Academic literature on the topic 'Variants structurels'

La variation génomique au sein d'une espèce constitue la base de la variation phénotypique héréditaire sur laquelle agit la sélection naturelle. Cependant, explorer le rôle des variants structurels (SV, plus de 50 paires de bases) sur la variation des traits reste un défi en raison de la difficulté à les détecter. Cette thèse vise à étudier l'impact phénotypique de ces variants en utilisant une population de plus de 1000 isolats de la levure Saccharomyces cerevisiae. La construction du pangénome à l'aide d'assemblages quasi-télomères-à-télomères a permis la création d'un catalogue complet de variants génomiques. Des études d'association avec plus de 8 000 caractères ont révélé l'impact relativement plus important des SV sur la variation des caractères, ainsi que des divergences entre l’architecture génétique de différents types de caractères. L'ensemble de ces travaux met en évidence le large impact phénotypique des grands variants génomiques au niveau de l'espèce<br>Genomic variation within a species provides the basis for the heritable phenotypic variation upon which natural selection acts. However, exploring the role of structural variants (SVs, more than 50 base pairs) on trait variation remains a challenge due to the difficulty of detecting them. This thesis research aims to address the phenotypic impact of such variants by leveraging a natural population of over a thousand isolates of the budding yeast Saccharomyces cerevisiae. Pangenome construction using near telomere-to-telomere assemblies enabled the creation of a comprehensive catalog of genomic variants. Association studies with more than 8,000 molecular and organismal traits revealed the relatively higher impact of SVs on traits variation, and discrepancies in the genomic basis of different types of traits. Together, this work highlights the strong phenotypic effect of large genomic variants at the species level

Orientadores: Maria de Fatima Sonati, Munir Salomão Skaf<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-23T23:13:39Z (GMT). No. of bitstreams: 1 Jorge_SusanElisabethDominguesCosta_D.pdf: 8714965 bytes, checksum: 3191d67be1e9be2f9782ce3483bcfd3a (MD5) Previous issue date: 2013<br>Resumo: O resumo poderá ser visualizado no texto completo da tese digital<br>Abstract: The complete abstract is available with the full electronic document<br>Doutorado<br>Ciencias Biomedicas<br>Doutora em Ciências Médicas

Les variants de structure (SVs) sont des réarrangements génomiques de plus de 50 paires de base et restent encore aujourd'hui peu étudiés malgré les impacts importants qu'ils peuvent avoir sur le fonctionnement des génomes. Récemment, les technologies de séquençage de troisième génération ont été développées et produisent des données de longues lectures qui s'avèrent très utiles car elles peuvent chevaucher les réarrangements. À l'heure actuelle, les méthodes bioinformatiques se sont concentrées sur le problème de la découverte de SVs avec des données de longues lectures. Aucune méthode n'a cependant été proposée pour répondre spécifiquement à la question du génotypage de SVs avec ce même type de données. L'objectif du génotypage de SVs vise pour un ensemble de SVs donné à évaluer les allèles présents dans un nouvel échantillon séquencé. Cette thèse propose une nouvelle méthode pour génotyper des SVs avec des longues lectures et repose sur la représentation des séquences des allèles. Notre méthode a été implémentée dans l'outil SVJedi. Nous avons testé notre outil à la fois sur des données simulées et réelles afin de valider notre méthode. SVJedi obtient une précision élevée qui dépasse les performances des autres outils de génotypage de SVs, notamment des outils de détection de SVs et des outils de génotypage de SVs de lectures courtes<br>Structural Variants (SVs) are genomic rearrangements of more than 50 base pairs. Since SVs can reach several thousand base pairs, they can have huge impacts on genome functions, studying SVs is, therefore, of great interest. Recently, a new generation of sequencing technologies has been developed and produce long read data of tens of thousand of base pairs which are particularly useful for spanning over SV breakpoints. So far, bioinformatics methods have focused on the SV discovery problem with long read data. However, no method has been proposed to specifically address the issue of genotyping SVs with long read data. The purpose of SV genotyping is to assess for each variant of a given input set which alleles are present in a newly sequenced sample. This thesis proposes a new method for genotyping SVs with long read data, based on the representation of each allele sequences. We also defined a set of conditions to consider a read as supporting an allele. Our method has been implemented in a tool called SVJedi. Our tool has been validated on both simulated and real human data and achieves high genotyping accuracy. We show that SVJedi obtains better performances than other existing long read genotyping tools and we also demonstrate that SV genotyping is considerably improved with SVJedi compared to other approaches, namely SV discovery and short read SV genotyping approaches

Mestrado em Biomedicina Molecular<br>A azoospermia afeta aproximadamente 15% de todos os homens inférteis e é frequentemente causada por anomalias cromossómicas e microdeleções do cromossoma Y. No entanto, em aproximadamente 70% dos casos de azoospermia não-obstrutiva (NOA) as causas permanecem por identificar. Nos últimos anos, a descoberta de variantes genómicas de número de cópia (CNVs), como as causadas por deleções, revelou uma fonte de variação genómica que afecta a dosagem génica e que poderá resultar em haploinsuficiência. De facto, observa-se uma sobre-representação de CNVs raros (<1% na população), sobretudo de grandes deleções de novo, em pacientes com diferentes distúrbios do desenvolvimento, comparados com controlos saudáveis. Porém, uma possível contribuição, para a infertilidade masculina, de variantes estruturais ligados ao cromossoma X e aos autossomas foi ainda pouco explorada. Este estudo foca-se na validação de deleções encontradas apenas em pacientes inférteis, no cromossoma X e em 11p13, que contêm genes candidatos a participar na espermatogénese. Estas deleções, previamente identificadas por arrays de oligonucleótidos, de elevada densidade (Affymetrix 6.0 SNP Array), numa coorte de 171 pacientes Portugueses com disfunção severa da espermatogénese (NOA e oligozoospermia severa), foram agora confirmadas por técnicas convencionais de genética molecular. Adicionalmente, a caraterização dos locais de quebra nestas deleções foi realizada por aCGH. Ainda que não se tenham validado as deleções menos extensas (em Xq21.1, Xq25, Xp11.4, Xq22.1 e Xq26.3), confirmou-se a nulizigotia em Xq28 nestes indivíduos, que abrange genes candidatos com uma função sugestiva na espermatogénese: MAGE-A8, expresso em testículo e em alguns cancros e o microRNA hsa-miR-4330, envolvido na regulação pós-transcricional de vários genes com expressão na linha germinal. Foi ainda validada, por MLPA, uma deleção extensa num paciente infértil não-sindrómico da nossa coorte. Estes resultados apontam a haploinsuficiência de WT1 como a causa mais provável de azoospermia neste paciente, já que não foram detetadas mutações germinais no alelo restante. Mutações no gene WT1, que codifica um factor de transcrição muito conservado, crucial para o desenvolvimento e manutenção gonadal em mamíferos, geralmente interferem com a ligação desta proteína ao DNA e estão principalmente associadas a síndromes que envolvem anomalias reprodutivas. Motivados pela nossa descoberta de uma deleção de WT1 num homem infértil embora saudável, decidimos abordar a contribuição de mutações exónicas no gene WT1 para a azoospermia isolada. Testámos a hipótese de que mutações localizadas em domínios que não aqueles essenciais à ligação ao DNA pudessem resultar na disfunção não-sindrómica da espermatogénese. Assim, analisámos a sequência codificante de WT1 num subgrupo de 40 pacientes azoospérmicos. Como resultado, descrevemos uma nova variação missense c.185C>T (P130L; ENST00000332351) no primeiro exão de WT1, inserida no domínio proteico de auto-associação. A nova variante descrita deverá ter um impacto menos drástico na função da proteína WT1, comparativamente com as mutações descritas no mesmo exão até à data, as quais resultam em proteínas truncadas e fenótipos severos de disfunção gonadal, incluindo a formação de tumores renais. Estes resultados revelam novos genes candidatos a um papel na espermatogénese e sugerem que a haploinsuficiência de proteínas importantes para o desenvolvimento do sistema reprodutor masculino podem resultar em azoospermia. Estudos futuros poderão clarificar a utilidade dos nossos genes candidatos como biomarcadores da infertilidade masculina. A implementação de novos biomarcadores beneficiaria os doentes azoospérmicos através da melhoria do diagnóstico, aconselhamento genético e acompanhamento destes pacientes, podendo vir a limitar a necessidade de procedimentos invasivos.<br>Azoospermia affects approximately 15% of all infertile males and it is frequently caused by chromosomal abnormalities and Yq microdeletions. However, despite considerable research efforts in the last decades, in approximately 70% of the cases of non-obstructive azoospermia (NOA) the causes are yet to be identified. In the last years, the discovery of genomic copy number variants, such as those caused by deletions, revealed a source of genomic variation which impacts gene dosage and may result in haploinsufficiency. In fact, rare CNVs (<1% population), mainly large de novo deletions, are over-represented in patients with different developmental disorders, compared to healthy controls. However, a possible contribution of X-linked and autosomal structural variants to male infertility is still largely unexplored. This study focused on the validation of rare patient-specific deletions found on the X chromosome and at 11p13 of infertile patients, which harbor candidate spermatogenesis genes. These deletions had been previously identified by high density oligonucleotide arrays (Affymetrix 6.0 SNP Array), in a cohort of 171 Portuguese patients with severe spermatogenic impairment (non-obstructive azoospermia and severe oligozoospermia) and were now confirmed by conventional molecular genetics techniques. Additionally, breakpoint characterization was carried out by aCGH. In fact, even though the smaller deletions (at Xq21.1, Xq25, Xp11.4, Xq22.1 and Xq26.3) were not validated, we confirmed nullizygosity at Xq28 in two patients, spanning either MAGE-A8, a known cancer-testis antigen, or hsa-miR-4330, a microRNA involved in post-transcription regulation, both with a suggestive role in spermatogenesis pathways. We have also validated by MLPA a large deletion at 11p13, in a non-syndromic infertile patient from our cohort. These results support WT1 haploinsufficiency as the likely cause of azoospermia in this patient, as no other germline mutations were detected in the remaining WT1 copy. Mutations in WT1, an evolutionarily conserved transcription factor crucial for gonadal development and maintenance in mammals, typically interfere with the DNA-binding properties of the protein and are mainly associated with syndromes involving reproductive abnormalities. Motivated by our finding of a WT1 deletion in an infertile but otherwise healthy man we addressed the contribution of WT1 exonic mutations to isolated azoospermia. We reasoned that mutations located in domains not essential for DNA binding could result in non-syndromic spermatogenic impairment. Thus, we analyzed the WT1 coding sequence in a subgroup of 40 azoospermic patients. As a result of the exon screening, we report a novel c.185C>T (P130L; ENST00000332351) WT1 missense variant on exon 1, within the protein self-association domain. While all exon 1 mutations as yet reported result in truncated proteins and severe phenotypes, including the formation of renal tumors, this novel variant is expected to have a milder impact on WT1 function. These results reveal new candidate genes for a role in spermatogenesis and suggest that haploinsufficiency of proteins important for the development of the male reproductive system can lead to azoospermia. Further studies will clarify the utility of our candidate genes as biomarkers of male infertility. The implementation of new biomarkers would benefit azoospermic men by improving diagnosis, genetic counseling and patient care, eventually limiting the need for invasive procedures.

El genoma humà està constituït per 3 bilions de parells de bases, que inclouen aproximadament 20.000-25.000 gens, i presenta una variabilitat en forma de variants en la seqüència i variants estructurals. L’aparició de noves tecnologies moleculars han revelat l’existència d’una gran quantitat de variants en nombre de còpies (CNVs) que representen canvis de dosi (guanys i pèrdues de material) descrits en el 4,8%-9,5% del genoma. Tot i identificar-se en població sana, s’ha reconegut que tenen una contribució en l’expressió gènica, l’estructura proteica i l’estabilitat cromosòmica i, en conseqüència, ha incrementat l’interès per entendre el paper que tenen les CNVs en malalties com la discapacitat intel·lectual i els trastorns psiquiàtrics. La discapacitat intel·lectual afecta entre l’1-3% dels individus i les millores en el seguiment mèdic dels pacients han contribuït en una major supervivència fins a etapes adultes, en les que es posa de manifest una gran incidència de trastorns psiquiàtrics i de la conducta associats. Amb l’objectiu de determinar l’etiologia genètica del diagnòstic dual de discapacitat intel·lectual i trastorns psiquiàtrics i/o de la conducta en una cohort de 100 adults i identificar CNVs de susceptibilitat per aquesta patologia, s’ha aplicat una estratègia d’anàlisi genètica seqüencial. Inicialment es realitza un cariotip amb bandes G, un cribatge de la síndrome X fràgil i estudis moleculars dirigits a la confirmació de la sospita clínica d’una síndrome específica. Per aquells casos que són negatius, es realitza un cribatge de CNVs submicroscòpiques de les regions subtelomèriques mitjançant multiplex ligation dependent probe amplification i posteriorment un cribatge del genoma amb un array d’hibridació genòmica comparada(aCGH) d’alta resolució (400k). S’ha establert una elevada freqüència diagnòstica (38%) en la cohort d’adults amb diagnòstic dual. La co-morbiditat d’un segon trastorn psiquiàtric augmenta la probabilitat de causa genètica. S’ha determinat un alt rendiment diagnòstic del cariotip molecular, pel que es proposa l’aCGH com a primera tècnica per l’estudi del diagnòstic dual. Mitjançant la caracterització de les CNVs, s’han identificat gens candidats que predisposen a discapacitat intel·lectual i trastorns psiquiàtrics, majoritàriament implicats en les primeres etapes del desenvolupament, amb expressió a sistema nerviós i de localització sinàptica. Hi ha gens que participen en les vies glutamatèrgiques i de les ubiquitines i gens implicats en mecanismes oxidatius. La valoració del grau de discapacitat intel·lectual, dels trastorns psiquiàtrics, dels trastorns de la conducta i la dismorfologia presents en els pacients ha permès establir una correlació genotip-fenotip, identificant CNVs associades al diagnòstic dual en el 19% dels casos i CNVs en regions candidates: dup3q29 (FBXO45, PAK2), del7q31.1 (IMMP2L), del8p23.1 (MSRA), del8q21.13 (STMN2), dup9p24.2p24.1 (SLC1A1), del10q21.3 (CTNNA3), dup15q14q15.1 (SPRED1), del15q26.2 (MCTP2), dup17q24.1q24.2 (PRKCA). Es determina que la deleció 2p16.3 és un factor de risc per discapacitat intel·lectual i trastorns psiquiàtrics amb una expressivitat variable. Es descriu per primer cop un fenotip dismòrfic comú entre els adults afectats i l’avaluació clínica dels familiars portadors identifica un patró cognitiu i psiquiàtric comú amb diferents nivells de severitat a tots els portadors de la deleció. L’estudi d’una població adulta aporta nombrosos avantatges, tant als pacients com als familiars, per adequar el pronòstic, seguiment, tractament i consell genètic. A més a més, el coneixement obtingut en pacients adults amb trastorns psiquiàtric pot ser de gran utilitat pels infants amb discapacitat intel·lectual, ja que el diagnòstic precoç n’afavoreix la prevenció mitjançant un seguiment i tractaments específics.<br>ABSTRACT The human genome contains nearly 3 billion base pairs that include around 20.000-25.000 genes. There are two sources of genetic variation among individuals: single nucleotide variants and structural variants. The improvement of molecular technologies has revealed a large amount of copy number variants (CNVs), which represents dose changes (gains and losses) in about 4,8%-9,5% of the genome. The CNVs contribute to the gene expression, protein structure and chromosome stability even if they are found in healthy people. Consequently, there has been a significant increase in the interest to understand the role of CNVs in diseases, such as intellectual disability and psychiatric disorders. Intellectual disability affects between 1¬3% of human population. With improvement in paediatric care, patients are most likely to survive into adulthood, in which is revealed a high incidence of psychiatric and behaviouraldisorders associated. In order to identify the genetic aetiology of dual diagnosis of intellectual disability and psychiatric and/or behavioural disorders in a cohort of 100 adults and to identify CNVs of disease susceptibility, a sequential genetic test workflow was performed. Firstly, G-banded karyotype, Fragile X syndrome screening and specific molecular technologies targeted to confirm a clinical suspicious of a syndrome were applied. In those negative cases, subtelomeric region screening by multiplex ligation dependent probe amplification and then a whole genome screening by high resolution (400k) comparative genomic hybridization array (CGHa) were performed. A high genetic diagnosis frequency (38%) has established in the adult cohort with dual diagnosis. The co-morbidity of a second psychiatric disorder increases the likelihood of genetic cause. The CNV characterization has identified candidate genes for intellectual disability and psychiatric disorder, mostly involved in the early stages of development, high expression in nervous system and synaptic localization. Some genes identified are involved in glutamatergic and ubiquitin pathways or in oxidative status. The assessment of the intellectual disability degree, psychiatric/behavioural disorders and dismorphology allowed us to establish a genotype-phenotype correlation. It has been identified CNVs associated with dual diagnosis in 19% of cases and CNVs in candidate regions: dup3q29 (FBXO45, PAK2) del7q31.1 (IMMP2L) del8p23.1 (MSRA) del8q21.13 (STMN2) dup9p24.2p24.1 (SLC1A1) del10q21.3 (CTNNA3) dup15q14q15.1 (SPRED1) del15q26.2 (MCTP2) dup17q24.1q24.2 (PRKCA). The 2p16.3 deletion is an intellectual disability and a psychiatric disorder risk factor with variable expressivity. For the first time, it has been described a common dysmorphic phenotype on those patients affected by a 2p16.3 deletion in addition to a common cognitive and psychiatric profile with different levels of severity among all carriers. Studies in an adult population provide numerous advantages in both patients and family members. Genetic diagnosis allows to adequate the prognosis, monitoring, treatment and genetic counselling. Moreover, the knowledge obtained in adult patients with psychiatric disorders can be useful for children affected by intellectual disability. The early diagnosis promotes prevention through monitoring and specific treatments.

Submitted by Fernanda Rodrigues de Lima (fernanda.rlima@ufpe.br) on 2018-08-01T20:49:02Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Francisco do Nascimento Junior.pdf: 1104753 bytes, checksum: 794ee127f9a27d065eb71104d4849c0e (MD5)<br>Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-08-03T19:38:31Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Francisco do Nascimento Junior.pdf: 1104753 bytes, checksum: 794ee127f9a27d065eb71104d4849c0e (MD5)<br>Made available in DSpace on 2018-08-03T19:38:31Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Francisco do Nascimento Junior.pdf: 1104753 bytes, checksum: 794ee127f9a27d065eb71104d4849c0e (MD5) Previous issue date: 2017-02-17<br>CAPES<br>The importance of structural variants as a source of phenotypic variation has grown in recent years. At the same time, the number of tools that detect structural variations using Next- Generation Sequencing (NGS) has increased considerably with the dramatic drop in the cost of sequencing in last ten years. Then evaluating properly the detected structural variants has been featured prominently due to the uncertainty of such alterations, bringing important implications for researchers and clinicians on scrutinizing thoroughly the human genome. These trends have raised interest about careful procedures for assessing the outcomes from variant calling tools. Here, we characterize the relevant technical details of the detection of structural variants, which can affect the accuracy of detection methods and also we discuss the most important caveats related to the tool evaluation process. This study emphasizes common assumptions, a variety of possible limitations, and valuable insights extracted from the state-of-the-art in CNV (Copy Number Variation) detection tools. Among such points, a frequently mentioned and extremely important is the lack of a gold standard of structural variants, and its impact on the evaluation of existing detection tools. Next, this document describes a biclustering-based methodology to screen a collection of structural variants and provide a set of reliable events, based on a defined equivalence criterion, that is supported by different studies. Finally, we carry out experiments with the proposed methodology using as input data the Database of Genomic Variants (DGV). We found relevant groups of equivalent variants across different studies. In summary, this thesis shows that there is an alternative approach to solving the open problem of the lack of gold standard for evaluating structural variants.<br>A importância das variantes estruturais como fonte de variação fenotípica tem se proliferado nos últimos anos. Ao mesmo tempo, o número de ferramentas que detectam variações estruturais usando Next-Generation Sequencing (NGS) aumentou consideravelmente com a dramática queda no custo de seqüenciamento nos últimos dez anos. Neste cenário, avaliar corretamente as variantes estruturais detectadas tem recebido destaque proeminente devido à incerteza de tais alterações, trazendo implicações importantes para os pesquisadores e clínicos no exame minucioso do genoma humano. Essas tendências têm impulsionado o interesse em procedimentos criteriosos para avaliar os variantes identificados. Inicialmente, caracterizamos os detalhes técnicos relevantes em torno da detecção de variantes estruturais, os quais podem afetar a precisão. Além disso, apresentamos advertências fundamentais relacionadas ao processo de avaliação de uma ferramenta. Desta forma, este estudo enfatiza questões como suposições comuns à maioria das ferramentas, juntamente com limitações e vantagens extraídas do estadoda- arte em ferramentas de detecção de variantes estruturais. Entre esses pontos, há uma muito questão bastante citada que é a falta de um gold standard de variantes estruturais, e como sua ausência impacta na avaliação das ferramentas de detecção existentes. Em seguida, este documento descreve uma metodologia baseada em biclustering para pesquisar uma coleção de variantes estruturais e fornecer um conjunto de eventos confiáveis, com base em um critério de equivalência definido e apoiado por diferentes estudos. Finalmente, realizamos experimentos com essa metodologia usando o Database of Genomic Variants (DGV) como dados de entrada e encontramos grupos relevantes de variantes equivalentes em diferentes estudos. Desta forma, esta tese mostra que existe uma abordagem alternativa para o problema em aberto da falta de gold standard para avaliar variantes estruturais.