<p>The studies in this thesis describe the development of a microarray based minisequencing system and its application to highly parallel genotyping of single nucleotide polymorphisms. The technical developments included identification of a three-dimensional microarray surface coating with high binding capacity for oligonucleotides modified with amino groups as the most optimal one for the system. The system was also established for multiplexed, reproducible quantitative analysis of SNP alleles both on the level of DNA and RNA. The sensitivity of the system to distinguish SNP alleles present as a minority in a mixed sample was found to be 1-6%. </p><p>The microarray based minisequencing system was applied in a pharmacogenetic study on antihypertensive drug response. A panel of 74 SNPs located in candidate genes related to blood pressure regulation were genotyped in DNA samples from hypertensive patients that had been treated with the antihypertensive drugs irbesartan or atenolol. Multiple regression analysis of the genotype data against the reduction in blood pressure identified genotype combinations of four to five SNPs that explain 44-56% of the reduction in blood pressure in the two treatment groups. The genotypes of two individual SNPs in the angiotensinogen (AGT) gene and a SNP in the low density lipoprotein receptor (LDLR) gene appeared to be associated to reduced blood pressure after treatment with atenolol, while a SNP in the apolipoprotein B (APOB) gene was associated to blood pressure reduction after irbesartan treatment. The genotype of one SNP in the adrenergic alpha-2A-receptor gene (ADRA2A) was related to the reduction in left ventricular mass following atenolol treatment while the genotypes of two SNPs, one in the APOB gene and one in the AGT gene were related to the reduction in left ventricular mass in the patients treated with irbesartan.</p>

<p>BACKGROUND: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. CD40 ligand is another membrane molecule, which ligates to cell types associated with atherosclerotic plaques thereby mediating intraplaque inflammation and weakening of the fibrous cap. Acute coronary syndrome (ACS) is a multi-factorial disease in which TF and CD40 ligand have prominent roles. Single nucleotide poly-morphisms (SNPs) in the TF and CD40 ligand genes may influence the development, pro-gression and outcome in ACS. AIM: The aim of this thesis was to investigate the genetic and molecular control of TF expression in healthy individuals and in patients with ACS. More-over, the aim was to investigate whether SNPs in the TF and CD40L genes respectively were associated with risk and outcome in ACS and / or with plasma concentrations of these pro-teins. RESULTS: A real-time PCR method that allowed sensitive and dynamic quantification of TF mRNA was established and used for the identification of a high and low response phe-nomenon of TF mRNA. The TF high and low response correlated with the expression of toll-like receptor 4 (TLR-4) thus linking TF to innate immunity in a novel fashion. Investigation of several SNPs in the TF and CD40L genes led to the identification of the 5466 A>G in the TF gene and the -3459 A>G SNP in the CD40L gene. The 5466 G allele was associated with cardiovascular death in patients with ACS and increased TF procoagulant activity in human monocytes, which explained the clinical association. The -3459 G allele regulated the produc-tion of soluble CD40L but was not related with patient outcome. Soluble CD40L levels above median were associated with the risk of MI in patients with ACS. A prolonged treatment with dalteparin was more efficient in patients presenting with high levels of sCD40L, which further supports sCD40L as a marker of a prothrombotic state. CONCLUSIONS: The results of this thesis adds to our current knowledge of factors influencing TF expression and activity by demonstrating the effects of TF gene variants, cell signalling molecules, CD40 ligand protein and gene variation. All of these effects have the potential to modify the risk of development, progression and outcome in the acute coronary syndrome and exemplify the interplay between coagulation and inflammation, in which both TF and CD40 ligand are active.</p>

<p>Many Single Nucleotide Polymorphisms (SNPs) found in coding or regulatory regions within the human genome lead to phenotypic differences that make prediction of physical appearance, based on genetic analysis, potentially useful in forensic investigations. Complex traits such as pigmentation can be predicted from the genome sequence, provided that genes with strong effects on the trait exist and are known. Phenotypic traits may also be associated with variations in gene expression due to the presence of SNPs in promoter regions. In this project, the identification of genes associated with these physical traits of potential forensic relevance have been collated from the literature using a text mining platform and hand curation. The SNPs associated with these genes have been acquired from public SNP repositories such as the International HapMap project, dbSNP and Ensembl. Characterization of different population groups based on the SNPs has been performed and the results and data stored in a MySQL database. This database contains SNP genotyping data with respect to physical phenotypic differences of forensic interest. The potential forensicrelevance of the SNP information contained in this database has been verified through in silico SNP analysis aimed at establishing possible relationships between SNP occurrence and phenotype. The software used for this analysis is MATCH&trade / .</p>

BACKGROUND: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. CD40 ligand is another membrane molecule, which ligates to cell types associated with atherosclerotic plaques thereby mediating intraplaque inflammation and weakening of the fibrous cap. Acute coronary syndrome (ACS) is a multi-factorial disease in which TF and CD40 ligand have prominent roles. Single nucleotide poly-morphisms (SNPs) in the TF and CD40 ligand genes may influence the development, pro-gression and outcome in ACS. AIM: The aim of this thesis was to investigate the genetic and molecular control of TF expression in healthy individuals and in patients with ACS. More-over, the aim was to investigate whether SNPs in the TF and CD40L genes respectively were associated with risk and outcome in ACS and / or with plasma concentrations of these pro-teins. RESULTS: A real-time PCR method that allowed sensitive and dynamic quantification of TF mRNA was established and used for the identification of a high and low response phe-nomenon of TF mRNA. The TF high and low response correlated with the expression of toll-like receptor 4 (TLR-4) thus linking TF to innate immunity in a novel fashion. Investigation of several SNPs in the TF and CD40L genes led to the identification of the 5466 A&gt;G in the TF gene and the -3459 A&gt;G SNP in the CD40L gene. The 5466 G allele was associated with cardiovascular death in patients with ACS and increased TF procoagulant activity in human monocytes, which explained the clinical association. The -3459 G allele regulated the produc-tion of soluble CD40L but was not related with patient outcome. Soluble CD40L levels above median were associated with the risk of MI in patients with ACS. A prolonged treatment with dalteparin was more efficient in patients presenting with high levels of sCD40L, which further supports sCD40L as a marker of a prothrombotic state. CONCLUSIONS: The results of this thesis adds to our current knowledge of factors influencing TF expression and activity by demonstrating the effects of TF gene variants, cell signalling molecules, CD40 ligand protein and gene variation. All of these effects have the potential to modify the risk of development, progression and outcome in the acute coronary syndrome and exemplify the interplay between coagulation and inflammation, in which both TF and CD40 ligand are active.

Single Nucleotide Polymorphisms (SNPs) are the most common DNA sequence variations where only a single nucleotide (A, T, C, G) in the human genome differs between individuals. Besides being the main genetic reason behind individual phenotypic differences, SNP variations have the potential to exploit the molecular basis of many complex diseases. Association of SNPs subset with diseases and analysis of the genotyping data with clinical findings will provide practical and affordable methodologies for the prediction of diseases in clinical settings. So, there is a need to determine the SNP subsets and patients&rsquo / clinical data which is informative for the prediction or the diagnosis of the particular diseases. So far, there is no established approach for selecting the representative SNP subset and patients&rsquo / clinical data, and data mining methodology that is based on finding hidden and key patterns over huge databases. This approach have the highest potential for extracting the knowledge from genomic datasets and to select the number of SNPs and most effective clinical features for diseases that are informative and relevant for clinical diagnosis. In this study we have applied one of the widely used data mining classification methodology: &ldquo / decision tree&rdquo / for associating the SNP Biomarkers and clinical data with the Alzheimer&rsquo / s disease (AD), which is the most common form of &ldquo / dementia&rdquo / . Different tree construction parameters have been compared for the optimization, and the most efficient and accurate tree for predicting the AD is presented.

Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans. The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system. The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning. In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems. The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.

Small intestinal neuroendocrine tumor (SI-NET) is the most common small bowel tumor in Europe and USA, with an annual incidence of around 0.3-1.3/100000 persons. SI-NETs are the most common type of gastroenteropancreatic NETs (GEP-NETs), and they are known for their ability to produce hormones such as tachykinins and serotonin, as well as for their favorable long-term prognosis in comparison to gastrointestinal adenocarcinoma. The overall aim of the thesis was to investigate unknown or unclear aspects of SI-NET disease, in connection with prognosis, treatment and follow-up. Paper I confirmed several known negative prognostic factors and also showed, for the first time, that para-aortal lymph node metastases and peritoneal carcinomatosis were associated with worse survival by multivariable analyses. Locoregional surgery was associated with a low post-operative mortality, and a prolonged long-term survival by multivariable analysis. In Paper II we continued to investigate peritoneal carcinomatosis and found it be a risk factor not only for death, but also for emergency re-surgery. Furthermore, genetic analyses of samples from primary tumors in patients with and without peritoneal carcinomatosis showed a difference in the DNA between these two groups. In Paper III the outcome after liver surgery and/or radiofrequency ablation of liver metastases was investigated. To summarize, no difference in survival was seen in patients treated with surgery/radiofrequency ablation in comparison with matched controls. However, a superior radiological response of liver metasases and lower U-5-HIAA values were seen in patients subjected to liver surgery and/or radiofrequency ablation compared to matched controls. Paper IV compared ultrasonography, computed tomography and 11C-5HTP-PET in the follow-up after radiofrequency ablation of NET liver metastases. The study concluded that 11C-5HTP-PET depicted all residual tumors after RFA and that it, if used, should be combined with computed tomography for easier interpretation, as RFA areas are not clearly distinguishable with 11C-5HTP-PET alone. Paper V studied gallstone complications after somatostatin analog treatment in SI-NET patients, and concluded that there was a rather high risk to be subjected to a cholecystectomy due to biliary colic, cholecystitis, cholangitis or pancreatitis after primary surgery in somatostatin analog treated patients.

River buffalo are economically important to many countries and only recently has their genome been explored for the purpose of mapping genetic variation in traits of economic and biologic interest. The purpose of this research is to characterize the genetic and evolutionary profile of Toll-like receptor 5 (TLR5), which mediates the mammalian innate immune response to bacterial flagellin. This study is comprised of three parts: 1) generating a radiation hybrid (RH) map of river buffalo chromosome 5 (BBU5) where the TLR5 gene is located and building a comparative map with homologous cattle chromosomes; 2) conducting a single-nucleotide polymorphism (SNP) survey of the TLR5 gene to reveal variation within river buffalo and other species; and 3) performing an evolutionary study by inferring phylogenetic trees of TLR5 across multiple taxa and determining the possible evolutionary constraints within the TLR5 coding region. River buffalo chromosome 5 is a bi-armed chromosome with arms corresponding to cattle chromosomes 16 and 29. A BBU5 RH map was developed using the previously published river buffalo RH mapping panel and cattle-derived markers. The RH map developed in this study became an integral part of the first river buffalo whole genome RH map. Genetic variation of the TLR5 gene was evaluated in a small domestic herd of river buffalo. Sequencing of the TLR5 coding region and partial associated 5'- and 3'-untranslated regions yielded 16 novel SNPs. Six SNPs were identified as non-synonymous with one predicted to potentially code for a functionally altered product. For the evolutionary study of the TLR5 coding region, phylogenetic trees were inferred based on TLR5 variation across multiple orders and another for artiodactyla. Species that are closely related to river buffalo appear to have undergone negative selection in TLR5 while those that diverged from river buffalo earlier may be retaining alleles that river buffalo are removing from the population. In conclusion, putative chromosomal rearrangements were identified between river buffalo and cattle, the variation that was uncovered in the TLR5 coding region could potentially lead to differential immunity across species, and there appears be some evolutionary flexibility in the DNA sequence of the TLR5 coding region.

Analysis of genetic relatedness is of great importance in forensic casework such as immigration and identification cases. The conventional methods for relationship testing are not sufficient in the most complicated cases, because more genetic markers are required to obtain results with satisfactory statistical security. This study demonstrates that microarrays, which can be used to genotype thousands of single nucleotide polymorphisms (SNPs), could be a promising solution to this problem. The microarray technique used in this study performed very well on blood samples and also worked well in combination with whole genome amplification, but did not generate any results when used on severely degraded materials. Markers suitable for relatedness analysis were selected from the microarray and were successfully tested on families with known genetic relations. Although a maximum of 64 autosomal markers were used, there is a great potential of selecting the hundreds or thousands of markers that may be required in some cases of relatedness investigation.

In this thesis, different genetic tools are used to investigate pre- and postzygotic barriers to gene exchange and their role in speciation in the pied flycatcher (Ficedula hypoleuca) and the collared flycatcher (F. albicollis). This species complex consists of four genetically distinct clades that apparently diverged in allopatry (I). Sequencing of introns from autosomal and Z-linked genes from the two species reveals signs of selection on the Z-chromosome. Sexual selection acting on Z-linked genes might explain this pattern (II). By using large-scale genotyping of single nucleotide polymorphisms (SNPs), introgression is observed at autosomal- but not Z-linked loci, mostly from the pied- to the collared flycatcher. Male plumage characters and genes involved in hybrid fitness are largely mapped to the Z-chromosome (III). By studying mate choice of female hybrids I show that there is a link between female preferences and the Z chromosome (IV). The rate of introgression in island versus clinal hybrid zones is consistent with regional differences in hybrid fertility. Asymmetric gene flow from allopatry on the islands may oppose reinforcement, leading to introgression and a partial breakdown of postzygotic isolation. Adaptive introgression may explain the high rate of introgression observed at one of the genetic markers (V). For late breeding female collared flycatchers it appears to be adaptive to use pied flycatchers as social fathers but conspecific males as genetic fathers. Additionally, females in mixed species pairs may reduce hybridization costs by producing an excess of male hybrid offspring that are more fertile than females (VI). In conclusion, the Z-chromosome appears to play a major role in flycatcher speciation. Sexual selection and reinforcement are important mechanisms in the divergence of these birds. However, gene flow from allopatry, introgression of adaptive genes and adaptive hetrospecific pairing by late breeding collared flycatcher females may work in the opposite direction.