IDENTIFICATION OF RARE AND COMMON GENETIC VARIANTS ASSOCIATED WITH HEPATOCELLULAR CARCINOMA IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE: MECHANISMS AND CLINICAL IMPLICATIONS

Nonalcoholic fatty liver disease (NAFLD) affects roughly 30% of the general population and its prevalence is increasing worldwide, in particular in Western countries where it is projected to become the main cause of hepatocellular carcinoma (HCC) within 2025. Although the majority of NAFLD progressing towards HCC are individuals with advanced fibrosis, NAFLD-HCC frequently develops even in patients without cirrhosis. Family history and genetic factors play an important role in the pathogenesis of progressive NAFLD and of HCC. Our hypothesis is that both common and rare variants may have a role in influencing HCC predisposition. Aim of this study was (1) to evaluate whether the rs641738 C>T common genetic variant in the MBOAT7/TMC4 locus, the last genetic variant recently associated with NAFLD development and progression towards early phases of damage, also predisposes to HCC in patients stratified by the presence of severe fibrosis, and (2) to assess whether telomere attrition and inherited rare hTERT mutations associates with NAFLD-HCC development, since mutations in Telomerase gene have been linked to familial liver diseases. In the first part of the study, we found that the rs641738 T allele is associated with NAFLD-HCC independently of fibrosis severity and of several confounders (allelic OR 1.65, 95% confidence interval 1.08-2.55; n=765), particularly in patients without advanced fibrosis (p<0.001). The rs641738 risk T allele is associated with reduced MBOAT7 expression, evaluated by qRT-PCR on liver biopsy, specifically in patients without severe fibrosis, and was more strongly associated with HCC development in this specific subgroup (p=0.02). Moreover, the number of PNPLA3, TM6SF2, and MBOAT7 risk alleles is associated with NAFLD-HCC development independently of clinical factors (p<0.001), but it did not significantly improve their predictive accuracy. The independent association between the 5 MBOAT7 risk variant and increased HCC risk has also been confirmed in a combined cohort of chronic hepatitis C or alcoholic liver disease patients without cirrhosis (n=1121). In the second part of the study, we found that telomere length, evaluated by qRT-PCR, is progressively reduced in 40 NAFLD-HCC vs. 45 NAFLD-cirrhosis (p=0.048) and 64 healthy controls (p=0.0006), independently of age and sex. We detected an enrichment of rare germline coding mutations in hTERT in NAFLD-HCC patients vs. controls, even after considering 78 European primary liver cancers. No mutations were found in NAFLD-cirrhosis and local controls, and only one in 503 Europeans from the 1000Genomes Project. Thus, overall mutations frequency was 0.025 in cases vs. <0.001 in controls (p=0.0005 at Burden test). hTERT mutations occurred predominantly in females (p=0.03) and were predominantly located in the N-terminal template-binding domain of the gene (p=0.037 for specific enrichment). The frameshift Glu113Argfs*79 and the missense Glu668Asp damaging mutations co-segregated with liver disease in families. Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels in HEK-293 cells. These data suggest that telomere attrition and certain hTERT mutations are involved in the pathogenesis of NAFLD-HCC. In conclusion, the identification of new common prognostic marker, such as the MBOAT7 rs641738 variant, in NAFLD patients and the evaluation of hTERT rare mutations in NAFLD-HCC may improve HCC screening strategies, by assisting in the identification of subjects at risk even in the absence of severe fibrosis and of family members at high risk of progressive liver disease, respectively. In future, functional studies are needed in order to understand the exact role and the interplay of the several common and rare genetic variants which contribute to the molecular mechanisms that underlie the pathogenesis of NAFLD-HCC.