify these patients. Transcriptome analysis in the muscle of two affected individuals suggests satellite cell depletion, a finding that is also present in other muscular dystrophies linked to the Notch pathway like POGLUT1 or MEGF10 related muscular dystrophies. If the implication of our three novel candidate genes is confirmed, the diagnostic yield in the cohort would increase to 67%. The zebrafish has been widely used to validate the implication of novel genes of myopathy, mostly with morpholino or mutated mRNA injections. As the specificity of morpholinos and mutated mRNA injections had been questioned, we have decided to use CRISPR-Cas9 to model loss-of-function or gain-of-function by creating knockout or knockin lines. We generated a knockout line for mtm1 and a knockin line for the S619L gain-of-function mutation in dnm2a because these mutations were associated with severe myopathic phenotypes in humans and had already been modeled in zebrafish with morpholino or mutated mRNA injections. In contrast to the severe phenotype of morpholino/mRNA injected fish, we did not observe any structural or functional defect in the muscle of mtm1 mutants and dnm2a mutants. Off-target effects of morpholino or mRNA injections as well as genetic compensation mechanisms in mutant lines are possible mechanisms explaining these discrepancies. The absence of phenotype in a zebrafish mutant line is not sufficient to rule out the implication of a gene in a human phenotype. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Yes / Currently, out of the 82 US FDA-approved targeted therapies for adult cancer treatments, only three are approved for use in children irrespective of their genomic status. Apart from leukemia, only a handful of genomic-based trials involving children with solid tumors are ongoing. Emerging genomic data for pediatric solid tumors may facilitate the development of precision medicine in pediatric patients. Here, we provide an up-to-date review of all reported genomic aberrations in the eight most common pediatric solid tumors with whole-exome sequencing or whole-genome sequencing data (from cBioPortal database, Pediatric Cancer Genome Project, Therapeutically Applicable Research to Generate Effective Treatments) and additional non-whole-exome sequencing studies. Potential druggable events are highlighted and discussed so as to facilitate preclinical and clinical research in this area. / Seed Grant of Strategic Research Theme for Cancer, The University of Hong Kong of AKSC. VWY Lui is funded by the Research Grant Council, Hong Kong (#17114814, #17121616, General Research Fund; T12–401/13-R, Theme-based Research Scheme), and the Start-up Fund, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong. W Piao is funded by the Faculty Postdoctoral Fellowship Scheme, Faculty of Medicine, the Chinese University of Hong Kong.