Global ETD Search

1	Untersuchung des Gens PMS2 und des Pseudogens PMS2CL bei Patienten mit HNPCC Andres, Friederike 04 November 2022 (has links) Background: The hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) is the most frequent hereditary colorectal cancer syndrome. It has been associated with different types of cancers besides the early onset of the disease. To define HNPCC the Amsterdam or Bethesda criteria have to be met. Causative for the Lynch syndrome is a germline mutation of one of the four mismatch repair genes MLH1, MSH2, MSH6 and PMS2. The detection of pathogenic alterations is of vital importance for the patient as well as for their relatives. The analyses for the PMS2 gene have been severely complicated by the presence of multiple pseudogenes with high homologies to the gene especially in its 5 ́-region. The PMS2CL pseudogene has a high homology to the 3 ́-region of PMS2, in particular the exons 9 and 11- 15, which result in recombination events between the paralogues. Objectives: In this study, we established the newly developed technique of long-range-PCR for mutation detection within the PMS2 gene. Using this method we could clearly discriminate gene from pseudogene which makes it a significant advancement in the detection of pathogenic mutations in PMS2 compared to previously used methods. Until recently mutation detections had been performed with a method, which is now obsolete. It has been the aim of this study to implement the new method of long-range-PCR in the daily diagnostic routine. However, within the study we examined only exons 11-15 of PMS2 equal to the long-range-PCR product 3, because this region contains the highly homologous region to the PMS2CL pseudogene and is known to undergo recombination. Furthermore, it was the aim of this study to make a statement about occuring recombination events and their nature, frequency and consequences. The sequence of the pseudogene has been amplified as well. Methods: Patients were selected based on an isolated loss for PMS2 in the immunohisto- chemistry of their tumor. The long-range-PCR was performed on DNA from blood leukocytes. The amplicons of both the gene and pseudogene were confirmed on gel electrophoresis and used as template for ensuing exon-specific nested-PCR prior to sequencing. Multiplex ligation-depend amplification (MLPA) was performed to screen for deleterious alterations. Results: The study comprised 23 patients. We identified pathogenic mutations in 14 patients (61 %). Four of these mutations are located in the 5 ́-region (upstream exon 8), ten mutations were found in the 3 ́-region (downstream exon 9). Those mutations were nearly evenly distrib- uted over the exons 11-14, whereas two mutations in exon 14 were large deletions. There was no mutation found in exon 15. Moreover we identified one putative pathogenic mutation in exon 12. The second part of this study aimed at the analysis of recombination events between PMS2 and PMS2CL. We identified 27 paralogous sequence variants, which were classified by un- derlying recombination patterns. We found patients without any recombination as well as re- combination events encompassing the exons 13-15. In 14 of 23 patients (61 %) a recombination event could be confirmed occurring primarily in the exons 13-15. The overall number of recombination events are benign and consequences for cancer development are not clear, except one patient who presented with a malignant tumor and carried a sequence recombina- tion in exon 11, which had not been found in former studies. Conclusion: From now on the long-range-PCR should be gold standard for detection of path- ogene alterations in the PMS2 gene. The mechanism and the verification of recombination events encompassing the whole gene PMS2 should be point of interest in further studies. info:eu-repo/classification/ddc/610 ddc:610
2	Variación haploide en secuencias nucleares humanas: el pseudogén GBA Martínez Arias, Rosa 12 March 2001 (has links) Hemos analizado la variabilidad genética de una zona no codificante autosómica, el pseudogén homólogo al gen de la glucocerebrosidasa (psGBA). Parte del análisis se ha realizado desde la perspectiva de la genética de poblaciones humanas. Desde un punto de vista más genómico hemos establecido la dinámica de la región, a fin de entender las causas del espectro de variación. Hemos analizado el papel de la mutación, recombinación, conversión génica y, especialmente, selección. Por otra parte, psGBA es importante en la producción de alelos complejos GBA-psGBA, que provocan los tipos más severos de la enfermedad de Gaucher. Mostramos cómo el conocimiento de la variabilidad en psGBA ayuda al reconocimiento de estos alelos complejos. Finalmente, con los datos de variabilidad de dos regiones parálogas situadas en la misma región cromosómica (gen GBA / pseudogen psGBA) hemos comparado los patrones de mutación que presenta una misma secuencia bajo diferentes presiones selectivas. / We have analyzed the genetic variability in a non-coding autosomal region, the pseudogene homologous to the glucocerebrosidase gene (psGBA).Part of the analysis has been performed from the human populations point of view.From a more genomic perspective, we have established the region dynamics in order to understand the causes of the variability pattern. We have analyzed the role of mutation, recombination, gene conversion and, especially, selection.On the other hand, psGBA is important in the production of complex alleles GBA-psGBA, that lead to the most severe types of Gaucher disease. We show how the knowledge of psGBA variability helps to the identification of those complex alleles.Last, from the variability data from two paralogous regions located in the same chromosomal region (GBA gene /psGBA pseudogene) we have compared the mutation patterns shown by the same sequence under different selective pressures. selection mutation pattern autosomal haplotypes hitchhiking complex alleles barrido selectivo espectro de mutacion seleccion conversion genica alelos complejos gene conversion peudogene haplotipos autosomicos Pseudogen 575
3	Vyšetření rekombinací mezi genem a pseudogenem pro β-glukocerebrosidasu vedoucích ke vzniku patogenních alel / Detection of β-glucocerebrosidase gene/pseudogene recombination events leading to pathogenic alleles Peková, Barbora January 2017 (has links) This diploma thesis provides an overview of gene conversion, its role in the pathogenesis of human diseases and the use of methods based on next-generation sequencing (NGS) for detection rare variants of DNA sequence. Labeling of target DNA molecules by random nucleotides in primer and NGS were used for detection point mutations arising de novo in the β-glucocerebrosidase gene by gene conversion between it and its pseudogene in meiotic and mitotic cells of control subjects. Primers specific for the active gene were used to selectively amplify the ninth and tenth exon of the gene where "recombinant" variants occur most frequently. Sequences generated from 20 genomic DNA samples on Illumina MiSeq platform were quality filtered, sorted by unique labels and consensus sequences were created from alignments of sequences carrying the same DNA tag. The number of potential point mutations in the samples ranged between 12 and 48. The mutations were manually re-evaluated from the alignments. The number of alignments with unique labeling was in the range of 7-15 thousand per sample. Only three samples carried possible recombinant mutations, suggesting a lower frequency of conversion in the region than reported by other techniques. Analysis of unique sequences in primer indicated possible ways to improve the...
4	Stabil och antibiotikafri läkemedelsproduktion i rekombinant Escherichia coli Benevides, Kristina, Broström, Oscar, Elison Kalman, Grim, Swenson, Hugo, Vlassov, Andrei, Ågren, Josefin January 2017 (has links) Den här rapporten presenterar ett antibiotikafritt, stabilt och kromosombaserat expressionssystem för läkemedelsproduktion i Escherichia coli på beställning av företaget Affibody AB. E. coli-stammen BL21(DE3) valdes som värdorganism för expressionssystemet. Systemet består av en genkassett som innehåller en T7-promotor, en 5′-UTR från genen ompA och en terminatorsekvens från RNA-operonet rrnB. Fyra kopior av genkassetten ska integreras i pseudogenerna caiB, yjjM, hsdS och yjiV. En datormodell som modellerar det egentliga kopietalet i cellerna har skapats i mjukvaran MATLAB, vilket visar att det uppskattas vara maximalt 32 kopior av genkassetten per cell på grund av replikation av kromosomen. Ett högt pH i fermentorn; att använda fed-batch och blandade kolhydratkällor; och att använda stammen BL21(DE3) minskar acetatproduktionen i cellen. En lägre acetatproduktion kan leda till en högre produkthalt. En proteinutbytesmodell för mjukvaran MATLAB har konstruerats för att uppskatta koncentrationen av Affibody®-molekylen i en E. coli cell. copynumber copy-number copy number origin of replication rekombinant proteinexpression läkemedel E. coli antibiotikafri antibiotika expressionssystem expression system escherichia coli kromosom kromosombaserad kromosomal plasmidfri plasmid T7-promotor T7 promotor ompA rrnB autoinduktion auto induktion matlab kopietalmodell utbytemodell stabil pH pseudogen pseudo gen caiB yjjM hsdS yjiV fermentor batch fed-batch kontinuerlig fed batch fermentation Affibody affibody-molekyl affibody-molekyler affibodymolekyl affibodymolekyler bioreaktor acetat acetatbildning acetatproduktion acetatreduktion acetatreducering peptidläkemedel peptider peptid T7-system operator lac-operator operon 5'-UTR 3'-UTR sekundärstruktur mRNA T1 T2 terminator termineringssekvens optimering lac-operon repressor inducering BL21(DE3) BL21 B-stam B stam modell kopietal kopie tal OriC Pharmaceutical Biotechnology Läkemedelsbioteknik

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