Authors Alyce McClellan (1), Natalie Wallis (1), Brian Lam (2), Aqfan Jamaluddin (3), Katherine Kentistou(3), John Perry (2,4), Sadia Saeed (5), Caroline Gorvin (3), Eleanor Raffan (1)
Affiliations 1. Department of Physiology Development and Neuroscience, University of Cambridge 2. Wellcome-MRC Institute of Metabolic Science, University of Cambridge 3. Institute of Metabolism and Systems Research, University of Birmingham 4. MRC Epidemiology Unit, University of Cambridge 5. Department of Metabolism Digestion and Reproduction, Imperial College London
Presentation Type Talk
It is difficult to move from canine genome wide association studies (GWAS) and their candidate genes to mechanistic insights. Here we describe how comparative analyses and hypothesis driven molecular studies, provided in vitro evidence for a novel mechanism of obesity across multiple species. The most significant association (p= 5.54e-07) in an unpublished GWAS for obesity in Labrador retrievers, identified a 2.2Mb region harbouring 8 protein coding genes. The large effect top SNP (beta effect = +0.94 BCS) was directly over the gene DENN domain containing 1B (DENND1B), but no haplotype or causative variant could be determined. Comparative analysis with a finer mapped human GWAS, identified DENND1B as the likely causative gene of a significant association for BMI (UK Biobank). In GTEX eQTL data, this protective top SNP (rs6702421) showed a large effect reduction in DENND1B expression in brain tissues.
DENND1B is important for clathrin mediated endocytosis of membrane receptors and has a documented role in allergic disease by mediating trafficking of T cell receptor. This inspired a hypothesis that DENND1B might alter signalling of anorexigenic receptors in the hypothalamus, a key centre in the regulation of energy homeostasis. The most understood receptor for which signalling causes a decrease in appetite, is the G protein-coupled receptor (GPCR) Melanocortin 4 receptor (MC4R). We show robust co-expression of DENND1B with MC4R in murine and human hypothalamic single nuclei RNA-sequencing data.
In vitro, we tested DENND1B action on activation and internalisation of MC4R using a luciferase-based GloSensor cAMP assay. As predicted, DENND1B overexpression significantly reduced MC4R signalling, inversely, knock down increased signalling. Internalisation of SNAP labelled receptors was quantified from HILO images. Overexpression of DENND1B significantly increased MC4R internalisation to the endosomes and knock down reduced internalisation. In comparison, no significant signalling or internalisation effect was observed for orexigenic (increasing appetite) GPCR Growth hormone secretagogue receptor (GHSR) even though it was co-expressed with DENND1B in the hypothalamus at a similar percentage to MC4R. This suggests some receptor specificity.
In a cohort of severely obese individuals (SOPP), a single homozygous patient was identified for a rare, predicted deleterious missense variant in DENND1B. This arginine to cysteine mutation (A501C) sits within the β2-ear binding site of AP2 (clathrin machinery) and is predicted to disrupt DENND1B function and/or localisation to the vesicle. Work to test whether this mutation affects DENND1B function on MC4R internalisation is in progress. These data support a novel mechanism by which DENND1B regulates adiposity. We predict loss of DENND1B reduces MC4R internalisation, increases receptor at the membrane and in turn signalling to decrease food intake. Future investigations can explore the potential for therapeutics targeting this regulatory pathway. This study highlights the power of using orthogonal comparative data in humans and mice to support functional investigations of canine associations in complex diseases.