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Vrozených Srde Genetické Příčiny Vrozených Srdečních Vad Monika

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Vrozených Srde Genetické Příčiny Vrozených Srdečních Vad Monika MASARYKOVA UNIVERZITA Přírodovědecká fakulta Ústav experimentální biologie Oddělení genetiky a molekulární biologie Genetické příčiny vrozených srdečních vad Diplomová práce Monika Švarcová VEDOUCÍ PRÁCE : Mgr. Diana Nikulenkov Grochová, Ph.D. Brno 2015 Bibliografický záznam Autor: Bc. Monika Švarcová Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Název práce: Genetické příčiny vrozených srdečních vad Studijní program: Experimentální biologie Studijní obor: Molekulární biologie a genetika Vedoucí práce: Mgr. Diana Nikulenkov Grochová, Ph.D. Akademický rok: 2014/2015 Počet stran: 101 Klíčová slova: Vrozené srdeční vady; sekvenování nové generace; varianty DNA; Sangerovo sekvenování Bibliographic Entry Author: Bc. Monika Švarcová Faculty of Science, Masaryk University Department of Experimental Biology Title of Thesis: Genetic causes of congenital heart defects Degree programme: Experimental biology Field of Study: Molecular biology and genetics Supervisor: Mgr. Diana Nikulenkov Grochová, Ph.D. AcademicYear: 2014/2015 Number of Pages: 101 Keywords: Congenital heart defects; next generation sequencing; DNA variants; Sanger sequencing Abstrakt Vrozené srdeční vady (lat. vitium cordis congenitum , VCC) jsou nejčastější skupinou vrozených vad, postihují více než 1 % narozených dětí. Etiologie většiny případů není zcela známa. Příčiny VCC lze dělit na negenetické (zevní) a genetické faktory. VCC mohou být následkem různých genetických změn, jako jsou aneuploidie, polyploidie, strukturní aberace chromozomů, ale také mutace jednotlivých genů. Tyto VCC jsou často asociovány i s malformacemi jiných orgánů (tzv. syndromové VCC). Další skupinou jsou VCC izolované, tj. bez vady dalšího orgánu, a jejich příčiny jsou méně jasné. Část izolovaných vad je následkem mutací uvnitř různých genů. U zbylých případů izolovaných VCC se předpokládá multifaktoriální příčina. Publikace z posledních let popisují řadu genů, které mají přímou souvislost s rozvojem syndromových a také izolovaných VCC. Cílem této diplomové práce bylo zmapování genetické etiologie VCC u prenatálně zjištěných případů v Centru prenatální diagnostiky, s.r.o., dále výběr kandidátních genů a detekce jejich variant s využitím přístroje pro masivně paralelní sekvenování MiSeq ® (Illumina). Celkem bylo u 41 plodů analyzováno 139 typů variant DNA. Část z nich je možné dle dostupné literatury a databází řadit mezi benigní, resp. potenciálně benigní varianty. U jednoho plodu byla detekována varianta patogenní a u dalších čtyř plodů bylo nalezeno šest potenciálně patogenních variant. Většinu variant je nutno stále řadit do skupiny s nejasným významem. Vzhledem k přitažlivosti zkoumání příčin VCC lze očekávat, že se i tyto varianty dočkají jasné klasifikace. V závěru práce je prezentována kazuistika rodiny s rekurentním výskytem VCC, u které byla detekována patogenní varianta v genu TBX5 . Abstract Congenital heart defects (CHD) are the most common group of birth defects, affecting more than 1 % of births. Most of the cases don´t have a completely known etiology. Some causes of CHD can be divided into two groups - nongenetic (environmental) and genetic factors. CHD can be caused by various genetic changes such as an aneuploidy, a polyploidy, some structural aberrations of chromosomes or mutations in single genes. These CHD are often associated with some malformations of other organs (syndromic CHD). A next group is isolated CHD without any defects of other organs. Causes of isolated CHD are less clear. A part of isolated defects is an effect of some mutations within different genes. A multifactorial inheritance is supposed in some remaining cases of isolated CHD. Some recent publications describe a number of genes that are associated with a development of syndromal and isolated CHD. The main aim of this diploma thesis was to describe a genetic etiology of CHD in prenatally detected cases in Centrum prenatální diagnostiky, s.r.o., then to select candidate genes and detect their variants using a massively parallel sequencing by using MiSeq ® instrument (Illumina). Altogether 139 types of DNA variants were analyzed in 41 fetuses. Some of them belong into some benign, resp. potentially benign variants. One detected variant in one fetus is a pathogenic variant and six variants in four fetuses are potentially pathogenic variants. Most of the variants must be classed into a group of variants of an unknown significance. Due to an attractiveness of a mutation investigation there is a probability that also these variants will gain a clear significance in the future. The end of this thesis is focused on a case of a family with a recurrent occurrence of CHD where a pathogenic variant in a gene TBX5 was detected. Poděkování Na tomto místě bych ráda poděkovala Mgr. Dianě Nikulenkov Grochové, Ph.D., vedoucí této práce, a RNDr. Jitce Kadlecové, Ph.D., odborné konzultantce, za vedení, pomoc a cenné rady, jež mi věnovaly při řešení teoretické i experimentální části mé diplomové práce. Dále bych chtěla poděkovat Cytogenetické laboratoři Brno, s.r.o. za možnost zpracování práce na jejich pracovišti. Mé díky patří také všem jejím zaměstnankyním, které mi svým přijetím a radami usnadnily řešení zadané problematiky. Současně děkuji M.Sc. Fedoru Nikulenkovovi, Ph.D. za pomoc s grafickou úpravou. V neposlední řadě děkuji lékařům Centra prenatální diagnostiky, s.r.o., jmenovitě pak MUDr. Ilze Grochové, MUDr. Haně Jičínské, MUDr. Tomáši Freibergerovi a MUDr. Pavlu Vlašínovi, za konzultace a pomoc týkající se práce. Prohlášení Prohlašuji, že jsem svoji diplomovou práci vypracovala samostatně s využitím informačních zdrojů, které jsou v práci citovány. Brno 25. dubna 2015 ………………………. Bc. Monika Švarcová Obsah 1. Úvod a problematika .................................................................................................... 11 1. 1. Embryonální vývoj srdce ..................................................................................... 11 1. 2. Vrozené srdeční vady ........................................................................................... 12 1. 3. Příčiny vrozených srdečních vad ......................................................................... 13 1. 4. Sekvenování nové generace ................................................................................. 20 2. Cíle diplomové práce ................................................................................................... 23 3. Materiál a metody ........................................................................................................ 24 3. 1. Materiál ................................................................................................................ 24 3. 2. Metody ................................................................................................................. 25 4. Výsledky ...................................................................................................................... 38 4. 1. Studium databáze případů VCC, výběr vzorků pro mutační analýzu .................. 38 4. 2. Optimalizace návrhu sond pro obohacení vzorku o cílové oblasti genomu ........ 42 4. 3. Výsledky protokolu HaloPlex, příprava a úprava knihovny na sekvenování ...... 45 4. 4. Výsledné parametry kvality mutační analýzy ...................................................... 48 4. 5. Varianty DNA nalezené mutační analýzou .......................................................... 54 4. 6. Validované vybrané varianty DNA ...................................................................... 66 4. 7. Kazuistika - Holtové-Oramův syndrom ............................................................... 72 5. Diskuze ........................................................................................................................ 78 5. 1. Zhodnocení kvality mutační analýzy ................................................................... 78 5. 2. Zhodnocení výsledků mutační analýzy ................................................................ 80 5. 3. Zhodnocení kazuistiky - Holtové-Oramův syndrom............................................ 82 5. 4. Návrh dalšího průběhu studie .............................................................................. 83 6. Souhrn .......................................................................................................................... 84 7. Summary ...................................................................................................................... 86 8. Literatura ...................................................................................................................... 88 8. 1. Literární zdroje ..................................................................................................... 88 8. 2. Elektronické zdroje .............................................................................................. 98 Seznam zkratek Array CGH Komparativní genomová hybridizace na čipu (array comparative genomic hybridization) CNV Varianty v počtu kopií (copy number variations) HOS Holtové-Oramův syndrom (Holt-Oram syndrome) MLPA Multiplexová amplifikace závislá na ligaci sond (multiplex ligation dependent probe amplification) NGS Sekvenování nové (příští) generace (next generation sequencing) QF-PCR Kvantitativní fluorescenční polymerázová řetězová reakce (quantitative fluorescent polymerase chain reaction) SBS Sekvenování syntézou (sequencing by synthesis) UTR Nepřekládaná oblast (untranslated region) VCC Vrozené srdeční vady ( vitium cordis congenitum ) VOUS Varianta DNA
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