A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution.

Linkage in the 2q37 region was evaluated using microsatellite markers in multi-case families from Sweden, Iceland and Norway. Both the two-point and the multipoint linkage analysis show highly significant LOD scores (Z=4.51 and 6.03, respectively). Linkage disequilibrium mapping indicates that some association exists in this region. The PDCD1 gene was suggested as a candidate gene within the 2q37 locus due to its importance in immune regulation. Indeed, one haplotype, described by the presence of allele A of the PD1.3 SNP located within intron 4 of this gene, shows linkage to SLE in the Nordic families. The PD1.3A allele is also found to be strongly associated in familiar and sporadic cases of SLE in Europeans and Mexicans. Functional studies further support PD1.3A to be a susceptibility allele for SLE. The 1q23 region, containing the genes for the low affinity Fcγ receptors, was fine mapped using single- and multi- case families of various origins. Genetic variants of those genes were analysed and association is found to both the risk alleles of FcγRIIA and FcγRIIIA in all families. In these families, a single haplotype carrying both risk alleles is predominantly transmitted to patients with SLE, suggesting a presence of linkage disequilibrium between those two genes. FcγRIIA and FcγRIIIA are also found to be associated to SLE and lupus nephritis in a case-control cohort from Sweden. In the same cohort, the PD1.3A allele shows strong association to lupus nephritis. We suggest that there may be an additive effect between FcγRIIA and PDCD1, since having the disease-associated genotypes at both loci gives an increased risk for developing lupus nephritis. Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disorder with a complex multifactorial aetiology. Genetic studies suggest that several genes are involved in disease pathogenesis and that extended genetic heterogeneity is present.

Angiogenesis can be defined as the formation of new blood vessels from pre-existing ones. Angiogenesis is required for development and maintenance of our vascular system and thus of fundamental importance to our existence. The endothelial cells that line the inside of the vessels de-differentiate, migrate, proliferate and re-differentiate during angiogenesis. Angiogenesis is tightly regulated, controlled by several angiogenic factors of various classes that promote angiogenesis but also by anti-angiogenic factors that counteract the effect of the pro-angiogenic factors. We have examined three factors involved in angiogenesis regulation, Vascular endotelial growth factor (VEGFR) -3, the matrix protein laminin-1 and the collagen XVIII derived fragment endostatin. Five tyrosine phosphorylation sites in the cytoplasmic tail of VEGFR-3 were identified by phosphopeptide mapping (PPM). The data was confirmed by PPM using point-mutated receptors generated by site-directed mutagenesis. Laminin-1 was found to promote angiogenesis in the chicken chorioallantoic membrane assay and in a synergistic fashion together with suboptimal levels of fibroblast growth factor 2 (FGF-2) in embryoid bodies. Laminin-1 also promoted endothelial tubular morphogenesis in vitro, and upregulated the expression of the endothelial differentiation marker Jagged-1. Endostatin was shown to affect endothelial FGF-2-induced cell survival and morphogenesis. This was a result of direct binding to endothelial cells and induction of tyrosine phosphorylation of many proteins including the adaptor protein Shb. The apoptotic and morphogenic responses induced by endostatin was shown to be dependent on Shb. Further, endostatin inhibited endothelial migration and affected molecules implicated in migration. In particular, FGF-2 induced actin reorganization, and β-catenin regulation was modulated by endostatin.

Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The VMD2 gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD. In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a C. elegans protein family, expressed in retina, was identified as the VMD2 gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the VMD2 gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. VMD2 expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation. Analysis of mouse Vmd2 and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. Vmd2 expression levels were highest around birth.

The phylogeography of a wide ranging temperate species, the adder, Vipera berus, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders. The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of V. b. bosniensis. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia.

Mitochondrial DNA (mtDNA) has been a potent tool in studies of the evolution of modern humans, human migrations and the dynamics of human populations over time. The popularity of this cytoplasmic genome has largely been due to its clonal inheritance (in Man) allowing the tracing of a direct genetic line. In addition, a comparatively high rate of nucleotide substitution facilitates phylogenetic resolution among relatively closely related individuals of the same species. In this thesis, a statistically supported phylogeny based on complete mitochondrial genome sequences is presented which, for the first time, unambiguously places the root of modern human mitochondrial lineages in Africa in the last 200 thousand years. This conclusion provides strong support for the “recent African origin” hypothesis. Also, the complete genome data underline the problematic nature of traditional approaches to analyses of mitochondrial phylogenies. The dispersal of anatomically modern humans from the African continent is examined through single nucleotide polymorphism (SNP) and sequence data. These data imply an expansion from Africa about 57 thousand years ago and a subsequent population dispersal into Asia. The dispersal coincides with a major population division that may be the result of multiple migratory routes to East Asia. Also investigated is the question of a common origin for the indigenous peoples of Australia and New Guinea. Previous studies have been equivocal on this question with some presenting evidence for a common genetic origin and other proposing separate histories. Our data reveals an ancient genetic link between Australian Aborigines and the peoples of the New Guinea highlands.

The aim of this thesis is to study phylogeny, species-wide phylogeography and genetic diversity in Formica ants across Eurasia in connection with the history of biotic responses to Quaternary environmental changes. The mitochondrial DNA phylogeny of Palaearctic Formica species supported the subgeneric grouping based on morphological similarity. The exception was that F. uralensis formed a separate phylogenetic group. The mitochondrial DNA phylogeny of the F. rufa group showed the division into three major phylogenetic groups: one with the species F. polyctena and F. rufa, one with F. aquilonia, F. lugubris and F. paralugubris, and the third one with F. pratensis. West-east phylogeographic divisions were found in F. pratensis suggesting post-glacial colonization of western Europe and a wide area from Sweden to the Baikal Lake from separate forest refugia. In contrast, no phylogeographic divisions were detected in either F. lugubris or F. exsecta. Contraction of the distribution range to a single refugial area during the late Pleistocene and the following population expansion could offer a general explanation for the lack of phylogeographic structure across most of Eurasia in these species. Sympatrically distributed and ecologically similar species F. uralensis and F. candida showed clear difference in the phylogeographic structure that reflected difference in their vicariant history. Whereas no phylogeographic divisions were detected in F. uralensis across Europe, F. candida showed a well-supported phylogeographic division between the western, the central and the southern group. In socially polymorphic F. cinerea, the overall level of intrapopulation microsatellite diversity was relatively high and differentiation among populations was low, indicating recent historical connections. The lack of correspondence between genetic affinities and geographic locations of studied populations did not provide any evidence for differentiating between alternative hypotheses concerning the directions and sources of postglacial colonization of Fennoscandia.

Sex chromosomes are useful in elucidating the evolutionary factors affecting diversity and divergence. In particular, Y chromosome analyses may complement studies using mitochondrial DNA for inferring sex-specific population genetic processes. Y chromosome studies have been scarce due to limited access to genetic markers and the dynamic evolution of Y. Conserved Y-specific primers that could amplify a diverse set of mammalian species were developed from comparison of gametologous X and Y sequences. Y-specific sequence, generally more than one kb, was amplified for all 20 species examined. Intraspecific diversity on mammalian Y was found to be reduced even when male-biased mutation rate and effective population size were corrected for. A number of factors can cause this low variation on Y of which selection on a haploid chromosome seems most important. The field vole (Microtus agrestis), a common and well-studied small mammal in Eurasia, was examined for X and Y variability. Earlier studies on mtDNA had shown that the field vole is separated in two distinct lineages in Europe. The X and Y chromosome sequences confirmed the deep split and suggested that the two lineages of field vole should be reclassified as two separate species. Two distinct Y chromosome haplogroups were found in modern European cattle, distributed among breeds according to a north-south gradient. Ancient DNA analysis of European aurochsen showed the northern haplogroup to be the most common, possibly indicating local hybridization between domestic cows and wild aurochs bulls in Europe.

Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are complex autoimmune diseases affecting 0.05-2% of the population worldwide. Genetic studies detected linkage with SLE in the 2q37 region, and intensive family-based and case-control association studies in several populations identified that allele A of the SNP PD-1.3 located in the immunoreceptor PDCD1 (PD-1) gene, increases risk of the disease by 2.6-fold in Caucasians (p<0.00001) and by 3.5-fold in Mexicans (p=0.0009). The same allele was found to be a risk factor for lupus nephritis, a severe clinical manifestation of SLE. In Swedish and European-American females with SLE, patients with the allele A had nephritis 1.8 times (p=0.01) more often than patients with allele G . Moreover, the allele A was also found 1.8 times (p=0.005) more often in RA patients, negative for the known risk-factors, rheumatoid factor and the shared epitope, than in other groups of patients and controls. Functional studies demonstrated that the mechanism behind the SNP PD-1.3 is related to the disruption of the binding site for RUNX transcription factors in the regulatory region. Expression of the PD-1 and RUNX genes was altered in the activated T cells of SLE patients compared to controls. The Tumor Necrosis Factor Receptor 2 (TNFR 2) gene was studied as a second candidate gene for both SLE and RA. The results of our studies in SLE and RA patients and controls from Sweden and Mexico do not support the association of the polymorphism TNFR 2 M196R with these diseases. Other polymorphisms in this gene and other genes in this region should therefore be studied.

Glaucoma is one of the leading causes of visual impairment in the world. In glaucoma, the patient’s peripheral vision is lost due to progressive and irreversible deterioration of the retinal ganglion cells and atrophy of the optic nerve. The effect on the visual field is gradual and painless, and the progression so slow, that the patient may not notice until a substantial part of the visual field is lost. If left untreated, glaucoma can lead to blindness. In this thesis, genes associated to glaucoma have been analysed in Swedish patients with primary open angle and exfoliative glaucoma. The genes studied were MYOC, oculomedin, GSTM1 and OPTN. The coding sequence of MYOC was analysed and mutations were found in 1% of the primary open angle glaucoma patients. Additionally, a predisposing variant was found in 1% of the patients as well as in 0.5% of the controls. No disease-associated variation was found in the exfoliative glaucoma cases. Mutations were also found in two families affected by glaucoma. The coding sequence of oculomedin was analysed, but none of the variants found were classified as disease causing in either patient group. GSTM1 was analysed for its presence in the patients. No association could be found for either hetero- or homozygous deletions. The coding sequence and haplotype distribution of OPTN was analysed. None of the variants found were classified as disease causing and none of the haplotypes were associated to the disease in either patient group. There are just a few per cent of the Swedish primary open angle glaucoma patients with genetic variation associated to disease, in the genes analysed in this study. No association to exfoliative glaucoma was found. This indicates heterogeneity in the genetics of glaucoma when different subtypes and different populations are compared. Likely, there are genes still to be identified.