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Genome analysis techniques and their applications in elucidation of molecular underpinnings of rare genetic diseases.
Přistoupilová, Anna ; Kmoch, Stanislav (advisor) ; Sedláček, Zdeněk (referee) ; Pačes, Jan (referee)
Rare diseases represent a heterogeneous group of more than ~7000 different diseases, affecting 3,5-5,9% of the global population. Most rare diseases are genetic, but causal genes are known only in some of them. Many patients with rare diseases remain without a diagnosis, which is crucial for genetic counseling, prevention, and treatment. With the development of new methods of genome analysis, decreasing cost of sequencing, and increasing knowledge of the human genome, a new concept for identifying disease-causing genes was established. It is based on comparing the patient's genetic variability with the genetic variability of the general population. This dissertation describes next-generation sequencing technologies (NGS), bioinformatic analysis of acquired data and their applications in the elucidation of molecular underpinnings of rare genetic diseases. These procedures have led to the identification and characterization of causal genes and gene mutations in autosomal dominant tubulointerstitial kidney disease (SEC61A1, MUC1), autosomal dominant neuronal ceroid lipofuscinosis (CLN6, DNAJC5), neurodegenerative disease of unknown etiology (VPS15), Acadian variant of Fanconi syndrome (NDUFAF6) and spinal muscular atrophy (SMN1). The application of novel genome analysis techniques increased the...
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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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Genotype-phenotype correlation in selected rare disorders using molecular analysis of genome and gene variants
Vlčková, Markéta ; Sedláček, Zdeněk (advisor) ; Gaillyová, Renata (referee) ; Baxová, Alice (referee)
The work was focused on detailed analysis of patients with rare genomic and gene variants. We studied the impact of these variants on the phenotype of the patients. As the majority of our patients, both syndromic and non-syndromic, were reffered to the detailed analysis due to intellectual disability and/or autism spectrum disorder, the work was focused on these two clinical diagnoses. At the beginning we analyzed patients with aberrations detected using cytogenetic analysis, and the extent, gene content and mechanism of origin of the aberrations were refined using molecular genetic methods, most often high-resolution array CGH. Later we analyzed patients with rare or unique submicroscopic aberrations detected using aCGH or SNP array. Using these methodes we analysed in the project patients with deletions of Xp22.1-p22.3, 6q11-q13, 6q14-q16, Xq25, 1q21.1, Xp21.2-p21.3, 2p14-p15, 17q21.31, 9q21.3 a 2p15- p16.1, and a patient with an Xp21.2-p21.3 duplication. In the last years we proceeded to the analysis of syndromic cases using next generation sequencing. This led to the identification of point mutations in the HCFC1, KAT6B, SOS2 and KMT2D genes, which were further studied. The work contributed to the knowledge about the impact of the genome and gene variants identified on the phenotype of the...
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Use of massive parallel sequencing in determination of skewed X inactivation
Veselková, Tereza ; Dvořáková, Lenka (advisor) ; Sedláček, Zdeněk (referee)
Skewed X chromosome inactivation has been often studied as a possible factor that influences manifestation of X-linked diseases in heterozygous women. Yet the association between phenotype and degree of skewing stays unclear for most disorders. Current works rely mostly on methods that are based on methyl-sensitive restriction while determining the X inactivation pattern and mainly the HUMARA assay which investigates the methylation profile in the AR gene. However those methods have some known disadvantages and therefore we are still seeking new methodical approaches. We used DNA isolated from whole blood and in some cases also buccal swabs to asses X inactivation patterns in 54 women using methylation-based methods for loci AR, CNKSR2 and RP2. Transcription-based assay was utilized to evaluate skewing of X inactivation in 32 of those women, whose samples were available for RNA extraction, using massive parallel sequencing and polymorphisms LAMP2 c.156A>T, IDS c.438C>T and ABCD1 c.1548G>A. Partly thanks to almost no stuttering during PCR the RP2 locus was the most informative in our study (71 % of women) and approximately the same number of women (69 %) were informative for the HUMARA assay. However when comparing the results of those two methods we determined difference greater than 10 % in...
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Application of novel genomic techniques in studies of pathogenesis of selected rare inherited disorders
Nosková, Lenka ; Kmoch, Stanislav (advisor) ; Sedláček, Zdeněk (referee) ; Živný, Jan (referee)
Rare diseases are a heterogeneous group of disorders. Knowledge of their molecular basis is poor and till recently there were no appropriate methodical approaches due to a limited number of patients. Novel genomic techniques, especially the DNA array technology and the next generation sequencing emerging in last few years, enabled studies of these diseases even in small families and sporadic cases. This PhD thesis focuses on application of novel genomic techniques in studies of rare inherited diseases. It describes a use of DNA array technology in linkage analysis, analysis of differential gene expression, analysis of copy number variations and homozygous mapping, and a use of next generation sequencing technology. Combination of these methods was used for identification of molecular basis of adult neuronal ceroid lipofuscinosis, Rotor syndrome, isolated defect of ATP synthase and mucopolysaccharidosis type IIIC.
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Analysis of copy number variant (CNV) in genomes of patiens with mental retardation
Hančárová, Miroslava ; Sedláček, Zdeněk (advisor) ; Gaillyová, Renata (referee) ; Michalová, Kyra (referee)
Mental retardation (MR) is a very heterogeneous common neurodevelopmental disorder with a population prevalence of 2.5-3 %. The importance of genetic factors in the development of MR is high but in a significant number of cases the etiology remains unexplained. Recent studies using array methods pointed to frequent occurrence of copy number variants (CNVs) in patients with MR. Pathogenic CNVs were identified in 10-15 % patients with idiopathic MR and normal karyotype. The aim of our work was the analysis of genome-wide gains and losses of genetic material in a group of Czech patients with MR and a thorough bioinformatic analysis of the genetic changes identified aiming at the assessment of their clinical significance. We performed whole genome analysis using the HumanCytoSNP-12 BeadChips (Illumina) in 183 patients with idiopathic MR, normal karyotype and no FMR1 gene expansion. Data analysis was carried out using two independent programmes, GenomeStudio and QuantiSNP. The findings were subjected to two rounds of thorough bioinformatic analysis. Based on this analysis we classified the CNVs into 4 categories: pathogenic CNVs, probably pathogenic CNVs, CNVs with uncertain clinical significance and benign CNVs. With the exception of the benign variants, all CNVs were confirmed using an independent laboratory...
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Molecular Basis of Familial Hyperuricemic Nephropathies
Živná, Martina ; Kmoch, Stanislav (advisor) ; Jirsa, Milan (referee) ; Sedláček, Zdeněk (referee)
In 1960 Duncan and Dixon described family whth chronic tubulointerstitial kidney disease associated with juvenile onset of hyperuricemia and gout. Based on combination of these clinical symptoms they named the disease familial juvenile hyperuricemic nephropathy (FJHN) [1]. Disease with very similar clinical presentation but different age of onset and kidney histology was described as a medullary cystic kidney disease (MCKD) in 1977 [2]. Until recently the molecular basis and pathogenesis of this syndrome remained unknown. The long term aim of our research group is to elucidate the genetic basis of the disease and to solve pathogenetic mechanisms leading to the individual clinical and biochemical symptoms (e.g. hyperuricemia) and kidney damage in general. We systematically identify patients with this disease and healthy family members and collect relevant clinical information and samples for classification (urine, blood, tissue biopsies) and subsequent clinical, biochemical, molecular biology and cell pathology correlations. We [3, 4] and others [5-7] proved genetic heterogeneity of FJHN and defined four FJHN loci on chromosomes 1q21, 1q41, 16p11.2. and 17q21.3. Further research defined disease causing mutations in three genes - uromodulin (UMOD) [8], hepatonuclear factor 1-beta (HNF-1) [9] and renin (REN)...
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Molecular genetic analysis of the fragile X syndrome and myotonic dystrophy
Mušová, Zuzana ; Sedláček, Zdeněk (advisor) ; Fajkusová, Lenka (referee) ; Maxová, Alica (referee)
More than 20 human hereditary diseases have been linked to expansions of unstable simple nucleotide repeats. These disorders include several clinically heterogeneous neurological diseases such as the fragile X syndrome (FXS), Friedreich's ataxia (FRDA1), Huntington's disease (HD), multiple types of spinocerebellar ataxias (SCA), and myotonic dystrophy type 1 (DM1). The phenotype of these disorders shows wide variability ranging from mild symptoms with late onset to severe congenital forms. The pathogenic mechanism of these expansions depends mainly on the type, localization and length of the repeat. Expansions of tracts of CAG triplets in coding regions cause polyglutamine disorders with abnormal protein function. Expanded non-coding repeats can silence gene expression or produce toxic RNAs which can be engaged in abnormal interactions with RNA-binding proteins [reviewed in 1]. The fragile X syndrome is one of the most common causes of inherited mental retardation with estimated frequency of 1 in 4000-9000 males and 1 in 7000-15000 females [2]. The disorder is associated with the expansion of a polymorphic CGG repeat in the 5' untranslated region of the FMR1 gene in Xq27.3 [3-6]. Affected males with full mutations (over 200 CGG repeats) have mental impairment with dysmorphic features (elongated face,...
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