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Diplomová Práce PŘÍRODOVĚDECKÁ FAKULTA Diplomová práce Adéla Horáková Brno 2019 PŘÍRODOVĚDECKÁ FAKULTA Analýza genomických defektů podmiňujících klonální evoluci chronické lymfocytární leukémie Diplomová práce ADÉLA HORÁKOVÁ Vedoucí práce: Mgr. Karla Plevová, Ph.D. Interní hematologická a onkologická klinika LF MU a FN Brno Brno 2019 Bibliografický záznam Autor: Bc. Adéla Horáková Přírodovědecká fakulta, Masarykova Ústav experimentální biologie Název práce: Analýza genomických defektů podmiňujících klonální evoluci chronické lymfocytární leukémie Studijní program: Experimentální biologie Studijní obor: Molekulární biologie a genetika Vedoucí práce: Mgr. Karla Plevová, Ph.D. Akademický rok: 2018/2019 Počet stran: 80 Klíčová slova: Chronická lymfocytární leukémie, TP53, klonální evoluce, celoexomové sekvenování Bibliographic Entry Author: Bc. Adéla Horáková Faculty of Science, Masaryk University Department of Experimental Biology Title of Thesis: Analysis of genomic defects underlying clonal evolution in chronic lymphocytic leukemia Degree programme: Experimental Biology Field of Study: Molecular Biology and Genetics Supervisor: Mgr. Karla Plevová, Ph.D. Academic Year: 2018/2019 Number of Pages: 80 Keywords: Chronic lymphocytic leukemia, TP53, clonal evolution, whole-exome sequencing Abstrakt Chronická lymfocytární leukémie je nejčastější leukémií v západním světě a její průběh je velmi heterogenní. U části pacientů dochází během onemocnění k zisku aberací spojených se zhoršením nemoci a rozvojem rezistence k protinádorové léčbě. Z klinického hlediska má zásadní význam nalezení takových znaků leukemických buněk, např. genových mutací, které by umožnily identifikaci pacientů v riziku tohoto nepříznivého průběhu. V rámci diplomové práce byla u vybraného souboru pacientů provedena analýza dat z celoexomového sekvenování a označeny geny potenciálně zodpovědné za nepříznivou klonální evoluci chronické lymfocytární leukémie. Abstract Chronic lymphocytic leukemia is the most common leukemia in the western world and its clinical course is very heterogenous. In a subgroup of patients, a gain of aberrations connected with deterioration of the disease course occurs and resistence to anticancer treatmeant often develops. From the clinical point of view, identification of progression markers of leukemic cells, for example genetic mutations, which would allow distinguishing patients at risk of unfavourable course, has a crucial importance. In the scope of this diploma thesis, analysis of whole-exome sequencing data in a selected group of patients was performed with the aim to identify genes potentially responsible for unfavourable clonal evolution of chronical lymphocytic leukemia. Poděkování Na tomto místě bych chtěla především poděkovat své školitelce Mgr. Karle Plevové, Ph.D. za pomoc při vypracování této práce, cenné odborné rady, připomínky a věnovaný čas. Dále bych chtěla poděkovat své konzultance Mgr. Kamile Stránské, za odborný dohled a pomoc při zpracování experimentální části práce a za množství velmi praktických rad. V neposlední řadě bych ráda poděkovala všem laborantkám, bioinformatikům a dalšímu odbornému personálu, který mi pomohl ze všech úskalí, která se během zpracování této práce vyskytla. A nakonec bych chtěla poděkovat své rodině, bez jejíž podpory a pomoci by tato práce nevznikla. Prohlášení Prohlašuji, že jsem svoji diplomovou práci vypracovala samostatně s využitím informačních zdrojů, které jsou v práci citovány. Text práce jsem vypracovala podle pravidel a zodpovídám za jeho jazykovou správnost. Brno, 9. května 2019 …………………………………. Adéla Horáková Obsah: 1. Seznam použitých zkratek ................................................................................................... 9 2. Úvod a problematika ......................................................................................................... 10 2.1. Chronická lymfocytární leukémie ............................................................................ 10 2.1.1. Charakteristika CLL buněk ............................................................................ 10 2.1.2. Diagnostika ..................................................................................................... 11 2.1.3. Prognóza ......................................................................................................... 12 2.1.4. Terapie ............................................................................................................ 13 2.2. Způsoby detekce genomických změn ...................................................................... 14 2.2.1. Fluorescenční in situ hybridizace (FISH) ....................................................... 14 2.2.2. Analýza metafázních chromozomů (G pruhování). ....................................... 14 2.2.3. CGH a čipové technologie .............................................................................. 15 2.2.4. MLPA a techniky založené na polymerázové řetězové reakci ....................... 16 2.2.5. Sekvenování DNA .......................................................................................... 16 2.3. Genetické abnormality a klonální vývoj CLL.......................................................... 18 2.3.1. Delece lokusu 13q14 ...................................................................................... 18 2.3.2. Delece lokusu 17p13 a mutace TP53 ............................................................. 18 2.3.3. Delece lokusu 11q22-q23 ............................................................................... 19 2.3.4. Trisomie chromozomu 12 ............................................................................... 20 2.3.5. Translokace ..................................................................................................... 20 2.3.6. Komplexní karyotyp ....................................................................................... 20 2.3.7. Další strukturní a numerické aberace ............................................................. 21 2.3.8. Somatické genové mutace a změny v expresi genů ....................................... 22 2.3.9. Klonální vývoj ................................................................................................ 29 3. Cíle diplomové práce ......................................................................................................... 30 4. Materiál a metody .............................................................................................................. 31 4.1. Sběr materiálu .......................................................................................................... 32 7 4.2. Separace buněk z periferní krve ............................................................................... 32 4.3. Analýza chromozomových aberací metodou FISH ................................................. 33 4.4. Stanovení mutace TP53 ........................................................................................... 34 4.5. Izolace DNA ............................................................................................................ 34 4.5.1. Izolace nádorové DNA pomocí QIAcube ...................................................... 35 4.5.2. Izolace nenádorové DNA ............................................................................... 35 4.5.3. Kontrola kvality a kvantity DNA ................................................................... 35 4.5.4. Hodnocení kvality DNA pomocí gelové elektroforézy .................................. 36 4.6. Celoexomové sekvenování ...................................................................................... 37 4.6.1. Příprava knihovny na sekvenování pomocí Truseq kitu ................................ 37 4.6.2. Příprava knihovny na sekvenování pomocí Nextera kitu ............................... 37 4.6.3. Měření koncentrace a hodnocení kvality DNA fragmentů ............................. 37 4.6.4. Sekvenování ................................................................................................... 38 4.6.5. Analýza dat ..................................................................................................... 39 5. Výsledky ............................................................................................................................ 41 5.1. Hodnocení pacientů ................................................................................................. 41 5.1.1. Pacienti s neexpandující mutací TP53 ............................................................ 41 5.1.2. Pacienti s expandující mutací TP53................................................................ 42 5.2. Celoxomové sekvenování ........................................................................................ 44 5.2.1. Hodnocení kvality DNA v rámci přípravy knihovny ..................................... 44 5.2.2. Kontrola správnosti přiřazení vzorků ............................................................. 45 5.2.3. Analýza získaných dat .................................................................................... 46 6. Diskuze .............................................................................................................................. 53 7. Souhrn ............................................................................................................................... 56 8. Summary ........................................................................................................................... 57 9. Přehled literatury ..............................................................................................................
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