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Dizertační Práce PÍRODOVDECKÁ FAKULTA Dizertační práce MARKÉTA WAYHELOVÁ Brno 2019 PÍRODOVDECKÁ FAKULTA Analýza patogenních genetických variant u dtí s mentálními retardacemi pomocí metod komparativní genomové hybridizace na mikročipech (array-CGH) a sekvenování "nové generace" (NGS) Dizertační práce MARKÉTA WAYHELOVÁ Vedoucí práce: doc. RNDr. Petr Kuglík, CSc. Ústav experimentální biologie Brno 2019 1 Bibliografický záznam Autorka: Markéta Wayhelová Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Název práce: Analýza patogenních genetických variant u dětí s mentálními retardacemi pomocí metod komparativní genomové hybridizace na mikročipech (array-CGH) a sekvenování "nové generace" (NGS) Studijní program: Molekulární a buněčná biologie a genetika Vedoucí práce: doc. RNDr. Petr Kuglík, CSc. Akademický rok: 2019/2020 Počet stran: 164 + 19 Klíčová slova: Poruchy intelektuálního vývoje; array-CGH; varianty v počtu kopií; sekvenování „nové generace“; sekvenční varianty 2 Bibliographic entry Author: Markéta Wayhelová Faculty of Science, Masaryk University Department of Experimental Biology Název práce: The analysis of pathogenic genetic variants in children with disorders of intellectual development using methods of array-based comparative genomic hybridization (array-CGH) and „next-generation“ sequencing (NGS) Degree programme: Molecular and cell biology and genetics Supervisor: doc. RNDr. Petr Kuglík, CSc. Academic Year: 2019/2020 Number of pages: 164 + 19 Keywords: Disorders of intellectual development; array-CGH; copy-number variations; „next-generation“ sequencing; sequence variants 3 Abstrakt Poruchy intelektuálního vývoje (disorders of intellectual development, DID) se svojí průměrnou prevalencí u 1-3 % populace představují jedno z nejzávažnějších témat v oblasti zdravotní péče a každodenního života lidské společnosti. DID zahrnují soubor geneticky a fenotypově různorodých onemocnění s nástupem obvykle již v raném dětském věku, jež negativně ovlivňují život postiženého jedince ve všech aspektech života. I přes rozvinutý systém zdravotní a sociální péče o postižené jedince a jejich rodiny dosud ve většině případů zůstává neobjasněna příčina manifestace DID a asociovaných onemocnění. Celogenomové analýzy u jedinců s DID prokázaly klíčový význam genetických variant v v patogenezi DID a dalších neurovývojových onemocnění. Identifikace genetických příčin DID je podmíněna vývojem efektivního molekulárně diagnostického algoritmu, jež v sobě zahrnuje cytogenetické, molekulárně cytogenetické a molekulárně genetické metody. Metoda komparativní genomové hybridzace na DNA mikročipech (array-CGH) představuje současný „zlatý standard“ pro identifikaci patogenních submikroskopických přestaveb chromosomů (copy-number variations, CNVs). Do studie bylo vybráno 724 dětí s DID a dalšími neurovývojovými poruchami vyšetřených na Oddělení lékařské genetiky Fakultní nemocnice Brno bylo v letech 2011-2018. Metodou array-CGH byla objasněna genetická příčina uvedených patologií u 17,3 % dětí ve formě patogenních přestaveb chromosomů různého rozsahu. V 5,9 % případů byl upřesněn cytogenetický nález a u 12,3 % dětí s normálním karyotypem či s karyotypem s nálezem balancované chromosomové přestavby byla detekována alespoň 1 patogenní/pravděpodobně patogenní CNV (32 případů rekurentních mikrodelecí, 13 případů rekurentních mikroduplikací, 24 non-rekurentních mikrodelecí a 7 non-rekurentních mikroduplikací, 8 nálezů dvou klinicky významných CNVs). Dále byly v 5 % případů identifikovány varianty s nejasným vlivem na fenotyp a v 7,7 % případů byly nalezené strukturní varianty chromosomů klasifikovány jako pravděpodobně benigní. Jednotlivé varianty v daných skupinách byly dále charakterizovány z hlediska jejich původu, rozsahu a genového obsahu a získaná data byla statisticky zpracována. Byly prokázány významné rozdíly mezi velikostmi variant jednotlivých klasifikačních skupin. Další analýzou patogenních/pravděpodobně patogenních CNVs byla zjištěna vyšší četnost mikrodelecí oproti mikroduplikacím a také vyšší četnost de novo mikrodelecí oproti de novo mikroduplikacím, avšak nebyl nalezen významný rozdíl mezi jejich velikostmi. Na základě výsledků vyšetření karyotypu a array-CGH bylo v letech 2015-2018 vyšetřeno metodou cíleného sekvenování „nové generace“ (NGS) 35 dětí bez nálezu 4 patogenních přestaveb chromosomů, přičemž v rámci volby optimálního vyšetřovacího postupu byli současně s 19 dětmi vyšetřeni i jejich rodiče (trio-based NGS) a ve zbylých 16 případech bylo zvoleno Sangerovo sekvenování pro přímou verifikaci patogenní/pravděpodobně patogenních sekvenčních variant u rodičů. Pro strategii cíleného NGS byl vybrán komerční design ClearSeq Inherited Disease (Agilent Technologies, Santa Clara, CA, USA) pokrývající převážně kódující oblasti 2742 genů popsaných v patogenezi lidských vrozených onemocnění. Přístupem cíleného NGS byla identifikována příčina manifestace DID a asociovaných patologií u 25,7 % dětí (9/35), přičemž byly detekovány patogenní/pravděpodobně patogenní sekvenční varianty původu de novo v genech MED13L, ASXL3, SCN2A, TUBB3, ve stavu složené heterozygotnosti v genech TSEN54, SLC6A19 a familiální X-vázaná sekvenční varianta v genu IQSEC2 a homozygotní sekvenční varianta v genu BTD. Výsledky vyšetření metodami array-CGH, NGS a příslušných verifikací byly korelovány s fenotypem daných jedinců a předány ošetřujícícm lékařům pro účely genetického poradenství. V průběhu řešení disertační práce byla prokázána efektivita molekulárně diagnostického algoritmu a návaznost použitých metodik. Současně však s nárůstem detekce nových genetických variant jsou spojeny vyšší nároky na erudovanost kvalifikovaných pracovníků a vývoj bioinformatických sérií programů. V neposlední řadě je oblast genomických analýz spojena s právními a etickými aspekty, jež je třeba uvažovat v souladu s efektivním využití získaných informací, jejich interpretací vyšetřovaným jedincům a ochranou genomických dat před zneužitím. 5 Abstract Disorders of intellectual development and their average worldwide prevalence 1-3 % represent one of the most serious topics in the field of healthcare and everyday life of human society. They encompass a group of genetically and phenotypically heterogeneous diseases with an early onset, which negatively impact the quality of life of affected individuals. Despite the developed system of healthcare and social care for affected individuals and their families most of them do not obtain the molecular diagnosis to elucidate the cause of manifestation of DID and associated diseases. Genomic analyses in individuals with DID proved the key impact of genetic variants in the pathogenesis of DID and other neurodevelopmental disorders. The identification of genetic causes of DID is conditioned by the development of effective molecular diagnostic algorithm, including cytogenetic, molecular-cytogenetic and molecular-genetic approaches. The method of comparative genomic hybridisation on DNA microarrays (array-CGH) represents a current „gold standard“ for the identification of causative chromosomal copy- number variations (CNVs). Our study included 724 children with DID and other neurodevelopmental disorders examined at the Department of Medical Genetics of University Hospital Brno in the period from 2011 to 2018. We identified the genetic cause of mentioned pathologies in 17.3% of children in the form of chromosomal rearrangements of various sizes. In 5.9% of cases the karyotype abnormalities were specified and in 12.3% of children with normal karyotype or with karyotype carrying balanced chromosomal rearrangements we identified at least 1 causative CNV (32 cases of recurrent microdeletions, 13 cases of recurrent microduplications, 24 cases of non-recurrent microdeletions, 7 cases of non- recurrent microduplications and 8 findings of two clinically relevant CNVs). We also detected variants of unclear significance in 5% of cases and the structural chromosomal variants were classified as likely benign in 7.7% of cases. The particular variants in the classification groups were subsequently characterized in aspects of their origin, size and gene content and the data were statistically evaluated. We proved the significant differences between the CNV sizes of particular classification groups. Further analysis of causative CNVs showed the higher frequency of microdeletions than microduplications and higher frequency of de novo microdeletions than de novo microduplications. Nevertheless the significant differences between their sizes were not found. 6 Based on the results of analyses of karyotype and array-CGH 35 children without pathogenic chromosomal rearrangements were examined using targeted „next-generation“ sequencing (NGS) in the period from 2015 to 2018. To find the optimal strategy, trio-based NGS with 19 children and their parents was selected as an approach for one part of the project. The remaining 16 children were examined using targeted NGS and causative sequence variants were verified in them and their parents by Sanger sequencing. We used commercial capture design ClearSeq Inherited Disease (Agilent Technologies, Santa Clara, CA, USA) targeted to predominantly coding regions of 2742 genes involved in the pathogenesis of human inherited diseases. We identified the genetic cause of the pathological phenotype of DID and associated pathologies in 25.7% of children (9/35). We detected causative sequence variants of de novo origin in the MED13L, ASXL3, SCN2A, TUBB3 genes, compound heterozygous variants in the TSEN54 and SLC6A19 genes and finally,
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