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Characterization of germline alterations affecting genes influencing development and prognosis of specific adult cancers
Jelínková, Sandra ; Kleibl, Zdeněk (advisor) ; Hlaváč, Viktor (referee) ; Hájková, Nikola (referee)
In my dissertation, I studied the genetic predisposition of selected types of cancer that have not been systematically studied in the Czech Republic. We used next-generation panel sequencing to identify germline pathogenic variants. Analysis of 1333 patients with ovarian cancer, 527 patients with endometrial cancer, and 334 patients with hepatocellular carcinoma included sequencing using the CZECANCA panel. A specific CZMELAC panel was prepared for the analysis of 264 melanoma patients. We focused on the identification of pathogenic variants in known predisposition genes. We also evaluated candidate genes and phenotypic characteristics in carriers of pathogenic variants. Analysis of high-risk melanoma patients revealed pathogenic variants in melanoma associated genes in 9/264 (3.4%) patients, and an additional 22 (8.3%) patients carried a pathogenic variant in one of the other predisposition genes. The odds of carrying a pathogenic variant were increased in probands with multiple melanomas and in the presence of melanoma in relatives. The incidence of germline pathogenic variants was highest in ovarian cancer, where pathogenic variants were found in 427/1332 (32.0%) patients, with a predominance of mutations in BRCA1, BRCA2, followed by alterations in other ovarian predisposition genes. Breast and...
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Advanced Computational Methods for CNV Detection in Bacterial Genomes
Jugas, Robin ; Bystrý, Vojtěch (referee) ; Šafránek,, David (referee) ; Vítková, Helena (advisor)
Hlavní pozornost v oblasti strukturálních variací je zaměřena na lidské genomy. Detekce změny variace počtu kopií (CNV) u bakterií je tedy méně rozvinutou oblastí. Běžně používané metody detekce CNV neberou v úvahu specifika bakteriálních kruhových genomů a obecně existuje prostor pro zlepšení metrik výkonnosti. Tato práce představuje metodu detekce CNV nazvanou CNproScan zaměřenou na bakteriální genomy. CNproScan implementuje hybridní přístup kombinující signály hloubky čtení a párů čtení. Bere v potaz všechny vlastnosti bakterií a využívá pouze sekvenační data. Na základě výsledků ze srovnání dosáhl CNproScan velmi dobrých výsledků v různých podmínkách. Pomocí informací z párových čtení jsou CNV klasifikovány do několika kategorií. Ve srovnání s jinými metodami může CNproScan také detekovat mnohem kratší CNV. Vzhledem k nutnosti slučovat nejen signály různých přístupů, ale také výsledky různých algoritmů, dizertační práce také představuje pipelinu nazvanou ProcaryaSV vyvinutou k detekci CNV s využitim pěti nástrojů a slučování jejich výsledků. ProcaryaSV se stará o celý postup od kontroly kvality čtení, ořezávání konců čtení, zarovnání čtení až k detekci CNV.
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Differential Gene expression using a negative binomial model
Janáková, Tereza ; Tkacz, Ewaryst (referee) ; Abo Khayal, Layal (advisor)
Hlavním cílem této diplomové práce je analýza diferenciální exprese genů na základě negativního binomického modelu. Úvodní část je věnována teoretickému základu, pojednává o sekvenování RNA, sekvenování nové generace, výhodách a možném využití, formátu fastQ aj. Následující část už se zabývá samotnou praktickou částí, zde byl vybrán vhodný set genů, které budou později analyzovány a příslušná data byla stažena. Tato data byla zarovnána k lidskému genomu verze 37 Burrowsovou-Wheelerovou transformací s využitím bowtie mapovače, byly tak vytvořeny soubory ve formátu SAM. Toto soubory dat byly později setříděny pomocí nástroje SAMtools. Následně byly v programovém prostředí Matlab (verze R2013b) vytvořeny anotované objekty genů s využitím služby Ensembl´s BioMart. Dále byla určena genová exprese a byly odhadnuty faktory velikosti knihovny. Na závěr byly odhadnuty parametry negativního binomického rozložení a byla vyhodnocena diferenciální exprese genů.
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Developmental disorders of the lens and anterior segment of the eye
Jedličková, Jana ; Ďuďáková, Ľubica (advisor) ; Šafka Brožková, Dana (referee) ; Cvekl, Ales (referee)
The anterior segment of the eye includes the eyelids, eyelashes, tear film, cornea, conjunctiva, iris, pupil, ciliary body, chamber angle, and lens. Cataracts and anterior segment dysgenesis are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance. The aim of this thesis was molecular genetic analysis of 51 patients from 23 families with congenital and early onset cataracts and/or anterior segment dysgenesis, using a modern as well as conventional methods, such as next-generation sequencing, direct sequencing, bioinformatic and functional analyses including the use of artificial splicing vectors. We identified 24 causal variants in the coding, non-coding and regulatory regions of the PAX6, FTL, FOXC1, PITX2, FOXE3, and CHRDL1 genes. We confirmed the role of the n.37C>T variant in MIR204 in the development of chorioretinal dystrophy variably associated with iris coloboma, early-onset cataracts and congenital glaucoma. Splicing assay confirmed pathogenic effect of two mutations on pre-mRNA splicing of the PAX6 gene. Establishing molecular genetics diagnosis improves patient counselling and their relatives in terms of the prognosis and risk of developing secondary glaucoma Key words: molecular-genetic diagnosis, anterior segment dysgenesis, next-generation sequencing,...
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Molecular genetic analysis of rare ocular disorders in Roma population from the Czech Republic
Rysková, Natálie ; Lišková, Petra (advisor) ; Klímová, Aneta (referee)
The Roma are the largest and most widespread transnational ethnic community. The Czech Republic estimates their 2% representation in its population. Due to the high level of endogamy, the spectrum and frequency of hereditary diseases in the Roma population differ from the majority population. Hereditary eye diseases are one of the most common causes of blindness in younger adults and thus represent a real socio-economic burden. The aim of the thesis was to perform molecular genetic analysis in individuals of Roma origin suffering from hereditary diseases affecting vision, including dual impairments and syndromes, and to determinate the frequency of the detected pathogenic variants in this population. Molecular genetic analysis of 17 families was performed using direct and whole exome sequencing. Within the framework of an international collaboration, the degree of their mutual kinship was calculated using the PLINK program. The frequency of selected variants was determined in a control dataset comprising 156 Roma exomes and genomes. The spectrum of analyzed diseases included various retinal dystrophies, primary congenital glaucoma, Usher syndrome, neuronal ceroid lipofuscinosis, Noonan syndrome, nanophthalmos and congenital cataract, facial dysmorphism and neuropathy. The results of the thesis...
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Sequence variants in metastatic colorectal cancer and their evolution during the disease course and treatment
Jandová, Eliška ; Boublíková, Ludmila (advisor) ; Vymetálková, Veronika (referee)
A deeper understanding of the molecular background of colorectal cancer (CRC) can help explain the development of the disease and its resistance to treatment, predict disease progression, and improve treatment prognosis. Some minimal molecular testing has been incorporated into standard clinical management to determine if a particular patient will benefit from a particular therapy, but more and more new genetic alterations are being discovered that appear to be associated with the development of resistance. Tissue biopsy of the tumor is the gold standard in terms of molecular testing, but there is an increasing demand for more non- invasive methods such as liquid biopsy. Using targeted next-generation sequencing, we analyzed sequence variants present in primary tumor, metastases, and cell-free tumor DNA - ctDNA of patients with metastatic CRC. The objectives were to analyze sequence variants of the primary tumor and identify possible pathogenic variants, to analyze differences between DNA of the primary tumor and metastases, to evaluate the use of ctDNA as a diagnostic tool, and to identify potential tumor-specific markers in ctDNA that can be used to monitor disease progression. Our results suggest the feasibility of using ctDNA for diagnostic purposes or even to monitor disease progression, but...
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Identification of hereditary factors influencing the formation of a pancreatic carcinoma and other solid tumours
Král, Jan ; Janatová, Markéta (advisor) ; Hojný, Jan (referee) ; Šeborová, Karolína (referee)
Pancreatic adenocarcinoma (PDAC) has one of the worst prognoses out of all cancers worldwide. Endometrial carcinoma (EC) is the most common gynecological cancer. Genetic background of tumors is highly heterogenous and differs among populations. We have analyzed DNA of 226 PDAC patients and 527 EC patients using panel next- generation sequencing. Targeted genes were divided into main predisposition genes (11 for PDAC, 19 for EC) and other candidate genes. EC patients were categorized based on meeting the indication criteria for germline genetic testing. Two sets of population-matched controls were used (controls with negative cancer history, and general population controls). Germline pathogenic variants (PV) in main predisposition genes were identified in 18 (8.0%) PDAC patients. The most mutated gene was BRCA2 (50% of carriers). PDAC risk was significantly elevated in carriers of PV in BRCA1 (OR = 10.4, p = 0.04), BRCA2 (OR = 6.4, p = 0.0009), and CHEK2 (OR = 17.5, p = 0.003). Germline mutations in genes participating in homologous recombination processes were associated with improved overall survival of patients. Among EC patients there were 60 (11.4%) carriers of PV in main predisposition genes. Carriers of PV in Lynch syndrome (LS) genes had markedly elevated risk of developing EC (OR = 22.4, p...
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Pathogenetic mechanisms of immune dysregulation and hematopoietic disorders
Svatoň, Michael ; Froňková, Eva (advisor) ; Filipp, Dominik (referee) ; Čermák, Jaroslav (referee)
Pathogenetic mechanisms of immune dysregulation and hematopoietic disorders Abstract The aim of my work was to introduce whole exome sequencing (WES) and a routine algorithm for data analysis in pediatric patients with suspected inborn error of immunity (IEI) or hematopoiesis. A clear molecular diagnosis based on genetic testingwas achieved in 25% of patients. Additionally, novel mutations not previously described in the pathogenesis of IEI or hematopoietic failure were revealed in 9% of the patients. Validation experiments were designed to confirm the causality of the identified mutations in the pathogenesis of the disease, based on literature review and known mechanisms of immune system development and regulation in these patients. Thanks to WES, we were able to elucidate the cause of two new congenital diseases - megaloblastic anemia due to a homozygous mutation of the SLC19A1 gene and immune dysregulation syndrome with autoimmune and autoinflammatory manifestations due to an X-linked mutation of the TLR8 gene in monozygotic twins with chronic autoimmune hemolytic anemia (AIHA). In five additional patients, novel mutations in the STAT3, SPTA1, SAMD9 and XIAP genes were identified and their causality in the pathogenesis of hematopoietic failure or IEI was demonstrated. In one patient with an atypical...
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Molecular genetic approaches used in preimplantation genetic analysis of human embryos in assisted reproduction
Limbergová, Jana ; Machoň, Ondřej (advisor) ; Šimoník, Ondřej (referee)
Preimplantation genetic testing is one of the main clinical procedures that take place in assisted reproduction centres around the world. It is sought after by couples for many different reasons. The causes of problems may relate to hereditary diseases that potential parents do not wish to pass on to their offspring, or may include the risk of transmission of chromosomal alterations. In addition, infertility may be caused by an increased risk of embryo aneuploidy, which correlates with mother's increased age and other factors that this bachelor thesis discusses. The bachelor thesis also focuses on up-to-date approaches to cytogenetic examinations for reproductive genetics and discusses recent clinical molecular methods that are used to improve the diagnosis and therapy of infertile couples. These are, for example, microarray methods, quantitative real-time PCR or next-generation sequencing and methods of whole-genome amplification. This work also summarizes methods that are gradually being abandoned, e.g. fluorescent in situ hybridization, and compares their advantages and disadvantages. Key words: preimplantation genetic testing, aneuploidy, structural rearrangements, monogenic diseases, biopsy, fluorescent in situ hybridization, microarray methods, comparative genomic hybridization, quantitative...
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