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Exome Sequencing in Gastrointestinal Food Allergies Induced by Multiple Food Proteins

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Exome Sequencing in Gastrointestinal Food Allergies Induced by Multiple Food Proteins Alba Sanchis Juan Department of Biotechnology Universitat Politècnica de València Supervisors: Dr. Javier Chaves Martínez Dr. Ana Bárbara García García Dr. Pablo Marín García This dissertation is submitted for the degree of Doctor of Philosophy September 2019 If you know you are on the right track, if you have this inner knowledge, then nobody can turn you off... no matter what they say. Barbara McClintock Declaration FELIPE JAVIER CHAVES MARTÍNEZ, PhD in Biological Sciences, ANA BÁRBARA GARCÍA GARCÍA, PhD in Pharmacy, and PABLO MARÍN GARCÍA, PhD in Biological Sciences, CERTIFY: That the work “Exome Sequencing in Gastrointestinal Food Allergies Induced by Multiple Food Proteins” has been developed by Alba Sanchis Juan under their supervision in the INCLIVA Biomedical Research Institute, as a Thesis Project in order to obtain the degree of PhD in Biotechnology at the Universitat Politècnica de València. Dr. F. Javier Chaves Martínez Dr. Ana Bárbara García García Dr. Pablo Marín García Acknowledgements Reaching the end of this journey, after so many ups and downs, I cannot but express my gratitude to all those who supported me and helped me through this challenging but rewarding experience. First and foremost, I would like to thank my supervisors, Dr. Javier Chaves Martinez, Dr. Ana Barbara García García and Dr. Pablo Marín García. They gave me the opportunity to work with them when I was still an undergraduate student, and provided me the opportunity to contribute to several exciting projects since then. They also encouraged me into the clinical genomics field and provided me guidance and advice throughout the last four years. I am also grateful to my mentor Prof. José Gadea Vacas for his support during this time. Thank you to the additional members of the Genotyping and Genetic Diagnostics Unit from the INCLIVA research foundation. I very much enjoyed being part of this big team, and learned a lot from it. Thank you to the Garmitxa organisation for generously funding this work and the first year of my PhD. I am also grateful to all patients who participated in the study, without whose generosity none of this work would have been possible. I am extremely grateful to my superiors at University of Cambridge, Prof. Lucy Raymond and Dr. Keren Carss. They provided a huge viii amount of support during my project, and were very understanding with me combining my PhD with my job at University of Cambridge. They encouraged, mentored and inspired me throughout my career. I have been very fortunate to work with them. Thank you to Dr. Courtney French for her most valuable comments to this dissertation. I am also grateful to all my colleagues from University of Cambridge and Universitat Politècnica de València, specially Dr. Detelina Grozeva, Dr. Beatriz Ballester Llobell and Dr. Marcia Hasenahuer, for their motivation and support especially in the most difficult moments. I thank Prof. Willem Ouwehand and the NIHR BioResource for providing me the opportunity to work in this stimulating group. I am grateful for all the opportunities that had arisen during this time. Last, but not least, I would like to thank my parents and my sister for their unconditional support, for their encouragement and for believing in me. I also thank my friends, but especially I am grateful to my endlessly supportive partner Mateus Patricio, who motivated me to go every weekend to the library, rain or shine, to make this thesis possible. Agradecimientos Llegando al final de este viaje, después de tantos altibajos, nopuedo dejar de expresar mi gratitud a todos aquellos que han estado conmigo a lo largo esta dura pero gratificante experiencia. En primer lugar, me gustaría agradecer a mis supervisores, el Dr. Javier Chaves Martínez, la Dra. Ana Barbara García García y el Dr. Pablo Marín García, por abrirme las puertas a su grupo cuando aún era estudiante. Gracias por introducirme en el campo de la genómica clínica y por brindarme orientación y asesoramiento durante los últimos cuatro años. Asimismo, me gustaria agradecer a mi tutor, el Profesor José Gadea Vacas, por todo su apoyo durante este tiempo. Gracias a los compañeros de la Unidad de Genotipado y Diagnóstico Genético de la fundación de investigación INCLIVA.He disfrutado de ser parte de este gran grupo y he aprendido mucho de todos vosotros. Gracias a la organización Garmitxa por financiar generosamente este trabajo y el primer año de mi doctorado. También quiero agradecer a todos los pacientes que han participado en el estudio, sin cuya generosidad no hubiera sido posible. Gracias a mis supervisoras en la Universidad de Cambridge, la Profe- sora Lucy Raymond y la Dr. Keren Carss, por su enorme apoyo durante mi proyecto y por ser tan comprensivas conmigo compaginando mi doc- x torado con mi trabajo en la Universidad de Cambridge. Me animaron, me orientaron y me inspiraron a lo largo de mi carrera. He sido muy afortunada de trabajar con ellas. Gracias a la Dr. Courtney French por los valiosos comentarios en el desarrollo de esta tesis. También estoy agradecida a todos mis com- pañeros de la Universidad de Cambridge y de la Universitat Politècnica de València, especialmente a la Dra. Detelina Grozeva, a la Dra. Beatriz Ballester Llobell y a la Dra. Marcia Hasenahuer, por su motivación y apoyo, especialmente en los momentos más difíciles. Gracias al Profesor Willem Ouwehand y al NIHR BioResource por brindarme la oportunidad de trabajar en este estimulante grupo. Estoy agradecida por todas las oportunidades que han surgido durante este tiempo. Por último, y más importante, me gustaría agradecer a mis padres y a mi hermana por su apoyo incondicional y por creer en mí. También agradezco a mis amigos, pero especialmente a Mateus Patricio, que me apoyó incesantemente y me motivó a ir todos los fines de semana ala biblioteca, llueva o truene, para hacer posible esta tesis. Abstract The study of genetics has been making significant progress towards understanding the causes of rare and common disease during the past decades. Across a wide range of disorders, there have been hundreds of associated loci identified and associated with multiple disorders. Now, with the advent of next-generation sequencing technologies, we are able to interrogate the contribution of high and low frequency variation to disease in a high throughput manner. This provides an opportunity to investigate the role of rare variation in complex disease risk, potentially offering insights into disease pathogenesis and biological mechanisms. In this thesis, it has been assessed the use of whole-exome sequenc- ing technology to investigate the role of rare variation in a complex disease, gastrointestinal food allergy induced by multiple food proteins. For that, a cohort of 31 individuals (eight affected and 23 non-affected) from seven different families was whole exome sequenced. Data ob- tained from multiple sequencing systems and libraries were analysed, and a workflow was developed, focusing on a comprehensive quality control to maximise the number of real positive calls. Different types of genome variations were investigated, including single nucleotide vari- ants, insertions/deletions, copy number variants and HLA haplotypes. By approaching different methods of variant filtering, a set of rare variants xii that could be associated with the disease was identified. The possible role of these candidate variants in the pathogenesis of gastrointestinal food allergies was also discussed. These results reveal important insights into the genetic architecture of gastrointestinal food allergies and lead to additional lines of investigation that will be required in order to fully understand the genetic basis of this disease. Resumen Durante las últimas décadas, se han realizado importantes avances en el estudio de las causas genéticas de enfermedades raras y comunes, donde un gran número de variantes han sido identificadas y asociadas a múltiples enfermedades. Con las tecnologías de secuenciación de nueva generación, hoy en día somos capaces de investigar, con un alto rendimiento, la contribución de variantes de alta y baja frecuencia a dis- tintos tipos de enfermedades, permitiéndonos así estudiar su importancia en el desarrollo de las mismas. En ésta tesis se ha utilizado la secuenciación del exoma como tec- nología para el estudio de variantes raras en una enfermedad compleja, la alergia gastrointestinal inducida por múltiples alimentos. Para ello, se realizó la secuenciación del exoma completo de una cohorte de 31 indi- viduos (ocho afectados y 23 no afectados) provenientes de siete familias diferentes. Se desarrolló un flujo de trabajo para procesar los datos gen- erados a partir de diferentes librerías e instrumentos de secuenciación, así como un control de calidad exhaustivo con el fin de maximizar el número de variantes de alta calidad. Diferentes tipos de mutaciones fueron investigadas, incluyendo polimorfismos de nucleótido único, in- serciones/deleciones, variantes del número de copia y haplotipos HLA, y se realizaron diferentes métodos de filtrado para su interpretación. xiv Finalmente, se encontraron una serie de mutaciones que podrían estar asociadas con la enfermedad y se describe su posible papel en la patogénesis de las alergias gastrointestinales. Los resultados de esta tesis suponen importantes avances en el estudio de la compleja arqui- tectura genética de las alergias gastrointestinales y abren las puertas a futuras líneas de investigación, que serán necesarias para entender completamente las bases genéticas de esta enfermedad. Resum Durant les últimes dècades, s’han realitzat importants avanços en l’estudi de les causes genètiques de malalties rares i comunes, on un gran nombre de variants han sigut identificades i associades a múltiples malalties. Amb les tecnologies de seqüenciació de nova generació, avui en dia som capaços d’investigar, amb un alt rendiment, la contribució de variants d’alta i baixa freqüència a diferents tipus de malalties, permetent-nos així estudiar la seva importància en el desenvolupament de les mateixes.
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