PROGRAMA DE DOCTORADO EN BIOMEDICINA (B11.56.1) RARE ALLELIC VARIANTS IN MENIERE’S DISEASE FROM FAMILIAL TO SPORADIC CASES VARIANTES ALÉLICAS RARAS EN LA ENFERMEDAD DE MENIERE DE LOS CASOS FAMILIARES A LOS CASOS ESPORÁDICOS INTERNATIONAL PhD THESIS TESIS DOCTORAL CON MENCIÓN INTERNACIONAL ALVARO GALLEGO MARTINEZ February 2019 GRANADA Editor: Universidad de Granada. Tesis Doctorales Autor: Álvaro Gallego Martínez ISBN: 978-84-1306-154-2 URI: http://hdl.handle.net/10481/55469 Index Contents Index .............................................................................................................................................. 1 Grants and funding ........................................................................................................................ 3 Abstract ......................................................................................................................................... 5 Resumen ........................................................................................................................................ 6 Abbreviations ................................................................................................................................ 7 Introduction ................................................................................................................................... 9 Anatomy of the inner ear ........................................................................................................ 11 2. Meniere’s Disease ........................................................................................................... 14 3. Epidemiology ................................................................................................................... 17 4. Pathophysiology .............................................................................................................. 18 5. Human genomics ............................................................................................................. 19 6. Variants ............................................................................................................................ 21 7. Next-generation sequencing technologies in HL and MD. .............................................. 27 8. Genetics ........................................................................................................................... 28 Hypothesis ................................................................................................................................... 31 Goals ............................................................................................................................................ 35 Methods ...................................................................................................................................... 39 1. Familial MD analysis ............................................................................................................ 41 1. Diagnosis of cases ............................................................................................................ 41 2. Familial samples .............................................................................................................. 41 3. Whole-exome sequencing (WES) .................................................................................... 42 4. Pipeline testing for candidate SNV priorization .............................................................. 42 5. Benchmarking procedures .............................................................................................. 45 6. Statistical analysis ............................................................................................................ 46 7. Bioinformatics tools for rare SNV selection .................................................................... 46 8. Control datasets to filter by Spanish population variants ............................................... 47 9. Minor allelic frequency filtering ...................................................................................... 47 10. Prioritization ................................................................................................................ 47 11. Linkage analysis ........................................................................................................... 47 12. Validation by Sanger Sequencing ................................................................................ 47 13. RNA extraction from cochlea and semicircular canals ................................................ 48 14. Expression analysis in tissue ........................................................................................ 49 15. Protein 3D modelling ................................................................................................... 49 16. Variant submission ...................................................................................................... 49 2. Sporadic MD analysis........................................................................................................... 50 1. Sporadic samples ............................................................................................................. 50 2. DNA extraction ................................................................................................................ 50 3. Selection of target genes ................................................................................................. 51 4. Preparation of pools ........................................................................................................ 51 5. Haloplex protocol (capture, enrichment, barcoding) ...................................................... 51 6. Data generation pipelines ............................................................................................... 52 7. Positive control SNV validation ....................................................................................... 53 8. Selection and priorization of pathogenic SNV ................................................................. 53 9. Validation of candidate pathogenic SNV ......................................................................... 54 10. Population statistics .................................................................................................... 54 11. Position of variants in significant enriched genes ....................................................... 55 Results ......................................................................................................................................... 59 1. Prioritizing variants in exome datasets ........................................................................... 59 2. Comparison of prioritizing strategies with FMD exome datasets ................................... 59 3. Benchmark in exome datasets containing variants described in AD-SNHL and CNM genes ....................................................................................................................................... 61 4. Families study .................................................................................................................. 64 5. Sporadic cases study ....................................................................................................... 71 6. Rare variants analysis ...................................................................................................... 75 7. Mitochondrial rare variants ............................................................................................. 78 8. Gene burden analysis ...................................................................................................... 82 Discussion .................................................................................................................................... 92 1. Candidate variant selection in singletons and small families .......................................... 92 2. Familial MD ...................................................................................................................... 94 3. Sporadic MD .................................................................................................................... 98 Conclusions ............................................................................................................................... 106 Bibliography ............................................................................................................................... 108 Supplementary data 2 Grants and funding Alvaro Gallego Martinez work and this thesis was feasible thanks to the following funds: Grant from ISCII PI13/01242 Grant from Meniere’s Society, UK Grant from Luxembourg National Research Fund INTER/Mobility/17/11772209 during three months in the Bioinformatics Core of the University of Luxembourg. 3 4 Abstract Meniere’s diseases [MD; MIM 156000] is a chronic disorder characterized by attacks of vertigo associated with sensorineural hearing loss (SNHL) involving low to medium frequencies. Although its etiology remains unknown, its prevalence is about 0.5 to 1 / 1000 individuals, affecting more to familial cases than sporadic cases. MD shows a high clinical heterogeneity and incomplete phenotypic forms that complicate its diagnosis. The goal of this thesis is to obtain a better and comprehensive image of the genetics surrounding MD disease and to characterise its sporadic and familial forms. Therefore, the first goal is to increase what we know about familial