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.

Ribosomal proteins are components of the ribosome, the protein synthesis machinery. The ribosomal protein S19 gene (RPS19) is mutated in Diamond-Blackfan anemia, DBA, which is a rare congenital anemia with absence or reduction of erythroid precursors in bone marrow. In this thesis, the role of RPS19 in erythropoiesis is investigated. A genetic analysis of RPS19 in 24 DBA cases was performed. Four novel RPS19 mutations were identified with evidence of wide clinical expression of the disease. Due to the clinical overlap in Transient Erythroblastopenia of Childhood, TEC, and DBA, the two diseases may be caused by a common genetic factor. In a study of seven TEC families, all affected shared at least one parental haplotype in the RPS19 gene region. Coding exons of RPS19 were normal for all affected, although mutations in intronic and regulatory sequences are not excluded. This indicates a genetic factor behind TEC and a possible association between RPS19 and TEC. To investigate the role of RPS19 in erythropoiesis in a mammal, we created a mouse model for the targeted disruption of the homologue Rps19 on the C57BL/6J genetic background. Null mutants are embryonic lethal prior to implantation. The Rps19+/- mice, however, are viable with normal development including the hematopoietic system. The Rps19 transcript level in Rps19+/- mice is normal. Accordingly, RPS19 protein levels are similar in Rps19+/- and Rps19+/+ mice. This argues for a transcriptional up-regulation to compensate for the loss of one Rps19 allele. Peripheral blood is normal in Rps19+/- mice also on the FVB/NJ strain which argues against strain-specific effects of the Rps19 disruption. Preliminary results indicate a reduced erythroid proliferation in response to erythropoietin in Rps19+/- mice, suggesting the requirement of both Rps19 alleles for normal erythroid proliferation under stress. This would support a mechanism by which haplo-insufficiency for RPS19 causes DBA.

Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) VH genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated VH genes display superior survival compared to unmutated cases. Biased VH gene usage has also been reported in CLL which may reflect antigen selection. We performed VH gene analysis in 265 CLL cases and confirmed the prognostic impact of the VH mutation status. Preferential VH gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular VH gene, VH3-21, displayed poor outcome despite that two-thirds showed mutated VH genes. Many of the VH3-21 cases expressed λ light chains, rearranged a Vλ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the VH3-21 subset comprises an additional CLL entity. To further explore the B-cell receptors in CLL, we analyzed the VH gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted VL gene usage as in the VH3-21-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. 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. 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 VH genes. 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.

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. 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 (RORa1) 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. 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. 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 (PIK3C2G). 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.

Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the PDGFRA gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called Sequence Allocator, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.

By comparing sequence diversity and divergence on sex chromosomes one can study how the rate of evolution in affected by mutation and/or selection. The rate of mutation in male biased, meaning that relatively more mutations are created in the male germ line than in the female. Since the male mutation bias (αm) most likely is a consequence of the difference in cell divisions between male and female germ lines, life history characters that affect this difference should covary with αm. Indeed, we found a positive correlation between estimates of αm and increased generation times and increased intensity of sperm competition. We have also found that estimates of αm varied significantly between gametologous introns located on the sex chromosomes. This could be a consequence of the variation in substitution rates between loci. Population genetics theory predicts that both positive and negative selection reduce genetic diversity around a selected locus at a distance determined by the rate of recombination. Consequently, a non-recombining chromosome, like the female specific W chromosome in birds, selection is expected to have a large impact on sequence diversity. Indeed, in a large sequence screening we found only one segregating site among 7643 base pairs sequenced in 47 chicken females. Furthermore, we also found that deleterious substitutions are fixed in a higher rate for W- than Z-linked sequences, which is in agreement with the lack of recombination and strong genetic drift due to the low effective population size. Rarely non-synonymous mutations are beneficial for an individual, but when it happens, the mutation is positively selected and rapidly reaches fixation in a population. We have found that positive selection has been acting on the female reproductive protein, zona pellucida c in birds. This rapid evolution is likely a mechanism to prevent hybridisation.

The aim with this thesis has been to identify genes involved in the pathogenesis of Systemic Lupus Erythematosus (SLE). SLE is a systemic autoimmune disorder, most likely caused by both several genetic and environmental factors. In order to identify susceptibility loci for the disease we performed linkage analyses on data from 70 families of various ethnic origins. Significant linkage was found in two regions. One region (chromosome 17p12-q11) was linked to SLE in a set of Argentine families. Since the same region had been previously identified in several linkage studies on Multiple Sclerosis patients, we propose that this locus may contain a genetic variant that affects not only SLE, but also autoimmunity in general. The second locus is located on chromosome 4p14-13 and has only been identified in a set of Icelandic families. We suggest that this locus contains a mutation that has been enriched in the Icelandic population due to its population history. The BCL2 gene has been suggested as a candidate gene for SLE. Three markers in this gene were investigated for association with the disease in two different populations. However, no association could be found with any of the markers or when these markers were analysed together as a haplotype. We conclude that the BCL2 gene is not associated with SLE in our material. This result contradicts previously published results of an association between BCL2 and SLE. We suggest that the PD-1 pathway (involved in inhibition of T- and B-cell responses) is an important component in SLE pathogenesis. A regulatory variant in the PD-1 gene had previously been associated with SLE and here we show strong association (p<0.0001) to a haplotype containing SNPs in both PD-L1 and PD-L2. Our results indicate that SLE is a disease caused by several genetic variations that differ between families and populations.

No description available.

Genomic imprinting is a phenomenon through which a subset of genes are epigenetically marked during gemtogenisis. This mark is maintained in the soma to often manifest parent of origin-specific monoalleleic expresson patterns. Genetics evidence suggests that gene expression patterns in mprinted genes, which are frequently organised in clusters, are regulated by the imprinting control regions (ICR). This thesis is mainly focused on the mechanisms through which the ICRs control the imprinting in the cluster, containing the Kcnq1, Igf2 and H19 genes, located at the distal end of mouse chromosome 7. The H19 ICR, located in the 5' flank of the H19 gene represses paternal H19 and maternal Igf2 expression, respectively, but has no effect on Kcnq1 expression, which is controlled by another ICR located at the intron 10 of the Kcnq1 gene. This thesis demonstrates that the maternal H19 ICR allele contains several DNase I hypersensitive sites, which map to target sites for the chromatin insulator protein CTCF at the linker regions between the positioned nucleosomes. The thesis demonstrates that the H19 ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the H19 ICR and CTCF. The Kcnq1 ICR function is much more complex, since it horbours both lineage-specific silencing functions and a methylation sensitive unidirectional chromatin insulator function. Importantly, the thesis demonstrates that the Kcnq1 ICR spreads DNA methylation into flanking region only when it is itself unmethylated. Both the methylation spreading and silencing functions map to the same regions. In conclusion, the thesis has unraveled and unrivalled complexity of the epigenetic control and function of short strtches of sequences. The epigenetic status of these cis elements conspires to control long-range silencing and insulation. The manner these imprinting control regions can cause havoc in expresson domains in human diseases is hence emerging.

Darmstadt, Techn. Univ., Diss., 2002.