A comprehensive molecular and phenotypic analysis of congenital and childhood cataractRachel L. Gillespie; The University of Manchester, Doctor of Philosophy, 2015Congenital and childhood cataract (CCC) is estimated to affect 3.5-6 per 10,000 children under 16 years in developed countries - a major cause of lifelong visual impairment. It is estimated that 25-50% of CCC cases are caused by genetic mutations. CCC demonstrates extreme heterogeneity with more than 100 associated genes, and may occur as an isolated anomaly of the eye (non-syndromic) or as a manifestation of a multisystem condition (syndromic). Limitations of conventional sequencing technologies have precluded precise genetic diagnosis and limited understanding of the epidemiological basis of the condition. Next generation sequencing (NGS) technologies have revolutionised the approach to the study of human disease. The aim of this research was to conduct a comprehensive molecular and phenotypic analysis of CCC using NGS.A disease-targeted NGS assay was designed to screen, in parallel, 115 genes associated with all forms of CCC. DNA from 36 patients, randomly selected from the study cohort, underwent cataract-targeted NGS. Putative cataract-causing variants were identified in 75% of individuals; 85% of non-syndromic patients and 63% of syndromic CCC patients. Cataract-targeted NGS was able to efficiently delineate disease sub-type and in some cases identified rare syndromic forms of the condition. These findings were envisaged to alter care and management of CCC patients demonstrating the potential clinical utility of the test. In a subset of cases, NGS identified CCC was a manifestation of an inborn error of metabolism. A number of these conditions were eligible for preventative treatment emphasizing the importance of early diagnosis.A strategic approach to the identification of novel recessive causes of CCC was also undertaken. Affected children from seven consanguineous families underwent pre-screening by cataract-targeted NGS to delineate those with mutations in known genes. Mutation negative patients underwent autozygosity-guided whole exome sequencing (WES) analysis. This strategic approach to disease gene discovery led to the identification three novel cataract-causing candidate genes, TRIM8, CCDC13 and GRWD1. It also led to the association of EIF2B2, known to cause adult-onset leukoencephalopathy with vanishing white matter (VWM) disease featuring pre-senile onset cataract, with childhood-onset cataract. This work demonstrated that cataract-targeted NGS offers an efficient and unbiased means of pre-screening, however, causation is difficult to assign to novel disease genes in the absence of experimental evidence. Correspondingly, in vitro analysis of a missense variant in HMX1 demonstrated the deleterious effect of the mutation on protein function. This work confirmed HMX1 as the cause of a rare oculoauricular phenotype and expanded the class of disease-causing mutations in this gene.In conclusion, this study has demonstrated that NGS is effective in the study of CCC and has provided a platform for future studies in to the genetic aetiology of the condition, as well as the molecular mechanisms underlying lens transparency and human development. The work adds to the increasing body of evidence that augurs an era of personalised genomic medicine in ophthalmology that will foresee improved patient outcomes attributable to the implementation of a stratified approach to medicine.