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Analysis of gonosomal mosaic using FISH method
ŠUTKOVÁ, Jana
In my bachelor thesis I dealt with the analysis of gonosomal mosaics. Mosaicism can be described as a condition in which two or more distinct cell lines originating from a single zygote are found in one organism. In women, the most common mosaic forms are Turner syndrome and triple X syndrome or superfemale. In the male population, Klinefelter syndrome and XYY syndrome (supermale) are most commonly detected. Mosaicism can be investigated by fluorescence in situ hybridization. In the theoretical part of my bachelor's thesis I focused on congenital chromosomal mosaicism, especially on its variants such as Klinefelter syndrome, Turner syndrome, triple X and XYY syndrome. Furthermore, in this part I dealt with fluorescence in situ hybridization. I described fluorescence per se and then its application in clinical practice, where several types of fluorescence in situ hybridization are used. In the practical part I examined the number of gonosomal chromosomes using fluorescence in situ hybridization. I prepared this part in the laboratory of the Faculty of Health and Social Sciences of the University of South Bohemia in České Budějovice. I examined 40 anonymized samples from 20 probands. Capillary blood samples and buccal swabs were taken from each of them. After hybridization of samples with centromeric probe for X and Y chromosomes microscopy was performed. In the last part of this bachelor thesis I dealt with the processing of the results obtained by fluorescence microscopy. Thus, a total of 6 cases of mosaicism were detected in women (the percentage of mosaic cells was above the admissible limit). In males there were a total of 2 cases of increased percentage of mosaic cells.
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Role of Next Generation Sequencing in Diagnostics and Prognosis of X-linked Lysosomal Storage Disorders
Řeboun, Martin ; Dvořáková, Lenka (advisor) ; Fajkusová, Lenka (referee) ; Krejčí, Jan (referee)
The characterisation of the molecular-genetic etiology of monogenic diseases includes not only the identification of the pathogenic variant, but also the description of its effect on the RNA structure and stability. Additionally, the X-inactivation plays an important role in X-linked diseases. In the presented thesis, we applied the methods of next generation sequencing to study three lysosomal disorders (mukopolysacharidosis type II, MPS II; Danon disease, DD; Fabry disease, FD). Two methodological approaches have been used: 1) panel sequencing with hybridization probes for identification of single nucleotide variants, small deletions/duplications and structure variants (CNVs) 2) amplicon sequencing for analysis of somatic mosaicism and allele specific expression. The panel sequencing enabled us to confirm the molecular-genetic basis of DD in two patients with the identification of two exons duplication and five exons deletion in LAMP2, respectively. The somatic mosaicism was being analyzed by the amplicon sequencing in families with DD, MPS II. We could identify the first case of somatic mosaicism in a patient with DD. The allele specific expression has enlarged the group of methods used in X-inactivation analysis. Its impact has been proved particularly in minimizing misinterpretation of XCI...
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Molecular-cytogenetic diagnostics of marker chromosomes
Tesner, Pavel ; Kočárek, Eduard (advisor) ; Zemanová, Zuzana (referee) ; Šubrt, Ivan (referee)
Supernumerary marker chromosomes (sSMCs) are a relatively rare cytogenetic phenomenon. Their laboratory examination is often difficult, and the clinical interpretation is even more challenging. The main reason is that most sSMC carriers have no clinical manifestations. The chromosome origin and exact range of the aberration are very important, as well as the fact that sSMCs are often found in mosaics that can strongly influence both the phenotype and the interpretation of result. Prenatal sSMC finding is one of the most challenging situations in both clinical and laboratory genetics. This work deals with the investigation process of sSMC carriers using molecular cytogenetic techniques, especially fluorescence in situ hybridization (FISH). We investigated a total of 67 families collected both prospectively and retrospectively, and we found 70 unique sSMCs in a total of 74 individuals. Six cases were familial and in three cases two sSMCs were found in one individual. According to the initial karyotype finding, the cases were divided into two groups, sSMCs supernumerary to a normal karyotype (group A) and sSMCT s supernumerary to the Turner karyotype (group B). The chromosomal origin was successfully determined in 88,6 % sSMCs. In group A the most common findings were sSMCs derived from chromosome 15,...
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Analysis of mosaic chromosomal aberrations using various methods
Oroszová, Karin ; Drábová, Jana (advisor) ; Šolc, Roman (referee)
Mosaicism is represented by two or more chromosomally different cell lines in an individual. Mosaics are most often caused by chromosome malsegregation during mitosis, resulting in the gain or loss of chromosomes, known as aneuploidy, but structural aberrations can also occur in mosaic form. The problem is the limitation of detection with standart cytogenetic methods. The present study was carried out to compare the efficiency of FISH, array CGH and cytogenetic techniques in detection of mosaicism. In the practical part the results of 45 patients with mosaicisms of aneuplody of gonosomes (26 patients) and mosaicisms of autosomes (19 patients) were compared. The data show that we have different peripheral blood karyotype and FISH results in 23 of 37 patients (62%). There was a case of failure of detection of the mosaicism on the karyotype and the FISH method revealed a abnormal cell lines with a percentage of less than 5%. The array CGH method confirmed the karyotype and FISH results in 10 out of 12 patients (83%) in peripheral blood tests. The work also dealt with artificially made mosaics. From the results, it is obvious that the FISH method has a more accurate percentage of mosaic capture compared to the karyotype. The results indicate that using the techniques in parallel allow in clinical...
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Polyspermy produces viable mosaics in sturgeon
IEGOROVA, Viktoriia Barativna
Chapter 1 (General Introduction), Chapter 2 (Polyspermy produces viable haploid/diploid mosaics in sturgeon), Chapter 3 (First evidence of viable progeny from three interspecific parents in sturgeon), Chapter 4 (General discussion, English Summary, Czech Summary, Acknowledgements, List of Publications, Training and Supervision Plan during Study, Curriculum Vitae). The thesis is written in English, contains 71 pages. The results were published in two scientific journals. The actuality of work: The majority of sturgeons are critically endangered and at the same time they are producers of the most expensive food product - black caviar. Presently, sturgeon restoring and conservation are depended on artificial reproduction in hatcheries. However, the way of fertilization is not clear: monospermy, physiological or pathological polyspermy? Moreover, during last decades it was believed that under artificial fertilizations, can appear atypically divided embryos like 3 or 6 cells, which die prior hatching stage or sometimes develop into malformed shape fry with soon death due to deformations. Atypically divided embryos, their origin and the reason of appearance was not studied well. Investigation of fertilization aspects in economically significant and endangered fishes is extremely important. Requires attention and studies a hybridization abilities in sturgeons, as hybridization plays an important role in sturgeon evolution. The purposes of the study were: to find a way of fertilization in sturgeons (monospermy/polyspermy); to understand the reasons of atypical division of cells during artificial breeding; to investigate if atypically divided embryos are able to develop beyond the feeding stage, to analyze their ploidy; to classify the types of atypically divided embryos and their frequency; to investigate sturgeon hybridization plasticity, to produce first interspecific hybrid from three parents. Novelty and scientific originality: In this research two ways of fertilization were found in sturgeons: physiological polyspermy (fertilization typical for caudate amphibians) and karyogamy with an additional plasmogamy, which results in production of viable multiple-sperm mosaics with atypical division on the 2-4 cell stage and mosaic ploidy). Conducted research is important for avoiding of negative effect on sturgeon propagation programs, due to uncontrolled fertilization and releasing of multiple-sperm mosaics into the wild, which can cause appearance of sturgeons with irregular ploidy and induce a detrimental genetic effects on sturgeon populations. However, multiple-sperm mosaics, which were discovered in this study and their easy way of production can be used as a beneficial tool for a rapid manufacture of isogenic strains in sturgeons. In this study was shown that applying of just one part of body (blood, tail, etc.) for ploidy determination is not giving a full view of a real ploidy of the studied individual. A huge ability for hybridization and plasticity were described in sturgeon. It was possible to generate a first viable hybrids from three interspecific parents.
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Molecular-cytogenetic diagnostics of marker chromosomes
Tesner, Pavel ; Kočárek, Eduard (advisor) ; Zemanová, Zuzana (referee) ; Šubrt, Ivan (referee)
Supernumerary marker chromosomes (sSMCs) are a relatively rare cytogenetic phenomenon. Their laboratory examination is often difficult, and the clinical interpretation is even more challenging. The main reason is that most sSMC carriers have no clinical manifestations. The chromosome origin and exact range of the aberration are very important, as well as the fact that sSMCs are often found in mosaics that can strongly influence both the phenotype and the interpretation of result. Prenatal sSMC finding is one of the most challenging situations in both clinical and laboratory genetics. This work deals with the investigation process of sSMC carriers using molecular cytogenetic techniques, especially fluorescence in situ hybridization (FISH). We investigated a total of 67 families collected both prospectively and retrospectively, and we found 70 unique sSMCs in a total of 74 individuals. Six cases were familial and in three cases two sSMCs were found in one individual. According to the initial karyotype finding, the cases were divided into two groups, sSMCs supernumerary to a normal karyotype (group A) and sSMCT s supernumerary to the Turner karyotype (group B). The chromosomal origin was successfully determined in 88,6 % sSMCs. In group A the most common findings were sSMCs derived from chromosome 15,...
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Molecular cytogenetic analysis of mosaic chromosomal abnormalities
Cinkajzlová, Anna ; Kočárek, Eduard (advisor) ; Král, Jiří (referee)
The focus of this diploma thesis is on mosaic numerical and structural chromosomal aberrations. In its theoretical part, general problems of mosaicism, its phenotypic effect, mechanisms of origin, related epigenetic modifications, and diagnostic options are described. The methodical part of the thesis then primarily refers to fluorescence in situ hybridization (FISH) and its application in the diagnostics of mosaicism. This method was used in the examination of 29 patients with numerical as well as structural abnormalities of autosomes or gonosomes with proven or suspected mosaicism. On the basis of this analysis, possible errors of measurement were determined and data for statistic evaluation were retrieved. For the examinations of three patients an alternative of the comparative genomic hybridization, the array CGH technique, was applied. The FISH method, although being based on random selection and human factor, proved sufficient sensitivity as well as specificity in the field of low-frequency mosaicism diagnostics. The main critical factors responsible for potential misinterpretation of the data arose from inherent characteristics of the biological material, incorrect targeting of the analysis, probe instability, bleed through effect and absence of mitosis during the structural aberrations analysis.
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Possibilities of assessment and the relevance of the results of FISH analysis of chromosomal abnormalities in mosaics
Neužilová, Linda ; Kočárek, Eduard (advisor) ; Panczak, Aleš (referee)
This bachelor thesis deals with mosaicism and its detection. Mosaicism is defined as the presence of two or more cell lines with different karyotype in the patient's body. An aspect of particular importance for the resulting phenotype is the overall ratio in which the individual cell lines are represented. It is often found in Turner and Klinefelter syndrome. Turner syndrome is caused by the loss of a part of, or the whole of the X chromosome in females and it belongs among the most common chromosomal constitutions, found in spontaneous abortions. However, it is estimated that only about half of the women with Turner syndrome have karyotype 45,X; the others are mosaics or have other abnormalities of the chromosome X. The main aim of the practical part of this thesis was to evaluate the possibilities of detecting mosaicism using fluorescence in situ hybridization (FISH) and to consider the possible factors affecting the accuracy of the examination. The objectives of the assessment were the individual variability between the evaluators and also the differences caused by using two differently labelled probes. As a result, the thesis confirms that the FISH method enhances the accuracy of the results of conventional cytogenetic examination and it is suitable for analysis of mosaicism thanks to the possibility of...
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