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Analysis of pre-mRNA alternative splicing products and their importance in breast cancer oncogenesis.
Hojný, Jan ; Kleiblová, Petra (advisor) ; Malík, Radek (referee) ; Boušková, Veronika (referee)
Breast cancer is the most common tumor disease diagnosed in women worldwide. The hereditary character of this disease is observed in 5-10 % of all cases, and it is usually caused by a pathogenic mutation in one of the predisposition genes. Although a variety of pathogenic mutations in the coding sequences of these genes was described, the cause of the disease is still unknown in many familial cases (> 50%). A great number of identified pathogenic mutations were localized in the consensus splicing sites, which results in the formation of aberrant mRNA splicing variants and their damaged protein isoforms. However, little is known about mutations affecting regulatory splicing sites, which can result in the translation of similarly affected mRNAs. In this work, we proposed a method for indirect detection of mutations affecting the natural splicing pattern of any gene of our interest based on multiplex PCR and NGS with high sensitivity. Verification of this method on the BRCA1 model gene revealed the presence of the total of 94 splicing variants in peripheral leucocytes and healthy breast and adjacent fat tissues. This is the most detailed catalogue of physically occurring BRCA1 mRNA variants thus far. The most commonly occurring variants, maintaining open reading frame, were quantified by RT-qPCR which...
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Analysis of genetic factors of breast cancer
Chmelařová, Žaneta ; Kleiblová, Petra (advisor) ; Brynychová, Veronika (referee)
The thesis The analysis of genetic factors of breast cancer by NGS deals with the current serious problematics of breast cancer from the perspective of genetic predisposition. Breast cancer is one of the most common tumors in women. Every year more than 7000 women are diagnosed with this disease and the mortality rate in the Czech Republic is nearly 2000 cases. Of the total number of patients diagnosed with breast cancer, approximately ten percent of patients have congenital mutations in one of the predisposing genes that cause a significantly increased risk of developing a cancer. More than half of these mutations occur in germline mutations of the BRCA1 or BRCA2 genes, others include a number of other genes, eg tp53, CDH1, PTEN, STK11, ATM, PALB2, CHEK2.Early diagnosis and identification of persons with increased risk of developing breast cancer is of key importance for their inclusion in preventive programs. Therefore, the thesis aims to testing genes that can cause a breast cancer. In the thesis, 219 known and candidate predisposition genes were analyzed in a group of 263 non-selected breast cancer patients using a targeted panel NGS, the Illumina platform. Selected identified suspect variants were further confirmed by Sanger sequencing. The aim of this work was also a mutational analysis of...
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Principles of the next gen sequencing and its application for studies of human malignant diseases
Chmelařová, Žaneta ; Kleiblová, Petra (advisor) ; Koudelková, Lenka (referee)
Modern technologies, including the next-gen sequencing (NGS), increase the speed for genetic data acquisition in analyses of the genetic susceptibility to various human pathologies. This, for example, entirely changes the field of tumor biology research and treatment because malignant tumor could be also considered as a genetic disease. This work aims to describe development of the DNA sequencing techniques with a focus on NGS. We summarize the basic scientific achievements stimulating the NGS development and its chemical, physical, and biological principles. Further, we overview the main steps in sequencing data acquisition and analysis, and principles used by the software tools that are required for the prediction of functional importance in identified genetic variants. The importance of NGS for current oncogenetics is illustrated using selected examples, including characterization of somatic genetic changes in sporadic tumors and identification of germinal pathogenic variants in case of hereditary cancer syndromes, that demonstrate the rapid implementation of NGS technologies into the clinical practice. Key words: DNA, PCR, next-gen sequencing, 3rd generation sequencing, gene mapping, gene annotation, hereditary tumors, cancer
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název v anglickém jazyce není uveden
Kleiblová, Petra ; Calda, Pavel (advisor) ; Dostálová, Ivana (referee) ; Čechák, Petr (referee)
23 7 SUMMARY This work deals with the role of adipose tissue-derived hormones - adipokines in the development of insulin resistance in pregnant women with gestational diabetes mellitus (GDM). The study consists of two parts. In the first one, the characterization of changes in concentrations of circulating adiponectin - the most abundant adipokine - during menstrual cycle in healthy women was performed. The second part was focused on the changes of gene expression in pregnant women with GDM relative to healthy pregnant controls (14 vs. 13 subjects). Specificial attention was focused on adipokines and their receptors (ADIPOQ, ADIPOR1, ADIPOR2, LEP, RETN, TNF , CFD a PAI1) as well as other genes potentially contributing to the etiopathogenesis of GDM in the samples of placenta, subcutaneous, and visceral adipose tissues obtained during surgical delivery. The changes of gene expression were assessed by the two approaches: Firstly, the gene expression of insulin signaling pathway regulators was characterized using expression arrays in a limited subset of samples from GDM and control subjects. Secondly, the expression of genes that differed most significantly in GDM vs. control group in arrays, together with other genes of involved in the regulation of insulin sensitivity, were analyzed using qPCR in the entire...
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