<p>A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. </p><p> With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. </p><p> Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations.</p><p> A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.</p>

Labor may perhaps be the most painful a woman might experience, although characterized by large inter-individual variability. The perceived pain during labor is the result of diverse factors, i.e. her previous pain experiences, the analgesia she receives and maybe also her genes. The overall aim of this thesis was to investigate biological and psychological mechanisms underlying inter-individual differences in labor pain related behaviors. The mechanisms that characterize endogenous pain relief during labor are not fully understood, though it is known to be partly explained by the effects of β-endorphin (BE). BE plasma levels were followed longitudinally in a cohort of pregnant women and were found to remain unchanged between early and late pregnancy, although with a nadir in the beginning of the third trimester. Furthermore, women with low levels of BE in plasma at the end of the third trimester, required second line labor analgesia to a significantly higher extent than women with normal levels. In a population-based sample of 814 pregnant women we investigated if inter-individual differences in labor pain related behavior was influenced by the pain-protective single nucleotide polymorphism (SNP) combination of guanosine triphosphate cyclohydrolase (GCH1) and the opioid receptor µ-1 gene (OPRM1) A118G SNP. We identified a possible association between the pain-protective SNP combination of GCH1 and use of second line analgesia. No association was found between the OPRM1 and use of analgesia or labor pain related behavior. The association between self-rated antenatal depressed mood and anxiety in relation to pain behaviors and self-reported pain during labor was investigated. We found that depressed mood during pregnancy is associated with early arrival to the delivery department, whereas antenatal anxiety is associated with increased self-rated pain prior to labor analgesia.  In conclusion, although an increasing number of studies strongly suggest that genetic predisposition plays an important role in pain and pain-related mechanisms, GCH1 and OPRM1 has little to offer in terms of individual counseling on labor analgesia. To enable the future use of genetic variability for pre-labor testing and counseling, a number of different genes reflecting pain mediation pathways, involving biological and psychological mechanisms, need to be analyzed in combination.

Major recent improvements in biotechnology have led to an accelerated production of DNA sequences. The completion of the human genome sequence, along with the genomes of more than two hundred other species, has marked the arrival of the genome era. The ultimate goal is to understand the structure and function of genomes and their genes. This thesis has focused on the computational analysis of complementary DNA (cDNA) sequences. These are copies of mRNA transcripts that correspond to the coding regions of genomes. Studying the expression patterns of genes is essential for understanding gene function. Many gene expression profiling techniques generate short sequence tags that derive from transcripts. A pilot study was performed to assess the feasibility of using the pyrosequencing platform for gene expression analysis. The sequences generated by pyrosequencing in most cases (≈ 85%) were long enough (&gt; 18 nucleotides) to uniquely identify the corresponding transcripts through database searches. Aspects of transcript identification by short sequence tags were further investigated in a number of public databases, revealing that a tag length 16-17 nucleotides was sufficient for unique identifi- cation. Longer transcript representations are obtained from expressed sequence tag (EST) sequencing. Method development for the analysis and maintenance of large EST data sets has been performed on data from poplar, which is a tree of commercial interest to the forest biotechnology industry. In 2003 a large ESTsequencing project reached &gt; 100 000 reads, providing a unique resource for tree biology research. ESTs have been grouped into clusters and singletons that represent potential genes. Preliminary analyses have estimated gene content in Populus to be very similar to that of model organism Arabidopsis thaliana. EST data collections provide a rich source for mining polymorphisms. A software application was developed and applied to EST data from two Populus species, and candidate single nucleotide polymorphisms (SNPs) were recorded. A study of genetic variation between the species revealed a striking similarity, with orthologous pairs being &gt; 98% identical on the protein level. Keywords: cDNA, EST, gene expression, SNP, SAGE, polymorphism, assembly, clustering, DNA sequencing, pyrosequencing, mRNA transcript, orthology, tree biotechnology, restriction enzyme

In spite of the fact that prostate cancer is the most common male cancer in both Sweden and many other countries in the developed world, little is known of risk factors and predisposing conditions. The only well recognized risk factors are age, race and familial aggregation. More knowledge about risk factors could lead to better preventive measures together with better treatments. One way to evaluate this is to study second primary cancers; the connection between two different cancers can give valuable insight in etiology or clues to shared risk factors. This thesis aims at evaluating risk factors for prostate cancer. We constructed a cohort of 135,713 men diagnosed with prostate cancer and reported to the Swedish Cancer Registry 1958-1996. The cohort was followed for second primary cancers and a doubled risk of male breast cancer was found. We also noted increased risks for small intestine cancers and melanoma. As a follow-up on the increased risk of male breast cancer, we performed a nested case – control study. Included cases were men with first prostate and then breast cancer (n = 41) matched to men with only prostate cancer (n =81). For these men, we collected medical records and extracted data regarding treatment. Furthermore, all men diagnosed with both prostate and breast cancer irrespective which came first (n = 83) were used as probands. To both these sets of cases with breast and prostate cancer, we identified first degree relatives and grandchildren from parish offices throughout Sweden. Linking to the Cancer Registry retrieved all cancer diagnoses amongst relatives. Results from this study show a relation between estrogen treatment of prostate cancer and the risk of developing breast cancer. We also found that a small part of the cases with both cancers appeared in families with inheritance patterns possibly attributed to BRCA2. As estrogen treatment seemed involved in increased risk of breast cancer after prostate cancer, we wanted to investigate the newly discovered Estrogen receptor β and the relation to prostate cancer risk. Previous reports have shown that ERβ acts as a negative regulator of proliferation. ERβ expression occurs mainly in prostatic epithelial cells and the expression gradually diminishes when cancer develops and aggravates. We used a single nucleotide polymorphism (SNP) association study approach to evaluate genetic variation in ERβ as a risk factor for prostate cancer. One SNP, located in the promoter region associated with a small increased risk of prostate cancer whereas variation in the rest of the gene did not. In the last paper, we investigated trans-urethral resection (TURP) of the prostate due to benign prostate hyperplasia (BPH) as a risk factor for later development of prostate cancer. Evidence has gathered that both BPH and prostate cancer are associated to inflammation. By comparing incidence and mortality in a cohort of 7,901 men with the general population there appeared to be an increased risk of prostate cancer but decreased mortality. Analyzing this increased risk further, we conducted a nested case - control study with men extracted from the cohort. Cases had a TURP and later developed prostate cancer and controls just had a TURP. We then evaluated the specimens from TURP regarding extent of inflammation, degree of androgen receptor down regulation and expression of p53, all factors previous associated with prostate cancer. None of these parameters differed between cases and controls and they can therefore not explain the increased risk. Decreased mortality but increased risk might be explained by surveillance bias, which means more medical attention to these patients, resulting in diagnosing clinically non-significant cancers. In summary, our results show a doubled risk of male breast cancer following prostate cancer. A risk that can be attributed to the use of estrogen to treat prostate cancer or to some extent a possible mutation in BRCA2. We also propose that a SNP change in the ERβ promoter confer a small increased risk of prostate cancer. A small risk elevation of prostate cancer following TURP most probable could depend on surveillance bias.

Advancements in prognostication have improved the subdivision of chronic lymphocytic leukaemia (CLL) into diverse prognostic subgroups. In CLL, IGHV unmutated and IGHV3-21 genes are associated with a poor-prognosis, conversely, IGHV mutated genes with a favourable outcome. The finding of multiple CLL subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has suggested a role for antigen(s) in leukemogenesis. Patients belonging to certain stereotyped subsets share clinical and biological characteristics, yet limited knowledge exists regarding the genetic and epigenetic events that may influence their clinical behaviour. This thesis aimed to, further investigate Swedish IGHV3-21-utilising patients, screen for genetic and DNA-methylation events in CLL subgroups/subsets and study DNA methylation over time and within different CLL compartments. In paper I, IGHV gene sequencing of 337 CLL patients from a Swedish population-based cohort revealed a lower (6.5%) IGHV3-21 frequency relative to previous Swedish hospital-based studies (10.1-12.7%). Interestingly, this frequency remained higher compared to other Western CLL (2.6-4.1%) hospital-based cohorts. Furthermore, we confirmed the poor-outcome for IGHV3-21 patients to be independent of mutational and stereotypy status. In paper II, genomic events in stereotyped IGHV3-21-subset #2, IGHV4-34-subset #4 and subset #16 and their non-stereotyped counterparts were investigated via SNP arrays (n=101). Subset #2 and non-subset #2 carried a higher frequency of events compared to subset #4. A high frequency of del(11q) was evident in IGHV3-21 patients particularly subset #2 cases, which may partially explain their poor-prognosis. In contrast, the lower prevalence of aberrations and absence of poor-prognostic alterations may reflect the inherent low-proliferative disease seen in subset #4 cases. In papers III and IV, differential methylation profiles in IGHV mutated and IGHV unmutated patients were identified using DNA-methylation microarrays. CLL prognostic genes (CLLU1, LPL), tumor-suppressor genes (TSGs) (ABI3, WISP3) and genes belonging to TGF-ß and NF-kB/TNFR1 pathways were differentially methylated between the subgroups. Additionally, the re-expression of methylated TSGs by use of methyl and deacetyl inhibitors was demonstrated. Interestingly, analysis of patient-paired diagnostic/follow-up samples and patient-matched lymph node (LN) and peripheral blood (PB) cases revealed global DNA methylation to be relatively stable over time and remarkably similar within the different compartments. Altogether, this thesis provides insight into the aberrant genomic and DNA methylation events in divergent CLL subgroups. Moreover this thesis helps distinguish the extent to which DNA methylation changes with respect to time and microenvironment in CLL.

The concept of the gene has been constantly challenged by new discoveries in the life sciences. Recent challenging observations include the high frequency of alternative splicing events and the common transcription of non-protein-coding-RNAs (ncRNAs) from the genome. The latter has long been considered noise in biological systems. Multiple lines of evidence from genomic studies indicate that alternative splicing and ncRNA play important roles in expanding proteome diversity in eukaryotes. Here, the aim is to find the link between alternative splicing and ncRNAs by studying the expression profile of the spliceosomal snRNAs (U snRNA). Spliceosomal snRNAs are essential for pre-mRNA splicing in eukaryotes. They participate in splice site selection, recruitment of protein factors and catalyzing the splicing reaction. Because of this, both the abundance and diversity of U snRNAs were expected to be large. In our study we deeply analyzed the U snRNA population in primates using a combination of bioinformatical, biochemical and high throughput sequencing approaches. This transcriptome profiling has revealed that human, chimpanzee and rhesus have similar U snRNA populations, i.e. the vast majority of U snRNAs originate from few well-defined gene loci and the heterogeneity observed in U snRNA populations was largely due to the presence of SNPs at these loci. It seems that the gene loci that could potentially encode a significantly heterogeneous population of U snRNAs are mostly silent. Only few minority transcripts were detected in our study, and among them three U1-like snRNAs might play a role in the regulation of alternative splicing by recognizing non-canonical splicing sites. Mutations of U snRNA have been shown to impact the splicing process. Therefore, our study provides a reference to study the biological significance of SNPs in U snRNA genes and their association with diseases.

<p>Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community.</p>

A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations. A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.

Wheat grain quality has a complex genetic architecture heavily influenced by the growing environment. Consistency in wheat quality not only affects the efficiency of milling and baking but also the quality of end-use products. The objectives of this study were to 1) analyze the different wheat quality parameters in Recombinant Inbred Lines (RILs) grown under different environments, and 2) to identify Quantitative Trait Loci (QTLs) associated with quality stability in RILs grown under different environments. A set of 180 RILs derived from two spring wheat lines 'Halberd' and 'Len' were grown at Uvalde and College Station TX, in the 2009/2010 growing season and at Chillicothe and College Station TX, in 2010/2011 growing seasons. The experiment was laid out in Randomized Complete Block Design (RCBD) with four replications within each location. Each line was tested for multiple quality traits that included grain hardness, protein content, dough mixing properties and bread baking quality using Single Kernel Characterization System (SKCS), Near-Infrared Reflectance Spectrometry (NIRS) analysis, mixograph and the Sodium Dodecyl Sulfate Sedimentation (SDSS) test. Genetic linkage map construction was carried out with 116 single nucleotide polymorphism (SNP) markers in the RILs. Then composite interval mapping was carried out to identify QTLs associated with quality traits. The SDSS column height was positively correlated across four environments. Similarly, it was found to have significant positive correlation with mixing tolerance and peak time within and also across locations. However, the SDSS was negatively correlated with the hardness index. The protein percent was not significant with any of the quality traits within and across environments. We were able to detect many QTLs for different quality traits but most of them were site specific. Only a few QTLs were consistent across environments. Most of the QTLs for quality traits i.e., SDSS, peak time, mixing tolerance and hardness index were identified on chromosome 1B. We were able to detect overlapped QTLs for SDSS column height and mixing tolerance on chromosome 1B. Furthermore, overlapping QTLs for mixing tolerance and peak time were detected on an unknown chromosome. We also detected overlapping QTLs for hardness index on chromosome 1B. We identified one stable QTL for SDSS column height on chromosome 4B. This QTL was detected based on the coefficient of variation (CV) for SDSS in four different environments.

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.