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University of Southampton Research Repository University of Southampton Research Repository Copyright © and Moral Rights for this thesis and, where applicable, any accompanying data are retained by the author and/or other copyright owners. A copy can be downloaded for personal non‐commercial research or study, without prior permission or charge. This thesis and the accompanying data cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content of the thesis and accompanying research data (where applicable) must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holder/s. When referring to this thesis and any accompanying data, full bibliographic details must be given, e.g. Thesis: Author (Year of Submission) "Full thesis title", University of Southampton, name of the University Faculty or School or Department, PhD Thesis, pagination. Data: Author (Year) Title. URI [dataset] UNIVERSITY OF SOUTHAMPTON Faculty of Medicine Human Development & Health The identification of causal variants in nystagmus and primary open-angle glaucoma patients through analyses of next-generation sequencing data Author: Luke O’Gorman Supervisory Team: Prof Sarah Ennis, Dr Jane Gibson, Ms Angela Cree, Mr Jay Self and Prof Andrew Lotery July 2019 i Abstract UNIVERSITY OF SOUTHAMPTON Faculty of Medicine Human Development & Health Doctor of Philosophy The identification of causal variants in nystagmus and primary open-angle glaucoma patients through analyses of next-generation sequencing data by Luke O’Gorman Background As next-generation sequencing (NGS) becomes more feasible and accessible, its applica- tion in medical genetics becomes increasingly prevalent. Ophthalmic diseases including primary open-angle glaucoma (POAG), oculocutaneous albinism and nystagmus have a strong underlying genetic basis. Understanding the genetic basis is therefore important to facilitate early diagnoses, and aid clinicians in directing targetted treatment to pro- vide best outcome for patients. The molecular basis remains undetermined for many patients. However, high-throughput sequencing and development of bioinformatic tools to process such data has enabled a robust capacity to identify likely causal variants in disease. Aims By analysing targetted and whole-exome sequencing (WES) data on patients individu- ii ally and across cohorts, this thesis aims to interrogate candidate genes which are known or predicted to be causal in POAG, albinism and nystagmus. From these analyses, much needed further insight into causal variants underpinning these ophthalmic diseases can be provided and, importantly, a molecular diagnosis for the patients involved in this work could be ascertained. Outcomes The work presented demonstrate herein the importance of TYR tri-allelic genotype in oculocutaneous albinism. With the realisation of its importance in molecular diagnosis of oculocutaneous albinism, it was demonstrated how the tri-allelic TYR genotype could be successfully incorporated into a diagnostic workflow. Furthermore, we were also able to demonstrate that by employing a similar workflow across consanguineous Pakistani ophthalmic disease patients, comparably high diagnostic yields were returned. This thesis confirms that 3.07% of British patients with POAG had likely causal variants in the coding region of the most common causal POAG gene, myocilin (MYOC ). There- fore, genetic testing for patients in MYOC coding sequences (namely exon 3) remains the most efficient plan of action when screening this gene. Other known Mendelian-like genes in POAG were able to further account for POAG in 1.17% of the UK POAG cohort. Functional assessment and larger samples sizes are required to test new hy- potheses implicating molecular pathologies and other genes as Mendelian-like. Conclusion This thesis has expanded on the known molecular genetics underpinning ophthalmic diseases. Importantly, this work has informed clinicians and geneticists on diagnostic workflows and the variants which should be considered in order to achieve successful molecular diagnoses in patients. Crucially, for many of the cases detailed in this work, a molecular diagnosis has successfully been made which will ultimately help ensure the iii best possible care for the patients involved. iv CONTENTS Contents Abstract ii Contents xiii List of Figures xvi List of Tables xxi Acknowledgements xxii Ethics Approval xxiii Funders xxiv Declaration of Authorship xxv Publications xxvi Abbreviations xxviii 1 Background1 1.1 Prevalence of ophthalmic diseases.....................1 1.2 Nystagmus..................................4 1.2.1 Description.............................4 1.2.2 Diagnosis..............................5 1.2.3 Genetic cause............................5 1.2.4 Treatment..............................9 1.3 Primary open-angle glaucoma....................... 10 1.3.1 Description............................. 10 1.3.2 Diagnosis of POAG......................... 12 v CONTENTS 1.3.3 Genetic causes of POAG...................... 13 1.3.4 Myocilin............................... 14 1.3.5 Treatment.............................. 15 1.4 Methods of genetic analysis........................ 16 1.4.1 Genetic linkage........................... 16 1.4.2 Genome wide association studies................. 18 1.4.3 DNA sequencing.......................... 20 1.4.3.1 Sanger sequencing.................... 20 1.4.3.2 Next (second) generation sequencing technology.... 22 1.4.3.3 Next (third) generation sequencing technology..... 25 1.4.3.4 NGS pipelines....................... 26 1.4.3.5 Whole exome sequencing................. 28 1.4.3.6 Whole genome sequencing................ 29 1.4.3.7 Somatic mutations.................... 30 1.4.3.8 Custom targeted sequencing............... 30 1.5 Aims..................................... 33 2 Targeted sequencing and analysis of the myocilin gene (MYOC ) in a selected cohort of primary open-angle glaucoma patients 35 2.1 Synopsis................................... 35 2.2 Background................................. 35 2.3 Aim..................................... 38 2.4 Methods................................... 39 2.4.1 Patient selection.......................... 39 2.4.2 Target selection........................... 39 2.4.3 Quality control on kit design.................... 40 2.4.4 Targeted sequencing........................ 41 vi CONTENTS 2.4.5 Bioinformatic pipeline....................... 41 2.4.6 Quality control of NGS data.................... 42 2.4.7 Variant contextualisation...................... 43 2.4.8 Prioritisation of variants...................... 44 2.5 Results.................................... 45 2.5.1 Patient clinical traits........................ 45 2.5.2 Quality control........................... 47 2.5.2.1 Quality control on kit design.............. 47 2.5.2.2 Quality control of NGS data............... 47 2.5.3 MYOC variant contextualisation................. 51 2.5.4 Exonic variants........................... 53 2.5.5 Non-coding variants........................ 57 2.6 Discussion.................................. 59 3 Next-generation sequencing analysis of 66 POAG genes across a se- lected cohort of severe primary open-angle glaucoma patients 65 3.1 Synopsis................................... 65 3.2 Background................................. 65 3.2.1 Mendelian-like genes........................ 66 3.2.2 POAG genes identified through GWAS.............. 69 3.2.3 Paucity of associations in POAG GWAS............. 70 3.2.4 Database of disease-associated variants.............. 71 3.2.5 NGS to identify causal genes and variants............ 72 3.2.6 Aim................................. 72 3.3 Methods................................... 72 3.3.1 Patient selection.......................... 72 3.3.2 Gene selection............................ 73 vii CONTENTS 3.3.3 Target gene capture and sequencing................ 73 3.3.4 Bioinformatic pipeline and filtering parameters.......... 73 3.3.5 Filtering of variants......................... 73 3.3.6 Interacting genes.......................... 75 3.3.7 Whole-gene pathogenicity..................... 76 3.4 Results.................................... 77 3.4.1 Patient clinical traits........................ 77 3.4.2 Gene selection for customised panel................ 78 3.4.3 Capture kit selection........................ 78 3.4.4 Quality control........................... 82 3.4.5 Filtering of variants......................... 82 3.4.5.1 Standard filtering (Mendelian-like genes, n=7)..... 83 3.4.5.2 Stringent filtering of complex POAG genes (coding genes, n=56)........................... 86 3.4.5.3 Filtering of non-coding genes (ncRNA, n=3)...... 88 3.4.6 Interacting proteins......................... 88 3.4.6.1 Indirect protein-protein interactions.......... 88 3.4.6.2 Direct protein-protein interactions........... 89 3.4.7 Whole gene pathogenicity scores.................. 90 3.4.7.1 Batch effect........................ 91 3.4.7.2 Overview of whole gene pathogenicity scores across the POAG cohort....................... 92 3.4.7.3 Comparing POAG whole gene pathogenicity scores against a control cohort...................... 96 3.4.8 Copy Number Variants (CNVs).................. 100 3.5 Discussion.................................. 102 viii CONTENTS 3.5.1 The seven Mendelian-like genes.................. 102 3.5.2 Assumed complex genes...................... 105 3.5.3 Limitations............................. 110 3.6 Conclusion.................................. 111 4 Nystagmus whole exome
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