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Neurological manifestation of lysosomal storage disorders
Májovská, Jitka ; Magner, Martin (advisor) ; Fajkusová, Lenka (referee) ; Aulická, Štefania (referee)
Objective: Lysosomal storage disorders (LSD) represent a rare cause of neurologic impairment in childhood and adulthood. The aim of our study was to characterize patients with late-onset form of Tay-Sachs disease (LOTS) and alpha-mannosidosis (AM) and to identify typical brain MRI findings. Methods: Patients with a genetically or enzymatically confirmed diagnosis of LOTS or AM and at least one brain MRI examination were included in the study. Results: We have characterized the clinical manifestation in a unique cohort of 14 Czech patients with LOTS. As results of international cooperation, we also published analysis of neuroradiological findings in 16 patients with LOTS disease and 14 patients with AM. Patients with LOTS clinically manifested by cerebellar symptoms, progressive motor neuron disease and psychiatric symptoms. A novel pathogenic variant c.754C˃T in HEXA gene was described in two brothers. Disease was outlined by the slower disease course, milder weakness of lower limbs, milder cerebellar symptomatology and normal cognitive function in them. The hallmark of neuroradiological findings was the cerebellar atrophy in both LOTS and AM. It was the pontocerebellar atrophy in LOTS, the finding which is also typical for spinocerebellar ataxia or multiple system atrophy. The concurrent presence...
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Analysis of genomic variants in patients with nerodevelopmental diseases with a focus on epilepsy.
Zůnová, Hana ; Vlčková, Markéta (advisor) ; Zemanová, Zuzana (referee) ; Fajkusová, Lenka (referee)
Epilepsy is a chronic neurological disease affecting millions of people worlwide. The etiology of epilepsy is heterogenous. Published studies confirm a strong genetic basis. While the genetic causes play a huge role in the development of epilepsy, the etiology remains unclear in many patiens. We present a cohort of 400 patients with epilepsy, who underwent whole genome testing focused on the detection of copy number variants (CNVs). The main criterion for inclusion into the group was manifestation of epilepsy in isolated form or in combination with other neurodevelopmental disorder or congenital anomalies. Genome-wide analysis was performed using two different platforms of array CGH (array comparative genome hybridization): SurePrint G3 CGH ISCA platform 4x180K and 8x60K (Agilent Technologies). For evaluation of clinical impact of detected CNVs different databases (DGV, ClinVar, OMIM, DECIPHER) and relevant articles were used. In our cohort we have detected 2730 CNVs in total and 86 of them (detected in 76 individuals), were evaluated as possibly clinically relevant - 82 CNVs were evaluated as a possible cause of epilepsy and 4 of them were evaluated as secondary finding without relationship to the patients' phenotype. Regarding to the current classification, 21/86 CNVs have been refered as...
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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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Analysis of the spectrum of genetic variants associated with development of Parkinson's disease
Stočesová, Lucie ; Hirschfeldová, Kateřina (advisor) ; Fajkusová, Lenka (referee)
Parkinson's disease (PD) is one of the most common neurodegenerative disease in humans. It affects all age categories and the number of patients with this disease is still growing. However, the genetic cause of PD is not yet very clear and new and new candidate genes are constantly being discovered. The aim of the thesis is to perform a mutation analysis in a group of patients and controls from the Czech population and thus find possible genetic causes of parkinsonism in a cohort of researched patients. The second aim is to evaluate data correlation obtained by different methods. Next generation sequencing was used for this purpose. The results of this sequencing were verified with methods such as MLPA (Multiplex Ligation-Dependent Probe Amplification), analysis of short tandem repeats and Sanger sequencing. Using these methods, we obtained a wide range of possible genetic causes of parkinsonism in the studied group of patients. Patogenic or risk variants were found not only in classical candidate genes typical for PD (called PARK), but also in genes associated with other neurodegenerative diseases. For less than half of the patients (42,64 %), the genetic cause of parkinsonism was not found. Using several methods, we found that next generation sequencing is a very precise method, that can well...
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Analysis of the LMNA gene and the SH3TC2 gene among Czech patients with hereditary neuropathy Charcot-Marie-Tooth type 1 and 2
Laššuthová, Petra ; Seeman, Pavel (advisor) ; Martásek, Pavel (referee) ; Fajkusová, Lenka (referee)
My PhD thesis can be devided into two parts: 1. Hereditary motor-sensory neuropathies (HMSN) 2. Selected muscle disorders The main emphasis was on the first part - hereditary motor and sensory neuropathies. Research was focused on autosomal recessive forms - demyelinating type CMT4C and axonal type CMT2B1. Most of the results obtained are related to these disorders. Data, which were obtained, are unique and were published in international journals with impact factor. Results obtained from CMT4C study are accepted for publication in Clinical Genetics. Results obtained in LMNA study (CMT2B1) were published in Journal of Human Genetics. The author performed and validated these new methods and original results, which are due to be used in genetic molecular testing of patients with hereditary neuropathies and muscle disorders: 1. Sequencing of all coding exons of the SH3TC2 gene. First mutations in the SH3TC2 gene in Czech HMSN I patients were found. 2. The prevalent mutation among Czech CMT4C patients was proven to be p.Arg954Stop. 3. Real-time PCR assay targeted at detection of the prevalent mutation p.Arg954Stop in the SH3TC2 gene was validated and is now used in our lab on a daily basis as a quick and efficient screening. 4. Molecular genetic testing of the SH3TC2 gene was introduced into the routine...
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Molecular syndromology: molecular genetic causes of rare diseases illustrated with Kabuki and Kabuki-like syndromes
Paděrová, Jana ; Macek, Milan (advisor) ; Baxová, Alice (referee) ; Fajkusová, Lenka (referee)
Kabuki syndrome (KS) is a dominantly inherited rare disease caused mainly by de novo pathogenic variants (henceforward mutations) in the KMT2D (formerly MLL2) and KDM6A genes. It is rare multisystemic syndrome characterized by intellectual disability (ID) and typical facial dysmorphism. KS is clinically heterogeneous, which complicates its clinical diagnosis. The first aim of this thesis was to introduce mutation testing of the two known KS causative genes in KS by Sanger DNA sequencing and by MLPA (Multiple Ligation Probe Amplification) at the Department of Biology and Medical Genetics of 2nd Medical Faculty of Charles University and University Hospital Motol, Prague followed by identification of underlying genetic mutations in KMT2D/KDM6A genes in 43 patients with phenotype typical for KS, who were indicated for this molecular genetic analysis by several collaborating genetic departments in the Czech Republic. We aimed to confirm or disprove of patient's clinical diagnosis, establish spectra of KMT2D/KDM6A mutations in the Czech population, render phenotype-genotype correlations and evaluate the phenotypic "MLL2-score" (published by Makrithanasis et al., 2013) utility as prediction tool for selection of cases for KMT2D sequencing. Mutations in the KMT2D gene were detected by Sanger DNA sequencing...
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Genetic and molecular basis of neurodegenerative and neuropsychiatric diseases
Jedličková, Ivana ; Kmoch, Stanislav (advisor) ; Fajkusová, Lenka (referee) ; Laššuthová, Petra (referee)
Next-generation (NGS) and third-generation (TGS) sequencing methods have played a key role in strategies of disease genes identification. Especially the exome sequencing increased the efficiency of causal variants identification up to tens of percent in study cohorts. Rare neurodegenerative diseases are clinically and genetically heterogeneous and show a broad differential diagnostics. NGS and TGS technologies have been crucial in our understanding of the pathomechanism of rare neurodegenerative diseases. NGS and TGS, used by research laboratories, have been essential for many patients to determine a correct diagnosis, provide genetic counselling and reach an adequate treatment. This thesis focuses on molecular mechanisms of selected rare neurodegenerative diseases, namely adult neuronal ceroid lipofuscinosis (ANCL), spinal muscular atrophy (SMA) and neuronal intranuclear inclusion disease (NIID). Modern DNA sequencing methods led to identification of causal lesions in ANCL suspect patients. We provide a concept of genetic testing for SMN1 negative SMA patients and present a method for validation of tandem repeat expansion in NIID. Key words: adult neuronal ceroid lipofuscinosis, spinal muscular atrophy, neuronal intranuclear inclusion disease, next-generation sequencing methods, DNAJC5
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Molecular syndromology: molecular genetic causes of rare diseases illustrated with Kabuki and Kabuki-like syndromes
Paděrová, Jana ; Macek, Milan (advisor) ; Baxová, Alice (referee) ; Fajkusová, Lenka (referee)
Kabuki syndrome (KS) is a dominantly inherited rare disease caused mainly by de novo pathogenic variants (henceforward mutations) in the KMT2D (formerly MLL2) and KDM6A genes. It is rare multisystemic syndrome characterized by intellectual disability (ID) and typical facial dysmorphism. KS is clinically heterogeneous, which complicates its clinical diagnosis. The first aim of this thesis was to introduce mutation testing of the two known KS causative genes in KS by Sanger DNA sequencing and by MLPA (Multiple Ligation Probe Amplification) at the Department of Biology and Medical Genetics of 2nd Medical Faculty of Charles University and University Hospital Motol, Prague followed by identification of underlying genetic mutations in KMT2D/KDM6A genes in 43 patients with phenotype typical for KS, who were indicated for this molecular genetic analysis by several collaborating genetic departments in the Czech Republic. We aimed to confirm or disprove of patient's clinical diagnosis, establish spectra of KMT2D/KDM6A mutations in the Czech population, render phenotype-genotype correlations and evaluate the phenotypic "MLL2-score" (published by Makrithanasis et al., 2013) utility as prediction tool for selection of cases for KMT2D sequencing. Mutations in the KMT2D gene were detected by Sanger DNA sequencing...
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