The adipocyte-secreted hormone leptin is considered a keyplayer in the regulatory mechanisms underlying obesity, since, among other functions, it induces satiety and thereby controls energy homeostasis. The mechanisms of leptin’s effects, as well as the regulation of its synthesis, remain the subject of recent studies. Several publications point to the involvement of long non-coding RNAs (lncRNA) in the regulation of leptin synthesis. Furthermore, in 2016 a genome-wide association study (GWAS) identified a single nucleotide polymorphism (SNP), rs10487505, in the proximal promoter region of the Leptin gene in humans, which was associated with decreased leptin levels and increased BMI. We analyzed an as yet uninvestigated lncRNA (termed LncR) and tested the hypothesis that LncR expression in subcutaneous and visceral adipose tissue (SCAT and VAT) affects Leptin mRNA expression, circulating leptin levels and other metabolic and anthropomorphic parameters. We also analyzed whether the SNP rs10487505 is associated with the above-mentioned parameters as well as with the LncR and Leptin gene expression levels. We report that lncR negatively correlates with circulating leptin levels in women. LncR did not correlate with Leptin mRNA expression, therefore, we assume that its regulatory mechanism might act downstream of the protein synthesis process. Since the effect was specific to women, we stratified the female patients into a pre- and postmenopausal group. We did not find any evidence that the effect on circulating leptin levels was influenced by female hormonal status. Furthermore, LncR correlated with parameters of obesity and inflammation. The effects were highly gender-specific and dependent on the fat depot in which LncR was expressed. We confirmed the leptin-depleting effect of the C allele of the SNP rs10487505 in women, as described in the 2016 GWAS. However, we found a BMI decreasing effect of the C allele in women in our highly obese cohort, in contrast to the mainly population-based cohort of the GWAS. We suggest a model in which the leptin depletion via rs10487505 enhances weight gain in normal weight and low obese individuals but prevents leptin resistance and further dysregulation of appetite and satiety in morbidly obese patients. The BMI-decreasing effect seems to be a postmenopausal phenomenon. Further, the leptin-depleting allele of rs10487505 was associated with parameters of obesity and inflammation, such as decreased FPI, gGT and IL-6 in women, and decreased body fat, FPI and increased adiponectin in men. In general, these results point to a rather protective role of the leptin-depleting allele in our cohort.:List of Figures III List of Tables IV Index of Abbreviations V 1. Introduction 1.1. Overweight and obesity 1.1.1. Definition 1.1.2. Epidemiology 1.2. Adipose tissue 1.2.1. Adipose tissue in energy homeostasis 1.2.2. Adipose tissue as endocrine organ 1.2.3. Body fat distribution 1.3. Leptin 1.3.1. Neuroendocrine effects 1.3.2. Metabolic effects of leptin 1.3.3. Leptin deficiency vs. leptin resistance 1.3.4. Regulation of leptin expression 1.3.5. Leptin regulation by a long non-coding RNA 2. Aim of this study 3. Material and Methods 3.1. Methods 3.1.1. Study participants 3.1.2. Defining the LncR gene locus 3.1.3. Analysis of LEP and LncR expression in human adipose tissue 3.1.4. Genotyping for SNP analysis of rs10487505 3.1.5. Statistical analyses 4. Results 4.1. Gene expression measurement 4.1.1. Characteristics of LEP gene expression 4.1.2. Characterization of LncR gene expression 4.1.3. Gender-specific correlation of LEP and LncR gene expression with anthropomorphic and metabolic parameters 4.1.4. The LncR/LEP ratio influences circulating leptin 4.1.5. Correlation analysis in pre- and postmenopausal women 4.2. SNP-Analysis 4.2.1. Effects of rs10487505 in pre- and postmenopausal women 5. Discussion 5.1. LncR expression in AT 5.2. LncR expression affects circulating leptin but only in women 5.3. LncR expression affects the metabolic and inflammatory state in women 5.4. Correlation of LncR with circulating leptin levels is not dependent on menopausal state 5.5. Rs10487505 affects circulating leptin levels but not via LEP or LncR gene expression 5.6. Rs10487505 C allele decreases BMI in a highly obese cohort 5.7. Rs10487505 correlates with obesity-related and inflammatory parameters 5.8. Conclusion 6. Summary 7. References 8. Supplements 8.1. LEP and LncR gene expression 8.2. SNP-Analysis 9. Selbstständigkeitserklärung 10. Danksagung

Yes / Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission. / Funding was received from the Research Foundation Flanders (FWO, grants 1501413N and 1101614N) and the European DG Health and Food Safety (SANTE). We thank the Center of Medical Genetics at the University of Antwerp for hosting the NGS facility.

京都大学 / 新制・課程博士 / 博士(人間・環境学) / 甲第25390号 / 人博第1132号 / 新制||人||263(附属図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 西川 完途, 教授 市岡 孝朗, 教授 瀬戸口 浩彰, 教授 本川 雅治 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Première cause de décès par cancer chez la femme, le cancer du sein développe souvent une résistance à la chimiothérapie pouvant impliquer des transporteurs ABC (ATP Binding Cassette). Ils transportent les médicaments hors de la cellule et diminuent leur efficacité thérapeutique. Nous nous sommes intéressés à la protéine ABCC11 ou MRP8 (Multidrug Resistance Protein 8), exprimée dans le sein et responsable de l’efflux de certains anticancéreux (5FdUMP et méthotrexate). Nous avons démontré que l’expression d’ABCC11 était dépendante des voies de signalisation impliquant ER (Récepteur aux œstrogènes) ou PR (Récepteurs à la Progestérone). De plus, le tamoxifène (antagoniste d’ER) et la dexaméthasone (activateur de PR), utilisés en association avec la chimiothérapie, induisent l’expression d’ABCC11 et influenceraient négativement la réponse aux traitements anticancéreux à base de substrats d’ABCC11. L’expression d’ABCC11 a été positivement corrélée à celles d’ER et PR dans des cancers du sein. En parallèle, nous avons généré 2 modèles in silico en conformation ouverte vers l’intracellulaire ou vers l’extracellulaire et identifier des acides aminés potentiellement critiques dans l’architecture de la protéine ainsi que dans la liaison avec certains substrats (5FdUMP et GMPc). Nous avons également généré les outils moléculaires permettant l’étude de l’impact de 13 SNP (Single Nucleotide Polymorphism) non synonymes d’ABCC11. En raison d’une instabilité des lignées cellulaires, l’étude n’a pu être menée à son terme. Notre travail a ainsi contribué à une meilleure caractérisation d’ABCC11 et souligne sa potentielle valeur pronostic et prédictive dans le traitement du cancer du sein. / Leading cause of woman death by cancer, breast cancer can unfortunately develops chemotherapy resistance involving ABC (ATP Binding Cassette) transporters. They transport drugs out of cells and decrease their therapeutic efficiency. We studied one ABCC sub-family member: ABCC11 or MRP8 (Multidrug Resistance Protein 8), expressed in breast and responsible for anticancer agent efflux (5FdUMP and methotrexate). We have demonstrated that ABCC11 expression was associated with ER (Estrogen Receptor) and PR (Progesterone Receptor) signaling pathways. Furthermore, tamoxifen (ER antagonist) and dexamethasone (PR activator), used in association with chemotherapy, increased ABCC11 expression and would negatively influence the response of ABCC11 substrate based anticancer treatments. Moreover, ABCC11 expression was positively correlated to ER and PR expression in breast cancer. In parallel, we have generated two in silico models obtained by homology. They represent two different spatial conformations: intracellular-facing (ready to bind substrate) or extracellular-facing (ready to release substrate). This has allowed us to identify amino acid residues potentially essential for the protein architecture and for substrate binding (5FdUMP and cGMP). In order to analyze SNP (Single Nucleotide Polymorphism) impact on ABCC11 expression and function, we generated vectors coding a wild-type or a mutated ABCC11 with 13 nonsynonymous SNPs. But, we did not succeed to create stable expressing cell lines to make a complete study of those SNPs. In conclusion, our work led to ABCC11 better characterization and underlined its putative prognostic and predictive value in breast cancer treatment.

A obesidade se tornou uma das maiores preocupações de saúde pública. É um distúrbio neuroendócrino, no qual fatores ambientais e genéticos agem em conjunto, levando ao excesso de armazenamento de energia na forma de gordura corporal. A síndrome de Prader-Willi (PWS) é a mais freqüente das síndromes que possui a obesidade como uma de suas características, com incidência de 1:25.000 nascimentos. É caracterizada por hipotonia neonatal com dificuldade de sucção, atraso do desenvolvimento neuropsicomotor (DNPM), hiperfagia, obesidade, baixa estatura em adolescentes, mãos e pés pequenos, hipogonadismo, distúrbios do sono, características faciais dismórficas, deficiência intelectual leve a moderada e comportamento obsessivo-compulsivo. Pacientes com atraso do DNPM e/ou dificuldade de aprendizado, distúrbios de comportamento, obesidade e/ou hiperfagia, com teste negativo para PWS, foram estudados com plataformas de SNP array, “The GeneChip® Mapping 500K Set” da Affymetrix, ou array-CGH, CytoSure ISCA 4x180k da OGT, para identificar genes relacionados a obesidade e hiperfagia, assim como, novas regiões genômicas implicadas na etiologia de síndromes genéticas associadas à obesidade. Dentre os 31 pacientes estudados, oito apresentaram variações de número de cópias (CNVs) em seu genoma: deleção em 1p22.1p21.2; deleção em 3q25.33q26.1 e deleção em 13q31.2q32.1; duplicação em 7q36.2; deleção em 8p23.3p23.1 e duplicação em 12p13.33p13.31; duplicação 16p13.11p12.3; duplicação em 17q11.2; deleção em 20p12.1; duplicação em 21q22.13. Duas dessas alterações foram herdadas de pais fenotipicamente normais. Algumas dessas CNVs sobrepõem regiões genômicas previamente relacionadas com obesidade, incluindo a microdeleção de 1p21.3 e as duplicações dos cromossomos 12 e 21. Identificamos genes anteriormente descritos como associados à obesidade (PTBP2, DPYD, MIR137, GNB3 e PPM1L), ou possivelmente envolvidos com este fenótipo (HTR5A e KCNJ6), mapeados em várias dessas CNVs. Além disso, os genes RNF135, NF1, DPP6, GPC5, DYRK1A e MACROD2 são os prováveis causadores da deficiência intelectual, atraso do desenvolvimento neuropsicomotor, dificuldades de aprendizagem, distúrbios de comportamento e outras características clínicas encontrados nos pacientes. O diagnóstico e prognóstico dos pacientes e o Aconselhamento Genético aos pais e familiares é fornecido / Obesity has become a major concern for public health. It is a neuroendocrine disorder, in which genetic and environmental factors act together, leading to excessive storage of energy as fat. Prader-Willi syndrome (PWS) is the main obesity-related syndrome with a birth incidence of 1:25,000. It is characterized by neonatal hypotonia, poor sucking, developmental delay, hyperphagia, obesity, short stature in adolescents, small hands and feet, hypogonadism, sleep disturbance, dysmorphic facial features, mild to moderate intellectual disability and obsessive-compulsive behavior. Patients with psychomotor developmental delay and/or learning disabilities, behavior disorders, obesity and/or hyperphagia, who tested negative for PWS, were studied by chromosomal microarray analysis, including the SNP-based platform “The GeneChip® Mapping 500K Set” (Affymetrix), and the array-CGH platform “CytoSure ISCA 4x180k (OGT)”, to identify genes related to hyperphagia and obesity, as well as new genomic regions implicated in the etiology of genetic syndromes associated with obesity. Of 31 patients studied, eight had copy number variants (CNVs) in the genome: 1p22.1p21.2 deletion; 3q25.33q26.1 deletion and 13q31.2q32.1 deletion; 7q36.2 duplication; 8p23.3p23.1 deletion and 12p13.33p13.31 duplication; 16p13.11p12.3 duplication; 17q11.2 duplicaton; 20p12.1 deletion; 21q22.13 duplication. Two of these CNVs were inherited from an unaffected father. Some of these CNVs overlap genomic regions that have previously been related to obesity, including the 1p21.3 microdeletion and the duplications of chromosomes 12 and 21. Furthermore, we identified genes previously described as associated with obesity (PTBP2, DPYD, MIR137, GNB3 and PPM1L), or possibly involved with this phenotype (HTR5A and KCNJ6), mapped to several of these CNVs. In addition, the genes RNF135, NF1, DPP6, GPC5, DYRK1A and MACROD2 are likely implicated in intellectual disability, developmental delay, learning disabilities, behavioral disorders and other clinical features found in patients. The diagnosis and prognosis of patients and genetic counseling to parents and families is provided

Les variations en nombre de copies (CNV) sont des gains ou pertes d’une séquence d’ADN et peuvent avoir un rôle dans la susceptibilité à certaines maladies. Les CNVs peuvent être détectés par les puces de SNPs de haute résolution en analysant les intensités des allèles avec des algorithmes de détection des CNVs tels que CNV partition, PennCNV et QuantiSNP. Dans cette thèse, nous avons évalué les performances de ces outils pour la détection des CNVs au niveau pangénomique et pour les tests d'association. Nous avons également étudié des stratégies d'association combinant les informations de l'allèle et du nombre de copies pour des SNP situés dans des CNV. Nous avons appliqué ces outils pour mener une étude d’association pan-génomique avec les CNV en utilisant les données de l'étude espagnole du cancer de lavessie (SBC)/EPICURO générées par la puce Illumina 1M.Nos résultats montrent une faible fiabilité et une faible sensibilité des algorithmes de détection des CNV. Dans la région du gène GSTM1 où un CNV très fréquent existe qui est associé au risque de cancer de la vessie, nous avons constaté que les algorithmes de détection des CNV ont de faibles performances. Néanmoins, l’utilisation de la mesure d'intensité des allèles dans les tests d'association peut alors être une alternative intéressante car cela nous a permis de détecter cette association connue. Pour les SNPs situés dans des CNVs, nous avons étudié plusieurs stratégies de tests d'association et nous avons montré que la plus puissante était d’utiliser un modèle avec deux termes correspondant respectivement à la somme et à la différence du nombre de copies des deux allèles. Finalement, en appliquant ces stratégies à l'étude (SBC)/EPICURO, nous avons identifié des CNVs potentiellement associés au risque de cancer de la vessie, ainsi que des SNP dont l'allèle et le nombre de copies pourraient être impliqués dans le risque de cancer de la vessie. / Copy number variations (CNVs) are losses or gains of DNA sequences that may play a role in specific disease susceptibility. CNVs can be detected by high-resolution SNP-arrays through the analysis of allele intensities with CNV calling algorithms such as CNVpartition, PennCNV and QuantiSNP. In this thesis, we identified and assessed the performances of available tools for CNV calling and for association testing, at the genome-wide level. We also investigatedassociation strategies that combine information on both the allele and the number of copies for SNPs located in CNV regions. We applied these tools to conduct a genome-wide association study with CNV using data from the Spanish Bladder Cancer (SBC)/EPICURO Study generated by the Illumina 1M SNP-array. Our results showed a low reliability and a low sensitivity of the investigated CNV calling algorithms applied to SNP-array data. The GSTM1 locus shows a very frequent CNV that is associated with bladder cancer (BC) risk. We reported that the calling algorithms performed very poorly in identifying this CNV. We proposed using allele intensity measures (LRR) as a screening step to assess association as it allowed the detection of the GSTM1 CNV association with BC. To combine the allele and the number of copies for SNPs located in CNV regions, we investigated several strategies of association testing and we showed that the more powerfulone used a two-term model with the sum and the difference of the number of copies of both alleles. Finally, by applying these strategies to the (SBC)/EPICURO Study, we identified CNV regions potentially associated with BC risk, as well as SNPs for which both the allele and the number of copies could be involved in BC risk.

Les strongles gastro-intestinaux, dont Haemonchus contortus constituent un problème majeur pour l'élevage des ovins allaitants. Ils entrainent des pertes de production et le recours aux anthelminthiques est remis en question par l'apparition de souches de vers résistantes. La sélection d'ovins plus résistants fait partie des stratégies complémentaires de lutte les plus sérieuses. Cependant sa mise en oeuvre requiert une meilleure compréhension des mécanismes sous-jacents. Cette thèse vise à identifier les régions du génome ovin impliquées dans la résistance aux strongles gastro-intestinaux. Une analyse statistique d'association entre des marqueurs génétiques et des mesures de résistance d'un troupeau d'ovins croisés Martinik Black-belly x Romane a mis en évidence un nombre limité de régions d'intérêt. Parmi celles-ci, un segment du chromosome 12 a été choisi pour effectuer des accouplements raisonnés et valider son rôle dans la résistance à H. contortus. L'effet de cette région a été validé chez les descendants issus d'accouplements assistés par marqueurs génétiques. Cette région semble limiter fertilité des vers femelles tout en contribuant à une réponse immunitaire plus forte. Le rôle d'une région du chromosome 21 dans la variation de concentration plasmatique en pepsinogène, un marqueur de lésions abomasales, a également été confirmé. Un gène candidat sous-jacent est en cours de séquençage et l'analyse des polymorphismes devrait contribuer à la validation de son rôle. Deux autres gènes très proches pourraient également être impliqués et mériteraient une considération future. Ces travaux illustrent à la fois la variation génétique disponible pour les caractères de résistance à H. contortus et la complexité des mécanismes mis en jeu. Des études complémentaires de séquençage et d'étude d'expression par séquençage devrait contribuer à une meilleure compréhension des fonctions des gènes impliqués et de leurs interactions. / Gastro-intestinal nematodes, among which Haemonchus contortus are a major threat to the meat sheep industry. They are responsible for production losses and the apparition of worm populations resistant to drugs limits their use as worm control strategy. Breeding more resistant sheep is among the most practicable alternative strategy. However its implementation requires a deeper understanding of underlying mechanisms. This PhD aims at identifying regions of the ovine genome affecting resistance to gastro-intestinal nematodes. A statistical analysis of existing associations between genetic markers and resistance traits of a Martinik Black-belly x Romane cross-bred sheep flock unraveled a limited number of key players. Among these, a fragment of the chromosome 12 was chosen to perform marker-assisted matings and to validate its role in resistance to H. contortus. The effect of this region was validated in the progenies born from matings. It seems this chromosomic fragment limits female worms fertility and is associated to a stronger immune response. The putative role played by a fragment of the chromosome 21 in plasmatic pepsinogen concentration (a biomarker of abomasal lesions) was also confirmed in this work. A candidate gene underlying this region has been sequenced and the analysis of the detected polymorphisms should confirm its role. Further, two other genes in its vicinity could also play a role in this biological phenomenon and they should also deserve future considerations. This work illustrated both the existing genetic variation for resistance to H. contortus and the associated complexity of underlying mechanisms. Additional sequencing and gene expression sequencing studies should help understanding gene functions and interactions.

Apesar de altamente estável, o DNA sofre milhares de alterações em sua estrutura diariamente, sejam essas espontâneas ou pela exposição a agentes mutagênicos. A maior parte dessas alterações é prontamente removida por um conjunto de eventos de reparo de DNA. A via de reparo por excisão de nucleotídeos (NER) é a mais versátil e flexível lidando com uma variedade de lesões que podem gerar distorções das hélices do DNA. Esses danos resultam em alterações características que, caso não reparadas, podem gerar mutações ou morte celular e, consequentemente, câncer e envelhecimento. Algumas síndromes, nas quais os pacientes são sensíveis à luz solar, estão relacionadas à deficiência no processo de NER, como a Xeroderma Pigmentosum (XP), síndrome de Cockayne (CS) e Tricotiodistrofia (TTD). Indivíduos brasileiros, incluindo pacientes com diagnóstico clínico de XP e membros das famílias, passaram por um processo in silico para a identificação variantes em genes relacionados aos processos de reparo do DNA após o sequenciamento do DNA por plataformas de nova geração (NGS: plataforma ABI 5500XL SOLiD e MiSeq Illumina) e análises de Bioinformática. Para cada paciente, foram selecionados os melhores valores de parâmetros para se realizar a busca por variantes considerando a qualidade de alinhamento e a taxa de cobertura das bases alvo. SNPs já depositados no banco de dados do projeto 1000genomes foram removidos de nossos dados. O restante das variantes foi analisado para encontrar potenciais candidatos que poderiam explicar o diagnóstico clínico do paciente. Em muitas amostras foi possível determinar pelo menos uma variante (mutação) com uma elevada possibilidade de ser responsável pelos sintomas XP. Para alguns pacientes, a má qualidade do sequenciamento ou eventos não esclarecidos durante este, dificultou a identificação de candidatos à mutação patogênica. Potenciais mutações não sinônimas foram analisadas com os programas SIFT e PROVEAN, que identificaram a potencial capacidade deletéria da alteração de aminoácido na proteína. Finalmente, foi desenvolvida uma interface de domínio público amigável, a Human Variantes do Finder Interface (http://www.varfinderhg.com.br), que visa facilitar a identificação de variantes em dados gerados por NGS. / Although highly stable, DNA molecule undergoes thousands of damage in its structure every day, due to spontaneous lesions or exposure to various mutagens. Most of these lesions are readily removed by a number of cellular DNA repair processes. The process of nucleotide excision repair (NER) is the most versatile and flexible dealing with a variety of lesions that can lead to distortions of the DNA strands. Ultraviolet irradiation induced DNA damage are the main substrates for NER. These DNA damage, if not repaired, can generate mutations or cell death causing several diseases, including cancer and aging. Some syndromes, sensitive to sunlight, are related to deficiencies in the NER process, such as Xeroderma Pigmentosum (XP), Cockayne syndrome (CS) and Trichothiodystrophy (TTD). Brazilian individuals, including patients with clinical diagnosis of XP and family members, went through in silico process for the identification of variants in genes related to DNA repair processes after DNA sequencing by next generation sequencing (NGS in the platforms ABI 5500XL SOLiD and MiSeq Illumina) and dedicated Bioinformatics pipelines. For each patient the best search pattern of variant calling was used considering the alignment quality and coverage rate of bases in target. SNPs already deposited at the 1000genomes project database were removed from the data. The remaining variants were analyzed to find potential candidates that could explain the clinical diagnosis. In many samples, it was possible to determine at least one variant (mutation) with a high possibility of being responsible for the clinical XP. For some patients, the poor quality of the sequencing or unclear events during sequencing hampered the identification of clear mutation candidates. Potential nonsynonymous mutations were analyzed with SIFT and PROVEAN softwares, which identified the potential deleterious capacity of the amino acid change in the protein. Finally, we developed a user-friendly public domain interface, the Human Variants Finder Interface (http://www.varfinderhg.com.br), which, we expect, will facilitate the identification of variants in data generated by NGS.

Ziel der vorliegenden Arbeit war die Entwicklung einer SNP-Genotypisierungsmethode mit auf Mikroarrays immobilisierten PCR-Produkten. Für die Analyse wurde ein faseroptischer Affinitätssensor bzw. ein Durchfluss-Biochip-Scanner mit integrierter Fluoreszenzdetektion verwendet. An den immobilisierten Analyten (PCR-Produkten) wurde eine Fluoreszenzoligonukleotidsonde hybridisiert und anschließend die Dissoziation der Sonde im Fluss verfolgt. Die Diskriminierung von Wildtyp- und Mutanten-DNA erfolgte durch die kinetische Auswertung der Dissoziationskurven sowie durch die Analyse der Fluoreszenzintensität. <br> <br> Die Versuche am faseroptischen Affinitätssensor zeigten, dass DNA-DNA-Hybride sowohl von Oligonukleotiden als auch von PCR-Produkten ein typisches Dissoziationsverhalten aufweisen, wobei fehlgepaarte Hybride eine signifikant schnellere Dissoziation zeigen als perfekt passende Hybride. Dieser Geschwindigkeitsunterschied lässt sich durch den Vergleich der jeweiligen kinetischen Geschwindigkeitskonstanten kD quantitativ erfassen. </p> <p>Da die Kopplung des Analyten an der Chipoberfläche sowie die Hybridisierungs- und Dissoziationsparameter essentiell für die Methodenentwicklung war, wurden die Parameter für ein optimales Spotting und die Immobilisierung von PCR-Produkten ermittelt. Getestet wurden die affine Kopplung von biotinylierten PCR-Produkten an Streptavidin-, Avidin- und NeutrAvidin-Oberflächen sowie die kovalente Bindung von phosphorylierten Amplifikaten mit der EDC/Methylimidazol-Methode. Die besten Ergebnisse sowohl in Spotform und -homogenität als auch im Signal/Rausch-Verhältnis wurden an NeutrAvidin-Oberflächen erreicht. </p> <p>Für die Etablierung der Mikroarray-Genotypisierungsmethode durch kinetische Analyse nach einem Hybridisierungsexperiment wurden Sondenlänge, Puffersystem, Spotting-Konzentration des Analyten sowie Temperatur optimiert. Das Analysensystem erlaubte es, PCR-Produkte mit einer Konzentration von 250 ng/µl in einem HEPES-EDTA-NaCl-Puffer auf mit NeutrAvidin beschichtete Glasträger zu spotten. In den anschließenden Hybridisierungs- und Dissoziationsexperimenten bei 30 °C konnte die Diskriminierung von homocygoter Wildtyp- und homocygoter Mutanten- sowie heterocygoter DNA am Beispiel von Oligonukleotid-Hybriden erreicht werden. </p> <p>In einer Gruppe von 24 homocygoten Patienten wurde ein Polymorphismus im SULT1A1-Gen analysiert. Sowohl durch kinetische Auswertung als auch mit der Analyse der Fluoreszenzintensität wurde der Genotyp der Proben identifiziert. Die Ergebnisse wurden mit dem Referenzverfahren, der Restriktionschnittstellenanalyse (PCR-RFLP) validiert. Lediglich ein Genotyp wurde falsch bestimmt, die Genauigkeit lag bei 96%. </p> <p>In einer Gruppe von 44 Patienten wurde der Genotyp eines SNP in der Adiponectin-Promotor-Region untersucht. Nach Vergleich der Analysenergebnisse mit denen eines Referenzverfahrens konnten lediglich 14 der untersuchten Genotypen bestätigt werden. Ursache für die unzureichende Genauigkeit der Methode war vor allem das schlechte Signal/Rausch-Verhältnis.</p> <p> Zusammenfassend kann gesagt werden, dass das in dieser Arbeit entwickelte Analysesystem für die Genotypisierung von Einzelpunktmutationen geeignet ist, homocygote Patientenproben zuverlässig zu analysieren. Prinzipiell ist das auch bei heterocygoter DNA möglich. Da nach aktuellem Kenntnisstand eine SNP-Analysemethode an immobilisierten PCR-Produkten noch nicht veröffentlicht wurde, stellt das hier entwickelte Verfahren eine Alternative zu bisher bekannten Mikroarray-Verfahren dar. Als besonders vorteilhaft erweist sich der reverse Ansatz der Methode. </p> <p>Der hier vorgestellte Ansatz ist eine kostengünstigere und weniger hoch dimensionierte Lösung für Fragestellungen beispielsweise in der Ernährungswissenschaft, bei denen meist eine mittlere Anzahl Patienten auf nur einige wenige SNPs zu untersuchen ist. Wenn es gelingt, durch die Weiterentwicklung der Hardware bzw. weiterer Optimierung, eine Verbesserung des Signal/Rausch-Verhältnisses und damit die Diskriminierung von heterocygoter DNA zu erreichen, kann diese Methode zukünftig bei der Analyse von mittelgroßen Patientengruppen alternativ zu anderen Genotypisierungsmethoden verwendet werden. / The aim of this thesis was the development of a SNP genotyping method involving PCR products immobilised on microarrays. For the analysis a fibre optic affinity biosensor and a flow-through biochip scanner were used. Fluorescent probes were hybridized with the immobilised PCR products. In order to start the dissociation process the surface was rinsed with buffer and the fluorescence intensity was measured. <br><br> Two different cases were studied: First, the full-matched DNA hybrid (wildtyp single strand with complementary wildtype single strand), second the mis-matched hybrid (wildtype single strand and mutant single strand). After determinating the reaction rates (kD) as kinetic parameter the kD values of both cases were compared. The experiments showed a significant difference in the kD value of the full- and the mis-match hybrids. Therefore, mutant and wildtype DNA were discriminated by kinetic analysis of the dissociation process and analysis of the fluorescence intensity. <br><br> To set up the complete analysis process the reaction parameters like coupling of the PCR products had to be optimised. Both affininty coupled (streptavidin, neutravidin, avidin - biotin) and covalent methods (EDC/methylimidazol) were carried out. Best results in spot homogeinity and spot appearance were obtained with coupling of biotinylated PCR products on neutravidin coated chip surfaces. Additionally, the length of the probe, the spotting concentration, the spotting buffer and the reaction temperature were optimised. In the optimised analysis PCR products (250 µg/µl) were spotted onto neutravidin coated surfaces. The hybridisation <br><br> and dissociation processes were carried out at 30°C. A HEPES-EDTA-NaCl buffer was used for spotting, diluting of the fluorescent probe and rinsing the microarray surface. A fluorescent probe was used with 13 nucleotides in length. The mis- or full-matching base indicating the polymorphism was located in the center position of the probe. <br><br> The analysis system was tested with the genomic DNA of a group of 24 homocygote individuals with a SNP in the SULT1A1 gene region. The hybridisation and dissociation processes were carried out and the reaction rates were determinated. Subsequently after the analysis in the flow-through biochip scanner the fluorescence intensity of the <br><br> spots were measured. The results showed very good comparability with results of a PCR-RFLP analysis (one false genotype). Additionally, a group of 44 heterocygote DNA samples with one SNP in the adiponectin promotor region were also genotyped. Compared to a reference method only 14 genotypes were correctly determined. This was mostly due to a low signal-noise-ratio and needs to be further investigated. <br><br> Besides the problem in analysing heterocygote DNA samples the developed analysis system is very useful for genotyping SNP in homocygote DNA samples. The successful analysis of heterocygote sample is principally possible and with further investigations/optimisation, a better analysis should be possible. <br><br> The most important advantage of the developed method is the reverse approach of binding PCR products at the surface instead of oligonucleotides. This allows the parallel genotyping of several individuals. Other advantages include low costs and medium sized dimensions in terms of throughput.

Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations. The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease. An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available. The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene. Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect.