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Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type
Neupauerová, Jana ; Laššuthová, Petra (advisor) ; Vlčková, Eva (referee) ; Vasovčák, Peter (referee)
Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type For my PhD thesis I chose to work with patients with axonal form of CMT, because at that time axonal forms were less likely to be clarified by classical methods of molecular genetics. For further examination in patients with unclear cause of the axonal CMT, the genes DNM2, GARS and TRPV4 were selected. The aim was to determine the significance of pathogenic mutations in these genes as the cause of CMT2 in Czech patients. In the course, we identified causal variants in the genes MORC2 and SOD1 with WES. Therefore, we have tested additional CMT2 patients for the presence of these variants. Using Sanger sequencing, I examined a representative set of patients for the DNM2 (37), GARS (10) and TRPV4 (24) genes without finding a causal mutation, then we investigated genes SOD1 (43 patients) and MORC2 (161 patients). The cohort (50 patients) was also subjected to MLPA analysis using a P406-A1 CMT2 duplication and deletion detection kit for genes RAB7A, GARS, HSPB1, HSBP8 and SPTLC1 (kit P406-A1 CMT2). At that time, massively parallel sequencing (MPS) was becoming important. We compared the cost of classical sequencing versus MPS, and accordingly, we decided that the genes DNM2, GARS, MORC2, TRPV4...
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The analysis of immunoglobulin and T-cell receptor gene rearrangements using next generation sequencing
Hašek, Daniel ; Froňková, Eva (advisor) ; Javorková, Eliška (referee)
DNA sequencing is a molecular genetic method that results in data about sequence and type of nucleotides present in a given sample of deoxyribonucleic acid (DNA), a molecular carrier of genetic information. These data are frequently of a crucial value for many fields; research, medicine, industry, criminalistics or others. During a long period of time almost all the sequencing was performed using a method invented by Frederick Sanger in the 70's, a technique that uses modified nucleotides that once incorporated into a DNA strand prevent this from further elongation. DNA synthesis in presence of such nucleotides leads to a formation of a mixture of fragments of different lenght that are electrophoretically separated by lenght and the sequence is read from the resulting gel. Since the principle of this method entails some inherent drawbacks (e.g. low throughput and coverage) a significant effort is made lately to develop alternative sequencing approaches. These methods colectively refered to as next-generation sequencing (NGS) use several technologies in order to overcome the limitations of the Sanger sequencing. This thesis discusses the most important NGS methods and focuses on their possible application for sequencing of immunoglobulin and T-cell receptor gene rearrangements, an area of undisputable...
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Construction and quality assessment of the genome assemblies
Korená, Lucie ; Leontovyč, Roman (advisor) ; Vorel, Jiří (referee)
Detailed information of the genome of the studied organism is crucial for many fields of modern research. Actual sequencing technologies are not able to read the whole DNA molecule at once therefore only fragments of the genetic information are obtained, which are not sufficiently informative on their own. The goal of the genomic-bioinformatic approach is to assemble these fragments into complete original information - genome assembly. The process of the genome assembly is demanding in terms of computational power, software equipment and expert staff. Many assemblers - programs for genome assembly are available differing in performance, size of the analyzed genome or target organism. The quality of final assembly is fully dependent on assembler and setting of inner parameters. In practice, multiple assemblies are constructed and their quality evaluated according to the technical and biological parameters. The presented thesis describes current high throughput sequencing technologies, different approaches and algorithms for genome assembly and methodology for their quality assessment. The practical part is focused on assembly and its quality assessment using Illumina data of the bird fluke Trichobilharzia szidati.
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Gene expression of mutant alleles and X inactivation pattern in patients with selected X-linked disorders
Černá, Alena ; Dvořáková, Lenka (advisor) ; Sedláček, Zdeněk (referee)
In comparison to men, the number of X-linked genes is doubled in women as they have two chromosomes X while men are hemizygotes for X-linked genes. This imbalance is compensated by X inactivation (XCI) process, also known as primary X-inactivation, occurring in the early stage of embryogenesis. X inactivation is a random process and females are mosaics of two cell populations. The ratio of expressed alleles in women can be random (50:50) or skewed (≥80:20). The skewed X inactivation may occur due to selection when one of the alleles is preferentially inactivated (secondary X inactivation). In this study XCI status in heterozygous females with various severity of phenotypic symptoms and traits in selected X linked inherited metabolic diseases is analysed, with the focus being Fabry disease - the deficiency of the enzyme alpha-galactosidase A encoded by GLA gene. Moreover, XCI in one family with X linked agammaglobulinemia is examined. Mutant alleles and XCI status based on various loci, different methodical approaches and different tissues is subjected to examination. For the first time, the direct analysis of GLA gene transcript to detect the allele ratio was used alongside with the single-nucleotide polymorphisms in the IDS and LAMP genes for allele-specific expression (ASE) and the AR, RP2 and...
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Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type
Neupauerová, Jana ; Laššuthová, Petra (advisor) ; Vlčková, Eva (referee) ; Vasovčák, Peter (referee)
Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type For my PhD thesis I chose to work with patients with axonal form of CMT, because at that time axonal forms were less likely to be clarified by classical methods of molecular genetics. For further examination in patients with unclear cause of the axonal CMT, the genes DNM2, GARS and TRPV4 were selected. The aim was to determine the significance of pathogenic mutations in these genes as the cause of CMT2 in Czech patients. In the course, we identified causal variants in the genes MORC2 and SOD1 with WES. Therefore, we have tested additional CMT2 patients for the presence of these variants. Using Sanger sequencing, I examined a representative set of patients for the DNM2 (37), GARS (10) and TRPV4 (24) genes without finding a causal mutation, then we investigated genes SOD1 (43 patients) and MORC2 (161 patients). The cohort (50 patients) was also subjected to MLPA analysis using a P406-A1 CMT2 duplication and deletion detection kit for genes RAB7A, GARS, HSPB1, HSBP8 and SPTLC1 (kit P406-A1 CMT2). At that time, massively parallel sequencing (MPS) was becoming important. We compared the cost of classical sequencing versus MPS, and accordingly, we decided that the genes DNM2, GARS, MORC2, TRPV4...
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The analysis of immunoglobulin and T-cell receptor gene rearrangements using next generation sequencing
Hašek, Daniel ; Froňková, Eva (advisor) ; Javorková, Eliška (referee)
DNA sequencing is a molecular genetic method that results in data about sequence and type of nucleotides present in a given sample of deoxyribonucleic acid (DNA), a molecular carrier of genetic information. These data are frequently of a crucial value for many fields; research, medicine, industry, criminalistics or others. During a long period of time almost all the sequencing was performed using a method invented by Frederick Sanger in the 70's, a technique that uses modified nucleotides that once incorporated into a DNA strand prevent this from further elongation. DNA synthesis in presence of such nucleotides leads to a formation of a mixture of fragments of different lenght that are electrophoretically separated by lenght and the sequence is read from the resulting gel. Since the principle of this method entails some inherent drawbacks (e.g. low throughput and coverage) a significant effort is made lately to develop alternative sequencing approaches. These methods colectively refered to as next-generation sequencing (NGS) use several technologies in order to overcome the limitations of the Sanger sequencing. This thesis discusses the most important NGS methods and focuses on their possible application for sequencing of immunoglobulin and T-cell receptor gene rearrangements, an area of undisputable...
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Molecular characterisation of novel subtype of Acute lymphoblastic leukemia with lineage switch during early phase of treatment
Dobiášová, Alena ; Fišer, Karel (advisor) ; Novotný, Marian (referee)
Leukemia is the most common malignant disease in children patients. In our laboratory (CLIP) a novel subtype of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) with lineage switch during early phase of treatment towards myeloid lineage (swALL) was recently documented. SwALL incidence is almost 4 % of all BCP-ALLs (Slámová et al., 2014). DNA methylation (presence of 5-methylcytosine) is together with post-translational histone modifications and non- coding RNAs an epigenetic mechanism which regulates gene expression without changes of genetic code. DNA methylation is easily detected by bisulphite conversion and subsequent sequencing. The aim of this work was to compare genome-wide DNA methylation patterns between patients with swALL and control BCP-ALLs. The first step in achieving that was revision and improvement of bioinformatic processing protocol for eRRBS data from massive parallel sequencing. To improve the sequence adapter trimming I tested four bioinformatic tools - FAR, cutadapt, Trimmomatic and fastx_clipper. I implemented the fastest and most effective - Trimmomatic into the processing protocol. As a next step I analysed the data with improved protocol and extended the analysis in R programming environment where the comparison of studied groups was performed. The comparison of...
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