Generating the First Emirati iPSCs as an in-vitro Disease Model of Adipogenesis to investigate an FTO Obesity-associated Genetic Variant

Abstract

According to the World Health Organization (WHO) 2016 global statistics, more than 1.9 billion adults are overweight, of which 650 million are obese. On a national level, the UAE reported an obesity prevalence of 31.7% among adults, highlighting it among the top 20 obese countries worldwide in 2016. Obesity results in reduced life expectancy due to its associated comorbidities including diabetes mellitus, cardiovascular diseases, Alzheimer’s disease and ovarian cancer. Obesity is a multifactorial disorder which results from the intricate interplay between genetic and environmental factors. The fat mass and obesity-associated (FTO) gene was identified as the first gene harboring the strongest genetic association with common polygenic obesity. Genome-wide association studies (GWAS) in 2007 identified single-nucleotide polymorphisms (SNPs) within FTO in association with obesity. Interestingly, a common FTO variant (rs9939609) was reported in association with overweight/obesity among several populations including European, Hispanic, East Asian, Emirati, Kuwaiti and Egyptian populations; making it the most commonly replicated variant in association with obesity. Notably, FTO is the first reported demethylase of N6-methyladenosine (m6A) in mRNA, where m6A is the most abundant mRNA internal modification, which modulates several cellular processes including alternative splicing, mRNA stability, degradation, and expression. GWAS represent a tool in establishing connections between FTO variants and obesity phenotypical features. Nevertheless, translating such associations with FTO in adipogenesis on a molecular level remain to be fully elucidated.

This thesis aims to investigate the possible molecular association of the common FTO obesity-susceptibility variant (rs9939609) with adipogenesis via the use of Emirati-specific human induced-pluripotent stem cells (hiPSCs) as the first in-vitro model of obesity in the UAE. First, FTO molecular association with obesity, as an m6A demethylase, is critically reviewed and a literature review is presented. Second, this thesis presents the generation of the first Emirati-specific hiPSC line KUSTi001-A using a thorough multistep characterization workflow. Using the same detailed workflow, a repository of Emirati hiPSCs has been established in this thesis, with detailed validation and characterization tests presented.

Third, Emirati hiPSCs from three donors were utilized for adipogenic differentiation experiments as the first in-vitro model of adipogenesis in the UAE. Validation of adipogenic differentiation is presented on gene and protein levels. hiPSC-derived beige adipocytes are utilized for comparative analysis of FTO expression levels between lean and obese subjects as well as adipogenic markers expression, in association with subjects’ phenotypes and genotypes. Our preliminary findings suggest that FTO expression levels are upregulated during adipogenesis and inversely correlate with total RNA m6A levels, in agreement with previous studies. Furthermore, data implies that higher FTO expression is observed in hiPSCs and hiPSC-derived beige adipocytes from obese/overweight AA risk genotype carriers, with parallel lower total m6A levels. In addition, our results suggest a potential association of FTO TT WT genotype with a protective anti-obesogenic effect via potentially generating ‘more efficient’ beige adipocytes that express higher thermogenic transcript levels, have higher mitochondrial mass and lower lipid droplet accumulation. This work needs to be further validated by including more Emirati biological replicates into our adipogenic differentiation future experiments.

Work established in this thesis paves the way for further adipogenesis and disease-modelling in-vitro investigations, as well as regenerative and precision medicine applications in the UAE.

Date of Award	Dec 2022
Original language	American English
Supervisor	Abdulrahim Sajini (Supervisor)