We aimed to assess whether the breast-cancer intermediate-risk genes CHEK2, ATM ant TP53 were subject to differential allelic expression (DAE) in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified.We implemented an assay based on high-resolution melting curve analysis (HRM) of single labeled fluorescent probes to detect allelic expression imbalance. The method relies on the distinction of the two alleles of an exonic marker SNP in heterozygous individuals with a fluorescent signal correlated to the relative abundance of each transcript. We developed an analysis tool for HRM data processing, specifically dedicated to DAE assessment. In our series, we found evidence for DAE for CHEK2, in carriers of the truncating mutation 1100delC. When combining mutation-screening data and assessment of DAE, we did not identify functional regulatory variant located in cis of the studied genes that would lead to DAE, in the transcriptional regulatory milieu of freely proliferating LCLs. Our results support that HRM is a method with high sensitivity and accuracy that can be used for DAE assessment. This approach can be applied to study breast and blood tissue samples. The latter would be of great interest for high-throughput mutation screening projects aiming to identify dysfunctional regulatory variants in candidate genes.

One of the main biological goals of recent years is to determine the genes in the human genome that cause disease. Recent technological advances have realised genome-wide association studies, which have uncovered numerous genetic regions implicated with human diseases. The current approach to analysing data from these studies is based on testing association at single SNPs but this is widely accepted as underpowered to detect rare and poorly tagged variants. In this thesis we propose several novel approaches to analysing large-scale association data, which aim to improve upon the power offered by traditional approaches. We combine an established imputation framework with a sophisticated disease model that allows for multiple disease causing mutations at a single locus. To evaluate our methods, we have developed a fast and realistic method to simulate association data conditional on population genetic data. The simulation results show that our methods remain powerful even if the causal variant is not well tagged, there are haplotypic effects or there is allelic heterogeneity. Our methods are further validated by the analysis of the recent WTCCC genome-wide association data, where we have detected confirmed disease loci, known regions of allelic heterogeneity and new signals of association. One of our methods also has the facility to identify the high risk haplotype backgrounds that harbour the disease alleles, and therefore can be used for fine-mapping. We believe that the incorporation of our methods into future association studies will help progress the understanding genetic diseases.

The human ribosomal RNA genes are critically important for cell metabolism and viability. They code for the catalytic RNAs which, encased in a housing of more than 80 ribosomal proteins, link together amino acids by peptide bonds to generate all cellular proteins. Because the RNAs are not repeatedly translated, as is the case with messenger RNAs, multiple copies are required. The genes which code for the human ribosomal RNAs (rRNAs) are arranged as clusters of tandemly repeated sequences. Three of four catalytic RNAs are spliced from a single transcript. The genes are located on the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22). The genes for the fourth rRNA are on chromosome 1q42, also arranged as a cluster of tandem repeats. The repeats are extremely similar in sequence, which makes them ideal for misalignment, non‐allelic homologous recombination (NAHR), and genomic destabilization during meiosis , replication, and damage repair. In this dissertation, I have used pulse‐field gel electrophoresis and in‐blot Southern hybridization to explore the physical structure of the human rRNA genes and determine their stability and heritability in normal, healthy individuals. I have also compared their structure in solid tumors compared to normal, healthy tissue from the same patient to determine whether dysregulated homologous recombination is an important means of genomic destabilization in cancer progression. Finally, I used the NCI‐60 panel of human cancer cell lines to compare the results from the pulsed‐field analysis, now called the gene cluster instability (GCI) assay, to two other indicators of homologous‐recombination-mediated genomic instability: sister chromatid exchange, and 5‐hydroxymethyl‐2’deoxyuridine sensitivity.

Durch eine Erhöhung der Diversität in Kandidatengenen für Ölgehalt könnten sich neue Ansätze zur Erhöhung des Ölgehalts ergeben (Osborn et al., 2007; Würschum et al., 2013). Im Rahmen dieser Arbeit wurde die allele Diversität auf der Basis von DNA-Sequenzen in einer großen Anzahl von Kandidatengenloci für Ölgehalt in exotischem Rapsmaterial (Sommerrapssorten, chinesische Rapssorten und Resynthesen) im Vergleich zu Winterraps untersucht. Viele Allele wurden exklusiv in den exotischen Genotypen aufgefunden (neue Allele). Die höchste Anzahl an neuen Allelen wurde in der Resynthese “MOY4“ (Brassica rapa var. trilocularis x Brassica montana) entdeckt. Viele der Allele wiesen SNP, die zu Aminosäureaustauschen führen, sowie InDel im kodierenden Bereich der untersuchten Kandidatengenloci auf. Einige dieser Polymorphismen konnten sogar Bereichen von Proteindomänen zugeordnet werden. Im Großen und Ganzen konnte in der durchgeführten Diversitätsstudie gezeigt werden, dass die untersuchten exotischen Genotypen genutzt werden können, um die allele Diversität in Kandidatengenen für Ölgehalt zu erhöhen. Zudem weist die relativ hohe Anzahl an nicht stillen Polymorphismen in den kodierenden Bereichen der verschiedenen Kandidatengenloci darauf hin, dass einige aufgefundene Allele den Ölgehalt beeinflussen könnten. Um einen möglichen positiven Einfluss von neuen Allelen in Kandidatengenen für Ölgehalt auf den Ölgehalt zu testen, wurden diese in spaltenden F2-Populationen an jeweils drei verschiedenen Standorten untersucht. In diesem Versuch wurden neue Allele, die in der durchgeführten Diversitätsstudie entdeckt wurden, sowie neue Allele, welche bereits in dem vorigen GABI BRIDGE Projekt aufgefunden wurden, mittels Varianzanalyse auf eine Auswirkung auf den Ölgehalt getestet. In einzelnen Populationen konnten signifikante Geneffekte auf den 87 Ölgehalt an drei verschiedenen Kandidatengenloci ermittelt werden: S13 (Kandidatengen KAS III), K48 (Kandidatengen PKP2), L65 (Kandidatengen LEC2). Einzel-Locus-Genotyp x Umweltinteraktionen mit einem signifikanten Effekt auf Ölgehalt wurden an sechs Kandidatengenloci inklusive S13 von KAS III aufgefunden. Diese aufgefundenen signifikanten Effekte auf den Ölgehalt können als deutlicher Hinweis auf die Wichtigkeit der untersuchten Loci beziehungsweise des Alleles auf den Ölgehalt interpretiert werden.

<p>T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.</p><p>The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.</p><p>Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, <italic>Tcrb</italic> alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However <italic>Tcrb</italic> alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of <italic>Tcrb</italic> alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.</p><p>In search of the mechanism by which recombination is suppressed on peripheral <italic>Tcrb</italic> alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral <italic>Tcrb</italic> alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.</p> / Dissertation

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2016-09-27T14:16:41Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Raque Bartz Kneib.pdf: 709774 bytes, checksum: ed9b7474064e09348586e51c9221a9fa (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-28T17:06:46Z (GMT) No. of bitstreams: 2 Dissertação Raque Bartz Kneib.pdf: 709774 bytes, checksum: ed9b7474064e09348586e51c9221a9fa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-28T17:06:46Z (GMT). No. of bitstreams: 2 Dissertação Raque Bartz Kneib.pdf: 709774 bytes, checksum: ed9b7474064e09348586e51c9221a9fa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A batata é o terceiro principal alimento produzido no mundo, e é um dos cultivos mais promissores para suprimir a fome em países em desenvolvimento. Cerca de 40 viroses infectam a cultura da batata, entre estas, o Potato virus Y (PVY) é, atualmente, o que causa maior impacto negativo na cultura. O PVY é transmitido de diversas formas, todas com controle e manejo deficitários, tornando a resistência genética a medida de controle mais eficaz. Esta resistência é controlada pelos genes Ry encontrados em espécies silvestres de batata. Marcadores do tipo SCAR (Sequence Characterized Amplified Region), os quais amplificam a região que contém o gene Ry, foram desenvolvidos e estão disponíveis para uso pelos programas de melhoramento de batata. Este trabalho foi realizado com o objetivo de aumentar a eficiência dos programas de melhoramento de batata em relação à resistência ao PVY fazendo uso da seleção assistida por marcadores moleculares. A dissertação foi dividida em dois capítulos. No primeiro, visando estimar a dosagem alélica do gene Ry e selecionar clones com múltiplas cópias do gene Ry para serem usados como genitores no programa de melhoramento de batata, foram genotipadas sete famílias clonais, envolvendo cinco genitores com resistência ao PVY, utilizando os marcadores SCAR RYSC3 e M45. Para o gene Ryadg, com base no marcador RYSC 3, dos cinco genitores resistentes ao PVY avaliados, quatro, C1883-22-97, C2372-02-02, C2388-01-02, e MB9846-1, são simplex, e o genitor C2389-01-02 é duplex. Para o gene Rysto, com base no marcador M45, dois genitores, C2388-01-02 e MB9846-1, são simplex, e o genitor C2389-01-02 é duplex. Para os genitores C1883-22-97 e C2372-02-02, as frequências observadas de presença do gene Rysto, nas progênies avaliadas, não se adequaram estatisticamente a nenhuma das constituições genotípicas propostas. No segundo capítulo, com objetivo de identificar germoplasma de batata com resistência extrema ao vírus Y associada aos componentes da aparência e do rendimento de tubérculos, além verificar as associações entre esses caracteres, foram avaliados clones portadores do gene Ry, oriundos de oito combinações genéticas distintas. As combinações genéticas C2389-01-02 x Asterix, e, BRS Ana x C1883-22-97, mostraram maior potencial para serem exploradas visando agregar a resistência ao vírus Y aos caracteres desejáveis de rendimento e de aparência de tubérculo. O grau de correlação entre caracteres indica fortíssima e direta associação entre aparência geral e aspereza de tubérculo; forte e direta associação da intensidade da cor de película com aspereza e aparência geral de tubérculo, entre número e massa média de tubérculo, entre massa média e cor da película, e entre massa total e número total de tubérculos; e forte e inversa associação de formato com cor da polpa, cor da película e massa média de tubérculos. Os resultados encontrados no nosso estudo mostraram que os marcadores moleculares RYSC3 e M45 são uma importante ferramenta para ser usada pelos programas de melhoramento de batata visando acelerar o processo de desenvolvimento de cultivares com resistência ao PVY. / Potato is the third main food crop in the world, and is one of the most promising crop to suppress hunger in developing countries. From the 40 viruses which infect potato growing fields, Potato virus Y (PVY) is currently considered the most important one. PVY is transmitted by several ways, all of them with limited control, begin genetic resistance the main way to control this important disease controlled by Ry genes. Molecular markers, which amplify the region containing Ry gene have been developed and are available to be used by potato breeding. This work was developed with the aim of increasing the efficiency of potato breeding programs for resistance to PVY making use of molecular assisted selection. The dissertation is divided into two chapters. In the first chapter, in order to estimate allelic dosage of Ry genes in clones used as genitors in the breeding program, progenies from seven families were genotyped with the markers RYSC3 and M45. For Ryadg gene, based on the marker RYSC, from the five PVY resistant parents analysed, four are simplex, C1883-22-97, C2372-02-02, C2388-01-02 and MB9846-1, and one is duplex, C2389- 01-02. For Rysto gene, based on the marker M45, both parents, C2388-01-02 and MB9846-1, are simplex, and the parent C2389-01-02 is duplex. For the C1883-22-97 and C2372-02-02, it was not possible to estimate the allelic dosage of the Rysto gene. In the second chapter, in order to identify germplasm with extreme resistance to PVY associated to desirable agronomic traits, clones carrying the Ry gene, belonging from eight different genetic combinations, were evaluated for characteres related to tuber appearance and yield. The genetic combinations C2389-01-02 x Asterix and BRS Ana x C1883-22-97 showed good potential to be exploited in order to add resistance to virus Y to traits related to yield and tuber appearance. The correlation between traits indicates very strong and direct association between tuber general appearance and roughness of tuber; strong and direct association of the intensity of the periderm color and tuber general appearance; between number of tubers per plant and average tuber weight; between average tuber weight and intensity of the periderm color and between the total mass of tubers per plant and total number of tubers per plant. Also, strong and inverse associations between tuber shape and flesh color and between periderm color and average tuber weight were detected. The results of our study showed that the molecular markers RYSC3 and M45 are important tools to be used by potato breeding programs to accelerate the development of cultivars with resistance to PVY.

Le domaine Dlk1-Dio3 est l’un des rares domaines imprimés contrôlés par une région de contrôle d'impression méthylée sur le chromosome paternel, nommée IG-DMR. Dans l’embryon, au niveau du domaine Dlk1, Rtl1 et Dio3 les gènes codant pour des protéines sont exprimés à partir du chromosome paternel, tandis que les ARNs non-codants dont Meg3, les snoRNAs à boite C/D et les micro-ARNs sont exprimés à partir du chromosome maternel.Il a été montré que la copie maternelle de l'IG-DMR est nécessaire pour l'expression des gènes imprimés de ce domaine et que les ARNs de types enhancer (de la même région) activent la transcription des ARNs non-codants. Cependant, les mécanismes qui régulent l'expression imprimée de gènes codant pour des protéines restent indéterminés. Dans ce projet, nous avons cherché à élucider les mécanismes qui contrôlent l'expression spécifiquement paternelle des gènes codant pour des protéines ainsi que le rôle possible des ARNs non-codants dans ce processus.Pour nos études alléliques, nous avons utilisé des cellules ES hybrides qui ont été obtenues en croisant des lignées de M. musculus domesticus et M. musculus molossinus. Ces cellules ont été différenciées in vitro dans des lignées neurales. Dans les cellules ES, l'expression Dlk1 est détectée à partir des deux chromosomes parentaux à des niveaux très bas. Lors de la différenciation, l'allèle paternel de Dlk1 devient actif tandis que le niveau d'expression de l'allèle maternel reste faible. Nos études de la chromatine ont montré que cette surexpression est due à l’activation de la chromatine sur l'allèle paternel de Dlk1.L'un de nos objectifs était d'explorer le rôle de Meg3 (un long ARN non-codant) dans la régulation de l’empreinte de Dlk1. A cet effet, nous avons généré des cellules souches embryonnaires déficientes en Meg3. Dans toutes les lignées déficientes, de suppressions maternelles ou bi-alléliques, nous avons constaté une perte d’expression de tous les ANRs non-codants. De plus, l’expression de Dlk1 devient bi-allélique dans ces cellules. Pour élucider le mécanisme de l'empreinte de ce gène, nous avons décidé d'étudier les caractéristiques de la chromatine au niveau du promoteur Dlk1 dans les cellules déficientes en Meg3. Nous avons examiné les modifications activatrices et répressives des histones ainsi que l'occupation de l'ARN Pol II. Nous avons observé l'acquisition des marques d’une chromatine active sur les deux chromosomes ainsi que le recrutement bi-allélique de l'ARN Pol II.Bien que nous n’ayons pas pu détecter une perte de la marque répressive H3K27me3 suite à la surexpression de Dlk1, nous avons observé un gain d'acétylation sur ce résidu lysine. Afin de comprendre davantage le rôle de la marque H3K27me3 sur l’empreinte de Dlk1, nous avons généré des cellules ES dépourvues de EZH2, la méthyltransférase de H3K27. L’expression de Dlk1 dans les cellules différenciées dépourvues de H3K27me3 est bi-allélique.Enfin, ces données suggèrent que l'expression des ARNs non-codant empêche l'activation de Dlk1 sur le chromosome maternel via l’activité de EZH2 au cours du développement. / The Dlk1-Dio3 imprinted domain is one of the few imprinted domains that are controlled by a paternally methylated imprinting control region, IG-DMR. Protein-coding genes of the domain, Dlk1, Rtl1 and Dio3 are expressed from the paternal chromosome, and non-coding RNAs (ncRNAs) including Meg3, C/D box snoRNAs and microRNAs are expressed from the maternal chromosome exclusively in the embryo. Maternal copy of the IG-DMR is required for the imprinted gene expression at this domain. Enhancer RNAs transcribed from this region are involved in activation of ncRNA expression on the maternal chromosome. However, the regulation of imprinted expression of protein-coding genes remains unknown. In this project, we aimed to elucidate the mechanisms controlling the paternal specific expression of protein-coding genes and a possible role of ncRNAs in this process.For our allelic studies, we made use of hybrid ES cells that were obtained by crossing M. musculus domesticus and M. musculus molossinus strains. These cells were differentiated in vitro into neural lineages. In ES cells, Dlk1 expression is detected from both parental chromosomes at very low levels. Upon differentiation, paternal allele of Dlk1 gets activated while low level of expression is detected from maternal allele. Our chromatin studies showed that this upregulation is through the acquisition of active chromatin on the paternal allele of Dlk1.One of our aims was to explore the role of Meg3 long non-coding RNA (lncRNA) in the regulation of Dlk1 imprinting. For this purpose, we generated ES cells deficient in Meg3. In all maternal or biallelic deletion lines, we observed complete loss of all ncRNA expression. Interestingly, in these cells Dlk1 expression becomes biallelic. To elucidate the mechanism of imprinting of this gene, we set out to study the chromatin features at the Dlk1 promoter in Meg3 deficient cells. We looked into active and repressive histone modifications and RNA Pol II occupancy. We observed acquisition of active chromatin marks on both chromosomes as well as biallelic recruitment of RNA Pol II.Although we could not detect a loss of repressive mark H3K27me3 upon Dlk1 upregulation on the paternal allele, we observed gain of acetylation on this lysine residue. To further investigate the role of H3K27me3 mark on Dlk1 imprinting, we generated ES cells that lack functional EZH2, the H3K27 methyltransferase. Dlk1 is biallelically expressed in the differentiated cells that are devoid of H3K27me3.Combined, these data suggest a model in which non-coding RNA expression prevents the developmental activation of Dlk1 on the maternal chromosome by a process that also requires the activity of EZH2.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23083号 / 医博第4710号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中島 貴子, 教授 近藤 玄, 教授 小杉 眞司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The human forkhead box O1A (FOXO1A) gene on chromosome 13q14.1 is a key transcription factor in insulin signaling in liver and adipose tissue and plays a central role in the regulation of key pancreatic β-cell genes including IPF1. We hypothesized that sequence variants of FOXO1A contribute to the observed defects in hepatic and peripheral insulin action and altered β-cell compensation that characterize type 2 diabetes (T2DM). To test this hypothesis, we screened the three exons, 3′ untranslated region, and 5′ flanking region for sequence variants in Caucasian and African-American individuals with early onset (<45 years) T2DM. We identified only six variants; none altered the coding sequence, and except for one variant in the 3′ untranslated region, they were rare or absent in Caucasians. To increase coverage of the gene, we selected seven additional variants in the large first intron and 5′ flanking region, thus providing 13 variants that spanned 116.4 kb. Based on frequency and linkage disequilibrium patterns in a subset of individuals, we selected eight SNPs to type in a Caucasian population comprising 192 unrelated nondiabetic control individuals and 192 individuals with T2DM, and 10 SNPs to type in 182 controls and 352 diabetic individuals of African-American ancestry. No variant was associated with T2DM (African-Americans, p > 0.08; Caucasians, p > 0.09). Of the 8 Caucasian SNPs, six comprised a single haplotype block spanning over 100 kb and including most of the large first intron. In contrast, no block was observed among SNPs typed in African-Americans. No haplotype was associated with T2DM. FOXO1A variation is rare and is unlikely to contribute to T2DM in either Caucasian or African-American populations.

When investigating the developmental roles of genes on phenotypic expression it may seem reasonable to assume that a mutation would result in consistent phenotypic change. However, increasing evidence has shown this is not often the case, and the “wild-type” genetic background of an individual plays a large role in phenotypic expression of mutations and severity of genetic mediated diseases. Previous work has demonstrated that degree of genetic background effects shows a non-linear relationship with severity of mutational effects. This relationship is characterized by alleles of moderate phenotypic expressivity showing the relatively greatest degree of background dependence and between genotype variability in comparison with alleles of severe and modest phenotypic expressivity. Our previous work has shown this relationship for Drosophila melanogaster wing size through a scalloped (sd) allelic series crossed to naturally derived strains from the Drosophila Genetics Reference Panel (DGRP). I explored these effects with a miniature (m) allelic series where the results from our experiment suggest a vastly different response. m when compared to sd is characterized by a more linear relationship, whereby alleles of moderate phenotypic effect do not show increased background dependence nor increased variability within and between strains. Furthermore, our results suggest a strong correlation across DGRP strains with respect to m mutational severity and that the effect m has on wing shape is not largely due to wing size. Our working hypotheses for why this might be occurring is due to the increased interaction of sd with other aspects of wing development relative to that of m, the differences in when the genes are playing active roles in wing development, or the effects the mutations have on the wing to affect size. To add to our previous results employingutilizing sd, I am beginnings to elucidate the non-linear relationship of genetic background effects with severity of mutational effects at a gene expression level. I am accomplishing this through crossing autilizing a sd allelic series crossed to six naturally derived DGRP strains used in previous experiments involving wing size. Preliminary results agree with previous work on genetic background effects, displaying a non-linear relationship with the severity of mutational effect. I aim to continue to explore this relationship including more genotypes and investigating more genes to better elucidate the mechanistic causes of genetic background effects. / Thesis / Master of Science (MSc) / When investigating the roles of genes on phenotype it may seem intuitive that a mutation affecting gene function would display a consistent change in phenotype. Increasing evidence has asserted that this may not always be the case and genetic background effects may affect the genotype-phenotype relationship affecting experimental design, disease treatment and evolutionary trajectories. Here, we investigate the mechanisms involved in these genetic background effects utilizing Drosophila melanogaster wing tissue. We outline a change from the typically observed non-linear relationship between genotype and phenotype and for the first time quantify shape change effects by the miniature mutation.