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Recombination between the gene and pseudogene for glucocerebrosidase as a mechanism of mutation generation in Gaucher disease
Peková, Barbora ; Hřebíček, Martin (advisor) ; Schierová, Michaela (referee)
Gaucher disease is an autosomal recessive disorder caused by the deficiency of β-glucocerebrosidase. Some Gaucher patients carry in their β-glucocerebrosidase genes complex mutations which apparently arose by a recombination with the non-functional β-glucocerebrosidase pseudogene. Recombination between genes and their corresponding pseudogenes plays a role in the development of other hereditary human diseases. Mutant alleles formed in male and female meiosis are a source of these variations in the gene pool. The study of frequency and scope of recombination events in human disease-associated genes in the gametes is of importance for evaluation of the disease burden in the population. The evaluation of the scope of single recombination events in the β-glucocerebrosidase gene in human gametes is technically challenging. Novel technologies such as next-generation sequencing, nanopore sequencing or droplet digital PCR may have advantages over previously used techniques in this application. Key words: recombination, gene conversion, pseudogene, β-glucocerebrosidase, complex alleles, Gaucher disease
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Meiotic homologous recombination and hybrid sterility
Gergelits, Václav ; Forejt, Jiří (advisor) ; Macholán, Miloš (referee) ; Munclinger, Pavel (referee)
(English) Meiotic homologous recombination, homologous chromosomes synapsis, and F1 hybrid sterility (enabling formation of species) are mutually interconnected phenomenons, one being the prerequisite to the latter. In the present thesis, these phenomenons were investigated on a genetic and mechanistic level using a mouse subspecies as a model. Noncrossovers (NCOs, gene conversions), 90% prevalent resolution of Prdm9- determined meiotic double-strand breaks (DSBs), were uniquely identified and characterized on a chromosome-wide level. The mean gene conversion tract length, based on 94 NCOs events, was calculated to be 32 bp. On a local level, the NCOs overlapped the known hotspots of PRDM9-controlled histone trimethylation and DSB formation, indicating their origin in the standard meiotic DSB repair pathway. On chromosome-wide level, NCO and CO distributions differed, in particular COs being relatively preferred over NCOs in subtelomeric regions. A specific subset of nonparental/asymmetric NCOs and COs was underrepresented in our datasets, proposing their problematic repair, hypothetically enabled by sister chromatids, and thus not contributing to indispensable homologous synapsis. Genome-wide crossover (CO) rates, genetically and mechanistically crucial ~10% of DSB repair, were proven to be...
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Detection of β-glucocerebrosidase gene/pseudogene recombination events leading to pathogenic alleles
Peková, Barbora ; Hřebíček, Martin (advisor) ; Schierová, Michaela (referee)
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...
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Recombination between the gene and pseudogene for glucocerebrosidase as a mechanism of mutation generation in Gaucher disease
Peková, Barbora ; Hřebíček, Martin (advisor) ; Schierová, Michaela (referee)
Gaucher disease is an autosomal recessive disorder caused by the deficiency of β-glucocerebrosidase. Some Gaucher patients carry in their β-glucocerebrosidase genes complex mutations which apparently arose by a recombination with the non-functional β-glucocerebrosidase pseudogene. Recombination between genes and their corresponding pseudogenes plays a role in the development of other hereditary human diseases. Mutant alleles formed in male and female meiosis are a source of these variations in the gene pool. The study of frequency and scope of recombination events in human disease-associated genes in the gametes is of importance for evaluation of the disease burden in the population. The evaluation of the scope of single recombination events in the β-glucocerebrosidase gene in human gametes is technically challenging. Novel technologies such as next-generation sequencing, nanopore sequencing or droplet digital PCR may have advantages over previously used techniques in this application. Key words: recombination, gene conversion, pseudogene, β-glucocerebrosidase, complex alleles, Gaucher disease
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