FUNCTIONAL CHARACTERIZATION OF A NOVEL GENETIC VARIANT PREDISPOSING TO ADVANCED FIBROSIS AND HEPATOCELLULAR CARCINOMA DEVELOPMENT IN NONALCOHOLIC FATTY LIVER DISEASE

Functional characterization of a novel genetic variant predisposing to advanced fibrosis and hepatocellular carcinoma development in nonalcoholic fatty liver disease
Introduction
Nonalcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in the Western countries and represent an emerging cause of liver cirrhosis and hepatocellular carcinoma (HCC). Several studies underlined the importance of heritability in modifying the susceptibility and progression of NAFLD. However, the specific determinants of NAFLD heritability remain largely unkonwn.
Aims
In the hypothesis that rare genetic variants with a strong impact on protein activity account for a fraction of NAFLD missing heritability, the first aim of the current study was to identify by Whole Exome Sequencing (WES) novel rare genetic variants determining an alteration of protein activity associated with advanced stage NAFLD. As we detected an association with a variant in Interferon regulatory protein 3 (IRF3) and hepatocellular carcinoma (HCC) related to NAFLD, we next examined the specific regulation of IRF3 isoforms in patients at risk of NAFLD in relation to liver damage, and tried to understand IRF3 regulation and to model the impact of IRF3 alternative transcripts downregulation by exploiting CRISPR/Cas9 gene editing approach in in vitro models. Finally, we tested the impact of IRF3 pathway inhibition by amlexanox, a TBK1-IRF3 axis inhibitor under study for the treatment of obesity related complications, on the proliferation of hepatoma cells (HepG2).
 
Patients and methods
• Variants discovery was performed by WES in 72 Italian NAFLD-HCC patients and 50 healthy individuals. HCC patients were compared to those in the European population (Exome Aggregation Consortium Non-Finnish Europeans, N=33,370).
• Validation was performed in NAFLD HCC patients (N=105) and in 211 patients with Advanced fibrosis (N=211), further 270 Italian healthy individuals. Furthermore, a larger database was used to evaluate the European population (genome aggregation consortium, N=64,603).
• Transcriptomic analyses were performed on 125 severely obese individuals (Transcriptomic cohort) who underwent to percutaneous liver biopsy performed during bariatric surgery. Bulk RNA-Seq was performed on RNA extracted from flash-frozen liver biopsies.
• To evaluate the effect of IRF3-CL a somatic variant in the IRF3-CL specific exon 7 splicing acceptor was introduced. Briefly, a Doxycycline (Therm-Fisher, Waltham, US) inducible Cas9 expressing HepG2 cell line was produced by lentiviral infection exploiting was produced exploiting Edit-R Inducible Lentiviral Cas9 Nuclease vectors (Dharmacon, Lafayette, U.S.A.). Specific guide RNA was designed using CRISPR design tool (http://crispr.mit.edu/) and cloned into an sgRNA expression vector (Addgene #51133) and doxycycline treated cells were transfected with the expression vector. Single cell derived populations were obtained by limiting diluition method and presence of mutations in the specified locus was investigated by T7 nuclease assay (New England Biolabs, Ipswich, US) and further confirmed by Sanger sequencing.
 
Results
The rs141490768 SNP in IRF3 resulted enriched in HCCs compared with European population (OR=37.1, p=4.5*10-7). We validated this association in the replication cohort (N=211, p=0.049; OR=5.8; 95% CI = 0.7-21). In the overall series of patients with advanced NAFLD (N=388), the association remained significant (OR=11; 95% CI= 4-24; p=6.16*10-6). The rs141490768 encodes a loss-of-function variant (A418T) in the IRF3 inhibitory isoform IRF3-CL, suggesting that increased IRF3 activity may predispose to NAFLD-HCC. Furthermore, two non-coding transcripts of unknown function (referred as IRF3-NC1 an