Identifying novel genes associated with breast cancer susceptibility using differential allelic expression ratios

Breast Cancer (BC) is the most common cancer among women worldwide. However, the current knowledge of BC susceptibility only accounts for half of the familial cases. The few functional studies performed for genome-wide association studies (GWAS) loci revealed a role for cis-regulatory variation, suggesting that risk variants may be acting by regulating gene expression levels. Therefore, we hypothesise that the most efficient approach to tackle BC missing heritability is to focus susceptibility studies on variants with greater cis-regulatory potential. Hereby, we present an innovative approach to genetic association studies, using a quantifiable readout of the effect of cis-regulatory variants — differential allelic expression (DAE). To identify candidate risk genes for our study, we selected Single Nucleotide Polymorphisms (SNPs) weakly associated with BC risk in GWAS and in the iCOGS consortium and identified their proxy SNPs. The resulting 591 candidate risk variants were located in 92 different genes, of which 41 had evidence of being cisregulated in a DAE study of normal breast tissue. The clinical impact of these genes was assessed, for a diverse list of clinical variables (differential expression analysis, FDR ⩽ 1% and absolute fold-change ⩾1.5). A final list of 18 risk candidates cis-regulated and with clinical impact genes was identified. OCIAD1 and GRHL2 genes were selected to perform case-control association studies using DAE values. DAE of OCIAD1 was significantly associated with BC risk (p-value=0.002 and 0.008, in two independent experiments using blood samples), while DAE of GRHL2 needs further validation of association (pvalue= 0.014 and 0.096, in two independent experiments in breast tissue). This project proved that association studies using DAE as a quantifiable variable, together with the whole pipeline used to select the candidate genes, is an efficient approach to detect novel risk genes for BCO cancro da mama é o tipo de cancro mais diagnosticado em mulheres, tanto em países desenvolvidos como em países em vias de desenvolvimento, representando 25% de todos os cancros mais diagnosticados. É uma doença caracterizada pelo crescimento anormal de células da mama conduzindo à formação do tumor. Esta neoplasia pode ser classificada segundo a sua morfologia e histologia básica, mas também a nível molecular. Trata-se de uma doença complexa, com uma componente genética e ambiental. Os fatores de risco para desenvolvimento de cancro da mama podem ser modificáveis, como é o caso da diminuição do contacto com radiação ionizante, consumo de hormonas femininas exógenas, entre outros, mas também existem fatores não modificáveis como é o caso da genética e da herança familiar. Cerca de 10-30% dos cancros da mama estão relacionados com fatores hereditários e dentro destes, entre 5-10% dos casos têm uma forte componente herdada. Os alelos genéticos podem ser categorizados de acordo com o risco e com a sua frequência na população, em alelos de alto risco, como é o caso de mutações nos genes BRCA1/BRCA2, alelos de risco moderado, como as mutações em ATM e BRIP1 e em alelos que conferem baixo risco na população, como o caso dos polimorfismos genéticos identificados nos genes FGFR2 e TOX3. No entanto cerca de metade da componente de risco familiar para o cancro da mama permanece por identificar. Os estudos de associação do genoma (GWAS) permitiram a identificação de muitos alelos de risco sem conhecimento prévio da posição ou função do gene. Estes estudos em cancro da mama revelaram que os polimorfismos de variante única (SNPs) associados a risco para a doença estão presentes maioritariamente em regiões não codificantes e os poucos estudos funcionais realizados nestes loci mostraram que variantes cis-reguladoras causavam risco através da regulação da expressão genética. As variantes em cis alteram a síntese dos transcritos de forma específica para cada alelo, podendo estar localizadas em regiões promotoras do gene, enhancers, bem como a centenas de kilobases (kb) de distância. A medição do rácio de expressão do RNA entre os dois alelos permite a deteção direta do efeito destas variantes, designando-se por análise de expressão alélica diferencial (DAE). Posto isto, sugere-se que para identificar novas variantes associadas a risco para cancro da mama deve-se focar os estudos em variantes cis-regulatórias e propõe-se utilizar rácios de DAE como medida quantitativa em estudos de associação genética. Para selecionar os genes candidatos para serem testados nos estudos de associação usando níveis de DAE usou-se a seguinte abordagem: primeiro identificaram-se 608 variantes candidatas a conferir risco para cancro da mama, uma vez que mostraram evidência de estarem associadas com risco para cancro da mama, mas não atingiram significância estatística nos GWAS. Estas variantes e os seus proxy SNPs (r2 ≥ 0.8) estavam localizadas em 92 genes diferentes. Visto que se pretendia selecionar genes para validação no laboratório que tivessem cisregulados, filtrou-se os 92 genes candidatos a risco com os dados de DAE do grupo (que indicavam quais os genes com evidência de cis-regulação), ficando-se com 41 genes cis-regulados e com evidência de poderem contribuir para risco de desenvolver cancro da mama. Com o objetivo de se selecionarem genes com possível impacto clínico, realizaram-se análises de associação estatística entre expressão genética no genoma inteiro (derivada de microarrays de ácido desoxirribonucleico complementar (cADN) e diferentes variáveis clínicas (recetor de estrogénio, recetor de progesterona, grau, entre outras). Com esta análise de expressão diferencial verificou-se que 18 dos genes anteriores possuíam impacto clínico em pelo menos uma das análises consideradas (p-value≤0,01 (1% False Discovery Rate correction) e |Fold Change|≥ 1,5) e foram estabelecidos os genes finais de interesse. Destes 18 genes, selecionaram-se dois para a realização do estudo caso-controlo, com base nas evidências de DAE das análises efetuadas no grupo anteriormente, na significância estatística para o risco observada nas primeiras fases dos GWAS, no impacto clínico verificado nas análises de expressão diferencial, na frequência do alelo mais raro do SNP localizado nesse gene), na expressão total dos transcritos no tecido saudável da mama, bem como na presença de expression quantitative trait loci para o SNP transcrito (tSNP) de cada gene candidato. Após esta análise o gene OCIA domain containing 1 (OCIAD1), e nomeadamente o seu tSNP rs9997920 (alelos C/T (minor)), assim como o Grainyhead Like Transcription Factor 2 (GRHL2) e o seu tSNP rs6989650 (alelos C/T (minor)) foram selecionados para realização dos estudos caso-controlo, tendo sido o OCIAD1 testado em tecido da mama e no sangue e o GRHL2 apenas em mama. Para cada estudo caso-controlo foi incorporada uma curva de calibração com uma amostra heterozigótica de modo a quantificar-se de forma precisa o DAE e uma curva standard com amostras heterozigóticas com os dois alelos em diferentes proporções para servir de controlo positivo. Os estudos de associação revelaram que no gene OCIAD1 no sangue existe uma diferença significativa entre os níveis de DAE nos casos e dos controlos (p-value= 0.002 e 0.008 em duas experiências independentes), tendo o alelo menos frequente do rs9997920 uma maior expressão nos casos do que nos controlos. No tecido mamário não se verificou esta diferença nos níveis de DAE do OCIAD1 (p-value= 0.4 e 0.07 para a segunda experiência), o que nos leva a concluir que apenas o sangue poderá ser usado para inferir o risco conferido por este gene. No entanto, e uma vez que os coortes populacionais do sangue são diferentes dos do tecido, este estudo deveria ser repetido usando sangue e tecido das mesmas pessoas. O estudo de associação genética para o GRHL2 revelou que este é um potencial gene de risco para cancro da mama uma vez que apresentava níveis de DAE diferentes entre casos e controlos no tecido da mama, mas apenas numa das experiências realizadas (=0,014 e 0.096 para a segunda experiência). Verificou-se que o alelo comum do rs6989650 está mais expresso nos casos do que nos controlos o que nos leva a concluir que este possivelmente é o alelo de risco, mas mais estudo têm que ser feitos para garantir que realmente a presença desta variante leva a risco para o desenvolvimento da doença. Este estudo permitiu a identificação de potenciais novos genes associados a suscetibilidade para cancro da mama, e gerou uma lista de novos genes candidatos para serem testados no futuro para associação com cancro da mama, através da análise de DAE. Futuras repetições das experiências têm que ser realizadas para garantir que o OCIAD1 e o GRHL2 são genes de risco para cancro da mama. Ao confirmarem-se, mais estudos deverão ser feitos de modo a identificar as variantes causais bem como o mecanismo pelo qual estarão a provocar o risco

The Multigeneic _Rhg1_ Locus: A Model For The Effects on Root Development, Nematode Resistance and Recombination Suppression.

Soybean (Glycine max L. Merr.) resistance to populations (HgType) of _Heterodera glycines I._, the soybean cyst nematode (SCN), requires a functional allele at rhg1. An apoptosis-like response in the giant cells formed around the nematode results 24-48 h after feeding commences. This study aimed to identify the role of the three genes within the rhg1 locus, a receptor like kinase (RLK), a laccase and an ion anti-porter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles. Features of the rhg1 locus, the candidate genes and their nascent transcripts and proteins in roots were elucidated. First, evidence for a syntenic gene cluster was found and the effectiveness of SNP probes for distinguishing the homeolog sequence variant on linkage group (Lg) B1 from alleles at the rhg1 locus on Lg G was shown. Analysis of plant s heterozygous at rhg1 showed that the allele for resistance was dominant. The absence of recombination events among the NILs between the RLK and other 2 genes eliminated the possibility of a monogeneic rhg1 locus. Finally, an effect on root development was discovered. A model for multigeneic resistance based on developmental control of root growth including a mechanism for segregation distortion is presented

Einfluss von NOD2, intestinalem Mikrobiom und Vitamin D auf den klinischen Verlauf von chronisch-entzündlichen Darmerkrankungen

Der Krankheitsverlauf bei CED-Patienten lässt sich aktuell nur unzureichend vorhersagen, prädiktive Marker stehen nur eingeschränkt zur Verfügung. Die hier vorgestellten Arbeiten beschäftigen sich mit dem Zusammenhang zwischen Darmmikrobiom, Vitamin D und genetischen Risikofaktoren, wie Mutationen im NOD2 Gen bei CED. Aus den hier durchgeführten Untersuchungen resultiert u.a. ein besseres Verständnis darüber, wie sich die einzelnen Faktoren gegenseitig beeinflussen und eine Auswirkung auf den Krankheitsverlauf bei CED haben

New insights in the paternal genetic landscape of Southwestern Europe: dissection of haplogroup R1b-M269 and forensic applications

325 p.El cromosoma Y determina el sexo masculino y posee una naturaleza haploide que escapa a la recombinación, lo que hace que sólo esté presente en individuos varones y que se trasmita prácticamente sin cambios de padres a hijos, estableciendo linajes paternos. El estudio de los linajes paternos, mediante polimorfismos de un solo nucleótido (single nucleotide polimorphism, SNP) en el cromosoma Y (Y-SNP), permite la reconstrucción de la historia evolutiva de los linajes paternos de la especie humana. Los marcadores genéticos del cromosoma Y pueden ser de dos tipos, los anteriormente mencionados Y-SNPs, y los microsatélites Y-STRs (short tandem repeat, STR). Su estudio permite conocer cuáles son los linajes paternos característicos o presentes en cada población humana, permitiendo así diferenciar entre unas poblaciones y otras. Por ello, en los últimos años su estudio es relevante dentro del área de la Genética Forense y otras áreas afines como la genética de poblaciones, genealogía genética o la genética evolutiva.El estudio de los Y-SNPs ha revelado que las agrupaciones de linajes paternos, también llamadas haplogrupos, se distribuyen en áreas geográficas concretas a lo largo del mundo, tanto continental comoregionalmente. En el caso del oeste de Europa el haplogrupo más común es R1b-M269. La actual composición genética de Europa ha sido objeto de múltiples controversias centradas alrededor del origen de M269, ya que las estimaciones de edad obtenidas a partir de los distintos estudios genéticos realizados por distintos autores situaron el origen de este linaje paterno tanto durante el periodo Paleolítico, como en tiempos más recientes, en el Neolítico.El objetivo principal de este estudio se centra en la reconstrucción del escenario evolutivo más probable del principal linaje paterno europeo M269 en la Península Ibérica y el suroeste de Europa a través de la disección en sus subhaplogrupos, lo que permitirá caracterizar de manera detallada la distribución de los linajes paternos presentes en la Península Ibérica e inferir el papel de esta región en la historia evolutiva de Europa.Los resultados obtenidos han permitido caracterizar el paisaje genético paterno del suroeste de Europa, revelando, por un lado, que el origen más probable de M269 sea el Este de Europa y, por otro lado, que uno de los sublinajes principales de M269, R1b-S116, presenta un patrón de distribución distinto al propuesto anteriormente por otros autores. Además, el paragrupo de S116, S116*, fue prácticamente resuelto por la presencia del sublinaje R1b-DF27, que ha resultado ser casi específico de la Península Ibérica, haciendo de él un marcador de potencial interés forense para la determinación de la ancestralidad biogeográfica paterna. Las estimaciones de edad de DF27 indican que se originó hace 4.000-4.200 años, durante la transición entre el Neolítico y la Edad de Bronce, siendo su lugar más probable de origen la región noreste de la Península Ibérica. Por otro lado, fruto de este estudio también se han desarrollado dos paneles multiplex de Y-SNPs e Y-STRs para su uso en Genética Forense y de poblaciones.En conclusión, el presente trabajo de tesis doctoral ha proporcionado, por un lado, nuevas pistas sobre la historia evolutiva de acervo genético europeo actual y, por otro lado, dos nuevas herramientas de uso forense para el análisis multiplex de Y-SNPs e Y-STRs

Anthrax: Evolutionary approaches for genetic-based investigative tools

A TaqMan-minor groove binding assay designed around a nonsense mutation in the plcR gene was used to genotype Bacillus anthracis, B. cereus, and B. thuringiensis isolates. The assay differentiated B. anthracis from these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverse B. anthracis strains

Development of PCR-based methods for detection of African lyssaviruses

The etiological agent of rabies encephalitis belongs to the genus Lyssavirus in the Rhabdoviridae family. Lyssaviruses are negative sense, single stranded RNA viruses and cause an estimated 55 000 human deaths per year with 44% of these deaths occurring in Africa (WHO, 2005). With intense research effort and increased sequence information it is becoming evident that the Lyssavirus genus is much more diverse than initially thought and therefore diagnostic methods need to be modified accordingly. The African continent sustains a diverse variety of lyssaviruses, however, most countries in Africa do not have active surveillance or necessary diagnostic tools and therefore rabies-related lyssaviruses are underreported. Previous studies have indicated that real-time PCR has improved sensitivity and rapidity over conventional molecular diagnostic methods with the added advantage of allowing accurate estimations of viral load in a wide variety of samples. Several realtime PCR assays have been developed; however, none were specifically aimed at detection of lyssaviruses present on the African continent. This study was therefore aimed at evaluating certain molecular diagnostic methods for the detection of African lyssaviruses. Furthermore, the application of real-time PCR for various fields in lyssavirus research i.e. diagnostics, surveillance and pathogenicity studies were evaluated. This study revealed two different hemi-nested PCR assays capable of detecting representatives of African lyssaviruses. A real-time PCR was developed that was successful for the detection of African lyssaviruses. In addition, a quantitative assay and internal control was successfully employed for confirming ante-mortem human rabies diagnosis as well as post-mortem animal rabies diagnosis in formalin fixed brain material. As such the real-time PCR assay developed in this study could therefore be routinely used for ante-mortem diagnosis and as a confirmatory test for post-mortem diagnosis. The ability of this assay to detect and quantify all currently known African lyssaviruses not only offers improved surveillance capacity, but offers unique potential as a sensitive tool to track virus movement in pathogenicity studies. These aspects are important in our search for a better understanding of the complex epidemiological and viral characteristics of African lyssaviruses. CopyrightDissertation (MSc)--University of Pretoria, 2010.Microbiology and Plant Pathologyunrestricte

Disentangling hexaploid genetics : towards DNA-informed breeding for postharvest performance in chrysanthemum

DNA-informed selection can strongly improve the process of plant breeding. It requires the detection of DNA polymorphisms, calculation of genetic linkage, access to reliable phenotypes and methods to detect genetic loci associated with phenotypic traits of interest. Cultivated chrysanthemum is an outcrossing hexaploid with an unknown mode of inheritance. This complicates the development of resources and methods that enable the detection of trait loci. Postharvest performance is an essential trait in chrysanthemum, but is difficult to measure. This makes it an interesting but challenging trait to phenotype and detect associated genetic loci. In this thesis I describe the development of resources and methods to enable phenotyping for postharvest performance, genetic linkage map construction and detection of quantitative trait loci in hexaploid chrysanthemum. Postharvest performance is a complicated trait because it is related to many different disorders that reduce quality. One of these disorders in chrysanthemum is disk floret degreening, which occurs after long storage. In chapter 2, we show that degreening can be prevented by feeding the flower heads with sucrose, suggesting carbohydrate starvation plays a role in the degreening process. To investigate the response to carbohydrate starvation of genotypes with different sensitivity to disk floret degreening, we investigated the metabolome of sugar-fed and carbohydrate-starved disk florets by 1H-NMR and HPAEC. We show that the metabolome is severely altered at carbohydrate starvation. In general, starvation results in an upregulation of amino acid and secondary metabolism. Underlying causes of genotypic differences explaining variation in disk floret degreening in the three investigated genotypes remained to be elucidated, but roles of regulation of respiration rate and camphor metabolism were posed as possible candidates. In chapter 3, disk floret degreening was found to be the most important postharvest disorder after 3 weeks of storage among 44 white chrysanthemum cultivars. To investigate the inheritance of disk floret degreening, we crossed two genotypes with opposite phenotypic values of both disk floret degreening and carbohydrate content to obtain a population segregating for disk floret degreening. To phenotype the cultivar panel and the bi-parental population precisely and in a high throughput manner, we developed a method that quantified colour of detached capitula over time. This method was validated with visual observations of disk floret degreening during vase life tests. In a subset of the bi-parental population we measured carbohydrate content of the disk florets at harvest. The amount of total carbohydrates co-segregated with sensitivity to degreening, which shows that the difference in disk floret degreening sensitivity between the parents could be explained by their difference in carbohydrate content. However, the correlation was rather weak, indicating carbohydrate content is not the only factor playing a role. In order to develop resources for DNA-informed breeding, one needs to be able to characterize DNA polymorphisms. In chapter 4, we describe the development of a genotyping array containing 183,000 single nucleotide polymorphisms (SNPs). These SNPs were acquired by sequencing the transcriptome of 13 chrysanthemum cultivars. By comparing the genomic dosage based on the SNP assay and the dosage as estimated by the read depth from the transcriptome sequencing data, we show that alleles are expressed conform the genomic dosage, which contradicts to what is often found in disomic polyploids. In line with this finding, we conclusively show that cultivated chrysanthemum exhibits genome-wide hexasomic inheritance, based on the segregation ratios of large numbers of different types of markers in two different populations. Tools for genetic analysis in diploids are widely available, but these have limited use for polyploids. In chapter 5, we present a modular software package that enables genetic linkage map construction in tetraploids and hexaploids. Because of the modularity, functionality for other ploidy levels can be easily added. The software is written in the programming language R and we named it polymapR. It can generate genetic linkage maps from marker dosage scores in an F1 population, while taking the following steps: data inspection and filtering, linkage analysis, linkage group assignment and marker ordering. It is the first software package that can handle polysomic hexaploid and partial polysomic tetraploid data, and has advantages over other polyploid mapping software because of its scalability and cross-platform applicability. With the marker dosage scores of the bi-parental F1 population from the genotyping array and the developed methods to perform linkage analysis we constructed an integrated genetic linkage map for the hexaploid bi-parental population described in chapter 3 and 4. We describe this process in chapter 6. With this integrated linkage map, we reconstructed the inheritance of parental haplotypes for each individual, and expressed this as identity-by-descent (IBD) probabilities. The phenotypic data on disk floret degreening sensitivity that was acquired as described in chapter 3, was used in addition to three other traits to detect quantitative trait loci (QTL). These QTL were detected based on the IBD probabilities of 1 centiMorgan intervals of each parental homologue. This enabled us to study genetic architecture by estimating the effects of each separate allele within a QTL on the trait. We showed that for many QTL the trait was affected by more than two alleles. In chapter 7, the findings in this thesis are discussed in the context of breeding for heterogeneous traits, the implications of the mode of inheritance for breeding and the advantages and disadvantages of polyploidy in crop breeding. In conclusion, this thesis provides in general a significant step for DNA-informed breeding in polysomic hexaploids, and for postharvest performance in chrysanthemum in particular.</p