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.

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.

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.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009.

Inledning: När en äldre människa har ett stort omvårdnadsbehov finns möjligheten att flytta till ett särskilt boende. Då äldres vardag ser olika ut är det av yttersta vikt att omvårdnadspersonalen kan ge stöd och hjälp så att den äldre skall kunna anpassa sig till den nya situationen. Syfte: Syftet med denna studie är att belysa hur anhöriga upplever att deras närstående i särskilt boende får en god omvårdnad. Metod: En kvalitativ ansats med empiriskt inslag användes där anhörigas upplevelser av omvårdnad, delaktighet och bemötande insamlades med hjälp av intervjuer. Resultat: Tre olika kategorier Omvårdnad, Delaktighet och Bemötande med nio underkategorier. En betydelsefull del i omvårdnaden är att det skapas en god kontakt mellan anhöriga och omvårdnadspersonalen för att finna ett bra sätt att kommunicera på. Det framkom hur viktigt det är att som vårdtagare känna att de blir sedda för den de är och att de får vara delaktiga i de omvårdnadsåtgärder som beslutas av omvårdnadspersonalen. Diskussion: Resultatet kan bidra till en ökad förståelse för anhörigas upplevelser av hur omvårdnaden bedrivs i särskilt boende. När anhöriga göras mer delaktiga i omvårdnaden, kan det leda till en bättre omvårdnad för vårdtagaren i särskilt boende. Slutsats: Resultatet som författarna kom fram till skulle kunna användas i utbildningssyfte då omsorgen av äldre människor kräver att omvårdnadspersonalen ständigt förnyar sina kunskaper. Detta kan vara till gagn för sjuksköterskan, de anhöriga och de äldre som bor i särskilt boende. / Introduction: When a senior person has a large need for special care there is an option to relocate to a nursing home. The seniors every day varies there for it is of outmost importance the nursing care staff can support the senior that he maybe adapt to the new situation. Purpose: The purpose with this study is to enlighten how relatives experience their close ones in special nursing home receive good care treatment. Method: A quality approach with empirical elements is used where relatives experiences of care, being part of and recievment was collected with the help of interviews. Results: Three categories Care, Involvment and Recievment with nine sub categories. An important part in care is to create good contact between relatives and nursing care staff to evolve good ways for communication. It was revealed how important it is as a health care patient to feel they’re being looked upon for who they are and they be part of treatment measures and decisions made by nursing care staff. Discussion: The results can contribute to an increased understanding to how relatives experience care is being conducted in a special accommodation. When relatives are made more involved in care, may lead to a better care for care patient in a nursing home. Conclusion: The results which have been concluded could be used in educational purposes when the care of senior people demands that nursing care staff continuously renews their knowledges. This could be of use for the nurse, the relatives and the seniors living in a nursing home.

Malt lymphoma is a malignant disease that can arise in a variety of extra nodal sites. Previous studies indicate that tumour arise from more mature B-cells. Our purpose was to examine the presence of clonality and somatic hypermutation of immunoglobulin (IgVн) of MALT lymphomas. Paraffin-embedded tumour samples from13 MALT lymphoma were subjected to rearrangement analysis, by using PCR, heteroduplex gels and sequence analysis. Successful amplification was seen in 10/13 cases and sequences of IgVн genes were obtained in 6/13, all of them were mutated. The percentage of mutation compared to germline sequences was 1,1% to 8,6% monoclonal rearrangemang. It was demonstrated that 5 of 7 clones were derived from the Vн3 family, 2 from Vн1 and 1 from the Vн 4 family.

This thesis presents genetic studies aiming at enlarging our knowledge regarding the genetic factors underlying two immune-mediated diseases, hypothyroidism and autoimmune Addison’s disease (AAD), in dogs and humans, respectively. Genetic and environmental factors are indicated to contribute to canine hypothyroidism, which can be considered a model for human Hashimoto’s thyroiditis (HT). In Paper I we performed the first genome-wide association (GWA) study of this disease in three high-risk dog breeds (Gordon Setter, Hovawart and Rhodesian Ridgeback). Using an integrated GWA and meta-analysis strategy, we identified a novel hypothyroidism risk haplotype located on chromosome 12 being shared by the three breeds. The identified haplotype, harboring three genes previously not associated with hypothyroidism, is independent of the dog leukocyte antigen region and significantly enriched across the affected dogs. In Paper II we performed a GWA study in another high-risk breed (Giant Schnauzer) and detected an associated locus located on chromosome 11 and conferring protection to hypothyroidism. After whole genome resequencing of a subset of samples with key haplotypes, we fine mapped the region of association that was subsequently screened for the presence of structural variants. We detected a putative copy number variant overlapping with the upstream region of the IFNA7 gene, which is located in a region of high genomic complexity. Remarkably, perturbed activities of type I Interferons have been extensively associated with HT and general autoimmunity. In Paper III we performed the first large-scale genetic study of human AAD, a rare autoimmune disorder characterized by dysfunction and ultimately destruction of the adrenal cortex. We resequenced 1853 immune-related genes comprising of their coding sequences, untranslated regions, as well as conserved intronic and intergenic regions in extensively characterized AAD patients and control samples, all collected in Sweden. We identified BACH2 gene as a novel risk locus associated with AAD, and we showed its independent association with isolated AAD. In addition, we confirmed the previously established AAD association with the human leukocyte antigen complex. The results of these studies will hopefully help increasing the understanding of such diseases in dogs and humans, eventually promoting their well-being.

Stroke is a common disorder of later life with a complex etiology, including both environmental and genetic risk factors. The inherited predisposition is challenging to study due to the complexity of the stroke phenotype. Genetic studies in an isolated population have successfully identified a positional candidate gene for stroke, phosphodiesterase 4D (PDE4D). The aim of this thesis was to identify stroke susceptibility loci and positional candidate genes, taking advantage of low genetic variation in the northern Sweden population. All stroke cases were identified in a population-based stroke registry at the northern Sweden MONICA Centre. 56 families containing multiple cases of stroke and a follow up set of an additional 53 families were used for linkage studies. For association studies, 275 cases of first ever stroke together with 550 matched community controls were included. In paper I, we used a candidate region approach to investigate the PDE4D region on chromosome 5q. Linkage was obtained with a maximum allele-sharing LOD score of 2.06; P = 0.001. However, no significant association of ischemic stroke to the previously defined at-risk allele in PDE4D was observed. We next performed a genome wide linkage scan to explore new susceptibility loci for common forms of stroke (paper II). Non-parametric multipoint linkage analysis yielded allele-sharing LOD scores > 1.2 at nine locations; 1p34, 5q13, 7q35, 9q22, 9q34, 13q32, 14q32, 18p11, 20q13. The highest allele-sharing LOD score was obtained on chromosome 18p (LOD = 2.14). Fine mapping resulted in increased allele-sharing LOD scores for chromosome 5q13 and 9q22. In the follow up analysis of the nine regions, including all 109 families, the highest allele-sharing LOD scores were obtained on chromosomes 5q, 13q and 18p although none reached the initial genome wide values. In paper III, we focused on the chromosome 5q region, and further mapping and haplotype analysis in the families was performed. A common 1 cM haplotype was found to be shared among affected members of five families. In this region only the regulatory subunit 1 of phosphatidylinositol 3-kinase (PIK3R1) gene was located. Association of three single nucleotide polymorphisms in the PIK3R1 gene to common stroke was obtained in the case-control material. Finally, in paper IV, an extended pedigree containing seven families connected to common founders eight generations back was identified by genealogical analysis, and submitted to a separate genome wide scan analysis. A significant allele-sharing LOD score of 4.66 (genome wide P < 0.001) at chromosome 9q31-33 was obtained. Haplotype analysis identified a minimal common region of 3.2 cM, which was shared by four of the seven families. These four families contained all of the primary intracerebral hemorrhagic cases present in the extended pedigree. In conclusion we have replicated linkage of stroke susceptibility to the PDE4D region on chromosome 5q, but no significant association of ischemic stroke to PDE4D was observed. Linkage analysis of stroke did not identify any new major stroke loci, indicating that multiple minor susceptibility loci in addition to the previously known locus on chromosome 5q could contribute to the disease. In the chromosome 5q region a novel positional candidate gene for stroke was identified, the PIK3R1 gene. The PIK3R1 protein has several biological actions with potential roles in stroke susceptibility. Also a novel susceptibility locus for common forms of stroke at chromosome 9q was identified in a large pedigree, which may be of special importance for susceptibility to hemorrhagic stroke.

Lymphocytes are essential for the generation of specific immunity. Development of B cells in the bone marrow and T cells in the thymus have several analogous features, and are tightly regulated processes. Even though there is an increasing amount of information concerning lymphopoiesis, a lot of questions remain. The aim of this thesis has been to understand some of the molecular events that contribute to the control of lymphocyte development. Expression of the B cell receptor is an important checkpoint in B lymphocyte development. The Dµ protein is a truncated B cell receptor that can induce some of the signals elicited by full length µ, but cannot promote further B cell differentiation. In order to determine if this could stem from an impaired survival signal, we introduced Bcl-2 into RAG2 deficient Dµ transgenic mice. Analysis of these mice showed that Dµ could not support pre-B cell maturation despite extended survival of B cell precursors by Bcl-2. In addition, data from recombination competent Dµ transgenic mice demonstrated that the Dµ induced partial block is permissive for marginal zone B cell development, whereas the formation of follicular B cells is severely reduced. The bHLH family of transcription factors is known to be involved in the regulation of lymphocyte development. Whereas the roles of E2A and HEB have been well documented in both B- and T-lymphocytes, detailed knowledge concerning E2-2 is lacking. To address the role of E2-2 in B cell development, we have reconstituted mice, using E2-2 deficient fetal liver cells, and analysed the B cell compartments. We also measured mRNA expression patterns for the three E-proteins in wildtype mice. Resulting data show that, in addition to a role in B cell lineage entry, E2-2 is required for efficient expansion of pro-B cells, and also influences the follicular versus marginal zone decision. While focusing on assigning a role for E2-2 in T-cell development, we analyzed the expression of the E-proteins during this process and performed functional studies in fetal thymic organ cultures. E2-2 deficient mouse embryos were shown to display a partial block at the DN3 stage, which was not due to proliferation or apoptosis defects. In addition, analysis of expression levels of the pre-Talpha chain suggests that E2-2 may play a role in the regulation of transcription of pre-Talpha, and therefore in the assembly of the pre-T cell receptor.