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Novel Approaches for Molecular Diagnosis of Genetic Diseases by Next Generation Sequencing

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Novel Approaches for Molecular Diagnosis of Genetic Diseases by Next Generation Sequencing Novel Approaches for Molecular Diagnosis of Genetic Diseases by Next Generation Sequencing: Application to Breast Cancer and Retinitis Pigmentosa in the Clinical Practice Work performed at the Molecular Genetics Unit of the Terrassa Hospital (Barcelona, Spain), and partially funded by the Carlos III Health Institute, the Fund for Health Research (FIS) from the Ministry of Health and Consumption of Spain and the Fundació Joan Costa Roma, of the Consorci Sanitari de Terrassa. The cover is composed of three images. The background image is an aerial view of the Douro River, a river that connects Spain with Portugal. The second image corresponds to a DNA sequence alignment software representation and the third one, used as a background for the titles and writing, is an image of the PicoTiter Plate after a sequencing run. Cover design by Miguel Constantino Silva de Sousa Dias. English edited by Richard May. Novel Approaches for Molecular Diagnosis of Genetic Diseases by Next Generation Sequencing: Application to Breast Cancer and Retinitis Pigmentosa in the Clinical Practice A Thesis Presented to The Department of Biochemistry and Molecular Biology Universitat Autònoma de Barcelona In Fulfilment of the Requirements for the Degree Philosophiæ Doctor (PhD) in Biochemistry, Molecular Biology and Biomedicine Director: Dr. José Miguel Carballo Villarino Tutor: Dr. Jaume Farrés Vicén by Miguel Miranda de Sousa Dias December 2013 El Dr. José Miguel Carballo Villarino, cap de la Unitat de Genètica Molecular de l’Hospital de Terrassa, i el Dr. Jaume Farrés Vicén, catedràtic del Departament de Bioquímica i Biologia Molecular de la Universitat Autònoma de Barcelona, CERTIFIQUEN: Que Miguel Miranda de Sousa Dias ha realitzat sota la seva direcció i supervisió el treball d’investigació que s’exposa en la memòria titulada “Novel Approaches for Molecular Diagnosis of Genetic Diseases by Next Generation Sequencing: Application to Breast Cancer and Retinitis Pigmentosa in the Clinical Practice” per optar al grau de Doctor en Bioquímica, Biologia Molecular i Biomedicina per la Universitat Autònoma de Barcelona. Que aquest treball s’ha dut a terme a la Unitat de Genètica Molecular de l’Hospital de Terrassa. I perquè consti, signem el present certificat Dr. José Miguel Carballo Villarino Dr. Jaume Farrés Vicén Director de la Tesi Tutor de la Tesi Miguel Miranda de Sousa Dias Autor de la Tesi Terrassa, a 2 de Desembre de 2013 ii À minha mãezinha, ao meu grande pai e à maluca da minha mana, cliché ou não, esta é para vocês! iv "Nothing in Biology Makes Sense Except in the Light of Evolution" Genetics Theodosius Dobzhansky Acknowledgements Even though I am the only author signing this thesis a great number of people have left their personal touch on it. For that reason not to mention them would be extremely unfair. My heart is Portuguese and because the next words come from my heart they too are in Portuguese. Começo por quem fez tudo isto possível, o Doutor Miguel Carballo, chefe da Unidade de Genética do Hospital de Terrassa e meu director de tese. Obrigado por teres dado a este português de atitude “un poco pardilla” a oportunidade de aprender os (tantos) mistérios por trás da genética humana e de demonstrar que tem a capacidade para desenvolver todo um trabalho de investigação de qualidade contigo. À parte disso, te agradeço também o facto de teres sido um bom chefe de equipa, compreensível e sempre zelando pelos interesses de todos os membros do grupo de trabalho. Cientificamente sublime e com ideias e soluções a condizer, posso até afirmar que, dos nossos brainstormings, ideias potencialmente milionárias foram engendradas. Assim sendo, expresso também o meu sincero agradecimento pela orientação científica, pela revisão crítica desta dissertação e pelo teu contributo para o resultado final deste trabalho. Muitas vezes faltam os meios, mas Miguel e a sua varilha mágica são capazes de resolverem os mais variados obstáculos. Por essa razão também desejo manifestar a minha gratidão pela perseverança e determinação que permitiram criar as condições indispensáveis para a realização deste trabalho. Obrigado Miguel, pela honra de ser aprendiz do já chamado “Famoso Doutor Miguel Carballo”. Ao Professor Doutor Jaume Farrés, professor do departamento de Bioquímica e Biologia Molecular da Universidade Autónoma de Barcelona, apresento o meu sentido de agradecimento por ter aceitado ser meu tutor neste projecto e estender o seu apoio e disponibilidade, bem como pela revisão desta dissertação e as suas palavras de apreço generosamente dirigidas. À Professora Doutora María Carmen Martínez, professora do departamento de Bioquímica e Biologia Molecular da Universidade Autónoma de Barcelona, agradeço o facto de ter aceite fazer parte da comissão de seguimento desta tese doutoral, como presidente. Às minhas colegas do laboratório da Unidade de Genética Molecular, Alba Chacón (ainda que por pouco tempo), Beatriz Pascual, Begoña Mañé, Emma Borràs, Imma Hernan e Maria José Gamundi, que passados quase quatro anos – e depois de ter visto três novas vidas virem a este mundo – penso cá com os meus botões que este será o grupo de trabalho mais fértil onde alguma vez trabalharei. Brincadeira à parte, sem dúvida aprendi muito com cada uma de vocês nestas muitas horas partilhadas em laboratório e por esse motivo apresento o meu profundo agradecimento. vi À cantina das dez da noite e todas as pessoas responsáveis por ela, apresento também o meu agradecimento pelos pitéus tardios no Hospital de Terrassa e por manterem este espécime de grande necessidade calórica sempre bem nutrido, mesmo naqueles dias – e foram bastantes – em que o trabalho ou a escrita desta tese foram A prioridade. Ao Instituto de Salud Carlos III, ao Fondo de Investigación Sanitaria (FIS) e à Fundació Joan Costa Roma, gostaria de agradecer o apoio financeiro parcial dos projectos nos quais participei ao longo destes quatros anos de bolsa de investigação. Estendo também os meus agradecimentos ao Doutor Manel Balcells, o então director-gerente da Fundació Joan Costa Roma. Ao Alex Perez, especialista de aplicações da Roche Diagnostics, pelo fornecimento do óleo de emulsificação necessário para levar a cabo inúmeras experiencias e também pela excelente receptividade que sempre demonstrou ao longo do presente trabalho. Richard May, how could I not thank you in your mother language? My deepest thanks to you, for doing the English revision of this dissertation. Your advices were amusing and effective lectures improving both my written English and this thesis. Lorena, zemra ime, diz-se que a paciência é uma grande virtude e tu sempre tiveste tanta comigo. Obrigado por acreditares em mim, pelo teu amor e confiança desde sempre demonstrados, que nos momentos de maior esmorecimento foram tão importantes para mim. Faleminderit shume jeta ime! Por último, mas não menos importante agradeço a minha Família, nomeadamente os meus pais, irmã e os meus amigos chegados! Mãe, as despedidas doem e a saudade aperta, mas mesmo à distância consegues ser esta imensa força que me empurra para a frente. Pai, tenho muita sorte que a tua veia artística tocou esta dissertação mas é também pelo teu apoio incondicional que te agradeço. Mana, sempre disponível com opiniões válidas, agradeço-te por teres estado sempre disponível para o teu irmão predilecto e por me mostrares que afinal até não sou o “especial” da família (“Mom says I’m special!”). vii Contents Abbreviations ........................................................................................................ xii List of Tables ......................................................................................................... xv List of Figures .................................................................................................... xviii 1. Introduction ...................................................................................................... 1 1.1. Genetic Disorders ........................................................................................................ 3 1.1.1. Mendelian Inheritance Patterns .............................................................................. 4 1.1.2. Risk of Breast and Ovarian Cancer Genes, BRCA1 and BRCA2 ........................... 7 1.1.2.1. Other cancers linked to mutations in BRCA1 and BRCA2 genes .................................. 8 1.1.2.2. Patent implementation of BRCA1 and BRCA2 .............................................................. 8 1.1.2.3. Traditional clinical screening of BRCA1 and BRCA2 ..................................................... 9 1.1.3. Retinitis Pigmentosa ............................................................................................ 10 1.1.3.1. The Visual Cycle .......................................................................................................... 11 1.1.3.2. Inheritance patterns of Retinitis Pigmentosa ............................................................... 12 1.1.3.3. Molecular genetics behind adRP ................................................................................. 14 1.1.3.4. Current methods for molecular diagnosis of adRP ...................................................... 16 1.2. DNA Sequencing Technologies for Molecular Diagnosis ....................................... 17 1.2.1. The First Generation ............................................................................................ 17 1.2.2. The Next Generation ...........................................................................................
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