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Erweiterung Des Genetischen Mutationsspektrums

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Erweiterung Des Genetischen Mutationsspektrums ERWEITERUNG DES GENETISCHEN MUTATIONSSPEKTRUMS VERSCHIEDENER KRANKHEITSBILDER UND IDENTIFIZIERUNG NEUER KRANKHEITSRELEVANTER GENE IM MENSCHEN MITTELS WHOLE EXOME SEQUENZIERUNG Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Caroline Lekszas geboren in Speyer Würzburg, 2019 Eingereicht am: MITGLIEDER DER PROMOTIONSKOMMISSION: Vorsitz: Erstgutachten: Prof. Dr. Thomas Haaf Zweitgutachten: Prof. Dr. Thomas Dandekar Datum des Promotionskolloquiums: Doktorurkunde ausgehändigt am: EIDESSTATTLICHE ERKLÄRUNG nach §7 Abs. 2 Satz 3, 4, 5 der Promotionsordnung der Fakultät für Biologie Die vorliegende Arbeit wurde von März 2015 bis September 2019 am Institut für Humangenetik der Julius-Maximilians-Universität Würzburg unter der Betreuung von Univ.-Prof. Dr. Thomas Haaf angefertigt. Hiermit erkläre ich an Eides statt, die Dissertation: „Erweiterung des genetischen Mutationsspektrums verschiedener Krankheitsbilder und Identifizierung neuer krankheitsrelevanter Gene im Menschen mittels Whole Exome Sequenzierung“ eigenständig, d. h. insbesondere selbstständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen, als die von mir angegebenen Quellen und Hilfsmittel verwendet zu haben. Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat. Weiterhin erkläre ich, dass bei allen Abbildungen und Texten, bei denen die Verwertungsrechte (Copyright) nicht bei mir liegen, diese von den Rechtsinhabern eingeholt wurden und die Textstellen bzw. Abbildungen entsprechend den rechtlichen Vorgaben gekennzeichnet sind. Bei Abbbildungen, die dem Internet entnommen wurden, ist der entsprechende Hypertextlink angegeben. AFFIDAVIT The present work was conducted from March 2015 to September 2019 at the Institute of Human Genetics of the Julius-Maximilians-University Würzburg under the supervision of Prof. Dr. Thomas Haaf. I hereby declare that my thesis entitled: „Expansion of the genetic mutation spectrum of different pathologies and identification of new disease-relevant genes in humans by means of Whole Exome Sequencing” is the result of my own work. I did not receive any help or support from commercial consultants. All sources and/or materials applied are listed and specified in the thesis. Furthermore, I verify that the thesis has not been submitted as part of another examination process, neither in identical nor in similar form. Besides, I declare that if I do not hold the copyright for figures and paragraphs, I obtained it from the rights holder and that paragraphs and figures have been marked according to law. For figures taken from the internet, the hyperlink has been added accordingly. Ort, Datum Unterschrift Place, Date Signature INHALTSVERZEICHNIS 1. ABKÜRZUNGSVERZEICHNIS ..................................................................... I 2. ZUSAMMENFASSUNG ................................................................................. 1 3. SUMMARY ...................................................................................................... 2 4. EINLEITUNG .................................................................................................. 3 4.1. Wandel der molekulargenetischen Sequenziertechniken ................................ 3 4.2. Whole Exome Sequenzierung ....................................................................................... 4 4.3. Skelettdysplasien ............................................................................................................. 6 4.4. Neuropathien .................................................................................................................... 7 4.5. Zielsetzung ......................................................................................................................... 8 5. MATERIAL UND METHODEN ................................................................... 9 5.1. Material ............................................................................................................................... 9 5.1.1. Patientenproben ....................................................................................................................................... 9 5.1.2. Chemikalien, Medien und Reagenzien .......................................................................................... 11 5.1.3. Puffer- und Medien-Zusammensetzungen .................................................................................. 12 5.1.4. Bakterienstämme, Enzyme, Morpholinos, Plasmide, Zelllinien ......................................... 13 5.1.5. Kommerzielle Kits ................................................................................................................................. 13 5.1.6. Primer- und Morpholino-Sequenzen ............................................................................................. 14 5.1.7. Geräte und Hardware ........................................................................................................................... 15 5.1.8. Software und Datenbanken ............................................................................................................... 15 5.2. Methoden ......................................................................................................................... 17 5.2.1. Polymerase-Kettenreaktion und Gelelektrophorese .............................................................. 17 5.2.2. Sanger-Sequenzierung ......................................................................................................................... 18 5.2.3. Whole Exome Sequenzierung ............................................................................................................ 21 5.2.4. Homozygosity Mapping ........................................................................................................................ 22 5.2.5. In-vitro-Spleißanalyse mit Hilfe von Minigen-Konstrukten ................................................. 22 5.2.6. In-vivo-Spleißanalyse an Patientenproben ................................................................................. 30 5.2.7. Extraktion und separate Sequenzierung von Gelbanden ...................................................... 31 5.2.8. Quantitative real-time-PCR ................................................................................................................ 31 5.2.9. Untersuchung der Effekte von verschiedenen vivo Morpholinos auf das MIA3 prä- mRNA Spleißen in HeLa-Zellen ........................................................................................................ 32 6. ERGEBNISSE .............................................................................................. 34 6.1. Überblick über die Analyseergebnisse ................................................................. 34 I 6.2. Fälle mit pathogenen Mutationen in bekannten krankheitsassoziierten Genen ................................................................................................................................ 36 6.2.1. Familie IRN_1 | CPS1 ............................................................................................................................. 36 6.2.2. Familie IRN_9 | IMPAD1 ...................................................................................................................... 38 6.2.3. Familie IRN_10 | PYCR1 ....................................................................................................................... 39 6.2.4. Familie IRN_11 | DNMT3B .................................................................................................................. 41 6.2.5. Familie IRN_12 | PEX7 .......................................................................................................................... 44 6.2.6. Familie IRN_13 | INPPL1 ..................................................................................................................... 46 6.2.7. Familie IRN_15 | TRAF3IP1 ................................................................................................................ 48 6.2.8. Familie PAK_1 | GCH1 ........................................................................................................................... 49 6.2.9. Familie PAK_4 | CHST3 ........................................................................................................................ 51 6.2.10. Familie PAK_9 | MAP3K20 | NEB...................................................................................................... 54 6.2.11. Familie PAK_11 | UROC1 ..................................................................................................................... 57 6.2.12. Familie PAK_14 | SGCA ......................................................................................................................... 59 6.2.13. Familie PAK_15 | CAPN3 ..................................................................................................................... 60 6.2.14. Familie PAK_16 | MTMR2 ................................................................................................................... 63 6.2.15. Familie PAK_18 | GAN .......................................................................................................................... 64 6.2.16. Familie PAK_20 | ATM .........................................................................................................................
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