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Studium celogenomové variability lidského cytomegaloviru.
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Roubalová, Kateřina (referee)
This work is part of a project focused on the study of the variability of human cytomegalovirus (HCMV) among clinical isolates with the aim to map the geographical distribution of HCMV genotypes, reveal the relationships between genotypes and the severity of HCMV-associated diseases, and identify regions in the HCMV genome with a potential for use as diagnostic and therapeutic targets. Attention was paid to the development of the methodology for the preparation of the material for next-generation sequencing (NGS) from HCMV clinical isolates and evaluation of the obtained sequencing data. Blood and urine samples collected from hematopoietic stem cell transplantat recipients and congenitally infected children were analyzed. Samples suitable for NGS were sequenced by the Illumina platform and sequences were created by de novo assembly followed by mapping assembly. Urine samples in comparison to blood samples had higher yield of material for NGS. Of the samples positive for HCMV DNA (7 of 50) after amplification in the cell cultures, only one sample had high purity of the viral DNA (98%) while six samples had purity of less than 7%. The sample containing 98% of the viral DNA was fully sequenced and the sequence was compared to the sequences of other clinical isolates from Belgium in 11 polymorphic...
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Analysis of hereditary genetic variants predisposing to the development of familial forms of ovarian cancer.
Lhotová, Klára ; Soukupová, Jana (advisor) ; Mohelníková Duchoňová, Beatrice (referee) ; Weinberger, Vít (referee)
Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates preventive management for carriers of mutations in OC-susceptibility genes. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We analyzed 219 genes in 1333 Czech OC patients and 2278 population-matched controls (PMC) using next-generation sequencing. Altogether, 427/1333 (32%) patients and 58 /2278 (2,5%) PMC carried pathogenic mutations in 18 known/anticipated OC predisposition genes. Mutations in BRCA1, BRCA2, RAD51C, RAD51D, BARD1 and mismatch repair genes conferred a high OC risk (with OR>5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR ≥2 - <5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Results of this study demonstrate the high proportion...
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The relation between microbial diversity and biodegradation of organic pollutants in soils
Adámek, Michael ; Svobodová, Kateřina (advisor) ; Matyska Lišková, Petra (referee)
This work sums up the knowledge of the currently studied microorganisms capable of degradation of organic compounds contaminating soil and of the impact of biodiversity on biodegradation efficiency. As documented in many studies effective soil remediation can be achieved by mixed-species consortia isolated from polluted soils. However, use of these cultures for bioaugumentation requires further research on their influence on the biodiversity of autochthonic soil microflora. Though bioaugumentation provably affects bioremediation effectivity no ideal carrier for microbes has been found yet that would provide survival of the introduced organisms in the competitive soil environment. Next, selection of suitable bioaugumentation agents should be based on previous analyses of autochthonic microbes in the targeted contaminated soils. Further, this work shows that the presence of key species might be more important for the biodegradation efficiency than biodiversity of soil microflora. Biodiversity is more related to the functionality of soil ecosystems which can be affected by the presence of contaminants leading to positive selection of taxa capable of pollutant degradation. However, there are just a few studies on the relationship between biodiversity and degradation of pollutants. Its further research...
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Genetically determined progression factors of selected chronic nephropathies
Obeidová, Lena ; Reiterová, Jana (advisor) ; Skálová, Sylva (referee) ; Vodička, Radek (referee)
Polycystic kidney disease is a severe genetic disease occurring in both adult and pediatric patients. The basic characteristic of this disease is the development and progressive enlargement of renal cysts gradually replacing functional kidney tissue. This leads to renal failure in many patients. However, renal cysts may also occur in a number of other diseases, including multisystem syndromes. This complicates differential diagnosis in some patients. In our study, we first focused on the diagnosis and characterization of genotypic-phenotypic relationships in patients with polycystic disease arising in childhood, later we extended our study to adult patients and patients with unclear clinical diagnosis. At the same time, we expanded the portfolio of analyzed disorders to a number of diseases in which the phenotype of polycystic kidneys may occur, and noncystic diseases as well. During our project, massive parallel sequencing was used to analyze 149 patients - 128 with cystic and 21 with noncystic clinically diagnosed nephropathies. At the same time, the findings were verified by Sanger sequencing in 176 relatives of our probands. Mutation detection reached 59% in cystic patients, and 43% in non-cystic patients, respectively. In many patients, molecular genetic analysis revealed a different etiology...
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The analysis of immunoglobulin and T-cell receptor gene rearrangements using next generation sequencing
Hašek, Daniel ; Froňková, Eva (advisor) ; Javorková, Eliška (referee)
DNA sequencing is a molecular genetic method that results in data about sequence and type of nucleotides present in a given sample of deoxyribonucleic acid (DNA), a molecular carrier of genetic information. These data are frequently of a crucial value for many fields; research, medicine, industry, criminalistics or others. During a long period of time almost all the sequencing was performed using a method invented by Frederick Sanger in the 70's, a technique that uses modified nucleotides that once incorporated into a DNA strand prevent this from further elongation. DNA synthesis in presence of such nucleotides leads to a formation of a mixture of fragments of different lenght that are electrophoretically separated by lenght and the sequence is read from the resulting gel. Since the principle of this method entails some inherent drawbacks (e.g. low throughput and coverage) a significant effort is made lately to develop alternative sequencing approaches. These methods colectively refered to as next-generation sequencing (NGS) use several technologies in order to overcome the limitations of the Sanger sequencing. This thesis discusses the most important NGS methods and focuses on their possible application for sequencing of immunoglobulin and T-cell receptor gene rearrangements, an area of undisputable...
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Determining the genetic cause of short stature as a way to understand the pathophysiological mechanisms affecting human growth
Plachý, Lukáš ; Průhová, Štěpánka (advisor) ; Zapletalová, Jiřina (referee) ; Pomahačová, Renata (referee)
Determining the genetic cause of short stature as a way to understand the pathophysiological mechanisms affecting human growth Abstract Short stature is one of the most common disorders followed-up by a paediatric endocrinologist. Pathophysiologic mechanisms leading to growth disorders are complex, however, the exact cause is mostly unknown. Our study is the first to evaluate the aetiopathogenesis of familial short stature (FSS). Using next-generation sequencing (NGS) techniques, we aimed to describe the monogenic aetiology of growth impairment in a group of FSS families, and therefore to elucidate mechanisms leading to this specific growth disorder. In selected genetic diagnoses, we additionally aimed to describe the phenotype including GH treatment response. Within Motol University Hospital centre for GH therapy, we formed a group of 98 FSS families with clear height definition in ≤-2 SD in both the child height before GH therapy and in his shorter parent. Using NGS, the FSS aetiology was elucidated in 40/98 (41%) families; 32/40 had a genetic growth plate disorder. Within the cohort, three genetically homogeneous subgroups of families were described (collagenopathies - 10/98 [10.2%] families, SHOX deficiency - 6/98 [6.1%] families, and C type natriuretic peptide receptor disorder - 4/98 [4.1%] families)....
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Anterior segment dysgenesis disorders and their molecular genetic cause
Moravíková, Jana ; Lišková, Petra (advisor) ; Krulová, Magdaléna (referee)
Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...
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Detection of chimeras in amplicon sequencing
Heřmánková, Kristýna ; Jurečková, Kateřina (referee) ; Sedlář, Karel (advisor)
Chimeric sequences are the most common artifacts that can occur in sequencing data after the sample amplification using the polymerase chain reaction. The presence of these artifacts can negatively affect results of the analysis. Therefore, the detection and subsequent filtration of chimeric sequences is an important step in the computational processing of sequencing data. This work deals with the principle of chimera formation and the possibility of reducing their occurrence. The aim of this work is to implement an algorithm for chimeras detection in R language and testing its accuracy on data provided by the Veterinary Research Institute in Brno.
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Identification of hereditary alterations predisposing to breast cancer development using "next-gen" sequencing
Lhota, Filip
Summary: Breast cancer (BC) is the most frequent cancer type in female population of Europe. Approximately 5 - 10 % accounts for its hereditary form which is characterized by high penetrance, early onset, risen recurrence risk and development of other cancers. Mutational analyses of high risk patients identify a predisposing mutation in one of the most studied genes (BRCA1, BRCA2, TP53, ATM, CHEK2, NBS1, PALB2) only in less than one third of tested breast cancer patients. Lately, with the use of new methods of next-generation sequencing, a number of other susceptibility or candidate genes were characterized, but the incidence of their pathogenic alteration is often geographically different. A notable proportion of high risk patients from families with hereditary BC can represent carriers of population-specific, or private mutations. Most of the to date identified BC susceptibility genes codes for proteins involved in DNA repair, especially repair of double strand break DNA repair. Nevertheless the mutation analysis was conducted only on a small fraction of these DNA repair genes. We can expect that in the group of yet nontested genes coding for DNA repair proteins a rare, but clinically important genetic alterations predisposing to BC in affected families can be discovered. This work describes a...
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Analysis of hereditary genetic variants predisposing to the development of familial forms of ovarian cancer.
Lhotová, Klára ; Soukupová, Jana (advisor) ; Mohelníková Duchoňová, Beatrice (referee) ; Weinberger, Vít (referee)
Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates preventive management for carriers of mutations in OC-susceptibility genes. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We analyzed 219 genes in 1333 Czech OC patients and 2278 population-matched controls (PMC) using next-generation sequencing. Altogether, 427/1333 (32%) patients and 58 /2278 (2,5%) PMC carried pathogenic mutations in 18 known/anticipated OC predisposition genes. Mutations in BRCA1, BRCA2, RAD51C, RAD51D, BARD1 and mismatch repair genes conferred a high OC risk (with OR>5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR ≥2 - <5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Results of this study demonstrate the high proportion...
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