Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.

<p>Innate immunity has received impressive attention in the past decade owing to the discovery of the Toll like receptors (TLRs) and the NOD-like receptors (NLRs). While the TLRs specialize in fighting microbes at the cell surface, the NLRs complement by detecting and responding to intracellular microbes. Recently, the non-microbe sensing NLR called inflammasomes, have been identified, which senses metabolic stress as well as certain pathogenic microbes and elicits host’s inflammatory response. <strong></strong></p><p>The NLR, NALP3 (formerly known as cryopyrin) forms a large cytoplasmic complex called the ‘inflammasome’ when NALP3, activated by a stimuli, associates with the adaptor proteins ASC and CARD-8. This interaction leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the gene encoding NALP3, termed <em>NLRP3</em> can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under cryopyrin associated periodic syndromes (CAPS).</p><p>This thesis describes a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was found to be a heterozygous carrier of two common polymorphisms Q705K in <em>NLRP3 </em>and C10X in the <em>CARD-8</em>. Experimental studies showed elevated levels of caspase-1 and IL-1β in the patient, and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms combined, were found to occur in approximately 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. Therefore, a cohort of rheumatoid arthritis (RA) patients, where elevated IL-1β could be one of the reasons behind chronic inflammation, was investigated. We found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup of patients might benefit from IL-1β blockade. Additional studies are warranted to elucidate the functional effects of the two polymorphisms and to determine whether they identify a subgroup of patients that could benefit from IL-1 targeted therapy. Given the structural similarity of NALP3 to other NALPs, the possibility of involvement of the alternative, homologous genes cannot be eliminated.</p>

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Background: Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with EGFR mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with KRAS mutations do not benefit of such treatment. Methods: This study investigates the frequency of EGFR and KRAS mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes. Results: Five EGFR mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. KRAS mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations. Conclusions: This study confirms previous observations regarding the frequency of EGFR and KRAS mutations in non-small cell lung cancer. Mutations in EGFR and KRAS were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.

Innate immunity has received impressive attention in the past decade owing to the discovery of the Toll like receptors (TLRs) and the NOD-like receptors (NLRs). While the TLRs specialize in fighting microbes at the cell surface, the NLRs complement by detecting and responding to intracellular microbes. Recently, the non-microbe sensing NLR called inflammasomes, have been identified, which senses metabolic stress as well as certain pathogenic microbes and elicits host’s inflammatory response. The NLR, NALP3 (formerly known as cryopyrin) forms a large cytoplasmic complex called the ‘inflammasome’ when NALP3, activated by a stimuli, associates with the adaptor proteins ASC and CARD-8. This interaction leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the gene encoding NALP3, termed NLRP3 can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under cryopyrin associated periodic syndromes (CAPS). This thesis describes a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was found to be a heterozygous carrier of two common polymorphisms Q705K in NLRP3 and C10X in the CARD-8. Experimental studies showed elevated levels of caspase-1 and IL-1β in the patient, and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms combined, were found to occur in approximately 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. Therefore, a cohort of rheumatoid arthritis (RA) patients, where elevated IL-1β could be one of the reasons behind chronic inflammation, was investigated. We found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup of patients might benefit from IL-1β blockade. Additional studies are warranted to elucidate the functional effects of the two polymorphisms and to determine whether they identify a subgroup of patients that could benefit from IL-1 targeted therapy. Given the structural similarity of NALP3 to other NALPs, the possibility of involvement of the alternative, homologous genes cannot be eliminated.

Non-coding antisense RNAs have been implicated in the epigenetic silencing of individual gene as well as chromosomal domains. While silencing of the overlapping gene by antisense RNAs has been well investigated, their functional role in silencing of chromosomal domains remains enigmatic. To elucidate mechanisms underlying the non-coding RNA mediated epigenetic silencing of chromosomal domains, we have chosen an antisense non-coding RNA, Kcnq1ot1, as a model system. Previously, a functional role of Kcnq1ot1 RNA and/or its transcriptional process has been implicated in silencing of multiple genes in the Kcnq1 imprinted cluster. However, these studies could not rule out the mechanisms involving other than Kcnq1ot1 RNA. Furthermore, it was also unclear how the Kcnq1ot1 promoter escapes silencing when its encoded RNA is capable of silencing flanking genes in cis. We have shown that NF-Y transcription factor plays a central role in the Kcnq1ot1 promoter activity, and that mutation of the NF-Y binding sites not only resulted in loss of silencing of flanking genes but also the ability of the Kcnq1ot1 promoter to protect against repressive chromatin marks, indicating that NF-Y maintains transcription-competent chromatin at the promoter through resisting the strong silencing effects of Kcnq1ot1 RNA. The Kcnq1ot1 RNA is an RNA Polymerase II encoded 91 kb long moderately stable nuclear transcript. We have demonstrated that it is the RNA not the act of transcription responsible for silencing and that the degree of silencing was proportional to the length of Kcnq1ot1 RNA. The kinetics of heterochromatin formation in relation to Kcnq1ot1 transcription revealed that overlapping gene was silenced initially by occlusion of basal transcription machinery and heterochromatin formation, whereas nonoverlapping gene was silenced subsequently by Kcnq1ot1-mediated heterochromatin spreading. This transcriptional silencing by Kcnq1ot1 RNA is mediated by an 890 bp region through promoting its interaction with the chromatin. Interestingly, we show that Kcnq1ot1 RNA establishes heterochromatin structures in a lineage-specific fashion by interacting with chromatin and chromatin remodelling complexes such as G9a and PRC2 complexes. More importantly, one of the parental chromosomes comprising Kcnq1 domain always found in the vicinity of perinucleolar region. Based on these data we proposed a mechanism whereby Kcnq1ot1 RNA establishes transcriptional silencing through recruitment of chromatin remodelling machinery and the maintenance of silencing achieved via targeting to the perinucleolar region.

The aim of this thesis has been to identify genetic variants that increase the susceptibility for Systemic Lupus Erythematosus (SLE), an autoimmune disease caused by a complex interplay between various genetic and environmental factors. Five different candidate genes were selected through different strategies, and were analysed for association with SLE in an attempt to distinguish some of the underlying mechanisms of this disease. Two of these genes, PD-L1 and PD-L2, appeared not to contain any major risk factors for SLE in the analysed European and Latin American populations. In two other genes, CD24 and STAT4, there appeared to be population-specific effects. The A57V amino acid substitution in the CD24 gene, previously implicated with multiple sclerosis, was associated in a Spanish cohort, with a weak trend in German samples, and no association in Swedish. The previously reported and highly convincing association of the STAT4 transcription factor gene was confirmed in all our cohorts. Interestingly, the results indicate the presence of at least two independent risk variants: the first, represented by a previously reported SNP, was the strongest in individuals of Northern European ancestry, and the second was more pronounced in individuals from Southern Europe and Latin America. We also report the identification of a novel susceptibility gene. The BANK1 gene, encoding a scaffold protein involved in B-cell activation, contains functional variants affecting important domains, which are associated in all investigated cohorts from Europe and Latin America. These results confirm the existence of replicable associations between genetic variants and SLE, which are common and present in many populations. The results also illustrate a certain degree of heterogeneity, where some risk factors could have variable effect in different populations.

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a defect in red blood cell production. The disease is associated with growth retardation, malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. In a cellular model for DBA, siRNA knock-down of RPS19 results in a relative decrease of other ribosomal (r) proteins belonging to the small subunit (RPS20, RPS21, RPS24) when compared to r-proteins from the large ribosomal subunit (RPL3, RPL9, RPL30, RPL38). RPS19 mutant cells from DBA patients show a similar and coordinated down-regulation of small subunit proteins. The mRNA levels of the small subunit r-proteins remain relatively unchanged. We also show that RPS19 has an extensive number of transcriptional start sites resulting in mRNAs of variable 5’UTR length. The short variants are translated more efficiently. Structural sequence variations in the 5’UTR of RPS19 found in DBA patients show a 20%-30% reduced translational activity when compared to normal transcripts. Primary fibroblast from DBA patients with truncating mutations in RPS19 or RPS24 showed specific cell cycle defects. RPS19 mutant fibroblasts accumulate in the G1 phase whereas the RPS24 mutant cells show a defect in G2/M phase. The G1 phase arrest is associated with a reduced level of phosphorylated retinoblastoma (Rb) protein, cyclin E and cdk2 whereas the G2/M phase defect is associated with increased levels of p21, cyclin E, cdk4 and cdk6. RPS19 interacts with PIM-1 kinase. We investigated the effects of targeted disruptions of both Rps19 and Pim-1 in mice. Double mutant (Rps19+/-, Pim-1-/-) mice have increased peripheral white- and red blood cell counts when compared to the wild-type mice (Rps19+/+, Pim-1+/+). Bone marrow cells in Rps19+/-, Pim-1-/- mice showed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl2, Bcl-xl and Mcl-1 and reduced levels of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. In summary, this thesis clarifies several mechanisms in the pathogenesis of DBA. Mutations in RPS19 results in coordinated down-regulation of several small subunit r-proteins causing haploinsufficiency for the small ribosomal subunit. RPS19 have multiple transcriptional start sites and mutations in the RPS19 5’UTR found in DBA patients result in reduced translational activity. At the cellular level, mutations in RPS19 and RPS24 cause distinct cell cycle defects and reduced cell proliferation. Finally, PIM-1 kinase and RPS19 cooperates in the proliferation of myeloid cells.

Inherited retinal degenerations are a group of disorders characterised by great genetic heterogeneity. Clinically, they can be divided into two large groups of diseases, those associated with night blindness, e.g. retinitis pigmentosa (RP), and those with macular malfunction, e.g. cone/cone-rod dystrophy (COD/CORD). This thesis is focused on finding the genetic basis of disease in families with autosomal dominant COD, autosomal dominant RP, and Bothnia dystrophy (BD), a regional variant of RP.   A variant of COD was previously mapped to 17p12-p13 in a family from northern Sweden. One additional family originating from the same geographical area was included in fine mapping of this chromosome region. Using 12 microsatellite markers in linkage and haplotype analysis, the region was refined from 26.9 to 14.3 cM. A missense mutation, Q626H, in an evolutionarily conserved region of PITPNM3, phosphatidylinositol transfer membrane-associated protein, was identified. The mutation segregated with the disease in both families and was absent from normal control chromosomes. PITPNM3 is a human homologue of the Drosophila retinal degeneration (rdgB) protein, which is highly expressed in the retina and has been proposed to be required for membrane turnover of photoreceptor cells. With the intention of establishing the global impact that PITPNM3 has on retinal degenerations 165 DNA samples from COD and CORD patients were obtained from Denmark, Germany, the UK, and USA and screened for mutations. The Q626H mutation found in the Swedish families was also found in one British family and a novel Q342P variant was detected in a German patient. In addition, two intronic variants were identified: c.900+60C&gt;T and c.901-45G&gt;A. Thus, we concluded that mutations in PITPNM3 represent a rare cause of COD worldwide. In two large families from northern Sweden showing autosomal dominant RP with reduced penetrance, the disease locus was mapped using genome-wide linkage analysis to 19q13.42 (RP11). Since mutation screening of eight genes on 19q13.42 revealed no mutations, multiplex ligation-dependent probe amplification (MLPA) was used to screen for large genomic abnormalities in PRPF31, RHO, RP1, RPE65, and IMPDH1. A large deletion spanning 11 exons of PRPF31 and three genes upstream was identified. Using long-range PCR, the breakpoints of the deletion were identified and the size of the deletion was determined to encompass almost 59 kb. BD is an autosomal recessive type of RP with high prevalence in northern Sweden. The disease is associated with a c.700C&gt;T mutation in RLBP1. In a screening of recessive RP in northern Sweden, 67 patients were found to be homozygous for c.700C&gt;T and 10 patients were heterozygous. An evaluation with arrayed primer extension (APEX) technology revealed a second mutation, c.677T&gt;A, in RLBP1 giving rise to compound heterozygosity in these patients. In addition, a c.40C&gt;T exchange in CAIV was detected in a patient with BD and in 143 healthy blood donors. The c.40C&gt;T substitution in CAIV has been reported to cause autosomal dominant RP in South African families with European ancestry. However, in the population of northern Sweden it appears to be a benign polymorphism. In summary, a first mutation in PITPNM3, encoding a human homologue of the Drosophila retinal degeneration protein, was detected in two large families with COD. A large deletion in PRPF31 was discovered in two families with autosomal dominant RP showing reduced penetrance and in 10 patients BD was shown to be caused by two allelic mutations in RLBP1.