<p>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 <i>VMD2</i> gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD.</p><p>In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a <i>C. elegans</i> protein family, expressed in retina, was identified as the <i>VMD2</i> 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 <i>VMD2</i> 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. <i>VMD2</i> 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.</p><p>Analysis of mouse <i>Vmd2</i> 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. <i>Vmd2</i> expression levels were highest around birth.</p>

<p>The phylogeography of a wide ranging temperate species, the adder, <i>Vipera berus</i>, 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.</p><p>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 <i>V. b. bosniensis</i>. 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. </p>

<p>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.</p><p>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.</p><p>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.</p><p>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.</p>

<p>The aim of this thesis is to study phylogeny, species-wide phylogeography and genetic diversity in <i>Formica</i> ants across Eurasia in connection with the history of biotic responses to Quaternary environmental changes.</p><p>The mitochondrial DNA phylogeny of Palaearctic <i>Formica</i> species supported the subgeneric grouping based on morphological similarity. The exception was that <i>F. uralensis</i> formed a separate phylogenetic group. The mitochondrial DNA phylogeny of the <i>F. rufa </i>group showed the division into three major phylogenetic groups: one with the species <i>F. polyctena</i> and <i>F. rufa</i>, one with <i>F. aquilonia</i>, <i>F. lugubris</i> and <i>F. paralugubris</i>, and the third one with <i>F. pratensis</i>.</p><p>West-east phylogeographic divisions were found in <i>F. pratensis</i> 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 <i>F. lugubris </i>or<i> F. exsecta</i>. 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.</p><p>Sympatrically distributed and ecologically similar species <i>F. uralensis </i>and<i> F. candida</i> showed clear difference in the phylogeographic structure that reflected difference in their vicariant history. Whereas no phylogeographic divisions were detected in <i>F. uralensis</i> across Europe, <i>F. candida</i> showed a well-supported phylogeographic division between the western, the central and the southern group.</p><p>In socially polymorphic <i>F. cinerea</i>, 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.</p>

<p>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.</p><p>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.</p><p>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.</p><p>The field vole (<i>Microtus agrestis</i>), 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.</p><p>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.</p>

<p>Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are complex autoimmune diseases affecting 0.05-2% of the population worldwide. </p><p>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). </p><p>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 .</p><p>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. </p><p>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.</p><p>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.</p>

<p>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.</p><p>In this thesis, genes associated to glaucoma have been analysed in Swedish patients with primary open angle and exfoliative glaucoma. The genes studied were <i>MYOC</i>, <i>oculomedin,</i> <i>GSTM1</i> and <i>OPTN</i>.</p><p>The coding sequence of <i>MYOC</i> 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 <i>oculomedin</i> was analysed, but none of the variants found were classified as disease causing in either patient group. <i>GSTM1</i> 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 <i>OPTN</i> 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.</p><p>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.</p>

<p>Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) V_H genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated V_H genes display superior survival compared to unmutated cases. Biased V_H gene usage has also been reported in CLL which may reflect antigen selection.</p><p>We performed V_H gene analysis in 265 CLL cases and confirmed the prognostic impact of the V_H mutation status. Preferential V_H gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular V_H gene, V_H3-21, displayed poor outcome despite that two-thirds showed mutated V_H genes. Many of the V_H3-21 cases expressed λ light chains, rearranged a V_λ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the V_H3-21 subset comprises an additional CLL entity.</p><p>To further explore the B-cell receptors in CLL, we analyzed the V_H gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted V_L gene usage as in the V_H3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL.</p><p>Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. <2%, 2-5% and >5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. </p><p>A 1513A/C polymorphism in the P2X₇ receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated V_H genes.</p><p> In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.</p>

<p>Positional cloning is a method to identify genes from their position in the genome without prior knowledge about function. We used this approach to investigate the basis for three distinct genetic disorders; Obesity, Ichthyosis Prematurity Syndrome and Meniere's disease.</p><p>Obesity appears when energy intake exceeds energy expenditure which leads to an abnormal accumulation of fat in the adipocyte tissue. We have studied a family with a balanced chromosomal translocation t(4;15) segregating with severe obesity. The chromosomal breakpoints create a fusion gene involving the gene for isoform 1 of RAR-related orphan receptor A (<i>RORa1</i>) which is implicated in the regulation of adipogenesis and lipoprotein metabolism. We hypothesize that the obesity in this family is caused by haploinsufficiency of this gene or a gain of function of the fusion gene.</p><p>Ichthyosis prematurity syndrome (IPS) is a rare skin disorder belonging to a group of autosomal recessive congenital ichthyosis. We have mapped the locus for IPS to chromosome 9q34. Within the IPS locus, we identified a core haplotype with a high carrier frequency among affected, which indicate a possible founder mutation for the disease. The minimal shared region in affected patients contains seven genes which are candidates for IPS.</p><p>Meniere's disease (MD) is characterised by spontaneous attacks of vertigo, fluctuating sensorineural low frequency hearing loss, aural fullness, and tinnitus. We mapped the MD locus to chromosome 12p13 using three Swedish families. The linked region is 463 kb, containing only one gene, a phosphoinositide-3-kinase (<i>PIK3C2G</i>). Involvement of phosphatidylinositol 3-kinases (PI-3K) in the intra cellular signalling cascades of cells in mammalian balance epithelia makes this gene a good candidate gene for MD.</p>

<p>Medulloblastoma is a highly malignant cerebellar childhood tumor. As in many other brain tumors, expression of platelet-derived growth factor (PDGF) and its receptors has been shown in medulloblastoma. To reveal the importance of this growth factor in cerebellar development and tumorigenesis, analyses were performed on human medulloblastoma cell lines and on tissue from normal mouse brain at different stages of development. The <i>in vivo</i> effect of a forced expression of PDGF-B in the cerebellar primordium was examined in transgenic mice. </p><p>In the normal mouse embryo, we found PDGF receptor-α-positive cells in the early neuroepithelium and on neuronal precursors. In the postnatal cerebellum, cells in the external germinal layer and Purkinje cells expressed the receptor. In the medulloblastoma cells, expression of all the three PDGF isoforms and PDGF receptors was seen and correlated to neuronal differentiation. Endogenously activated, <i>i.e.</i> tyrosine phosphorylated, PDGF receptors were identified. To reveal the role of PDGF in normal cerebellar development, we established transgenic mice where a PDGF-B cDNA was introduced via homologous recombination into the engrailed-1 gene. Engrailed-1 is specifically expressed at the mid-/hindbrain boundary of the early neural tube, <i>i.e.</i> in an area from which the cerebellar primordium develops. The ectopic expression of PDGF-B caused a disturbance of cerebellar development. Midline fusion of the cerebellar primordium did not occur properly, which resulted in cerebellar dysplasia in the adult mouse.</p><p>In a parallel study, the expression pattern of a glial fibrillary acidic protein (GFAP)-<i>lacZ</i> transgene was followed in the embryonic mouse central nervous system. It was shown that the human GFAP promoter was already active by embryonic day 9.5 and as development proceeded, expression occured in different, independent cell populations. Among these cell populations were the radial glial cells in the neocortex.</p>