The role mitochondrial genetics in the development of type 2 diabetes.

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SYNOPSIS: Mitochondrial function impacts upon many biological processes that implicate type 2 diabetes (T2D). To date, however, human genetic association studies have identified only a few nuclear encoded mitochondrial genes that directly confer risk of T2D. This PhD thesis will systematically assess whether nuclear-encoded mitochondrial genes confer risk of T2D using cutting-edge genomic analytical methods and the most promising genes will be functionally analysed in vitro. In addition, mitochondrial DNA (mtDNA) next generation sequence pilot data generated from twin families (see below) will be utilised to investigate mitochondrial "genetic bottlenecks".Two independent primary hypotheses will be investigated during the course of the PhD:1) Mitochondrial dysfunction causally contributes to the development of T2D, with risk conferred by inherited genetic variants that regulate the expression levels of nuclear-encoded mitochondrial genes. 2) mtDNA heteroplasmy rates for germ-line and somatic mutations are influenced by heritable factors, implying active "quality control" mechanisms that reduce mitochondria mutational burden.The student will use a genomic catalogue of all T2D disease loci placed upon fine-scale genetic maps that includes disease loci that regulate adipose expression of approximately 70 identified nuclear-encoded mitochondrial genes. Candidate genes for functional investigation will be identified using a wide range of data mining and bioinformatics tools, in order to explore the genomic landscape, including chromatin marks and expression profiles. The function of these candidate genes and/or genomic regions will then be characterised using cellular systems, including differentiated human adipocytes from SGBS cells and mouse pancreatic cells, by means of over-expression and shRNA-mediated knockdown. Putative T2D genetic variants will be modelled using CRISPR/Cas9-mediated site-directed mutagenesis and their effect on cellular phenotype and function will be characterised. The mtDNA pilot data includes 12 families of four (including 7MZ and 5DZ female twin pairs) generated by Baylor College using HiSeq2000, which will be used to study the impact of genetic bottlenecks on mtDNA mutational load.