| 1. |
- Bin Kaderi, Mohamed Arifin, 1978-
(författare)
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Assessment of Novel Molecular Prognostic Markers in Chronic Lymphocytic Leukemia
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, which has prompted the search for biomarkers that can predict prognosis in this disease. The IGHV gene mutation status and certain genomic aberrations have been identified as reliable prognostic markers of clinical outcome for this disorder. However, the search for more feasible prognostic markers in CLL is still being pursued. Recently, certain single nucleotide polymorphisms (SNPs) in the GNAS1, BCL2 and MDM2 genes and the RNA expression levels of the LPL, ZAP70, TCL1, CLLU1 and MCL1 genes were suggested as novel prognostic markers in CLL. In papers I-III, we performed genotyping analyses of the GNAS1 T393C, BCL2 -938C>A and MDM2 SNP309 polymorphisms in 268-418 CLL patients and related the genotypes with clinical data. Association studies between the polymorphisms and established prognostic markers (i.e. IGHV mutation status, genomic aberrations, CD38 expression) were also performed. Our studies did not find any significant relationship between these SNPs with either clinical outcome or other known prognostic markers in CLL. In paper IV, we measured the RNA expression levels of LPL, ZAP70, TCL1, CLLU1 and MCL1 in 252 CLL cases and correlated these levels with clinical outcome. Here, we verified that high expression of all these RNA-based markers, except MCL1, were associated with an unfavourable prognosis. We also confirmed a close relationship between IGHV mutation status and the RNA-based markers, especially for LPL and CLLU1 expression. Among the RNA-based markers, multivariate analysis revealed LPL expression as the strongest independent prognostic marker for overall survival and time to treatment. Furthermore, the RNA-based markers could add further prognostic information to established markers in subgroups of patients, with LPL expression status giving the most significant results. In summary, data from papers I-III could not verify the GNAS1 T393C, BCL2 -938C>A and MDM2 SNP309 polymorphisms as prognostic markers in CLL. Future SNP markers must hence be confirmed in large, independent cohorts before being proposed as prognostic marker in CLL. In paper IV, we conclude that LPL expression appears to be the strongest among the RNA-based markers for CLL prognostication. Further efforts to standardize LPL quantification are required before it can be applied in the clinical laboratory to predict clinical outcome in this disease.
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| 2. |
- Delgado Vega, Angélica María, 1982-
(författare)
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Dissecting the Genetic Basis of Systemic Lupus Erythematosus : The Pursuit of Functional Variants
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Systemic lupus erythematosus (SLE) is a chronic and systemic autoimmune disease that primarily affects women during the childbearing years. SLE is characterized by the production of autoantibodies against nucleic acids and their interacting proteins. The exact molecular mechanisms leading to the breakdown of self-tolerance remain to a large extent unknown, but it is well established that they are influenced by both non-genetic (i.e. environmental and hormonal) and genetic factors. SLE is a complex, polygenic disease. Several susceptibility variants have been identified in SLE. However, the functional role in disease pathogenesis for the majority of them remains largely unknown.This thesis includes case-control association studies where the role of the genes TNFSF4 (Paper I), STAT4 (Paper II), CD226 (Paper III), and BLK (Papers IV and V) in the susceptibility of developing SLE was investigated. The primary focus was on the identification of the functional variants underlying the association. For each of these genes, fine mapping was performed using single nucleotide polymorphisms (SNPs), the linkage disequilibrium (LD) was characterized, and the association was narrowed down to specific haplotypes by means of several different statistical genetic strategies. Candidate variants were prioritized for further functional analysis on the basis of their potential effect on the gene function, their association, and/or biological plausibility. In Paper I, the association of TNFSF4 with SLE was validated and attributed to a risk haplotype tagged by SNPs rs1234317-T and rs12039904-T. Paper II provides evidence supporting the presence of at least two independent genetic effects within the STAT4 gene represented by rs3821236-A and rs7574865-A, which correlated with increased levels of gene expression. In Paper III, a functional allele in CD226 (rs727088-C) was identified, which was responsible for decreased levels in both mRNA and protein expression. In Paper IV, two independent genetic effects in the BLK gene were demonstrated. The first one comprised multiple regulatory variants in high LD that were enriched for NFκB and IRF4 binding sites and correlated with low BLK mRNA levels. The second was a low-frequency missense substitution (Ala71Thr) that decreased the BLK protein half-life. In Paper V, a genetic epistatic interaction between BANK1 rs10516487 (GG) and BLK rs2736340 (TT+TC) was demonstrated. Additional molecular analyses established that these molecules interact physically. These studies have contributed to the dissection of the genetic architecture of SLE. They highlight the allelic heterogeneity of the disease and provide functional links to the associated variants, which has significantly aided in the understanding of SLE disease pathogenesis.
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| 3. |
- Olsson, Malin, 1972-
(författare)
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Familial amyloidosis with polyneuropathy : studies of genetic factors modifying the phenotype of the disease
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Norberg, Maria, 1976-
(författare)
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In Vitro Drug Sensitivity and Apoptosis in Chronic Lymphocytic Leukemia
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy displaying varying clinical outcome, where molecular markers today can divide patients into prognostic subgroups. Despite the introduction of new agents for treatment, remissions are usually not sustained in CLL and resistance towards treatment can partly be explained by aberrant apoptosis. The aim of this thesis was to find new drugs for CLL patients resistant to conventional therapy and to analyze genes involved in apoptosis within different prognostic subgroups. In paper I-II, the in vitro activity of substances was investigated using the fluorometric microculture cytotoxicity assay (FMCA). When evaluating rapamycin (paper I), an inhibitor of mTOR, in 97 tumor samples from different entities, CLL was found to be one of the most sensitive tumor types. Combination experiments on patient CLL cells indicated that rapamycin acted synergistically with the CLL drugs vincristine and chlorambucil. An investigation of 20 anti-cancer agents in cells from 40 CLL patients (paper II) revealed that prednisolone and rolipram displayed high activity in poor-prognostic patients, in particular IGHV unmutated CLL. Furthermore, when used in combination these agents were found to produce a synergistic effect. In paper III, the anti-apoptotic BCL2 family member BFL1 was evaluated in 37 CLL cases. Levels of BFL1 were higher in fludarabine-resistant patients compared to fludarabine-sensitive patients. In addition, the high expression of BFL1 inversely correlated to fludarabine-induced apoptosis in CLL cells. A single nucleotide polymorphism in the anti-apoptotic BCL2 gene (-938C>A) has been suggested as a novel poor-prognostic marker in CLL. In paper IV, we investigated this BCL2 polymorphism in 268 CLL patients and correlated genotypes to clinical data. However, no association could be confirmed between this polymorphism and clinical outcome or established prognostic markers. In conclusion, this thesis has shown that rapamycin is a potential drug for treatment in CLL. Furthermore, prednisolone and rolipram were identified as interesting candidates for treatment of poor-prognostic patients. Finally, the anti-apoptotic protein BFL1 may contribute to chemoresistance and hence represents a potential therapeutic target in CLL, whereas from our data, the BCL2 -938C>A polymorphism does not appear to have any prognostic significance.
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| 5. |
- Nord, Helena, 1980-
(författare)
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Application of Genomic and Expression Arrays for Identification of new Cancer Genes
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Copy number variation (CNV) comprises a recently discovered kind of variation involving deletion and duplication of DNA segments of variable size, ranging from a few hundred basepairs to several million. By altering gene dosage levels or disrupting proximal or distant regulatory elements CNVs create human diversity. They represent also an important factor in human evolution and play a role in many disorders including cancer. Array-based comparative genomic hybridization as well as expression arrays are powerful and suitable methods for determination of copy number variations or gene expression changes in the human genome. In paper I we established a 32K clone-based genomic array, covering 99% of the current assembly of the human genome with high resolution and applied it in the profiling of 71 healthy individuals from three ethnic groups. Novel and previously reported CNVs, involving ~3.5% of the genome, were identified. Interestingly, 87% of the detected CNV regions overlapped with known genes indicating that they probably have phenotypic consequences. In papers II through IV we applied this platform to different tumor types, namely two collections of brain tumors, glioblastoma (paper II) and medulloblastoma (paper III), and a set of bladder carcinoma (paper IV) to identify chromosomal alterations at the level of DNA copy number that could be related to tumor initiation/progression. Tumors of the central nervous system represent a heterogeneous group of both benign and malignant neoplasms that affect both children and adults. Glioblastoma and medulloblastoma are two malignant forms. Glioblastoma often affects adults while the embryonal tumor medulloblastoma is the most common malignant brain tumor among children. The detailed profiling of 78 glioblastomas, allowed us to identify a complex pattern of aberrations including frequent and high copy number amplicons (detected in 79% of samples) as well as a number of homozygously deleted loci. These regions encompassed not only previously reported oncogenes and tumor suppressor genes but also numerous novel genes. In paper III, a subset of 26 medulloblastomas was analyzed using the same genomic array. We observed that alterations involving chromosome 17, especially isochromosome 17q, were the most common genomic aberrations in this tumor type, but copy number alterations involving other chromosomes: 1, 7 and 8 were also frequent. Focal amplifications, on chromosome 1 and 3, not previously described, were also detected. These loci may encompass novel genes involved in medulloblastoma development. In paper IV we examined for the presence of DNA copy number alterations and their effect on gene expression in a subset of 21 well-characterized Ta bladder carcinomas, selected for the presence or absence of recurrences. We identified a number of novel genes as well as a significant association between amplifications and high-grade and recurrent tumors which might be clinically useful. The results derived from these studies increase our understanding of the genetic alterations leading to the development of these tumor forms and point out candidate genes that may be used in future as targets for new diagnostic and therapeutic strategies.
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| 6. |
- Cahill, Nicola, 1983-
(författare)
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Molecular Genetic and DNA Methylation Profiling of Chronic Lymphocytic Leukaemia : A Focus on Divergent Prognostic Subgroups and Subsets
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
- Jiao, Xiang
(författare)
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Somatic Mutations in Breast Cancer Genomes : Discovery and Validation of Breast Cancer Genes
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Breast cancer is the most common cancer in women worldwide. However, the genetic alterations that lead to breast cancer are not fully understood. This thesis aims to identify novel genes of potential mechanistic, diagnostic or therapeutic interest in breast cancers by mutational analysis and whole-genome sequencing.In paper I, sequencing of 36 previously identified candidate genes in 96 breast tumors with patient-matched normal DNA determined the somatic mutation prevalence of these candidate genes and identified additional mutations in Notch, NF-κB, PI3K, and Hedgehog pathways as well as in processes mediating DNA methylation, RNA processing and calcium signaling.In paper II, comparison of massively parallel mate-pair sequencing results of a human genome before and after phi29-mediated multiple displacement amplification (MDA) revealed that MDA introduces structural alteration artifacts, with an emphasis on false positive inversions, and impairs the sensitivity to detect true inversions. Therefore, MDA has limited value in sample preparation for whole-genome sequencing for structural alteration detection.In paper III, massively parallel paired-end sequencing identified gene rearrangements in 15 hormone receptor negative breast cancers. Forty validated rearrangements were predicted to directly affect 30 genes, involved in epigenetic regulation, cell mitosis, signalling transduction and glycolytic flux. RNA interference-based assays revealed the potential roles in cell growth of some affected genes, among which DDX10 was implicated to be involved in apoptosis.In paper IV, a method for statistical evaluation of putative translocations detected by massively parallel paired-end sequencing was proposed. In an application of this method to analyse translocations detected by cancer genome deep paired-end sequencing, 76 putative translocations were classified into four categories, with the majority likely to be caused by mismapping due to repetitive regions.Taken together, this thesis provides insights into genes and pathways mutated in sporadic breast cancer genomes, which broaden our understanding of the genetic basis of breast cancer and may ultimately facilitate the diagnosis and treatment of this disease.
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| 8. |
- Zainuddin, Norafiza, 1978-, et al.
(författare)
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Quantitative evaluation of p16INK4a promoter methylation using pyrosequencing in de novo diffuse large B-cell lymphoma
- 2011
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Ingår i: Leukemia research. - : Elsevier BV. - 0145-2126 .- 1873-5835. ; 35:4, s. 438-443
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Tidskriftsartikel (refereegranskat)abstract
- The p16INK4a tumor suppressor gene can be inactivated by a variety of events including promoter hypermethylation. In diffuse large B-cell lymphoma (DLBCL), p16INK4a methylation has been associated with advanced disease stage and higher IPI. The prognostic impact of p16INK4a methylation in DLBCL remains unclear; however, it has been suggested to correlate with inferior outcome. To further investigate the clinical impact of p16INK4a methylation in DLBCL, promoter methylation of this gene was assessed quantitatively by pyrosequencing. Forty-two of 113 (37%) DLBCL patients with methylation level above 5% were categorized as methylated and subsequently divided into low, intermediate and high methylation categories. Overall, no association was shown between the extent of p16INK4a methylation and patients’ clinical characteristics, except disease stage (P=0.049). Moreover, we could not reveal any impact of p16INK4a methylation on lymphoma-specific survival. Although >25% of p16INK4a methylation correlated with a better progression-free survival (P=0.048), the significance of this finding, if any, needs to be further investigated. In conclusion, our finding questions the role of p16INK4a promoter methylation as a negative prognostic factor in DLBCL.
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| 9. |
- Janunger, Tomas, 1972-
(författare)
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The genetic contribution to stroke in northern Sweden
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Stroke is a common multi factorial cerebrovascular disorder with a large impact on global health. It is a disorder primarily associated with old age but environmental factors, lifestyle choices and medical history are all important for the risk of developing the disorder. It is also known that heritability is important for predisposition to the disorder. The aim of this work has been to identify genetic variations that increase the risk of being affected by stroke in the population of northern Sweden, a population well apt for genetic studies due to well kept church and medical records together with limited genetic diversity. In the first paper we used linkage analysis in families with early onset of stroke. By this approach we identified a region on chromosome 5q to be linked to an increased risk of developing stroke, a region previously identified as a susceptibility locus for stroke in the Icelandic population. In the second study we used genealogy to identify common ancestry and thereby identify common susceptibility to stroke. The seven families we connected showed significant linkage to the chromosome 9q31-33 region and four of the families shared a common haplotype over 2.1 megabases. In the third manuscript we investigated sequence variation of two candidate genes, TNFSF15 and TLR4. Sequencing of the TLR4 gene revealed previously identified variations in affected individuals from two of the families. Further SNP analysis showed five separate haplotypes among the investigated families and four haplotypes for TNFSF15. However none of these co-segregated with stroke among the investigated families. In the final paper we used a case-control stroke cohort to ascertain association for genetic variation in five genes and genetic regions previously suggested to be linked with stroke. Initial analyses showed association for the 9p21 chromosomal region and a variant in Factor 5 that showed protection against stroke, but after adjustments for common risk factors for stroke, the findings were no longer significant. In conclusion, by studying selected families we have been able to show linkage to two chromosomal regions, 5q and 9q31-33, that indicate genetic predisposition for developing stroke. Further we have shown that family based studies are still an important tool in deciphering the underlying mechanisms for complex disease.
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| 10. |
- Lindahl, Katarina, et al.
(författare)
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Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels : a Novel Therapeutic Approach in Osteogenesis Imperfecta
- 2013
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Ingår i: International Journal of Medical Sciences. - : Ivyspring International Publisher. - 1449-1907. ; 10:10, s. 1333-1343
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Tidskriftsartikel (refereegranskat)abstract
- Osteogenesis imperfecta, also known as "brittle bone disease", is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3'UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3'UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.
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