The transition to flowering is a major developmental switch in angiosperms, the timing of which is expected to be important for fitness. In this thesis the focus has been on identification of genes affecting natural variation in flowering time in Brassica nigra. The background for this thesis is an earlier QTL-mapping study in B. nigra. The genomic area with the greatest effect on flowering time in that study contained a homolog to the CONSTANS gene, which is known to affect flowering time in A. thaliana. When studied more closely this gene did not seem to affect flowering time variation in B. nigra. Near the B.nigra CO gene (BniCOa), however, we identified a homolog to the related CONSTANS LIKE 1 (COL1) gene. In A. thaliana COL1 has not been shown to be associated with induction of flowering but since the B. nigra homolog (BniCOL1) in the QTL area showed surprising amounts of variation between early and late flowering plants we set out to test if this variation was associated with flowering time variation. In the first paper we found a significant association between flowering time and one indel (Ind2) in the coding region. Motivated by the results in paper one, we searched for evidence of selection at the BniCOL1(paper two). In paper three the aim was to validate the results from the first paper in a larger sample of populations, and to check whether the association found in paper I could reflect linkage disequilibrium with areas outside of the gene. Finally, in paper four we investigate the general evolution of three CONSTANS-LIKE genes in B. nigra, namely BniCOL1, BniCOa and BniCOb.

Inherited retinal degenerations are a group of disorders characterised by great genetic heterogeneity. Clinically, they can be divided into two large groups of diseases, those associated with night blindness, e.g. retinitis pigmentosa (RP), and those with macular malfunction, e.g. cone/cone-rod dystrophy (COD/CORD). This thesis is focused on finding the genetic basis of disease in families with autosomal dominant COD, autosomal dominant RP, and Bothnia dystrophy (BD), a regional variant of RP.   A variant of COD was previously mapped to 17p12-p13 in a family from northern Sweden. One additional family originating from the same geographical area was included in fine mapping of this chromosome region. Using 12 microsatellite markers in linkage and haplotype analysis, the region was refined from 26.9 to 14.3 cM. A missense mutation, Q626H, in an evolutionarily conserved region of PITPNM3, phosphatidylinositol transfer membrane-associated protein, was identified. The mutation segregated with the disease in both families and was absent from normal control chromosomes. PITPNM3 is a human homologue of the Drosophila retinal degeneration (rdgB) protein, which is highly expressed in the retina and has been proposed to be required for membrane turnover of photoreceptor cells. With the intention of establishing the global impact that PITPNM3 has on retinal degenerations 165 DNA samples from COD and CORD patients were obtained from Denmark, Germany, the UK, and USA and screened for mutations. The Q626H mutation found in the Swedish families was also found in one British family and a novel Q342P variant was detected in a German patient. In addition, two intronic variants were identified: c.900+60C>T and c.901-45G>A. Thus, we concluded that mutations in PITPNM3 represent a rare cause of COD worldwide. In two large families from northern Sweden showing autosomal dominant RP with reduced penetrance, the disease locus was mapped using genome-wide linkage analysis to 19q13.42 (RP11). Since mutation screening of eight genes on 19q13.42 revealed no mutations, multiplex ligation-dependent probe amplification (MLPA) was used to screen for large genomic abnormalities in PRPF31, RHO, RP1, RPE65, and IMPDH1. A large deletion spanning 11 exons of PRPF31 and three genes upstream was identified. Using long-range PCR, the breakpoints of the deletion were identified and the size of the deletion was determined to encompass almost 59 kb. BD is an autosomal recessive type of RP with high prevalence in northern Sweden. The disease is associated with a c.700C>T mutation in RLBP1. In a screening of recessive RP in northern Sweden, 67 patients were found to be homozygous for c.700C>T and 10 patients were heterozygous. An evaluation with arrayed primer extension (APEX) technology revealed a second mutation, c.677T>A, in RLBP1 giving rise to compound heterozygosity in these patients. In addition, a c.40C>T exchange in CAIV was detected in a patient with BD and in 143 healthy blood donors. The c.40C>T substitution in CAIV has been reported to cause autosomal dominant RP in South African families with European ancestry. However, in the population of northern Sweden it appears to be a benign polymorphism. In summary, a first mutation in PITPNM3, encoding a human homologue of the Drosophila retinal degeneration protein, was detected in two large families with COD. A large deletion in PRPF31 was discovered in two families with autosomal dominant RP showing reduced penetrance and in 10 patients BD was shown to be caused by two allelic mutations in RLBP1.

Diabetes mellitus represents a group of metabolic disorders caused by both environmental and genetic factors. The two most common forms of diabetes are type 2 diabetes (T2D) and type 1 diabetes (T1D). T2D is associated with obesity and the disease is caused by insulin resistance and pancreatic b-cell dysfunction. T1D is an autoimmune disease in which the insulin- producing b-cells in the pancreas are destroyed by infiltration of lymphocytes. The aim of this thesis was to identify genes conferring susceptibility to diabetes. This was approached using genetic methods, both linkage and association studies, within the population of northern Sweden. The northern Swedish population is well suited for genetic studies of familial forms of disease, since an internal expansion of the northern Swedish population, coupled with a low frequency of immigration and a high frequency of consanguineous marriages, has resulted in a relatively homogeneous gene pool. This simplified genetic background increases the probability of identifying genes contributing to disease. The family-based material used for the type 2 diabetes studies (papers I and II) consisted of 231 individuals from 59 families originating in northern Sweden. The type 2 diabetes case-control material (papers I and II) consisted of 872 cases and 857 matched controls, all from northern Sweden. In paper I we performed a genome-wide linkage scan, seeking T2D susceptibility loci. Linkage to the previously identified Calpain-10 region was found, however, association studies in the case-control material revealed no association to the CAPN10 gene. Using both the family-based and the case-control material, we were able to confirm the association of polymorphisms in the TCF7L2 gene to T2D in the population of northern Sweden (paper II). CTLA-4 is a negative regulator of T cell activity, belonging to the CD28 co-stimulatory receptor family. Numerous reports, including our own, have associated CTLA-4 variants with T1D as well as other autoimmune diseases, such as autoimmune thyroid disease (AITD). Allelic variation in the 3ÚTR of the CTLA-4 gene was associated to human T1D and this variant has also been suggested to affect the level of mRNA encoding the soluble form of the molecule (sCTLA-4). We confirmed the association of allelic variation in the 3ÚTR of the CTLA-4 gene in a T1D/AITD case-control material from northern Sweden, consisting of 104 individuals with ATID, 149 individuals with T1D and 865 matched controls. However, we were unable to identify any correlation between allelic variants in the 3ÚTR of the CTLA-4 gene and expression of sCTLA-4 (paper III). Based on recently published genome-wide association (GWA) scans, 33 single-nucleotide polymorphisms (SNPs) located within 16 genes were selected for an association analysis in T1D/AITD families from northern Sweden. The T1D/AITD family-based material consisted of 253 cases and 206 healthy individuals from 97 northern Swedish families. Analysis revealed association to T1D for SNPs in PTPN22, COL1A2, IL-2Ra and INS. In addition, SNPs in CTLA-4, IL-2 and C12orf30 were shown to be associated to AITD (paper IV). Together, these results underpin the notion that the population of northern Sweden is well suited for the detection of genes involved in complex diseases. The use of our more restricted patient material, compared to materials used in published GWA scans, enables the discovery of disease associated genes in a more cost effective manner and show that our population is capable of detecting general susceptibility genes.

Diabetes or diabetes mellitus which is the correct medical term is a medical condition were the affected person lack the ability to regulate his or her blood glucose levels. This inability is directly due to the fact that the insulin producing cells, residing in the pancreas, can’t meet the body’s demand for insulin. It is estimated that close to 200 million people are suffering from diabetes today and this number is predicted to double within 20 years. Of the approximately 200 million people suffering from diabetes today approximately 20 million are in dependent on daily injections of insulin. Being dependent on exogenous insulin is not only an inconvenience it also increase the risk for several medical complications such as stroke, heart disorders, kidney failure, retinopathy, atherosclerosis and impaired wound healing. The major risk factor for all these complications is long periods of high blood sugar levels that is damaging to thin blood vessels and nerves.  Even in the best of situations the blood sugar levels of a diabetic with need for daily insulin injections can never be as well controlled as in a healthy individual. Increased understanding in the developmental processes behind the formation of the pancreas, and more specifically the insulin producing β-cells could result in new treatments for diabetics. By imitating the in vivo conditions generating pancreatic development scientist are now able to induce embryonic stem cells to differentiate into pancreatic progenitors as well as insulin producing β-cells in vitro. These in vitro generated pancreatic cells might in the future serve as a donor source for transplantations, thereby restoring the insulin producing capability of diabetic patients. An alternative approach to restore insulin production in diabetics is to influence cells in the pancreas to generate more insulin producing cells. To successfully achieve this, what cell types have the capacity to generate β-cells needs to be appreciated. In this thesis papers concerning in vitro differentiating of embryonic stem cells towards a pancreatic fate as well as in vivo studies in basic pancreas development are presented and discussed.

Stroke is a common multi factorial cerebrovascular disorder with a large impact on global health. It is a disorder primarily associated with old age but environmental factors, lifestyle choices and medical history are all important for the risk of developing the disorder. It is also known that heritability is important for predisposition to the disorder. The aim of this work has been to identify genetic variations that increase the risk of being affected by stroke in the population of northern Sweden, a population well apt for genetic studies due to well kept church and medical records together with limited genetic diversity. In the first paper we used linkage analysis in families with early onset of stroke. By this approach we identified a region on chromosome 5q to be linked to an increased risk of developing stroke, a region previously identified as a susceptibility locus for stroke in the Icelandic population. In the second study we used genealogy to identify common ancestry and thereby identify common susceptibility to stroke. The seven families we connected showed significant linkage to the chromosome 9q31-33 region and four of the families shared a common haplotype over 2.1 megabases. In the third manuscript we investigated sequence variation of two candidate genes, TNFSF15 and TLR4. Sequencing of the TLR4 gene revealed previously identified variations in affected individuals from two of the families. Further SNP analysis showed five separate haplotypes among the investigated families and four haplotypes for TNFSF15. However none of these co-segregated with stroke among the investigated families. In the final paper we used a case-control stroke cohort to ascertain association for genetic variation in five genes and genetic regions previously suggested to be linked with stroke. Initial analyses showed association for the 9p21 chromosomal region and a variant in Factor 5 that showed protection against stroke, but after adjustments for common risk factors for stroke, the findings were no longer significant. In conclusion, by studying selected families we have been able to show linkage to two chromosomal regions, 5q and 9q31-33, that indicate genetic predisposition for developing stroke. Further we have shown that family based studies are still an important tool in deciphering the underlying mechanisms for complex disease.

Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments. The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences. A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping. The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.

All contemporary taurine cattle originated some 10,000 years ago when their wild ancestor, the aurochs, was domesticated in the Near East. Although the aurochs was widespread also in Europe, there is no evidence for a local domestication. The aurochs has been extinct since 1627 and therefore little is known about its biology. Following domestication, cattle were selected for traits of interest to humans. All modern cattle breeds were developed in the 19th century and the only sources of information about prehistoric breeding practices, and breeds, come from a few ancient Roman Empire and medieval European written accounts. The aim for this thesis was to investigate the effects early selection may have had on the cattle genome and to investigate genetic variation in European aurochs. Using second-generation sequencing and coalescent simulation analyses of aurochs Y chromosomal DNA, I estimated effective population size to between 20,000-80,000 aurochs bulls, indicating that a large population was present when domestic cattle entered Europe. A Y chromosomal SNP revealed that the two male lineages present in modern cattle were also present in European aurochs, and that the frequency of these lineages in domestic cattle fluctuated over time. This indicates that cattle were mobile and that bottlenecks, possibly due to selective breeding, occurred. I used nuclear SNPs to trace genetic variation in North European cattle through time and show that when genetics is combined with archaeology and osteology, even small but notable changes in the use of cattle can be detected. There has been a significant decrease in genetic variation over time, with the most dramatic changes associated with the formation of breeds during the 19th century.

The aim of this thesis has been to identify genes and gene regions underlying four different disorders. In papers I-IV, positional cloning methods, such as linkage, association and haplotype analysis have been used for the identification of genomic regions associated with the ichthyosis prematurity syndrome (IPS), adult-onset autosomal dominant leukodystrophy (ADLD) and Kostmann disease. IPS is a rare autosomal recessive skin disorder, which includes a premature birth of the affected child. We mapped the IPS locus to a region on chromosome 9q34, and within this region a haplotype is shared by IPS patients, which suggests a strong founder effect. The haplotype spans 76 kb, which includes four known genes. No sequence or mRNA expression alterations could be detected for the four genes in IPS patients. A candidate region for an adult-onset leukodystrophy (ADLD) on chromosome 5 was investigated in a large Swedish family with ADLD. A significant multipoint LOD score of 9.45 was obtained for markers in the chromosome 5 region and fine-mapping of recombination events restricts a candidate gene region to 1.5 Mb. Kostmann disease is an autosomal recessive form of severe congenital neutropenia. We have identified a 1.2 Mb region on chromosome 1q22 associated with the disease in the original Kostmann family. The region contains 37 genes. In paper V, cDNA microarrays were used to asses the mRNA levels of 7,700 genes in lymphoblastoid cell lines derived from autistic and control samples. The SEMA5A gene, which is involved in axonal guidance, was found downregulated in the cells derived from autistic individuals, and this was confirmed by quantitative PCR. In summary, candidate genes or gene regions have been identified for all four disorders and further studies are needed to confirm their roles in the pathogenesis of the disorders.

To understand the etiology of psychopathic personality traits and thus in the long run to be able to develop successful prevention, a first step is to find out what role genetic and environmental effects play. A meta-review of 15 twin studies (N=26, 981), was conducted to estimate the magnitude of genetic and environmental influences on psychopathic personality traits. The results show that additive genetic (heritable) factors and non-shared environmental factors each explain 50% of the variance in psychopathic personality traits, while shared environmental factors were of no importance. Measure, informant, age, and sex were investigated as potential moderators showing that informant had an impact on the findings. This meta-analysis provides a structured synthesis of the relative genetic and environmental contributions in psychopathic personality traits through various stages of development and across sex. / För att förstå etiologin av psykopatiska personlighetsdrag och därmed i det långa loppet kunna utveckla framgångsrik prevention, är ett första steg att klargöra vilken roll genetiska och miljömässiga effekter spelar. En meta-översikt på 15 tvillingstudier (N=26,981), genomfördes för att uppskatta i vilken grad genetiska och miljömässiga faktorer påverkar psykopatiska personlighetsdrag. Resultaten visade att additiva genetiska (ärftliga) och unika miljömässiga faktorer förklarar 50% var av variansen i psykopatiska personlighetsdrag, medan delade miljömässiga faktorer inte var av betydelse. Mått, informant, ålder och kön undersöktes som potentiella moderatorer och visade att informant påverkade resultaten. Denna meta-analys ger en strukturerad syntes av de relativa genetiska och miljömässiga bidrag som påverkar psykopatiska personlighetsdrag genom olika utvecklingsstadier och mellan könen.

There are around 20.000 genes in the human genome all of which could potentially be expressed. However, it is obvious that not all of them can be active at the same time. Thus, there is a need for coordination achieved through the regulation of transcription. Transcriptional regulation is a crucial multi-component process involving genetic and epigenetic factors, which determine when and how genes are expressed. The aim of this thesis was to study two of these components, the transcription factors and the DNA sequence elements with which they interact. In papers I and II, we tried to characterize the regulatory role of repeated elements in the regulatory sequences of nitric oxide synthase 2 gene. We found that this type of repeat is able to adopt non B-DNA conformations in vitro and that it binds nuclear factors, in addition to RNA polymerase II. Therefore it is probable that these types of repeats can participate in the regulation of genes. In papers III-V, we intended to analyze the genome-wide binding sites for six transcription factors involved in fatty acid and cholesterol metabolism and the sites of an epigenetic mark in a human liver cell line. For this, we applied the chromatin immunoprecipitation (ChIP) method together with detection on microarrays (ChIP-chip) or by detection with the new generation massively parallel sequencers (ChIP-seq). We found that all of these transcription factors are involved in other liver-specific processes than metabolism, for example cell proliferation. We were also able to define two sets of transcription factors depending on the position of their binding relative to gene promoters. Finally, we demonstrated that the patterns of the epigenetic mark reflect the structure and transcriptional activity of the promoters. In conclusion, this thesis presents experiments, which moves our view from genetics to genomics, from in vitro to in vivo, and from low resolution to high resolution analysis of transcriptional regulation.