Mutations in genes of importance for cartilage development may lead to skeletal malformations, chondroskeletal dysfunction and increased susceptibility to degenerative joint disease. Characterization of these mutations and identification of molecular pathways for the corresponding gene products have contributed to our understanding of mechanisms regulating skeletal patterning, endochondral ossification and joint formation. A five generation family segregating autosomal dominant osteochondritis dissecans (OCD) was identified. Affected family members presented with OCD in knees, hips and elbows, short stature, and early osteoarthritis. A genome wide scan and a multipoint linkage analysis identified aggrecan (ACAN) as a prime candidate gene. DNA sequence analysis of the ACAN-gene revealed heterozygosity for a missense mutation (c.6907G>A) in affected subjects, resulting in a p.V2303M substitution in the aggrecan G3 domain C-type lectin. This domain is important for the interaction with other proteins in the cartilage extracellular matrix. To determine the effect of the V2303M substitution on secretion and interaction, we performed binding studies with recombinant mutated and wild type G3 proteins. We found decreased affinity or complete loss of interaction between V2303M aggrecan and fibulin1, fibulin2 and tenascin-R. Analysis of articular cartilage from an affected family member confirmed that V2303M aggrecan is produced and present. In search for gene mutations associated with multiple epiphyseal dysplasia (MED) we considered the ACAN-gene a likely candidate. The ACAN-gene was analysed in 39 individuals with MED and screened negative for mutations in six previously known MED genes. Sequence analysis revealed a heterozygous missense mutation (c.1448G>T) in one adult male and compound heterozygous missense mutations (c.1366T>C and c.836G>A) in a five year old boy with healthy parents, each of them carrier for one of the mutations. A large family segregating autosomal dominant brachymesophalangia and OCD in finger joints was characterised. The clinical presentation in six affected family members was consistent with the diagnosis Brachydactyly type A1, in this family characterized by shortening of the middle phalanges, short ulnar styloid process, flattening of the metacarpal heads and mild osteoarthritis. The condition may be caused by mutations in the Indian hedgehog gene (IHH) or a yet unidentified gene on chromosome 5p13. Sequence analysis of the IHH-gene in affected individuals revealed a novel C to T transition (c.472C>T) leading to a p.158Arg>Cys substitution. Residue 158 in IHH is highly conserved throughout evolution and molecular structure modelling of IHH suggests that the R158C substitution leads to a conformational change at the site of interaction with the IHH-receptor. This supports that the substitution causes Brachydactyly type A1 in this family. In summary, we report on the clinical, radiological and molecular genetic characteristics of the three skeletal disorders OCD, MED and BDA1. Our results provide a novel molecular mechanism in the pathophysiology of familial osteochondritis dissecans confirming the importance of aggrecan C-type lectin for cartilage function. We also show that ACAN-gene mutations may be associated with MED extending the spectrum of skeletal dysplasias associated with the aggrecan gene. Finally, we report on a novel missense mutation in a conserved region of the IHH-gene associated with BDA1.

Spinocerebellar ataxia type 7 (SCA7) is a hereditary neurodegenerative disorder affecting the cerebellum, pons and retina. SCA7 patients present with gait ataxia and visual impairment as the main symptoms. Anticipation, commonly observed in SCA7 families, is a phenomenon where an earlier age at onset and a more severe progression of disease is seen in successive generations. In order to identify the gene responsible for SCA7, we performed linkage analysis on a Swedish SCA7 kindred. Evidence for linkage of the SCA7 disease locus to a 32 cM region on chromosome 3p12-21.1, between markers D3S1547 and D3S1274, was established. A number of neurodegenerative disorders associated with anticipation are caused by expanded (CAG)n repeats in their respective disease genes. In order to isolate the SCA7 disease gene we, therefore, screened a human infant brain stem cDNA library for CAG repeat containing clones, mapping to chromosome 3. Four candidate clones were isolated and analysed, but could all be excluded as the SCA7 disease gene. In 1997, the SCA7 disease gene was identified and, as expected, shown to harbour a CAG repeat, expanded in SCA7 patients. Analysis of the SCA7 CAG repeat region in Swedish SCA7 patients demonstrated that CAG repeat size was negatively correlated to age at onset of disease. Furthermore, patients with larger repeats presented with visual impairment, whereas patients with smaller repeats presented with ataxia as the initial symptom. SCA7 is the most common autosomal dominant cerebellar ataxia in Sweden and Finland, but rare in other populations. In order to investigate if the relatively high frequency of SCA7 in these countries is the result of a founder effect in the region, a haplotype analysis was performed on all SCA7 families available. All 7 families shared a common haplotype of at least 1.9 cM surrounding the SCA7 locus. In addition, strong linkage disequilibrium was demonstrated for marker D3S1287 closely linked to the SCA7 gene, suggesting a founder effect for the SCA7 mutation in Sweden and Finland. The function of the SCA7 protein, ataxin-7, is not known and it does not show significant homologies to any previously known proteins. In order to gain insight into the function of ataxin-7 we analysed the expression of ataxin-7 in brain and peripheral tissue from SCA7 patients and controls. In brain, expression was found to be mainly neuronal with a nuclear subcellular localisation. Ataxin-7 expression was found throughout the CNS, not restricted to sites of pathology. We also confirmed previously reported findings of neuronal intranuclear inclusions (NIls) in the brains of SCA7 patients. Based on our findings, we conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression pattern in affected and non-affected tissue or the distribution pattern of aggregated protein.

Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans. The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system. The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning. In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems. The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.

The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome. In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes. In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors. In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

The unconventional myosin IC has previously been suggested to be a haploinsufficient tumour suppressor. The mechanism for this action has hitherto been unknown, however, and hence we decided to attempt to elucidate the genes involved. The first study involved knock-down of MYO1C using siRNA technology followed by whole transcriptiome microarray analysis performed on samples taken at different time points post transfection. This revealed a cornucopia of differential expressions compared to the negative control, among them we found an early up-regulation of the PI3K/AKT pathway and the pathway for prostate cancer. Among the down regulated pathways we found endometrial-, colorectal cancer and small cell lung cancer as well as the cell cycle pathway which was a little counter intuitive to the hypothesis that MYO1C suppresses cancer. For the next study six different genes (CCND1, CCND2, CDKN2B, CDKN2C, MYC, RBL1) important for the transitions into S-phase of the cell cycle were therefore chosen for validation using qPCR. These six genes and MYO1C were analysed on both the original time series and a new biological replicate as well as a well stratified set of endometrial carcinoma samples. We were able to verify the significant down-regulation of CCND2 in both time series indicating that this is caused by the depletion of MYO1C. In the tumour samples we saw a negative correlation between the expression of MYO1C and FIGO grade corroborating results previously found by our group when looking at protein expression.

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

Cystinuria is defined as an inherited disorder characterized by increased urinary excretion of cystine and the dibasic amino acids arginine, lysine and ornithine. The only clinical manifestation of cystinuria is renal cystine stone formation due to the low solubility of cystine in the urine. Cystinuria can be attributed to mutations in the SLC3A1 and SLC7A9 genes in the majority of all cases and it has been a common expectation that molecular genetic studies of cystinuria would aid in understanding of the varying clinical outcome seen in the disease. Besides human, the disease has been most extensively studied in the domestic dog. The present study was undertaken to investigate the molecular genetic basis of cystinuria in patients from Sweden and to correlate genetic findings with phenotypes produced regarding cystine and dibasic amino acid excretion. Further, attempts were made to elucidate the molecular genetics of cystinuria in the dog. The entire coding sequences of the SLC3A1 and SLC7A9 genes were analysed by means of SSCA and DNA sequencing in 53 cystinuria patients and genetic findings were related to urinary excretion of cystine and dibasic amino acids in a subset of the patient group. We detected a total number of 22 different mutations in the SLC3A1 and SLC7A9 genes, 18 of which were described for the first time. We have found a probable genetic cause of cystinuria in approximately 74 % of our patients and a possible contribution to the disease in another 19 %. Mutations in the SLC3A1 gene is the major cause of cystinuria in our group, with only a minor contribution of SLC7A9 mutations. The group of patients presenting SLC3A1 mutations in a heterozygous state or lacking mutations in both genes had higher values of total urinary cystine and dibasic amino acids compared to patients homozygous for SLC3A1 mutations. The reason for this discrepancy remains unclear, but the possible impact of medical treatment with sulfhydryl compounds on total cystine values was ruled out. Sequencing of the full-length canine SLC7A9 cDNA was accomplished using the RACE technology and results from mutation analyses of SLC7A9 and SLC3A1 in cystinuric dogs showed that only two out of 13 dogs have mutations with possible impact on protein function in these genes. DNA sequencing was used for all exons of both genes in the dog, and in human cystinuria patients, all samples lacking mutations or showing heterozygosity after SSCA screening were sequenced in both genes as well. This implies that all point mutations present have been detected, but the possibility of mutations escaping PCR based methods as well as mutations in regulatory parts of the SLC3A1 and SLC7A9 genes remains in cases lacking a full molecular genetic explanation of the disease. Finally, clinical and genetic data from our study of cystinuria both in man and dog exemplifies that manifestation and clinical severity of cystinuria is not determined by genetic alterations in the SLC3A1 and SLC7A9 alone. Environmental factors, congenital malformations and modulating genetic factors are all possible contributors to the clinical outcome of cystinuria.

Pain is a major public health issue throughout the world. Increased understanding of the different forms of pain and identification of susceptibility genes could contribute to improved treatments. The main aims of this thesis were to identify the underlying genetic causes of pain by studying two large families affected with migraine and pain insensitivity, respectively. Migraine is one of the most common neurovascular disorders, affecting over 12% of the western population. The genetic contribution to migraine is about 50% according to family and twin studies. To identify novel susceptibility loci for migraine, we performed a genome-wide screen in a large family with migraine from northern Sweden. Linkage analysis revealed significant evidence of linkage (LOD=5.41) on chromosome 6p12.2-p21.1. A predisposing haplotype spanning 10 Mb was inherited with migraine in all affected members of the pedigree. Further fine-mapping of multiple SNP markers restricted the disease critical region to 8.5 Mb. Nine candidate genes were sequenced, revealing no disease-associated polymorphisms in SLC29A1, CLIC5, PLA2G7, IL17, SLC25A27 and TNFRSF21, but rare novel polymorphisms segregating with the disease haplotype in EFHC1, RHAG and MEP1A. EFHC1 has recently been shown to be involved in epilepsy, which is interesting considering the link between migraine and epilepsy. However, association analysis of EFHC1 revealed no difference between patients and controls, suggesting that this gene is not a risk factor for migraine. The combination of the two polymorphisms in RHAG and MEP1A could, however, not be found in any control individuals, indicating that they might be involved in genetic predisposition to migraine in this family. Disorders with reduced pain sensitivity are very rare, since pain perception is essential for survival. A number of disorders have still been identified with pain insensitivity and peripheral nerve degeneration as major clinical signs, including the hereditary sensory and autonomic neuropathies (HSAN). In order to identify novel susceptibility genes for HSAN V, we performed a genome-wide screen in a large consanguineous pedigree from a small village in northern Sweden. A homozygous region identical-by-descent was identified on chromosome 1p11.2-p13.2 in the three most severely affected patients. Subsequent analysis of candidate genes revealed a missense mutation in a conserved region of the nerve growth factor beta (NGFB) gene, causing a drastic amino acid change (R211W) in the NGF protein. NGF is important for the development and maintenance of the sympathetic and sensory nervous system and is therefore likely to be involved in disease. Functional analysis revealed that mutant NGF failed to induce neurite outgrowth and cell differentiation in PC12 cells. Furthermore, almost no mutant NGF was secreted by COS-7 cells, indicating that the processing and/or secretion of the protein might be disrupted. In conclusion, these findings present a novel migraine locus on chromosome 6 and identification of two rare polymorphisms that might be risk factors for migraine. Furthermore, a mutation in NGFB was found to cause complete loss of deep pain perception, which represents a very interesting model system to study pain mechanisms.

Background. Familial Amyloidosis with Polyneuropathy (FAP) is an autosomal dominantly inherited systemic amyloid disease. The disease is caused by mutations in the transthyretin (TTR) gene, where close to 100 different amyloidogenic mutations have been identified. FAP is found worldwide, but endemic areas with a high frequency of patients are found in Portugal, Japan and northern Sweden. Cases from these endemic areas all share the same TTR c.148G>A, p.V50M ("V30M") mutation, but the phenotype of the disease varies between the areas, and also within the endemic areas. The mean onset of the disease is two decades earlier in Portugal and Japan compared to Sweden, but late as well as early age at onset cases occur within all the populations. Interestingly, the different populations all display a maternal anticipation, where an earlier onset is observed for those individuals who inherit the trait from their mother. Since substantial variation in the phenotype is observed for different populations, epigenetic/genetic and/or environmental factors must exert a significant impact on the penetrance of the disease. Amyloid formation is caused by conformational changes of proteins, which facilitates their assembly into fibrils, amyloid. Oxidative stress can mediate conformational changes of proteins and since the mitochondria regulate oxidative processes within the cell, mitochondrial function may affect amyloid formation. The mitochondrial DNA is a non-nuclear DNA, which is entirely maternally inherited, and therefore could be related to the observed maternal anticipation of the disease. In addition, differences within the surrounding regions of the TTR gene may have an impact on the transcription of the gene and thereby on the expression of the different alleles. Material and methods. DNA from early and late onset V30M cases and from non-carriers (the latter utilised as controls) from Swedish, French, Japanese and Portuguese populations were analysed. In addition, DNA from healthy Swedish V30M carriers was analysed. Conventional analytical methods were employed, such as PCR, sequencing and genotyping. Conventional statistical methods used were t-test, Chi-squared test and maximum likelihood. Results. The study of V30M carrier frequency in two counties (Lycksele and Skellefteå) within the Swedish endemic area revealed a carrier frequency of 2.14% and 2.54%, respectively. The mitochondrial haplogroup analysis showed that in populations with generally late onset (French and Swedish), the haplogroup distribution of late onset cases resembled that of the controls derived from the same area, whereas haplogroup distribution for early onset patients was significantly different. The most pronounced difference was for the rare haplogroup K, of which early onset cases had a higher frequency than the controls. Analysis of the Portuguese population, with predominantly early onset, showed that haplogroup distribution for early onset cases were similar to the Portuguese control group, which had a different distribution than the Swedish control group. By analysis of pedigrees from Swedish and Portuguese patients it could be shown that mitochondrial genetic variation entirely could explain maternal anticipation in the Portuguese patients, whereas for Swedish patients, an additional parent of origin effect is present. Our analysis of the TTR gene disclosed a polymorphism (rs62093482) in the 3'UTR region of the Swedish patients. This polymorphism was found in all V30M carriers, irrespective of symptoms. In addition, homozygous TTR V30M carriers were homozygous also for the polymorphism. Since Swedish patients share a common founder this polymorphism thus is localised on the V30M allele. This polymorphism was found in only 4% of the Swedish controls. French controls showed the same frequency, but none of the French V30M patients displayed the polymorphism. In the Japanese population the polymorphism was not present at all. Interestingly, this polymorphism generates a potential binding site for microRNA and thereby possibly could down-regulate the expression of the mutated TTR allele. Conclusions. The carrier frequency in the endemic area is remarkably high, above 2% in the Lycksele and Skellefteå areas. The prevailing haplogroup distributions in the different endemic areas are consistent between the general population and the patient group with the predominant phenotype of that area. Mitochondrial genetic differences may explain maternal anticipation in Portuguese patients, and have an influence in Swedish patients. A polymorphism in the 3'UTR regulatory region of the mutated TTR allele is found in all Swedish patients. This polymorphism may down-regulate TTR V30M expression and thereby contribute to the late onset of the disease noted in the Swedish population.

Tumour immunity is a balance between immune mediators that promote tumor progression versus mediators that promote tumor rejection. Infiltrating lymphocytes in human colorectal cancer tissues are independent prognostic factors for a better survival and a high number of cytotoxic CD8+ T-cells have been associated with a better prognosis in terms of a longer and disease free survival for the patient. In our syngeneic rat model we induce colon carcinoma subperitoneally by injecting a colon cancer cell line BN7005, a cell line expressing the epitope (Lewis Y) for the BR96 antibody. Tumours are dissected out and treated with different fixatives and then either frozen, snap-frozen or embedded in paraffin followed by sectioning. Immunohistochemistry using monoclonal antibodies against the tumour infiltrating leukocytes was performed on the tissue. The results were seen as an infiltration of different leukocytes in the tumours.