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Title: Approaches to the analysis of genetic variation in Mendelian disease genes in a population context : cardiovascular and respiratory applications
Author: Boustred, Christopher Robert
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2012
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Genetic variation underlies the molecular basis of disease and its identification is the focus of the research underpinning both risk prediction and disease treatment strategies. The investigation of Mendelian diseases has led to the discovery of aetiological mutations, improved understanding of disease mechanisms and novel treatments for affected individuals. Furthermore, these discoveries have provided insights into biological mechanisms underlying more common multi-factorial disorders. Common genetic variation does not appear to explain the heritability of common diseases investigated to date. However, associations between common variants in Mendelian disease gene regions and complex diseases support the hypothesis that rarer variants within these genes could contribute to complex disease risk. The aim of this thesis was to develop high-throughput, cost effective methods for genetic variant screening, and apply them to the detection of rare genetic variants. High-resolution melt curve analysis (HRMCA), next generation sequencing (NGS) and statistical imputation were investigated as methods to identify rare variants. Cost efficient methods were developed by combining NGS with multi-dimensional DNA pooling and the targeting of candidate genes. The sensitivity of HRMCA was shown to be poor for regions of high GC% content. Two-dimensional pooling in conjunction with targeted NGS of known Mendelian genes was shown to be a sensitive detection technique. Associations were identified between rare variants and ECG traits in the general population. Variants identified as having a causal role in long QT syndrome (e.g. ANK2, p .Glu1425Gly) were identified in the general population, highlighting the difficulties in determining causality of genetic variants. The utility of whole exome sequencing (WES) was investigated for identifying aetiological mutations in cases of recessive respiratory disease. A known causal mutation (RSPH9 p.Lys268del) was identified in a single individual and negative molecular diagnosis was established in another. An incidentalloss-of-function mutation (p.Arg427X) in EGFL6, a gene reported to have an essential role in early human development was identified in an 11 yr old and functionally characterized. Cost efficient variant screening with NGS can be achieved in large sample sizes through target emichment and multi-dimensional pooling techniques. Optimum methods for variant screening depend on budget, genetic heterogeneity, GC% and sample size. Interpreting the effects of genetic variants on phenotypes remains a significant challenge that requires development of statistical, computational, in vivo, in vitro and systems analysis methods.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available