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Fusarium verticillioides è l’agente responsabile della Fusariosi della Spiga del mais, contamina la granella con fumonisine, micotossine responsabili di diverse patologie umane e animali. Per la resistenza alla fusariosi e all’accumulo di fumonisine esiste variabilità tra genotipi diversi ma non sono ancora disponibili ibridi immuni. L’obiettivo di questo lavoro è stato quello di individuare marcatori associati alla resistenza a F. verticillioides. Mediante un bioassay è stato testato un association panel per la resistenza a F. verticillioides. Al fine di identificare i marcatori di resistenza sono stati applicati un approccio GWAS e uno per geni candidati. L’analisi GWAS è stata eseguita con 227K SNPs restituendo 206 marcatori significativi. Da un lavoro di RNASequencing sono stati individuati i geni coinvolti nella risposta a F. verticillioides mentre i geni R sono stati recuperati della letteratura scientifica. Genotipi resistenti (CO433 e CO441) e suscettibili (CO354 e CO389) sono stati scelti per individuare polimorfismi nei geni candidati da associare ai fenotipi rilevati mediante il bioassay. Quattro marcatori sono risultati significativi. Infine, la correlazione tra l’incidenza della fusariosi rilevata in campo e mediante bioassay è stata analizzata in una popolazione di 172 RIL derivanti da CO441 x CO354, tuttavia, non è stata individuata alcuna corrispondenza. / Fusarium verticillioides is the causal agent of Fusarium ear rot (FER) in maize and contaminates grains with fumonisin, a family of mycotoxins involved in several human and animal diseases. Quantitative genetic variation exists for resistance to FER and fumonisin contamination among genotypes, however, resistant maize hybrids are currently not available. The aim of this work was the identification of genetic markers associated to resistance against F. verticillioides. A bioassay was used to screen inbred lines of the maize association population for FER resistance, GWAS and candidate gene approaches were applied to identify markers. GWAS was performed using a 227K SNP matrix and resulting in 206 significant markers. Genes involved in F. verticillioides response in developing maize kernels were retrieved from a previous RNASequencing study while maize R genes were retrieved from scientific literature. Resistant (CO433 and CO441) and susceptible genotypes (CO389 and CO354) were selected to amplify and sequence candidate genes. Polymorphisms detected were used to find association with phenotypes scored using the bioassay. Four significant markers were found. Finally, the correlation between FER phenotypes scored in field experiments and bioassay phenotypes was investigated. A population of 172 RILs (CO441 x CO354), was tested. No correlation was found.

La drépanocytose est une maladie causée par une seule mutation dans le gène de la bêta-globine. Les complications liées à la maladie se manifestent sur le plan génétique, épigénique, transcriptionnel, et métabolique. Les approches intégratives des technologies de séquençage à haut-débit permettent de comprendre le mécanisme pathologique et de découvrir des thérapies en lien avec la maladie. Dans cette thèse, j’intègre divers jeux de données omiques et j’applique des méthodes statistiques pour élaborer de nouvelles hypothèses et analyser les données. Dans les deux premières études, je combine les résultats des études d'association pangénomique d'hémoglobine fœtale (HbF) et des globules rouges denses déshydratés (DRBC) avec l'expression génique, l'interaction chromatinienne, les bases de données relatives aux maladies et les cibles médicamenteuses sélectionnées par des experts. Cette approche intégrative a révélé trois nouveaux loci sur le chromosome 10 (BICC1), le chromosome 19 (KLF1) et le chromosome 22 (CECR2) comme régulateurs de l'HbF. Pour l’étude sur la densité de globules rouges, quatre cibles médicamenteuses (BCL6, LRRC32, KNCJ14 et LETM1) ont été identifiées comme des modulateurs potentiels de la sévérité. Dans la troisième étude, j’intégre la métabolomique à la génomique pour établir une relation causale entre la L-glutamine et les crises douleurs en utilisant la randomisation mendélienne. En outre, nous avons identifié 66 biomarqueurs pour 6 complications liées à la drépanocytose et le débit de filtration glomérulaire estimé (DFGe). Enfin, dans la dernière étude j’ai appliqué une approche de clustering aux métabolites que j’ai ensuite combiné aux données de génotype. J’ai découvert des changements métabolomiques mettant en évidence des familles de métabolites impliqués dans les dysfonctionnements rénaux et hépatiques, en plus de confirmer le rôle d'une classe d'acides gras dans la formation en faucille des globules rouges. Ce travail met en évidence l'importance des approches multi-omiques pour découvrir de nouveaux mécanismes biologiques et étudier les maladies humaines. / Sickle cell disease is a monogenic disorder caused by a point mutation in the beta-globin gene. The complications related to the disease are characterized by a broad spectrum of distinct genetic, epigenetic, transcriptional, and metabolomic states. Integrative high-throughput technologies approaches to sickle cell disease pathophysiology are crucial to understanding complications mechanisms and uncovering therapeutic interventions. In this thesis, I integrate various omics datasets and apply statistical methods to derive new hypotheses and analyze data. I combine genome-wide association studies results of fetal hemoglobin (HbF) and dehydrated dense red blood cells (DRBC) with gene expression, chromatin interaction, disease-relevant databases, and expert-curated drug targets. This integrative approach revealed three novel loci on chromosome 10 (BICC1), chromosome 19 (KLF1) and chromosome 22 (CECR2) as key modulators of HbF. For DRBC, four drug targets (BCL6, LRRC32, KNCJ14, and LETM1) were identified as potential severity modifiers. Using mendelian randomization, I integrated metabolomics with genomics in the third study to establish a potential causal relationship between L-glutamine and painful crisis. Additionally, we identified 66 biomarkers for 6 SCD-related complications and estimated glomerular filtration rate (eGFR). Finally, the last study applied a clustering framework to metabolites which I then combined with genotypes. I found specific metabolomics changes highlighting families of metabolites involved in renal and liver dysfunction and confirming the role of a class of fatty acids in red blood cell sickling. This work highlights the importance of multi-omics approaches to unearth new biology and study human diseases.

The case-control design is one of the most commonly used designs in genome- wide asociation studies. When we increase the sample size of either the controls or, more importantly, the cases, the power of whatever test we use will certainly increase. However increasing the sample size, means that addi- tional individuals need to be genotyped and this implies extra financial costs. However, nowadays with the emergence of genetic studies, a large number of genetic data are available at low or no extra cost. Even though those data may not be completely relevant to the current study, they can still be used to increase the probability to identify true associations. Furthermore, additional information, non-necessarily genetic, can also be used to improve the power of a method. In this thesis we extend the case-control design in order to take ad- vantage of such types of additional data and/or information. We discuss three designs; the case-cohort-control, the kin-cohort and the super-case– case–control–super-control designs. For each of these, we present methods that are adjusted or modified versions of standard case-control methods but we also propose novel ones developed with those extended designs in mind. Ultimately, we describe how those methods can be used in order to increase the power of association tests, especially compared to similar methods of the case-control design.

The aims of this study were to analyse known and potential biomarkers of common and genetically complex human disorders and to identify genetic and environmental variation associated with plasma biomarker concentrations. Two groups of protein biomarkers were analysed. First, plasma complement factor H (CFH) was selected as a potential biomarker for age-related macular degeneration (AMD), since common variants in the CFH gene show strong association with this disorder. Secondly, two isoforms of amyloid-β (Aβ40 and Aβ42) were selected as biomarkers for Alzheimer disease (AD) since Aβ deposits are major constituents of the amyloid plaques characteristic of this disorder. Physiological and anthropometric measurements and samples of human and genomic DNA were collected from a population sample of 1,021 individuals from the Croatian island of Vis. Quantitative determination of plasma Aβ40 and Aβ42 concentrations was performed using enzyme-linked immunosorbent assays. Heritabilities and significant covariate effects were estimated for each trait in the Croatian data set. Genome-wide linkage and association analyses were conducted for the biomarker traits. A novel finding was the genome-wide significant association between a CFH and several polymorphisms close to and within the CFH gene. The strongest association was with an intronic SNP within CFH, which explained 28% of the total trait variance (P < 10-50). The association was also replicated in a Dutch sample set. A SNP haplotype was identified which accounted for a higher proportion of the phenotypic variance. Conditional haplotype analysis showed that the effect of this haplotype on plasma CFH concentration was independent of the CFH Y402H variant, and significantly stronger than a deletion of the adjacent CFHR3/CFHR1 which was already known to affect AMD susceptibility. Genetic analysis of 382 AMD cases and 201 controls was consistent with the CFH Y402H variant being the strongest AMD susceptibility locus. Variation in plasma CFH concentration was found to explain up to 1.8% of the variation in susceptibility to AMD with an odds 2.1 (95% C.I. 1.3-3.4, P = 0.003). SNPs that were strongly associated with a CFH concentration also influenced AMD susceptibility (P < 0.05) independently of the CFH Y402H polymorphism. Functional analysis of genomic regions associated with plasma CFH is needed to identify the causal variants. Associations were observed between plasma Aβ40 concentration and several novel candidate loci, spanning regions of approximately 0.2 Mb, on chromosomes 9 and X. Similarly, novel associations with plasma Aβ42 were found in several regions, each spanning 0.2-0.4 Mb, on chromosomes 2, 5, 9, 15 and 20. The proportion of the phenotypic variance in plasma Aβ42 explained by these putative associations ranged between 1.8 and 2.8%. However, none of the associated SNPs was significant after correction for multiple testing, therefore replication is required. Finally, attempts were made to identify and quantitate new protein biomarkers of disease in human plasma using mass spectrometry. Development and optimisation of techniques was initially undertaken to deplete high-abundance plasma proteins and improve signal:noise ratio. This allowed the assessment of downstream proteomic approaches including MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and ion exchange chromatography (IEC), each with the potential for large-scale quantitation of plasma proteins. Although the analysis of single protein analytes, using CE and IEC proved promising, the results highlighted the difficulty associated with MALDI-TOF and protein ionisation techniques in analysing complex mixtures such as plasma.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Genetic association studies simplify genotype‐phenotype relationship investigation by considering only the presence of a given polymorphism and the presence or absence of a given downstream phenotype. Although such associations do not indicate causation, collections of phenotypes sharing association with a single genetic polymorphism may provide valuable mechanistic insights. In this thesis we explore such genetic pleiotropy with Deep Phenotype Association Studies (DeePAS) using data from the Age‐Related Eye Study 2 (AREDS2). We also employ a novel text mining approach to extract pleiotropic associations from the published literature as a hypothesis generation mechanism. Is it possible to identify pleiotropic genetic associations across multiple published abstracts and validate these in data from AREDS2? Data from the AREDS2 trial includes 123 phenotypes including AMD features, other ocular conditions, cognitive function and cardiovascular, neurological, gastrointestinal and endocrine disease. A previously validated relationship extraction algorithm was used to isolate descriptions of genetic associations with these phenotypes in MEDLINE abstracts. Results were filtered to exclude negated findings and normalize variant mentions. Genotype data was available for 1826 AREDS2 participants. A DeePAS was performed by evaluating the association between selected SNPs and all available phenotypes. Associations that remained significant after Bonferroni‐correction were replicated in AREDS. LitWAS analysis identified 9372 SNPs with literature support for at least two distinct phenotypes, with an average of 3.1 phenotypes/SNP. PheWAS analyses revealed that two variants of the ARMS2‐HTRA1 locus at 10q26, rs10490924 and rs3750846, were significantly associated with sub‐retinal hemorrhage in AMD (rs3750846 OR 1.79 (1.41‐2.27), p=1.17*10‐7). This associated remained significant even in populations of participants with neovascular AMD. Furthermore, odds ratios for the development of sub‐retinal hemorrhage in the presence of the rs3750846 SNP were similar between incident and prevalent AREDS2 sub‐populations (OR: 1.94 vs 1.75). This association was also replicated in data from the AREDS trial. No literature‐defined pleiotropic associations tested remained significant after multiple‐testing correction. The rs3750846 variant of the ARMS2‐HTRA1 locus is associated with sub‐retinal hemorrhage. Automatic literature mining, when paired with clinical data, is a promising method for exploring genotype‐phenotype relationships.

A hipertensão essencial, um problema de saúde pública mundial, é uma doença multifatorial, cujo componente genético varia entre 25 a 60%. A despeito da alta prevalência e incidência da hipertensão essencial em populações de ancestralidade africana, os estudos sobre o componente genético da hipertensão essencial têm sido primariamente realizados em populações de ancestralidade européia ou asiática. Dessa maneira, o objetivo geral deste trabalho foi investigar e quantificar potenciais fatores de risco genéticos associados à hipertensão essencial em populações dos remanescentes de quilombos do Vale do Ribeira SP, indivíduos afro-descendentes, oriundos de populações parcialmente isoladas, previamente bem caracterizadas do ponto de vista clínico, genealógico e genético-populacional. A amostra foi constituída de 759 indivíduos adultos com estudo clínico e antropométrico oriundos das seguintes populações quilombolas: Abobral, Galvão, São Pedro, Pedro Cubas, André Lopes, Nhunguara, Sapatu, Pilões, Ivaporunduva, Maria Rosa, Poça e Reginaldo, localizadas no Vale do Ribeira, no estado de São Paulo. Estimativas de ancestralidade (baseadas em 48 marcadores INDEL autossômicos) sugerem que a contribuição africana, européia e ameríndia na amostra estudada é de aproximadamente 39,7%, 39,0% e 21,3%, respectivamente (N=307), sendo igualmente distribuídas em normotensos e hipertensos (37,6%, 39,0% e 19,9% e 41,1%, 37,8% e 20,4%, P = 0,845). O estudo dos fatores de risco genéticos associados à hipertensão foi realizado por duas abordagens: (i) um estudo transversal com indivíduos não aparentados (N = 383) e (ii) um estudo de associação baseado em famílias (N total = 759, 79 famílias). Foram selecionados para estes estudos sete polimorfismos (I/D, Glu298Asp, C825T, G-350A, M235T, A/C, A486V) em seis genes candidatos com forte base funcional: ACE, NOS3, GNB3, AGT, ADD2 e GRK4. As análises do estudo transversal revelaram uma associação significativa entre o polimorfismo C825T do GNB3 e a hipertensão essencial (P=0,036), quando consideramos o fenótipo mais extremo (PAS maior igaul a 160mmHg e/ou PAD maior igual a 95mmHg, ou uso de medicamentos anti-hipertensivos). Análises haplotípicas do GNB3 (variantes G-350A e C825T) revelaram que cada cópia do haplótipo C825/G-350 aumenta em 1,73 a chance de hipertensão em relação ao haplótipo basal T825/G-350 (OR = 1,73; IC 95% = 1,05 2,84, P = 0,029). O haplótipo C825/G-350 estaria relacionado com um aumento na média esperada da PAS e da PAD (Delta = 6,07 mmHg; IC de 95%: 1,64 10,50 mmHg; P = 0,007 e Delta = 3.75 mmHg; IC de 95%: 1,16 6,35 mmHg; P = 0,005, respectivamente). Estudos de interação gene-gene também foram conduzidos pela técnica de redução de dimensionalidade de múltiplos fatores generalizada (GMDR). Os modelos de predição por GMDR revelaram interações de alta ordem entre as variantes avaliadas e o risco de hipertensão essencial. Os melhores modelos de interação foram: NOS3/GRK4, ACE/GNB3/AGT e ACE/GNB3/AGT/ADD2/GRK4, com uma acurácia média balanceada de teste de 60% (P=0,006), 59% (P =0,019), e 59% (P = 0,025), respectivamente. As análises de segregação em famílias indicaram uma associação significativa entre o polimorfismo G-350A do gene GNB3 e a hipertensão essencial, quando consideramos o fenótipo classificado de acordo com o critério da OMS (PAS maior igual a 140mmHg e/ou PAD maior igual a 90mmHg, ou uso de medicamentos anti-hipertensivos, P = 0,018), e também quando consideramos o fenótipo mais extremo (PAS maior igual a 160mmHg e/ou PAD maior igual a 95mmHg, ou uso de medicamentos anti-hipertensivos, P = 0,001). Finalmente, as estimativas da herdabilidade corroboram a noção de que o componente genético da pressão arterial é elevado nessas populações: herdabilidades (ajustadas para idade, gênero e IMC) de 36,1 e 42.9% para PAS e PAD, respectivamente. Apresentamos nesse trabalho uma investigação extensiva e original sobre o componente genético da hipertensão essencial em uma população brasileira de afrodescendentes (com características históricas, genéticas e ambientais peculiares), reforçando a hipótese de um mecanismo poligênico de regulação da pressão arterial nas populações remanescentes de quilombos do Vale do Ribeira - SP. / Essential hypertension is a worldwide public health problem. Although this pathology has a multi-factorial etiology, the heritability of blood pressure is high, ranging from 25 to 60%. Despite the high prevalence and incidence of essential hypertension among subjects of African ancestry, most genetic association studies has been carried out either in subjects with European descent or in Asian-derived individuals. Thus, the aim of this study was to investigate genetic risk factors putatively associated with essential hypertension in the quilombo populations, which are partially isolated African-derived populations, well characterized in terms of clinical, genealogical and population data, living in the Ribeira River Valley São Paulo, Brazil. A total of 759 adults with clinical and anthropometric data sampled from the 12 populations (Abobral, Galvão, São Pedro, Pedro Cubas, André Lopes, Nhunguara, Sapatu, Pilões, Ivaporunduva, Maria Rosa, Poça and Reginaldo) were studied. Ancestry estimates (based on 48 ancestry informative INDEL markers) suggested that the African, European and Amerindian contributions were 39.7%, 39.0% and 21.3% respectively (N = 307) to the studied sample. These proportions did not differ significantly between hypertensive and normotensive subjects(41.1%, 37.8% , 20.4% and 37.6%, 39.0% e 19.9%, respectively; P = 0.845). Two approaches were employed to investigate potential genetic risk factors for essential hypertension: (i) a cross-sectional study with unrelated individuals (N = 383) and (ii) a family-based association study (N = 759, 79 families). Seven polymorphisms (I/D, Glu298Asp, C825T, G-350A, M235T, A/C, A486V) in six candidate genes with strong functional basis (ACE, NOS3, GNB3, AGT, ADD2 and GRK4) were selected. The cross-sectional study revealed a significant association between the C825T GNB3 polymorphism and essential hypertension (P = 0.036) defined as a SBP > or = 160 mmHg and/or DBP > or = 95 mmHg, or use antihypertensive drugs). Haplotypic analyses of both G-350A and C825T GNB3 variants indicated that each copy of the haplotype C825/G-350 increases in 1.73 the odds of hypertension compared to the reference T825/G-350 haplotype (OR = 1.73, CI 95% = 1.05 - 2.84, P = 0.029). The haplotype C825/G-350 was also associated with augmented levels of both SBP and DBP (expected mean difference, Delta = 6.7, 95% CI: 1.64 10.50, P = 0.007 and Delta = 3.75, CI 95%: 1.16 6.35, P = 0.005, respectively) compared to the reference haplotype. Gene-gene interactions were also investigated by the use of the generalized multifactor dimensionality reduction (GMDR). GMDR prediction models revealed high-order interactions for hypertension between and among the studied variants. The best interaction models were NOS3/GRK4, ACE/GNB3/AGT, and ACE/GNB3/AGT/ADD2/GRK4, with an average testing balanced accuracy of 60% (P = 0.006) 59% (P = 0.019), and 59% (P = 0.025), respectively. The family-based association study showed a significant association of the GNB3 G-350 allele with essential hypertension (P = 0.018) when the diagnosis of essential hypertension was based on the WHO criteria (SBP > or = 140mmHg and / or DBP > or = 90 mmHg, or use of antihypertensive drugs, P = 0.018). Qualitatively analogous results were observed when a more extreme phenotype was considered (i.e. hypertension defined as a SBP > or = 160mmHg and / or DBP > or = 95 mmHg, or use of antihypertensive drugs, P = 0.001) Finally, heritability estimates corroborated the notion that the genetic component of blood pressure is high in these populations. Heritability (adjusted for age, gender and BMI) estimates of 36.1 and 42.9% for SBP and DBP, respectively, were obtained. We presented in this work an extensive and original investigation about the genetic component of essential hypertension in an African-Brazilian population (with peculiar historical, genetical and environmental characteristics). This study strengthens the hypothesis of a polygenic mechanism of regulation of blood pressure in the quilombos from Ribeira River Valley São Paulo.

Doctor of Philosophy / Genetics Interdepartmental Program / Jesse A. Poland / A central question in modern biology is to understand the genotype-to-phenotype (G2P) link, that is, how the genetics of an organism results in specific characteristics. However, prediction of phenotypes from genotypes is a difficult problem due to the complex nature of genomes, the environment, and their interactions. While the recent advancements in genome sequencing technologies have provided almost unlimited access to high-density genetic markers, large-scale rapid and accurate phenotyping of complex plant traits remains a major bottleneck. Here, we demonstrate field-based complex trait assessment approaches using a commercially available light-weight Unmanned Aerial Systems (UAS). By deploying novel data acquisition and processing pipelines, we quantified lodging, ground cover, and crop growth rate of 1745 advanced spring wheat lines at multiple time-points over the course of three field seasons at three field sites in South Asia. High correlations of digital measures to visual estimates and superior broad-sense heritability demonstrate these approaches are amenable for reproducible assessment of complex plant traits in large breeding nurseries. Using these validated high-throughput measurements, we applied genome-wide association and prediction models to assess the underlying genetic architecture and genetic control. Our results suggest a diffuse genetic architecture for lodging and ground cover in wheat, but heritable genetic variation for prediction and selection in breeding programs. The logistic regression-derived parameters of dynamic plant height exhibited strong physiological linkages with several developmental and agronomic traits, suggesting the potential targets of selection and the associated tradeoffs. Taken together, our highly reproducible approaches provide a proof-of-concept application of UAS-based phenomics that is scalable to tens-of-thousands of plots in breeding and genetic studies as will be needed to understand the G2P and increase the rate of gain for complex traits in crop breeding.

Recently, the pace of growth in the amount of data sources within Life Sciences has increased exponentially until pose a difficult problem to efficiently manage their integration. The data avalanche we are experiencing may be significant for a turning point in science, with a change of orientation from proprietary to publicly available data and a concomitant acceptance of studies based on the latter. To investigate these issues, a Network of Excellence (EMBRACE) was launched with the aim to integrate the major databases and the most popular bioinformatics software tools. The focus of this thesis is therefore to approach the problem of seamlessly integrating varied data sources and/or distributed research tools. In paper I, we have developed a web service to facilitate allergenicity risk assessment, based on allergen descriptors, in order to characterize proteins with the potential for sensitization and cross-reactivity. In paper II, a web service was developed which uses a lightweight protocol to integrate human endogenous retrovirus (ERV) data within a public genome browser. This new data catalogue and many other publicly available sources were integrated and tested in a bioinformatics-rich client application. In paper III, GeneFinder, a distributed tool for genome-wide association studies, was developed and tested. Useful information based on a particular genomic region can be easily retrieved and assessed. Finally, in paper IV, we developed a prototype pipeline to mine the dog genome for endogenous retroviruses and displaying the transcriptional landscape of these retroviral integrations. Moreover, we further characterized a group that until this point was believed to be primate-specific. Our results also revealed that the dog has been very effective in protecting itself from such integrations. This work integrates different applications in the fields of protein allergology, biotechnology, genome association studies and endogenous retroviruses. / EMBRACE NoE EU FP6