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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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Genomic imprinting and evolution of sexually dimorphic traits
Farkačová, Klára ; Kratochvíl, Lukáš (advisor) ; Munclinger, Pavel (referee)
Genomic imprinting is a process whereby expression of an allele differs depending upon its parent of origin. It can be found on autosomes and also on sex chromosomes. Basic hypothesis for the evolution of genomic imprinting is the hypothesis based on the existence of sexual conflict. It can be classified into interlocus sexual conflict and intralocus sexual conflict hypotheses. Under interlocus sexual conflict hypothesis we can diffferentiate parental conflict hypothesis and parent-offspring conflict hypothesis. These theories were historically proposed for the first two taxonomical groups, where genomic imprinting was discovered, namely for angiosperms and placental mammals. Theory of parental conflict proposes that genomic imprinting evolved because the paternally inherited alleles are more selfish to mothers than are the maternally inherited alleles. Parent-offspring conflict hypothesis proposes that genomic imprinting evolved because maternal genes try to regulate demands of paternally inherited alleles in embryos. More recently, genomic imprinting has been found also in other taxons and in alleles, which do not bring any advantage during embryonic development. The intralocus sexual conflict hypothesis is applicable for every trait under sexually-specific selection. It provides potential...
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Molecular genetic and biochemical studies of selected inherited metabolic disorders, development and applications of new methods
Mušálková, Dita ; Hřebíček, Martin (advisor) ; Adam, Tomáš (referee) ; Macek, Milan (referee)
Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...
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Genomic imprinting and evolution of sexually dimorphic traits
Farkačová, Klára ; Kratochvíl, Lukáš (advisor) ; Munclinger, Pavel (referee)
Genomic imprinting is a process whereby expression of an allele differs depending upon its parent of origin. It can be found on autosomes and also on sex chromosomes. Basic hypothesis for the evolution of genomic imprinting is the hypothesis based on the existence of sexual conflict. It can be classified into interlocus sexual conflict and intralocus sexual conflict hypotheses. Under interlocus sexual conflict hypothesis we can diffferentiate parental conflict hypothesis and parent-offspring conflict hypothesis. These theories were historically proposed for the first two taxonomical groups, where genomic imprinting was discovered, namely for angiosperms and placental mammals. Theory of parental conflict proposes that genomic imprinting evolved because the paternally inherited alleles are more selfish to mothers than are the maternally inherited alleles. Parent-offspring conflict hypothesis proposes that genomic imprinting evolved because maternal genes try to regulate demands of paternally inherited alleles in embryos. More recently, genomic imprinting has been found also in other taxons and in alleles, which do not bring any advantage during embryonic development. The intralocus sexual conflict hypothesis is applicable for every trait under sexually-specific selection. It provides potential...
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Molecular genetic and biochemical studies of selected inherited metabolic disorders, development and applications of new methods
Mušálková, Dita ; Hřebíček, Martin (advisor) ; Adam, Tomáš (referee) ; Macek, Milan (referee)
Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...
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