Understanding the genetics of obesity- from GWAS to function in a canine model

博士项目战略主题：生物科学促进对健康的综合了解肥胖是一个全球健康问题，其特点是发病率和死亡率高，从而对公共卫生系统产生负面影响。这种复杂的疾病在人类中具有高度的遗传性(40%-70%)。尽管如此，常见肥胖症的多基因性质限制了研究人员精确定位调节体重的重要基因和确定新的治疗靶点的能力。针对体重指数(BMI)和相关性状的全基因组关联研究(GWAS)已经在人类中发现了500个肥胖相关基因座，但仍然只解释了BMI变异的约4%。与人类肥胖有关的环境因素，如运动量减少和容易获得高能量食物，在他们的犬类伴侣动物中变得更加常见，导致大约59%的狗超重或肥胖。狗的品种为发现疾病基因提供了一个强大的遗传模型，因为遗传瓶颈导致了异质性的减少、广泛的连锁不平衡(LD)和疾病变异频率的增加。与人类相比，在种内GWA中需要更小的样本量和更少的遗传标记。Raffan团队之前已经对127只瘦或肥胖的哈巴狗进行了GWAS肥胖测试，多个基因座达到了基因组范围的意义，但这些基因座很大，很难准确定位原因突变。在我的博士项目中，通过将样本输入到更大的SNP密度，执行二次GWAS和精细定位基因座，将改进这一PUG GWAS。我们将利用生物信息学方法(如基因本体论(GO)浓缩、转录因子结合分析、DNA元件和组织特异性基因表达分析)、人工筛选和文献综述，在功能注释的基础上确定肥胖的候选风险变量。将基因座与突变和功能联系起来的最佳方法依赖于使用生物信息学和细胞研究的多因素方法。我们将根据候选基因进行相关实验，包括测量基因表达、蛋白质定位、启动子活性的技术，以及可能的基因组编辑技术，这些技术允许直接比较相同基因组背景中的突变。已经有几种生物途径参与了体重调节，包括下丘脑瘦素-黑素皮质素途径在控制能量摄入和消耗方面的中心作用。人类的严重肥胖有时是由于编码该途径成员的基因功能的突变引起的，这些基因包括瘦素及其受体POMC、负责POMC蛋白分解的酶和黑素皮质素受体。已经在狗身上发现并在一定程度上研究了黑素皮质素受体的三个突变。拉凡博士实验室未发表的工作表明，所有这三种突变都对黑素皮质素信号在刺激下的反应产生了显著影响。我们预测，这些功能差异将导致肥胖表型的可测量差异。我博士的这一目标将测量这些突变在30个品种以及混合品种狗中的频率。我们将通过测试这些突变与受影响品种的体重、肥胖和食物动机的关系来进行研究。

PhD project strategic theme: Biosciences for an integrated understanding of healthObesity is a global health problem characterised by high morbidity and mortality rates that consequently have a negative impact on public health systems. This complex disease has a high degree of heritability in humans (40-70%). Despite this, the polygenic nature of common obesity has limited researchers' ability to pinpoint genes important in the regulation of body weight and identify novel therapeutic targets. Genome wide association studies (GWAS) for body mass index (BMI) and related traits have led to the identification of >500 adiposity associated loci in humans, while still only explaining ~4% of the variation in BMI. The environmental factors implicated in human obesity such as reduced exercise and easy access to high-energy food have simultaneously become more common in their canine companion animals leading to approximately 59% of dogs being overweight or obese. Dog breeds provide a powerful genetic model for the discovery of disease genes as genetic bottlenecks have led to a reduction in heterogeneity, extensive linkage disequilibrium (LD) and increased frequency of disease-variants. Compared to humans, smaller sample sizes and fewer genetic markers are needed in within-breed GWAS. The Raffan group have previously carried out a GWAS for obesity in 127 lean or obese pug dogs with multiple loci reaching genome-wide significance, however the loci are large making it difficult to pinpoint the causal mutations. In my PhD project, this pug GWAS will be improved by imputing the samples up to a larger SNP density, performing a secondary GWAS and fine mapping the loci. We will identify candidate risk variants for obesity on the basis of functional annotation using bioinformatics approaches (e.g. gene ontology (GO) enrichment, transcription factor binding analysis, DNA element and tissue specific gene expression analysis), manual curation and literature review. The best approach to linking loci to mutations and then function relies on a multifactorial approach using bioinformatics and cellular studies. We will perform relevant experiments depending on candidate genes including techniques to measure gene expression, protein localisation, promoter activity and possibly genome editing techniques which allow direct comparison of mutations in identical genomic backgrounds.Several biological pathways have already been implicated in the regulation of body weight including a central role of the hypothalamic leptin-melanocortin pathway in controlling energy intake and expenditure. Severe obesity in humans is sometimes caused by mutations disrupting the function of the genes encoding members of this pathway including leptin, its receptor, POMC, the enzymes responsible for proteolytic cleavage of POMC, and melanocortin receptors. Three mutations in melanocortin receptors have been identified and studied to some extent in dogs. Unpublished work from Dr Raffan's lab demonstrated that all three mutations had significant effects on melanocortin signalling in response to stimulation. We predict that these functional differences will cause measurable differences in obesity phenotypes. This aim of my PhD will measure the frequency of these mutations in 30 breeds as well as mixed breed dogs. We will follow this by testing the association of these mutations to body weight, adiposity and food-motivation in affected breeds.