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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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Association study with the ADA gene and detailed characterization of two chromosome aberrations in autism
Vážna, Alžběta ; Sedláček, Zdeněk (advisor) ; Ferák, Vladimír (referee) ; Korabečná, Marie (referee)
Autism affects up to 1/150 children and represents therefore a serious social problem. It is a complex disorder with a clearly documented genetic component, but so far unexplained aetiology, which is currently a subject of intensive research. In the field of genetics various gene and chromosome defects are examined, as well as other mechanisms, including epigenetics, which could play a role in pathogenesis. In our work we tried to replicate the finding of association between the ADA*2 risk allele and autism in a sample of 385 Czech children. Our sample was larger than those originally published. We also focused on individual endophenotypes (types of autism, degree of mental retardation and co-morbidity). Our results did not confirm the association of autism with the ADA*2 allele in the complete sample or in any of the subsets. Chromosomal changes represent another finding in autistic patients. We performed the analysis of a ring chromosome 17 and a chromosome X deletion in two patients. Our studies represent an up to now unimaginable link between classical cytogenetics and molecular genetics at the DNA sequence level. As the first in the world, we described the structure of a human ring chromosome. Characterization of the defects allowed us to speculate on the impact of the genes involved in the phenotype...
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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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The alterations of MLL (mixed-lineage leukemia) gene and their clinical importance in pediatric patients with acute leukemia
Řezníčková, Leona ; Trka, Jan (advisor) ; Sedláček, Zdeněk (referee)
Presence of the MLL gene rearrangement at 11q23 is an important prognostic feature. Moreover, the rearrangements represent a suitable target for the minimal residual disease (MRD) monitoring in some subtypes of childhood acute leukaemias (AL). Currently more than 80 different translocations involving the MLL gene and more than 50 of those are characterised at the molecular leve\. Using multiplex-reverse transcriptase polymerase chain reaction (multiplex RT -PCR) and - in some cases - its combination with DNA analysis of the translocation breakpoint we examined a group of infants «1 year of age) diagnosed with acute lymphoblastic leukaemia (ALL), children with M4 and M5 subtypes of acute myeloid leukaemia (AML) and patients with secondary leukaemias. Moreover, we exaITŮned children with B-cell precursor fulfilling at least one of the ťollowing criteria: proB immunophenotype, cytogenetically confirmed MLL rearrangement and/or expression oťNG2 molecule shown by f10w cytometry. MultiplexRT PCR technique enables fast detection of the most frequent fusion partners of the :MLL gene (AF4, AF6, AF9, AFlO, ENL a ELL). We screened almost 80 patients diagnosed and treated in the Czech Republic between 1997 and 2007 and we found an MLL-fusion gene in 51 of them. Vast majority of rearrangements (92%) was detected by the...
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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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Application of whole-exome sequencing methods for the study of rare inherited diseases
Piherová, Lenka ; Kmoch, Stanislav (advisor) ; Sedláček, Zdeněk (referee) ; Tichý, Boris (referee)
Rare diseases (RD) are a heterogeneous group of diseases that affect about 5% of the world population. RDs represent more than 7.000 different phenotypes and many of them are genetically determined. RDs provide unique biological models for understanding the basic principles of molecular and cellular organization and function of human tissues and organs. Results of studies focused at pathogenesis of RDs are often used to diagnose and treat the affected patients. Significant progress in molecular genetic techniques, specifically the use of the next generation sequencing (NGS) in clinical practice, substantially facilitated and improved efficiency of RD laboratory diagnostics. Moreover, these novel testing algorithms identified the previously unknown molecular causes of many RDs. This thesis demonstrates the utility of NGS techniques and bioinformatics processing of obtained data in studies aimed at understanding molecular basis of selected RDs. These methods led to identification and characterization of causative pathogenic variants in the NDUFAF6 and PLD1 genes among patients affected by the Acadian variant of Fanconi disease and patients with a rare congenital heart defect, respectively. This approach was further used to analyze exomes of a large cohort of patients with different types of...
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Detection of submicroscopic chromosomal aberrations in phenotypically abnormal carriers of apparently balanced rearrangements using array CGH
Slámová, Zuzana ; Sedláček, Zdeněk (advisor) ; Michalová, Kyra (referee) ; Kuglík, Petr (referee)
Carriers of apparently balanced chromosomal aberrations (BCA) are usually phenotypically normal. However, it has been estimated that up to 27% of these BCA may be associated with an abnormal phenotype, most often caused by cryptic imbalances at the breakpoints, gene disruption by the breakpoint or via the position effect. In contrast to conventional karyotyping, molecular cytogenetic techniques enable more detailed BCA characterization and better correlation between genotype and phenotype of the patient. The aim of this thesis was to evaluate the presence of copy number variants (CNVs) at breakpoints or elsewhere in the genome in patients with abnormal phenotype who carry de novo or inherited BCA. 54 BCA were investigated using array CGH (20 de novo cases, 27 inherited and 7 cases of unknown origin) including 32 reciprocal translocations, 6 robertsonian translocations, 12 inversions and 4 complex chromosomal rearrangements. If possible, the parents were also examined to ascertain the inheritance of the relevant CNVs. In order to specify microarray findings or exclude gene disruption, FISH was used in selected patients. Among the patients included, in 31,5% (17/54) at least one (in 8 patients more than one) significant CNV was detected. Four cases carried cryptic imbalances only at the breakpoints,...
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