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Title: Functional study of waist-hip ratio associated loci and the WARS2 gene as modulators of fat distribution
Author: Muso, Milan
ISNI:       0000 0004 8507 3208
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Waist-hip ratio (WHR) is a risk factor for diabetes and cardiovascular disease. Genome-wide association studies identified 346 loci linked to increased WHR adjusted for Body Mass Index (WHRadjBMI) (Shungin et al., 2015; Pulit et al., 2018). To elucidate the genetic underpinning of WHRadjBMI, I aimed to identify causal SNPs in 4 of these loci and test the tryptophanyl-tRNA synthetase 2 (WARS2) gene, previously shown to severely affect adipose mass, as modulators of fat distribution and browning. To prioritise real signals, with colleagues, I selected 3 SNPs in EMILIN2, VEGFA, C5orf67 loci with posterior probability analysis (PPA) scores > 0.95 for functional analysis. In the more complex TBX15-WARS2 locus, we filtered 61 SNPs and selected 5 SNPs with a PPA score > 0.2 and further 8 top-scoring SNPs according to the epi-Phylogenetic Module Complexity Analysis (epi-PMCA). Using electrophoretic mobility shift assays (EMSAs) and luciferase assays in a differentiating human white adipose cell line, I showed that rs3936510 in the C5orf67 locus and rs3810068 in the EMILIN2 affected DNA-protein binding and identified enhancer/promoter activities in these loci. I demonstrated that rs3810068 altered the binding of CCAAT/enhancer-binding protein beta (CEBP/β). In the TBX15-WARS2 locus, I used luciferase assays, RNA degradation analysis and allele-specific qPCR to show that the rs2645294, lead SNP in the 3'UTR of WARS2, did not impact RNA stability. Wars2 (Wars2V117L/V117L) mice showed previously progressive tissue-specific pathologies including reduced adiposity and browning of white adipose tissue (GTEx-Consortium, 2013; Civelek et al., 2017; Agnew et al., 2018). I show that whilst heterozygous female mice (Wars2+/V117L and Wars2+/-) were unaffected, Wars2V117L/V117L mice failed to gain fat mass on a high-fat diet (HFD) in both sexes, had increased visceral:subcutaneous adipose ratio specifically in males on HFD and showed lower food intake in males. In both sexes, brown adipose tissue (BAT) showed loss of browning markers and mitochondrial dysfunction. In white adipose tissue (WAT), browning markers were upregulated, with a greater difference in the subcutaneous inguinal WAT compared to visceral gonadal WAT. In differentiated primary preadipocytes no difference in browning was observed. Plasma showed elevated levels of Fibroblast growth factor-21 (FGF21) and Growth/differentiation factor 15 (GDF15) In summary, a hypomorphic mutation in the mitochondrial gene WARS2 reduced fat mass irrespective of diet and altered fat distribution in a diet and sex-specific manner, likely due to differences in depot-specific browning and elevation of mitokines FGF21 and GDF15 from other affected tissues.
Supervisor: Minchole, Ana ; Rodriguez, Blanca Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Physiology ; Genetics