New Approaches to Use Genomics, Field Traits, and High-throughput Phenotyping for Gene Discovery in Maize (\u3ci\u3eZea mays\u3c/i\u3e)

Maize is one of major crop species over the world. With lots of genetic resources and genomic tools, maize also serves as a model species to understand genetic diversity, facilitate the development of trait extraction algorithms and map candidate functional genes. Since the first version of widely used B73 reference genome was released, independent research groups in the maize community propagated seeds themselves for further research purposes. However, unexpected or occasional contamination may happen during this process. The first study in this thesis used public RNA-seq data of B73 from 27 research groups across three countries for calling single nucleotide polymorphisms (SNP). Those SNPs were applied for investigating the distance of 27 maize B73 samples from the reference genome and three major clades were defined for determining their original sources. On the other side, maize is a plant with clear plant architecture. The second study was to employ the high-throughput plant phenotyping to dissect plant phenotypes using computer vision methods. A total of 32 maize inbreds distributed from the Genomes to Fields project were captured images in daily by 4 types of cameras (RGB, Hyperspectral, Fluorescence and Thermal-IR) for approximate 1 month. Differences between computer vision measurements and manual measurements about the plant fresh biomass were evaluated. Broad-sense heritability was estimated for extracted measurements from images. The expanded types of plant phenotype from the perspective of imaging provided a broader range of opportunities for connecting phenotypic variants with genetic variants. The third study utilized the phenome-wide variants in maize Goodman-Buckler 282 association panel to scan and associate with genetic variants of annotated genes along the maize genome. Genes detected by the proposed model, Genome-Phenome Wide Association Study (GPWAS), are significantly different from conventional GWAS detected genes. GPWAS genes tend to be more functionally conserved and more similar as classical maize mutants with known functions. Results from these researches assist to answer question about the genetic purity of same maize genotype. Methods developed in this thesis can also provide the valuable reference for trait discoveries from images and candidate functional gene identification using a broad set of phenotypes. Adviser: James C. Schnabl

Genetic factors involved in stroke susceptibility and in outcome at three months

Tese de doutoramento, Biologia (Genética), Universidade de Lisboa, Faculdade de Ciências, 2011Stroke is a significant cause of death and disability in developed countries. It is a multifactorial disease, resulting from the interplay between genes and well-known life-style/environmental risk factors. Numerous studies have attempted to identify the genetic risk factors predisposing to stroke, but few have investigated the genetic factors involved in stroke outcome. This work aimed at the identification of genes contributing to stroke and influencing patient’s outcome after three months. Four inflammatory genes (IL1B, IL6, MPO and TNF) and two genes involved in the nitric oxide metabolism (NOS1 and NOS3) were tested for association with stroke. The results suggest that the IL6 and MPO genes influence stroke susceptibility through independent effects and non-additive interactions. Furthermore, they provided novel evidence for the involvement of the NOS1 gene in stroke susceptibility. Several studies have shown the important impact of oxidative stress, inflammation, angiogenesis, neurogenesis, neurovascular damage and neurovascular remodeling for stroke-associated brain damage and/or stroke recovery. Association analyses were thus carried out to assess the role of candidate genes involved in inflammatory processes (IL1B, IL6, MPO and TNF) and oxidative stress (NOS1 and NOS3), as well as matrix metalloproteinase genes (MMP2 and MMP9) and growth factor genes (BDNF, FGF2 and VEGFA) in patient’s outcome at three months. MMP2 genetic variants were found associated with patient’s outcome, and the results also indicate that two epistatic interactions between the BDNF and FGF2 genes and between the FGF2 and VEGFA genes influence this phenotype. A genome-wide association study was performed in stroke outcome using DNA pooled samples to provide novel insights into the mechanisms involved in stroke recovery. The BBS9 and GLIS3 genes were found associated with patient’s outcome at three months. Taken together, these results suggest that stroke susceptibility and outcome are modulated by a combination of main gene effects and gene-gene interactions, independently of stroke risk factors and/or severity parameters, highlighting the complexity of mechanisms predisposing to stroke and influencing recovery afterwards.O Acidente Vascular Cerebral (AVC) é uma das principais causas de morte e incapacidade permanente nos países desenvolvidos, tendo importantes consequências económicas e sociais. O AVC é considerado uma doença complexa, que resulta de uma acção combinada entre genes e factores de risco ambientais ou de estilo de vida. Muitos estudos foram já levados a cabo com o intuito de identificar os factores genéticos de risco para o AVC, mas os resultados têm sido inconsistentes. A maioria desses estudos analisou o papel de genes candidatos; mais recentemente, foram também realizados estudos de associação ao nível de todo o genoma. Ao contrário da susceptibilidade, poucos estudos procuraram identificar quais os factores genéticos envolvidos na recuperação após um AVC. Este trabalho de doutoramento pretendeu assim identificar genes que contribuam para a susceptibilidade ao AVC e que influenciem o estado de incapacidade funcional (outcome) do doente ao fim de três meses de recuperação. Para isso, foram utilizadas duas estratégias distintas. A primeira estratégia consistiu na análise do papel de genes candidatos nos dois fenótipos referidos anteriormente, genes esses que tinham sido escolhidos com base na sua função, nos resultados de estudos de associação anteriores e/ou nos resultados de estudos com modelos animais de AVC, factores de risco ou condições médicas associadas. Foi ainda realizado um estudo de associação ao nível de todo o genoma com o objectivo de identificar factores genéticos que influenciem o outcome do doente. Nesta segunda estratégia não existia uma hipótese a priori relativamente ao papel de um determinado gene no fenótipo, correspondendo por isso a uma análise não enviesada. Vários estudos têm sugerido que a inflamação e o stress oxidativo desempenham um papel relevante na susceptibilidade ao AVC. Factores de risco já conhecidos, como aterosclerose, diabetes, obesidade e hipertensão, estão associados a um perfil inflamatório elevado. Além disso, é também conhecida a importância das enzimas sintase do óxido nítrico (NOS) para a aterosclerose e a regulação da pressão sanguínea. Atendendo a isso, foi testada a associação de quatro genes inflamatórios (IL1B, IL6, MPO e TNF) e de dois genes envolvidos no metabolismo do óxido nítrico (NOS1 e NOS3) com o risco de AVC. Foram encontradas associações de variantes genéticas nos genes inflamatórios IL6 e MPO com a doença, assim como de uma interacção epistática entre eles contribuindo para o risco. Isto sugere que os dois genes influenciam a susceptibilidade ao AVC através de efeitos independentes e de efeitos de interacção não aditivos. Os resultados mostram ainda uma associação entre o AVC e variantes genéticas no gene NOS1, que codifica uma das isoformas de NOS. A análise de genes candidatos sugere assim que efeitos independentes dos genes inflamatórios ou de stress oxidativo IL6, MPO e NOS1, e efeitos não aditivos resultantes de interacções entre os genes IL6 e MPO têm um impacto na susceptibilidade ao AVC. Estes resultados são compatíveis e reforçam as observações feitas em estudos in vitro e in vivo relativamente ao papel da inflamação e do stress oxidativo nesta doença. É igualmente reconhecido o importante impacto de stress oxidativo, inflamação, angiogénese, neurogénese, dano e remodelação neurovasculares na lesão cerebral associada ao AVC e/ou na recuperação funcional dos doentes. Assim, foram também levados a cabo estudos de associação para avaliar o papel de genes candidatos envolvidos em processos inflamatórios (IL1B, IL6, MPO e TNF) e de stress oxidativo (NOS1 e NOS3), assim como de genes das metaloproteinases da matriz (MMPs) (MMP2 and MMP9) e de genes de factores de crescimento (BDNF, FGF2 and VEGFA), no outcome do doente após três meses de recuperação. Relativamente ao gene MMP2, os resultados mostram a associação entre variantes genéticas deste gene e o outcome do doente após três meses de recuperação. Vários estudos anteriores demonstraram que a ruptura da barreira hemato-encefálica associada ao AVC está relacionada com a expressão e activação de MMPs, levando a hemorragia, edema e morte celular. No entanto, outros estudos sugerem que a actividade destas proteínas pode ser benéfica na angiogénese e na remodelação neurovascular em fases tardias de recuperação, o que poderá contribuir para a recuperação funcional do doente. Neste trabalho de doutoramento foram ainda identificadas duas interacções epistáticas entre os genes BDNF e FGF2 e entre os genes FGF2 e VEGFA em associação com o outcome do paciente. Estes três genes codificam factores de crescimento que são partilhados pelo sistema nervoso e vascular e que afectam a homeostasia e desenvolvimento dos dois sistemas. Os factores de crescimento têm importantes funções ao nível da angiogénese, neurogénese e protecção neuronal, influenciando o estado neurológico dos doentes e a recuperação dos mesmos após o AVC. Tendo em conta estes resultados, a análise de genes candidatos sugere que o gene MMP2 e interacções epistáticas entre os genes BDNF e FGF2, e entre os genes FGF2 e VEGFA têm um impacto no outcome do doente ao fim de três meses de recuperação. Estes resultados são também compatíveis com estudos in vitro e in vivo que tinham demonstrado previamente a importância das MMPs e dos factores de crescimento na recuperação após um AVC. Foi feito um rastreio genómico com o objectivo de identificar factores genéticos que influenciem o outcome do doente. Para isso foi testada a associação de mais de 250 mil polimorfismos, localizados ao longo de todo o genoma, com o outcome do doente. Uma das vantagens desta estratégia é a de possibilitar a descoberta de novos mecanismos envolvidos neste fenótipo. Para realizar este estudo de uma forma economicamente eficiente foram analisados conjuntos (pools) de amostras de DNA de doentes. Após a identificação dos marcadores mais importantes com base em quatro estratégias distintas, esses resultados foram validados por genotipagem individual. Este estudo permitiu a identificação de uma associação entre os genes BBS9 e GLIS3 e o outcome do doente. Sabe-se que pacientes com a síndrome Bardet-Biedl têm mutações no gene BBS9. A obesidade é uma das manifestações clínicas mais importantes desta síndrome e foi demonstrado que, após um AVC, os danos neurológicos e as lesões cerebrais são menores em ratinhos em regime de restrição calórica do que em ratinhos sem esta restrição, o que sugere que o excesso de calorias consumidas/obesidade poderá influenciar o outcome após AVC. Relativamente ao gene GLIS3, que codifica um factor de transcrição, tinham sido anteriormente reportadas associações deste gene com a diabetes. Esta doença é um factor de risco para o AVC e está também associada com maior risco de morte e incapacidade funcional de doentes quando o AVC ocorre. Apesar de não ser imediatamente perceptível qual o papel dos genes BBS9 e GLIS3 no outcome dos doentes, é possível que estes genes tenham uma influência indirecta nesse fenótipo através de um efeito na diabetes e obesidade. Em conclusão, os resultados obtidos durante este trabalho de doutoramento sugerem que tanto a susceptibilidade ao AVC como o outcome do doente são modulados por uma combinação de efeitos de genes independentes e de interacções entre genes. Isto indica que os mecanismos envolvidos na predisposição a esta doença e na recuperação posterior dos doentes poderão ser bastante complexos. É de salientar que, com este trabalho de doutoramento, o número de genes candidatos analisados, até ao momento, na área da genética do outcome praticamente duplicou. Além disso, pela primeira vez, foi realizado um estudo de associação ao nível de todo o genoma com o outcome do doente. No futuro, será desejável aumentar a dimensão da amostra, em especial dos doentes com informação sobre recuperação. Deverão, ainda, ser realizados estudos para identificar as variantes genéticas causais que estão na base das associações encontradas com a susceptibilidade ao AVC e com o outcome do doente

The XVth World Congress of Psychiatric Genetics, October 7–11, 2007: Rapporteur summaries of oral presentations

The World Congress of Psychiatric Genetics (WCPG) has become an annual event since the early 1990's sponsored by the International Society of Psychiatric Genetics (ISPG). Each year the latest published and unpublished findings are aired for discussion by representatives of the majority of research programs on this topic world-wide. The 2007 congress was held in New York City and attracted over 1000 researchers. The topics emphasized included results from whole genome association studies, the significance of copy number variation and the important contributions of epigenetic events to psychiatric disorders. There were over 20 oral sessions devoted to these and other topics of interest. Young investigator recipients of travel awards served as rapporteurs to summarize sessions and these summaries follow.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58040/1/30711_ftp.pd

TYK2 Protein-Coding Variants Protect against Rheumatoid Arthritis and Autoimmunity, with No Evidence of Major Pleiotropic Effects on Non-Autoimmune Complex Traits

Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3x10-21), A928V (rs35018800, OR = 0.53, P = 1.2x10-9), and I684S (rs12720356, OR = 0.86, P = 4.6x10-7). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6x10-18), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; Pomnibus = 0.005). Finally, in a phenomewide association study (PheWAS) assessing >500 phenotypes using electronic medical records (EMR) in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases

Genetic Architecture of Aluminum Tolerance in Rice (Oryza sativa) Determined through Genome-Wide Association Analysis and QTL Mapping

Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils, and rice has been demonstrated to be significantly more Al tolerant than other cereal crops. However, the mechanisms of rice Al tolerance are largely unknown, and no genes underlying natural variation have been reported. We screened 383 diverse rice accessions, conducted a genome-wide association (GWA) study, and conducted QTL mapping in two bi-parental populations using three estimates of Al tolerance based on root growth. Subpopulation structure explained 57% of the phenotypic variation, and the mean Al tolerance in Japonica was twice that of Indica. Forty-eight regions associated with Al tolerance were identified by GWA analysis, most of which were subpopulation-specific. Four of these regions co-localized with a priori candidate genes, and two highly significant regions co-localized with previously identified QTLs. Three regions corresponding to induced Al-sensitive rice mutants (ART1, STAR2, Nrat1) were identified through bi-parental QTL mapping or GWA to be involved in natural variation for Al tolerance. Haplotype analysis around the Nrat1 gene identified susceptible and tolerant haplotypes explaining 40% of the Al tolerance variation within the aus subpopulation, and sequence analysis of Nrat1 identified a trio of non-synonymous mutations predictive of Al sensitivity in our diversity panel. GWA analysis discovered more phenotype–genotype associations and provided higher resolution, but QTL mapping identified critical rare and/or subpopulation-specific alleles not detected by GWA analysis. Mapping using Indica/Japonica populations identified QTLs associated with transgressive variation where alleles from a susceptible aus or indica parent enhanced Al tolerance in a tolerant Japonica background. This work supports the hypothesis that selectively introgressing alleles across subpopulations is an efficient approach for trait enhancement in plant breeding programs and demonstrates the fundamental importance of subpopulation in interpreting and manipulating the genetics of complex traits in rice

TYK2 protein-coding variants protect against rheumatoid arthritis and autoimmunity, with no evidence of major pleiotropic effects on non-autoimmune complex traits

Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3 x 10(-21)), A928V (rs35018800, OR = 0.53, P = 1.2 x 10(-9)), and I684S (rs12720356, OR = 0.86, P = 4.6 x 10(-7)). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6 x 10(-18)), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; P(omnibus) = 0.005). Finally, in a phenome-wide association study (PheWAS) assessing \u3e500 phenotypes using electronic medical records (EMR) in \u3e29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases

Application of next generation sequencing in genetic and genomic studies

Genetic variants that spread along the human genome play vital roles in determining our traits, affecting development and potentially causing disorders. Most common disorders have complex underlying mechanisms involving genetic or environmental factors and the interaction between them. Over the past decade, genome-wide association studies (GWAS) have identified thousands of common variants that contribute to complex disorders and partially explain the heritability. However, there is still a large portion that is unexplained and the missing heritability may be caused by several factors, such as rare or low-frequency variants with high effect that are not covered by GWAS and linkage analysis. With the development of next generation sequencing (NGS), it is possible to rapidly detect large amount of novel rare and low-frequency variants simultaneously at a low cost. This new technology provides vast information on studying the association of genetic variations and complex disorders. Once the susceptibility gene is mapped, model organisms such as zebrafish (Danio rerio) are popular for further investigating the possible function of diseaseassociated gene in determining the phenotype. However, the genome annotation of zebrafish is not complete, which affects the characterization of gene functions. Accordingly, highthroughput RNA sequencing can be employed for identifying new transcripts. In our studies, pooled DNA samples were used for whole genome sequencing (WGS) and exome sequencing. In Paper I, we evaluated minor allele frequency (MAF) estimates using three variant detection tools with two sets of pooled exome sequencing and one set of pooled WGS data. The MAFs from the pooled sequencing data demonstrated high concordance (r = 0.88-0.94) with those from the individual genotyping data. In Paper II, exome sequencing implementing pooling strategy was performed on 100 idiopathic scoliosis (IS) patients for mapping susceptibility genes. After validating 20 candidate single nucleotide variants (SNVs), we did not find associations between them and IS. However, the previously reported common variant rs11190870 near LBX1 was validated in a large Scandinavian cohort. In Paper III, we analyzed WGS of pooled DNA samples performed on 19 affected individuals who shared a phenotype-linked haplotype in a dyslexic Finish family. Two of the individuals were sequenced for the whole genome individually as well. The screen for causative variants was narrowed down to a rare SNV, which might affect the binding affinity of LHX2 that regulated dyslexia associated gene ROBO1. In Paper IV, RNA sequencing (RNA-seq) data were analyzed for identifying novel transcripts in zebrafish early development using an inhouse pipeline. We discovered 152 novel transcribed regions (NTRs), validated more than 10 NTRs and quantified their expression in early developmental stages. In our studies, we evaluated and applied a pooling approach for identifying variants susceptible to disease using high-throughput DNA sequencing. Based on RNA sequencing data, we provided new information for genome annotation on model organism zebrafish, which is valuable for studying the function of disease causative genes. In summary, the whole series of studies demonstrate how NGS can be applied in studying the genetic basis of complex disorders and assisting in follow-up functional studies in model organisms