Elevated risk of cardiometabolic disease is magnified by variation in body fat distribution, in particular increased accumulation of visceral fat. Genome-wide association studies have mainly focused on anthropometric indices such as WHR and BMI to assess adiposity. They successfully identified over 100 loci highlighting for total fat mainly pathways in the brain involved in the regulation of energy expenditure and appetite and for fat distribution genes expressed in adipose and the periphery. Although genetic variants affecting localised fat deposition are known, the functional mechanisms of regional fat accumulation remain poorly understood. Here, we aimed to explore the genetic contribution to body composition to gain further mechanistic insight, and increase our understanding of the role of such genetic variants in metabolic health. We focused on the isolated population of Orkney. 1,301 participants from the Orkney Complex Disease Study, ORCADES underwent DXA scans allowing direct assessment of fat mass in various depots around the body. Genetic data imputed to the 1000 Genomes Project enabled the investigation of 35 million genetic variants. We first used univariate and bivariate analysis to quantify the contribution of genetic factors to the variation of body composition and establish genetic correlations with metabolic traits. We carried out genome-wide association analyses for body composition to identify new underlying genetic loci. We sought to replicate these findings in the Icelandic AGES cohort and the UK Biobank, with 3,219 and 1,575 participants with body composition analysis, respectively. We investigated the coding variation or the regulatory landscape around the associated variants to understand their functional impact. We further focused on one of the associated loci in greater detail. To establish a potential, causal gene for the associated variants and understand the impact of genetic variation on the regulatory elements, we carried out chromatin conformation studies around ENPP6 by. We then explored the role of causal gene candidate on body composition and metabolic health in an animal mouse model. Individual fat depots were moderately heritable with heritability estimates ranging from 35-50% in ORCADES. The genetic correlations with metabolic traits were highest with android, and visceral fat and the ratio of android and gynoid fat percentage: Insulin (0.68-0.75), HOMA-B (0.58-0.70), HOMA-IR (0.69-0.75), CRP (0.47-0.55) and DBP (0.49-0.58). Genome-wide association analysis identified three regions associated with body composition: VRTN, EXOC6B and ENPP6. Low frequency variants on chromosome 4, mapping within the ENPP6 gene associated with the ratio of android and gynoid fat (p= 4.5x10-10), which replicated in abdominal fat by CT in AGES (p=0.003). Per allele, variant carriers show a reduction in android fat by 3% and visceral fat of 140g as well as lowered diastolic blood pressure of 10mmHg. Due to this evidence ENPP6 was chosen as a focus for further mechanistic and functional studies. The lead SNPs map to an ENCODE-predicted DNase1 hypersensitivity site within the second intron of the ENPP6 gene, suggesting a role in genome regulation. Marking the areas with sequence-specific probes by 3D fluorescent in situ hybridisation confirmed that the association interval co-localised more frequently with the ENPP6 promoter than with other gene promoters within the same chromosomal region in SH-SY5Y neurons (p=0.01) but not human SGBS adipocytes. This indicates ENPP6 as a possible causal gene. Consistent with this ENPP6 mRNA levels were extremely low in human subcutaneous and visceral adipose tissue. ENPP6 expression is highest in the brain and kidney, suggesting a neuronal/renal mediated mechanism driving body composition. To model the impact of Enpp6 on adiposity in vivo, Enpp6-/- mice were generated and their metabolic profile investigated. Enpp6-/- mice showed a decrease in visceral fat depot and improved glucose tolerance (n= 24, pfat=0.002, pGTT=0.001). However, no difference was found with regards to their feeding or physical activity behaviour, suggesting an intrinsic alternative to maintaining an energy balance. Using the advantage of genetic drift in a population isolate and direct fat phenotyping we confirmed the contribution of genetic variants to variation in body composition and describe the involvement of three particular loci VRTN, EXOC6B and ENPP6. In particular, we describe ENPP6 as a likely neuronal mechanism underlying selectively visceral adiposity in humans and mice. This study sets a starting point for the investigation into ENPP6 as an anti-obesity and anti-diabetes therapeutic.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:738825 |
| Date | January 2017 |
| Creators | Schraut, Katharina Elfriede |
| Contributors | Morton, Nicholas ; Wilson, James ; Navarro, Pau |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28894 |
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