Human endogenous retroviruses (HERVs) represent approximately 7% of the human genome. This investigation was focused on one particular HERV, ERV3, with the main purpose of characterising its gene expression patterns and genomic distribution of ERV3-like sequences. Furthermore, this careful expression study should provide insights into the biological role of HERVs. The impact of HERVs in health and disease is not yet clarified. ERV3 is expressed as three envelope (env) transcripts, of which two also contain a cellular gene, H-plk (human proviral linked Krüppel). ERV3 env expression was mainly investigated at the RNA level. The gene expression of two other HERVs, HERV-K and HERV-E was analysed and compared with ERV3 activity. Real-time PCRs were developed and in combination with in situ hybridisation, it was found that ERV3 is expressed in a tissue- and cell-specific way. High levels of ERV3 mRNA (up to six times over Histone3.3) were demonstrated in placenta, sebaceous glands, foetal and adult adrenal glands, brown adipose tissue, corpus luteum, pituitary gland, thymus and testis. In monocytic cells including both normal monocytes and malignant U-937 cells, elevated mRNA levels were observed after retinoic acid (RA)-induced differentiation. ERV3-encoded Env protein was detected in selected cases, one following RA-treatment. In addition, several new ERV3-like sequences were discovered in the human genome. ERV3 was found to have conserved open reading frames in contrast to other ERV3-like sequences in the human genome. This suggests that ERV3 may be involved in important cellular processes such as differentiation, cell fusion, immunomodulation and protection against infectious retroviruses. The developed techniques and obtained results will allow further studies of HERV expression to better correlate HERV activity to both normal development and disease.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with unknown etiology. The aim of this thesis was to identify susceptibility regions through genetic mapping, using model-based linkage analysis on nuclear and extended SLE multicase families. In the first paper we performed a genome scan on 19 genetically homogenous Icelandic and Swedish families. One region at 2q37 was identified with a significant linkage with contribution from both populations (Z=4.24). Five other regions 2q11, 4p13, 9p22, 9p13 and 9q13 showed suggestive linkage (Z>2.0). In the second paper, 87 families from 10 different countries were analysed only for chromosome 1. One region at 1q31 showed significant linkage (Z=3.79) with contribution from families from all populations, including Mexicans and Europeans. Four other regions 1p36, 1p21, 1q23, and 1q25, showed levels of suggestive linkage. Linkage for most regions was highly dependent on what population was used, which indicated strong genetic heterogeneity in the genetic susceptibility for SLE. In the two last papers, we used the positional candidate gene strategy, in order to investigate candidate genes in two regions linked to SLE. For the Bcl-2 gene (at 18q21) we could not detect any association with SLE using three different markers. However, when we investigated the tightly linked low-affinity family of FcγR genes (at 1q23), we could find association for two risk alleles in the FcγRIIA and FcγRIIIA genes. The risk alleles were transmitted to SLE patients on one specific haplotype and therefore are not independent risk alleles. The results show that model-based linkage analysis is a strong approach in the search for susceptibility genes behind complex diseases like SLE.

Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma in situ.

The purpose of gene mapping is to understand the underlying genetics of simple and complex traits like plumage colour and growth. This thesis is based on a cross between the wild ancestor of the modern chicken, the red junglefowl, and a White Leghorn line selected for high egg mass. There are obvious phenotypic differences between these two breeds in several aspects such as growth, egg production and behaviour. These complex traits are often influenced by a number of genes or Quantitative Trait Loci (QTL) as well as environmental factors. Identification of QTL regions involves testing of association between genetic markers and the phenotype of interest. The QTL identified in this study explain most of the difference in adult body weight between the red junglefowl and the White Leghorn, but less of the difference at earlier age. By applying a different method for detection of QTL, including gene interactions, epistasis, we can understand more of the genetics behind early growth. The allele coming from the red junglefowl is generally associated with lower weight, egg production and food consumption. In this study we have also identified two genes explaining the difference in plumage colour in the cross. The Extension locus, encoded by the melanocortin receptor 1 (MC1R), controls the amount of pigment produced has shown to be associated with plumage colour. A mutation in the MC1R gene causes black pigmentation of the plumage. We have also found association between the PMEL17 gene, known to be involved in normal pigmentation, and the Dominant white phenotype present in the White Leghorn. After comparison of sequences from different alleles at the Dominant white locus, amino acid alteration caused by insertion and deletion in the transmembrane region of the PMEL17 protein has been revealed. These mutations are associated with alleles representing different plumage colour variants.

Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the NF2 gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the NF2 gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the NF2 locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the NF2 locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the GSTT1/CABIN1 locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the CABIN1 gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.

Microarray-based comparative genomic hybridization (array-CGH) has emerged as a versatile platform with a wide range of applications in molecular genetics. This thesis focuses on the development of array-CGH with a specific aim to approach disease-related questions through improved strategies in array construction and enhanced resolution of analysis. In <b>paper I</b>, we applied an array covering 11 Mb of 22q, encompassing the NF2 locus, for deletion detection in sporadic schwannoma. Hemizygous deletions and tumor heterogeneity were identified. Array-CGH was established as a reliable platform for detection of DNA dosage alterations. <b>Paper II</b> described the construction of the NF2 gene-specific microarray for high-resolution scanning of deletions in the NF2 locus. We report a novel PCR-based non-redundant strategy for microarray fabrication, which considerably improved the sensitivity and reliability of deletion detection. <b>Paper III</b> reported the first tiling-path array comprehensively covering a human chromosome. The usefulness of the 22q-array was demonstrated by applying it to detect DNA dosage-alterations in 22q-associated disorders. In <b>paper IV</b>, we optimized array-CGH protocols for deletion detection in 22q11 deletion-syndrome. We showed that genomic and cDNA clones are not optimal for analysis of 22q11 locus and that PCR-based non-redundant strategy is reliable for deletion detection in such regions. In <b>paper V</b>, we utilized the 22q-array for understanding the genetic basis of schwannomatosis. Two commonly deleted regions were identified within the IGL and the GSTT1/CABIN1 loci. Further investigations using high-resolution arrays, bioinformatic analysis and mutational screening were performed. Missense mutations, specific to the schwannomatosis- and NF2 samples, were identified in the CABIN1 gene. <b>Paper VI</b> described the first array-CGH study for comprehensive and high-resolution profiling of deletions spanning the 17q11 locus. Both typical and atypical deletions were identified in NF1 samples. Bioinformatic analysis revealed novel segmental duplications, which can potentially mediate 17q11 deletions.

The array-based form of comparative genomic hybridization (array-CGH) is a new methodology that has shown to be of significant importance. This thesis focuses on the development of array-CGH with the aim to define candidate regions/genes on chromosome 22 in a wide spectrum of cancer-related conditions. In <b>paper I</b>, we developed and applied the first comprehensive genomic microarray, representing human chromosome 22, for analysis of DNA copy number. Using this array-based approach, we identified gene copy number alterations, including heterozygous/homozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of imbalanced translocation, in several 22q-associated disorders. In <b>paper II</b>, we applied the same array to perform DNA copy number profiling of a series of ovarian carcinoma. cDNA arrays were also used in this study to correlate gene expression levels with DNA-copy number. In the course of this analysis, we determined a small 3.5 Mb candidate 22q telomeric region and suggested a number of specific candidate genes. <b>Paper III</b> described the comprehensive and high-resolution analysis of chromosome 22 in a large set of various stage breast cancers. Multiple distinct patterns of genetic aberrations were observed. The smallest identified candidate locus was 220 kb in size and mapped to a gene-rich region in the vicinity of telomere of 22q. Intriguing result of this study was the detection of high frequency (26.6%) of intra-tumoral clonal variation in gene copy number profiles, which should be viewed as a high number, considering that we study in detail only a single human chromosome. In <b>paper IV</b>, we profiled a series of 28 Wilms tumor samples using 22q-array in order to assess specific regions affected with DNA dosage-alterations. The distribution of aberrations defined a complex amplifier genotype and delimited two tumor suppressor/oncogene candidate loci. These results open up for several avenues for continued research of these tumor forms. These findings also demonstrate the power of array-CGH in the precise determination of minute DNA copy number alterations and strengthen the notion that further studies, preferentially in the context of the entire human genome, are needed.

Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) is the most common hereditary syndrome predisposing to colorectal cancer, accounting for 1-3% of all colorectal cancer. This multi-organ cancer predisposition syndrome is caused by mutations in the mismatch repair (MMR) genes, especially MLH1 and MSH2, and to lesser extents MSH6 and PMS2, which lead to widespread genetic instability and thus microsatellite instability (MSI). Hereditary cancer often manifests in two or more tumours in a single individual; 35-40% of Lynch syndrome patients have synchronous or metachronous tumours of the two major Lynch syndrome-related cancers: colorectal and endometrial. The main purposes of the work underlying this thesis were to identify persons at risk of Lynch syndrome or other types of hereditary colorectal cancer, to estimate the cancer risks associated with these predispositions and to identify the underlying genetic causes. A population-based cohort of 78 persons with double primary colorectal or colorectal and endometrial cancer was identified. Cancer risks in their 649 first-degree relatives were estimated in relation to tumour MSI status (positive or negative) and age at diagnosis (before or after 50 years of age) in the probands. The overall standardised incidence ratio was 1.69 (95% CI; 1.39-2.03). The highest risks for Lynch syndrome-associated cancers: (colorectal, endometrial, ovarian and gastric) were found in families with young MSI-positive probands, likely representing Lynch syndrome families. Importantly, no overall risk was found in families with old probands, irrespective of MSI status. Blood samples were available from 24 MSI-positive patients for mutation screening of MLH1, MSH2 and MSH6. Sequence variants or rearrangements predicted to affect protein function were found in 16 patients. Six novel variants were found: two large rearrangements, two truncating and two missense mutations. The missense mutations were found to segregate in the families. Studies of allele frequencies, MSI and loss of immunostaning in tumours from family members further supports the hypothesis that these missense changes play a role in Lynch syndrome, as do the non-conservative nature and evolutionary conservation of the amino acid exchanges. Five families had mutations in MLH1, five in MSH2, and six in MSH6. The unexpectedly large impact of MSH6 was in genealogical studies shown to be due to a founder effect. Cumulative risk studies showed that the MSH6 families, despite their late age of onset, have a high lifetime risk for all Lynch syndrome-related cancers, significantly higher in women (89% by age 80 years) than in men (69%). The gender differences are in part due to high endometrial (70%) and ovarian cancer risk (33%) in addition to the high colorectal cancer risk (60%). These findings are of great importance for counselling and surveillance of families with MSH6 mutations. Finally, in a large family with MSI-negative hereditary colorectal cancer for which the MMR genes and APC had been excluded as possible causes, a genome-wide linkage analysis was performed, resulting in a suggested linkage to chromosome 7. Conclusions: Relatives of probands with MSI-positive, double primary colorectal and endometrial cancer diagnosed before the age of 50 years have significantly increased risks of Lynch syndrome-related cancers. MSH6 mutations, which have unusually high impact in this study population due to a founder effect, confer high cumulative risks of cancer despite the generally late age of onset.

Gene expression differences between patients and controls can be used to find susceptibility genes and drug targets for a disease. High-resolution strategies are required because the differences between the investigated groups may be small and numerous factors may affect the mRNA quantity. This thesis is based on the use of real-time RT-PCR combined with a new statistical approach, developed to detect small differences between patients and controls and differences due to patient subgroups. Comparisons between human brain biopsy and autopsy samples showed that post-mortem tissue can be used to make conclusions on the relative mRNA levels in the living brain. Power analysis based on human brain mRNA expression from 14 genes adjusted with two reference genes, revealed that a sample size of 50 patients and 50 controls was required to detect a 2-fold difference with a power and a confidence of 95%. A similar study in rats revealed that approximately the same sample size was required for rat brain mRNA expression studies. The mRNA levels of several genes were studied in 55 schizophrenia and 55 control prefrontal brain autopsies, using a novel and more powerful statistical analysis. The serotonin receptor 2C gene (HTR2C) showed a significant 1.5-fold decrease in the patients as compared to controls, and the monoamine oxidase B gene (MAOB) a 1.2-fold increase. The mechanism behind the decrease of HTR2C mRNA levels was investigated by studying the correlation of drug treatment and HTR2C promoter polymorphisms to the HTR2C expression levels. The observed decrease was present in untreated patients, suggesting that the HTR2C mRNA decrease is correlated with the disease and not the treatment. There was no association between promoter polymorphisms and HTR2C expression levels. Thus, the molecular mechanism for the decreased expression remains unclear. Nevertheless, the results support a role for monoaminergic synapses in schizophrenia.

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&gt;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.