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

线粒体功能对多种生物学过程产生影响，从而导致2型糖尿病(T2D)的发生。然而，到目前为止，人类遗传关联研究只确定了几个直接导致T2D风险的核编码线粒体基因。这篇博士论文将使用最先进的基因组分析方法系统地评估核编码的线粒体基因是否会增加T2D的风险，并将在体外对最有希望的基因进行功能分析。此外，从双胞胎家族(见下文)产生的线粒体DNA(MtDNA)下一代序列引导数据将被用于研究线粒体的“遗传瓶颈”。在PHD的过程中将研究两个独立的主要假设：1)线粒体功能障碍是导致T2D发生的原因，其风险来自于调节核编码线粒体基因表达水平的遗传变异。2)胚系和体细胞突变的mtDNA异质性比率受遗传因素的影响，这意味着积极的“质量控制”机制，以减少线粒体突变负担。学生将使用放置在精细遗传地图上的所有T2D疾病基因的基因组目录，其中包括调节约70个已识别的核编码线粒体基因的脂肪表达的疾病基因。将使用广泛的数据挖掘和生物信息学工具来确定用于功能研究的候选基因，以便探索基因组图景，包括染色质标记和表达谱。这些候选基因和/或基因组区域的功能将利用细胞系统，包括从SGBs细胞分化的人脂肪细胞和小鼠胰腺细胞，通过过度表达和shRNA介导的敲除来表征。假定的T2D遗传变异将使用CRISPR/Cas9介导的定点突变来建模，并将表征它们对细胞表型和功能的影响。线粒体DNA先导数据包括由贝勒学院使用HiSeq2000生成的12个四个家庭(包括7MZ和5DZ女性双胞胎)，这些数据将用于研究遗传瓶颈对mtDNA突变载量的影响。

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.