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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...

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...

Turner syndrome (TS) is a genetic anomaly occurring in women with worldwide frequency 1:2,000. Turner syndrome's possible causes include X chromosome monosomy, mosaic karyotype (45,X/46,XX; 45,X/46,XY; 45,X/47,XXX), X isochromosome (46,X,i(Xq)), ring X chromosome (45,X/46,X,r(X)), and other X chromosome aberrations (deletion and translocation). Patients with Turner syndrome (with the exception of ring chromosome abnormalities) aren't diagnosed with mental retardation. Turner syndrome is closely related to the regulation of gene expression level, particularly with X chromosome inactivation. Genes escaping the X chromosome inactivation process in 45,X women are expressed in half the dose of a healthy woman. An example of such a gene is SHOX, which I decided to focuse on in this thesis. SHOX gene haploinsufficiency causes major Turner syndrome phenotype manifesting of short body and bone abnormalities. The research of other genes with possible roles in Turner syndrome is complicated by the absence of adequate model organism, which could be used for TS study with possibility to extrapolate the results to humans. In mice, both the inactivation process itself is different and the phenotypic manifestation of X monosomy (39, X) is also much milder than in 45,X women. This difference could be explained by...