DiMeN Doctoral Training Partnership: Mechanisms of genetic polymorphisms associated with non-alcoholic fatty liver disease (NAFLD) and its role in disease progression

Student learning: This project will allow the student to gain multidisciplinary skills including Super-Resolution microscopy, molecular and cell biology, biochemistry and patient data analysis.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, however mechanisms underlying its progression from a non-serious fatty liver to a more serious condition of steatohepatitis remain unknown and there are no specific licensed pharmacotherapies for its treatment. NAFLD is strongly associated with inflammation and cardiovascular disease mortality. Animal data have suggested that liver sinusoidal endothelial cell (LSEC) injury appears during steatosis. LSECs can produce a number of pro-inflammatory cytokines and pro-thrombotic factors and as inflammation is a driving factor in the progression of NAFLD from simple steatosis to fibrosis and cirrhosis it is likely that LSEC dysfunction plays a prominent role in NAFLD pathology.

Recently we demonstrated, that a novel Rab GTPase (Rab46) has important roles in the secretion of inflammatory and pro-thrombotic mediators. Notably, genome wide association studies have linked a polymorphism (rs887304) in the gene that encodes for Rab46 to the pathogenic progression of NAFLD. In endothelial cells Rab46 is expressed as a 95kD Rab GTPase whereas in T cells it is expressed in an additional 45kD non-Rab protein form, involved in ion channel modulation. Alternative splice site analysis predicts that rs887304 introduces a cryptic splice site into the gene, suggesting genotype-dependent alteration in expression of Rab46.

We therefore hypothesise that SNP rs887304 alters the expression of Rab46 with subsequent effects on vesicular trafficking and endothelial cell secretion. Investigating the effects of this polymorphism in endothelial cells under conditions where lipid metabolism is perturbed would produce new insight into the role of endothelial cells in NAFLD progression. In addition, we will investigate the association of rs887304 with other inflammatory diseases using data from the UK Biobank to present Rab46 as a major therapeutic target in inflammatory diseases.

Hypothesis: Functional expression of genetic polymorphisms in endothelial cells contribute to inflammation and disease progression in non-alcoholic fatty liver disease.
Aims:
1. Characterisation of genetic polymorphisms in endothelial cells
Utilizing molecular and cell biology techniques and under the supervision of the primary and secondary supervisor, the student will undertake genotyping in primary endothelial cells isolated from single donors using established methodology and ethics (expected allele frequency 30% in Europeans). Transcripts will be analysed at the protein level and compared to endogenous levels by western blotting.
Simultaneously, the expression of some common NAFLD related polymorphisms will be analyzed in endothelial cells and compared to hepatocytes.

2. Functional effect of polymorphisms in lipid loaded endothelial cells.
Utilizing super-resolution microscopy (Faculty of Biological Sciences) alongside traditional biochemical analysis, the student will explore the effects of the polymorphism(s) in lipid loaded endothelial cells (from patients) on endothelial vesicular trafficking. In addition, the contribution of polymorphisms on expression levels will be mimicked using siRNA and mutant overexpression.

3. Population analysis of EFCAB4B SNP rs887304 in NAFLD and inflammatory diseases.
In collaboration with the third supervisor in the Leeds Institute of Data Analysis the student will analyse associations between genotypes taken from patients with known inflammatory disorders (UK biobank) and rs887304.