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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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Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type
Neupauerová, Jana ; Laššuthová, Petra (advisor) ; Vlčková, Eva (referee) ; Vasovčák, Peter (referee)
Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type For my PhD thesis I chose to work with patients with axonal form of CMT, because at that time axonal forms were less likely to be clarified by classical methods of molecular genetics. For further examination in patients with unclear cause of the axonal CMT, the genes DNM2, GARS and TRPV4 were selected. The aim was to determine the significance of pathogenic mutations in these genes as the cause of CMT2 in Czech patients. In the course, we identified causal variants in the genes MORC2 and SOD1 with WES. Therefore, we have tested additional CMT2 patients for the presence of these variants. Using Sanger sequencing, I examined a representative set of patients for the DNM2 (37), GARS (10) and TRPV4 (24) genes without finding a causal mutation, then we investigated genes SOD1 (43 patients) and MORC2 (161 patients). The cohort (50 patients) was also subjected to MLPA analysis using a P406-A1 CMT2 duplication and deletion detection kit for genes RAB7A, GARS, HSPB1, HSBP8 and SPTLC1 (kit P406-A1 CMT2). At that time, massively parallel sequencing (MPS) was becoming important. We compared the cost of classical sequencing versus MPS, and accordingly, we decided that the genes DNM2, GARS, MORC2, TRPV4...
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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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The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms
Staněk, David ; Laššuthová, Petra (advisor) ; Halbhuber, Zbyněk (referee) ; Kemlink, David (referee)
8 Summary The thesis "The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms" is focused on processing the massively parallel sequencing (MPS) data from a gene panel, whole-exome sequencing (WES) and whole-genome sequencing (WGS). The aim of the study was to develop a suitable pipeline to evaluate at least 250 MPS gene panel data, 150 WES data and 20 WGS data in order to improve molecular genetic testing of rare neurogenetic disorders. Associated data management and database implementation is also described. Targeted gene panel sequencing A custom-designed gene panel consisting of ge- nes previously associated with the disease was used. In the Epileptic Encephalopathy (EE) panel, two prerequisites need to be met for inclusion into the panel: the gene has to have been published in at least two independent publications OR at least in one publication but in multiple independent families. In the case of the EE panel, 112 genes were included. The targeted gene panel sequencing was then performed on 257 patients with EE. Pathogenic or likely pathogenic (according to ACMG criteria) variants have been found in 28% of patients (72 out of 257). Further analysis of the pathogenic or likely pathogenic variants was performed (76 in total); the variants were grouped by...
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Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type
Neupauerová, Jana ; Laššuthová, Petra (advisor) ; Vlčková, Eva (referee) ; Vasovčák, Peter (referee)
Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type For my PhD thesis I chose to work with patients with axonal form of CMT, because at that time axonal forms were less likely to be clarified by classical methods of molecular genetics. For further examination in patients with unclear cause of the axonal CMT, the genes DNM2, GARS and TRPV4 were selected. The aim was to determine the significance of pathogenic mutations in these genes as the cause of CMT2 in Czech patients. In the course, we identified causal variants in the genes MORC2 and SOD1 with WES. Therefore, we have tested additional CMT2 patients for the presence of these variants. Using Sanger sequencing, I examined a representative set of patients for the DNM2 (37), GARS (10) and TRPV4 (24) genes without finding a causal mutation, then we investigated genes SOD1 (43 patients) and MORC2 (161 patients). The cohort (50 patients) was also subjected to MLPA analysis using a P406-A1 CMT2 duplication and deletion detection kit for genes RAB7A, GARS, HSPB1, HSBP8 and SPTLC1 (kit P406-A1 CMT2). At that time, massively parallel sequencing (MPS) was becoming important. We compared the cost of classical sequencing versus MPS, and accordingly, we decided that the genes DNM2, GARS, MORC2, TRPV4...
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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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