In a recent article published in Nature Medicine, researchers pursued evidence of whether the interaction between the patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant and the female sex at the genetic and molecular level explains why premenopausal women remain protected against fatty liver disease (FLD) while a rapidly progressive disease ensues in some peri- and postmenopausal women.
FLD is a metabolic dysfunction encompassing several liver pathologies, ranging from intracellular triglycerides accumulation to severe lipotoxicity and fibrosing steatohepatitis, all of which are leading causes of liver disease(s), liver transplantation, hepatocellular carcinoma (HCC) and mortality, especially in women.
It is also noteworthy that genetic predisposition and epigenetic modifiers synergize with environmental factors to induce liver diseases.
In premenopausal women, high estrogen (E2) levels confer protection against FLD; however, during and after menopause, high E2 levels are lost, increasing the susceptibility to developing FLD. Nonalcoholic FLD (NAFLD), a leading cause of cirrhosis and HCC, also occurs in women more than men, especially older postmenopausal women aged ≥55. Furthermore, studies have shown that modulation of estrogen receptor alpha (ER-α) activity increases the risk of steatohepatitis in vulnerable women.
Even though the p.I148M variant of PNPLA3 accounts for the highest proportion of FLD heritability, its genetic, molecular, experimental, and clinical aspects are understudied. In Europeans, it accounts for 27% and 16% of HCC and cirrhosis variance, respectively. Yet all in vitro and in vivo evidence of the mechanism(s) governing this genetic association in women are unclear or plain circumstantial.
About the study
In the present study, researchers recruited multiple clinical and general population-based cohorts to test the presence of an interaction between female sex and the PNPLA3 p.I148M variant and understand the mechanism determining the genetic predisposition to FLD.
The researchers used data from 1,861 European ancestry from the Liver Biopsy Cohort (LBC), who likely had nonalcoholic steatohepatitis (NASH). In addition, they age-stratified female patients to capture their reproductive stage and examine the effect of NASH grade as an FLD hallmark.
A subgroup of 125 severely obese individuals from the LBC provided material for extracting high-quality ribonucleic acid (RNA) for transcriptomic analysis of PNPLA3 gene expression.
The researchers also used an independent case–control cohort comprising 4374 individuals with severe metabolic FLD to confirm whether the presence of an interaction between the female sex and the PNPLA3 p.I148M variant determined the progression of severe FLD to advanced fibrosis or HCC.
Lastly, the team used the Liver-Bible-2022 cohort encompassing 1,142 healthy individuals aged 40–65 years to examine the role of genetic factors and other noninvasive NAFLD biomarkers to lay the framework to design precision medicine approaches to prevent cardiometabolic diseases associated with NAFLD.
Furthermore, the team used four types of cellular models: HepG2, LX-2, 293T, and HepaRG. They used the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 method to investigate the role of the estrogen response elements (EREs), especially PNPLA3-ERE1, in the overexpression of PNPLA3.
The results showed that the effect size of the PNPLA3 p.I148M variant on all liver damage outcomes was larger in women than in men, resulting in a higher relative FLD risk in women than men.
While women aged <45 years remained protected against steatosis, those aged 45–54 and ≥55 years were not. Consistently, the multiplicative interaction with the female sex was specific for the PNPLA3 p.I148M variant; thus, other gene variants contributing to FLD susceptibility, e.g., TM6SF2 p.E167K, did not contribute to FLD risk even in menopausal women. Moreover, reduced E2 levels in postmenopausal women often lead to insulin resistance (IR) and changes in adiposity, which also trigger the phenotypic expression of the PNPLA3 p.I148M variant. Together, these changes exacerbated the FLD risk.
In human hepatocytes and liver organoids, ER-α agonists induced PNPLA3. Chromatin immunoprecipitation and luciferase assays characterized an ER-α-binding site within a PNPLA3 enhancer, a DNA sequence that drove PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional (3D) multilineage spheroids with stellate cells. Thus, genetic deletion of PNPLA3-ERE1 completely abrogated ER-α-mediated PNPLA3 upregulation in HepG2 cells.
The study data suggested a variant-specific multiplicative interaction between the female sex and PNPLA3 p.I148M variant, which determined FLD risk, including risk for steatosis, fibrosis, and HCC in men and women; however, postmenopausal women were at the highest risk.
Regarding the mechanism underpinning this interaction, the authors observed that it involved E2-mediated upregulation of PNPLA3 via ER-α and ERE1, a specific DNA region in the PNPLA3 promoter. This DNA region mediated the accumulation of PNPLA3 on lipid droplets, increasing their numbers under the influence of ER-α agonists, e.g., tamoxifen. Hepatic PNPLA3 expression was higher in women than men, even in obese individuals.
Overall, this interaction governed the sexual dimorphism in FLD prevalence. Thus, study data could help design precision therapies to prevent and treat FLD in women, taking into account their genetic and hormonal profiles. In fact, menopausal women (≥55 years) appear to be the most relevant subset to target with a precision therapeutic approach for liver diseases.