Patatin-like Phospholipase Domain-containing Research Articles

Metabolomics biomarkers of hepatocellular carcinoma in a prospective cohort of patients with cirrhosis

Background & AimsThe effectiveness of surveillance for hepatocellular carcinoma (HCC) in patients with cirrhosis is limited, due to inadequate risk stratification and suboptimal performance of the screening modalities. MethodsWe developed a multicenter prospective cohort of cirrhotic patients undergoing surveillance with MRI, and applied global untargeted metabolomics to 612 longitudinal serum samples from 203 patients. Among them, 37 developed HCC during follow-up. ResultsWe identified 150 metabolites with significant abundance changes in samples collected prior to HCC (Cases) compared to samples from patients who didn’t develop HCC (Controls). Tauro-conjugated bile acids and gamma-glutamyl amino acids were increased, while acyl-cholines and deoxycholate derivatives were decreased. Seven amino acids including serine and alanine, had strong associations with HCC risk, while strong protective effects were observed for N-acetylglycine and glycerophosphorylcholine. Machine learning using the 150 metabolites, age, gender, patatin-like phospholipase domain-containing protein 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TMS6SF2) SNPs, identified 15 variables giving optimal performance. Among them, N-acetylglycine had the highest AUC in discriminating Cases and Controls. When restricting Cases to samples collected within 1 year prior to HCC (Cases-12M), additional metabolites including microbiota-derived metabolites were identified. The combination of the top 6 variables identified by machine learning (alpha-fetoprotein, 6-bromotryptophan, N-acetylglycine, salicyluric glucuronide, testosterone sulfate and age) had good performance in discriminating Cases-12M from Controls (AUC=0.88 [0.83-0.93]). Finally, 23 metabolites distinguished Cases with Liver Imaging Reporting and Data System (LI-RADS)-3 lesions from Controls with LI-RADS-3 lesions, with reduced abundance of acyl-cholines and glycerophosphorylcholine-related lysophospholipids in Cases-LR3. ConclusionsThis study identified N-acetylglycine, amino acids, bile acids and choline-derived metabolites as main biomarkers of HCC risk, and microbiota-derived metabolites as main contributors to HCC development. Impact and ImplicationsThe effectiveness of surveillance for hepatocellular carcinoma (HCC) in patients with cirrhosis is limited. There is an urgent need for improvement in risk stratification and new screening modalities, blood biomarkers in particular. Longitudinal collection of paired blood samples and MRI images from cirrhotic patients is particularly valuable in assessing how early blood and imaging markers become positive during the period when lesions are observed to obtain a diagnosis of HCC. We generated a multicenter prospective cohort of patients with cirrhosis under surveillance with contrast MRI, applied untargeted metabolomics on 612 serum samples from 203 patients and identified metabolites associated with risk of HCC development. Such biomarkers may significantly improve early stage HCC detection for cirrhotic patients undergoing HCC surveillance, a critical step to increasing curative treatment opportunities and reducing mortality.

PNPLA3 rs738409, age, diabetes, sex, and advanced fibrosis jointly contribute to the risk of major adverse liver outcomes in metabolic-associated steatotic liver disease.

The patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 variant is associated with Steatotic Liver Disease (SLD) and its progression. We examined the association between PNPLA3 and the development of major adverse liver outcomes (MALO) and how non-modifiable and modifiable conditions modify this relationship. 2,075 adults with biopsy-confirmed Metabolic-Associated SLD (MASLD) were enrolled in the MASLD CRN studies and followed prospectively until death, transplant, or withdrawal of consent. 104 MALOs were recorded during an average of 4.3 years. PNPLA3 G-allele (Adj. SubHazard ratio (sHR): 1.4, 95% confidence interval (CI): 1.07-1.8), advanced fibrosis (AF) (Adj. sHR: 7.8, 95% CI: 4.4-13.8), age >60 years (Adj. sHR: 2.9, 95% CI: 1.3-6.8), and type 2 diabetes mellitus (T2DM) (Adj. sHR: 2.8, 95% CI: 1.8-4.2) were associated with MALO. Among participants with AF, those carrying the G-allele displayed the highest cumulative incidence of MALO (85%) vs. noncarriers (53%), p=0.03, and p value for interaction<0.01. The strength of the association between PNPLA3 and MALO was statistically significantly greater among older than 60 years (sHR: 2.1, 95% CI: 1.5-2.8), women (sHR: 1.4, 95% CI: 1.1-1.9), and those with AF (sHR: 1.9, 95% CI: 1.5-2.4) or T2DM (sHR: 2.1, 95% CI: 1.5-2.8) as compared with their counterparts, p value for interaction between PNPLA3 and each factor<0.01. The deleterious effects of PNPLA3 rs738409 on the risk of MALO are significantly worsened by advanced fibrosis, age, T2DM, and sex.

The Role of PNPLA3_rs738409 Gene Variant, Lifestyle Factors, and Bioactive Compounds in Nonalcoholic Fatty Liver Disease: A Population-Based and Molecular Approach towards Healthy Nutrition.

This study aimed to investigate the impact of a common non-synonymous gene variant (C>G, rs738409) in patatin-like phospholipase domain-containing 3 (PNPLA3), leading to the substitution of isoleucine with methionine at position 148 (PNPLA3-I148M), on susceptibility to nonalcoholic fatty liver disease (NAFLD) and explore potential therapeutic nutritional strategies targeting PNPLA3. It contributed to understanding sustainable dietary practices for managing NAFLD, recently referred to as metabolic-dysfunction-associated fatty liver. NAFLD had been diagnosed by ultrasound in a metropolitan hospital-based cohort comprising 58,701 middle-aged and older Korean individuals, identifying 2089 NAFLD patients. The interaction between PNPLA3 and lifestyle factors was investigated. In silico analyses, including virtual screening, molecular docking, and molecular dynamics simulations, were conducted to identify bioactive compounds from foods targeting PNPLA3(I148M). Subsequent cellular experiments involved treating oleic acid (OA)-exposed HepG2 cells with selected bioactive compounds, both in the absence and presence of compound C (AMPK inhibitor), targeting PNPLA3 expression. Carriers of the risk allele PNPLA3_rs738409G showed an increased association with NAFLD risk, particularly with adherence to a plant-based diet, avoidance of a Western-style diet, and smoking. Delphinidin 3-caffeoyl-glucoside, pyranocyanin A, delta-viniferin, kaempferol-7-glucoside, and petunidin 3-rutinoside emerged as potential binders to the active site residues of PNPLA3, exhibiting a reduction in binding energy. These compounds demonstrated a dose-dependent reduction in intracellular triglyceride and lipid peroxide levels in HepG2 cells, while pretreatment with compound C showed the opposite trend. Kaempferol-7-glucoside and petunidin-3-rutinoside showed potential as inhibitors of PNPLA3 expression by enhancing AMPK activity, ultimately reducing intrahepatic lipogenesis. In conclusion, there is potential for plant-based diets and specific bioactive compounds to promote sustainable dietary practices to mitigate NAFLD risk, especially in individuals with genetic predispositions.

Molecular Genealogy of Metabolic-associated Hepatocellular Carcinoma.

This review examines the latest epidemiological and molecular pathogenic findings of metabolic-associated hepatocellular carcinoma (HCC). Its increasing prevalence is a significant concern and reflects the growing burden of obesity and metabolic diseases, including metabolic dysfunction-associated steatotic liver disease, formerly known as nonalcoholic fatty liver disease, and type 2 diabetes. Metabolic-associated HCC has unique molecular abnormality and distinctive gene expression patterns implicating aberrations in bile acid, fatty acid metabolism, oxidative stress, and proinflammatory pathways. Furthermore, a notable frequency of single nucleotide polymorphisms in genes such as patatin-like phospholipase domain-containing 3, transmembrane 6 superfamily member 2, glucokinase regulator, and membrane-bound O-acyltransferase domain-containing 7 has been observed. The tumor immune microenvironment of metabolic-associated HCC is characterized by unique phenotypes of macrophages, neutrophils, and T lymphocytes. Additionally, the pathogenesis of metabolic-associated HCC is influenced by abnormal lipid metabolism, insulin resistance, and dysbiosis. In conclusion, deciphering the intricate interactions among metabolic processes, genetic predispositions, inflammatory responses, immune regulation, and microbial ecology is imperative for the development of novel therapeutic and preventative measures against metabolic-associated HCC.

Abstract P181: Association Between Non-Alcoholic Fatty Liver Disease Risk SNP Variants and Lipid Metabolism

Introduction: The impact of non-alcoholic fatty liver disease (NAFLD) risk SNP variants on ipid metabolism in the general population has yet to be determined. We aimed to assess this association using a large prospective cohort study. Methods: A total of 1488 Japanese men and women (median age 62 years and median body mass index 24.7 kg/m2) free of cardiovascular disease were studied. Among NAFLD risk SNPs, variants of patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR), and neurocan (NCAN) were assessed. Plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (sdLDL-C), LDL-triglycerides (LDL-TG), high-density lipoprotein cholesterol (HDL-C), HDL3-C, triglycerides (TG), lipoprotein(a), and adiponectin were measured at both baseline and after a five-year follow-up. Univariate and multivariate analyses, Kruskal-Wallis, Wilcoxon’s rank sum, and paired t-tests were performed to examine the associations between NAFLD risk SNPs and lipoproteins. Results: All of the measured lipoproteins increased significantly over the 5 years. The TM6SF2 variant did not affect lipoproteins at baseline. However, after 5 years, the non-major allele group had significantly lower HDL-C and HDL3-C and significantly higher LDL-C, sdLDL-C, and LDL-TG than the major allele group. For the GCKR variant, TG, LDL-C, sdLDL-C, and LDL-TG were significantly higher in the non-major allele group at baseline; LDL-C, sdLDL-C, and LDL-TG were still significantly higher in the non-major allele group at 5 years than the CC group. The PNPLA3 and NCAN variants did not affect lipoproteins in this study. Conclusions: NAFLD risk SNP variants might influence atherogenic lipoprotein metabolism.

High red meat consumption among PNPLA3 polymorphism carriers is associated with NAFLD in a multi-center cross-sectional study.

Patatin-like phospholipase domain-containing 3 gene (PNPLA3) polymorphism has been implicated in susceptibility to non-alcoholic fatty liver disease (NAFLD), with evidence for potential interaction with nutrition. However, the combination of meat consumption with genetic polymorphism has not been tested. Therefore, this study aims to test the association between the joint presence of PNPLA3 rs738409 G-allele with high meat consumption and NAFLD in populations with diverse meat consumption. A cross-sectional study among Israeli screening and Brazilian primary healthcare populations. Food consumption was assessed by a food-frequency questionnaire. PNPLA3 polymorphism was defined as homozygous (GG) or heterozygous (GC). Inconclusive/probable NAFLD was defined as a fatty liver index (FLI) ≥ 30 and probable NAFLD as FLI ≥ 60. The sample included 511 subjects from the screening and primary healthcare populations (n = 213 and n = 298, respectively). Genetic polymorphism (homozygous GG or heterozygous GC) combined with high consumption of total meat, red and/or processed meat, unprocessed red meat, and processed meat was associated with the highest odds for inconclusive/probable NAFLD (OR = 2.75, 95%CI 1.27-5.97, p = 0.011; OR = 3.24, 1.43-7.34, p = 0.005; OR = 2.92, 1.32-6.47, p = 0.008; OR = 3.16, 1.46-6.83, p = 0.003, respectively), adjusting for age, gender, BMI, alcohol consumption, carbohydrate, and saturated fat intake. In addition, genetic polymorphism combined with high processed meat consumption was associated with the highest odds for probable NAFLD (OR = 2.40, 95%CI 1.04-5.56, p = 0.040). High red meat intake may confer a greater risk for NAFLD among PNPLA3 polymorphism carriers. Prospective studies are needed to confirm these findings and consider minimizing red and processed meat consumption among PNPLA3 polymorphism carriers.

17-Beta-Hydroxysteroid Dehydrogenase 13 Loss of Function Does Not Confer Protection to Nonalcoholic Fatty Liver Disease in Indian Population

A splice variant in HSD17B13 gene is demonstrated to protect against nonalcoholic fatty liver disease (NAFLD), and mitigate the effect of Patatin-like phospholipase domain-containing 3 (PNPLA3-I148M). It is being explored as a putative drug target and in polygenic risk scores. Based on whole exome sequencing (WES) in our cohort of biopsy proven NAFLD and limited data on the variant in our ethnicity, we sought to explore its role. This is a cross-sectional study that recruited 1,020 individuals with ultrasound/biopsy-confirmed NAFLD and matched controls. Liver enzymes and lipid profiles were estimated (Beckman coulter LX750/DXH800); WES was performed in NAFLD patients and controls (Illumina; HiSeqX); HSD17B13-A-INS/I148M-PNPLA3 variants were genotyped (sequencing/qR T-PCR); HSD17B13 protein expression was estimated (immunohistochemistry); the Student's t-test/Mann-Whitney U/Chi-square test and odds ratio (95% confidence interval) were used. There was no significant difference (Odds ratio=0.76; 95% CI -0.57 to 1.03; P=0.76) in the frequency of the rs72613567-A-INS between controls and patients (17.8% vs. 14.4%). No difference in the ALT (Alanine transaminase; 72.24±65.13 vs. 73.70±60.06; P=0.51) and AST levels (Aspartate aminotransferase; 60.72±55.59 vs. 61.63±60.33; P=0.91) between HSD17B13-wild and variant carriers were noted. Significantly elevated liver enzymes were seen in PNPLA3-148-variant/HSD17B13-wild compared with PNPLA3-148-variant/HSD17B13-variant (90.44±59.0 vs. 112.32±61.78; P=0.02). No difference in steatosis (P=0.51) between HSD17B13-wild and variant carriers was noted. No other variants in the intron-exon boundaries were identified. Although, protein expression differences were noted between wild and variant carriers, no difference in the extent of steatosis was seen. Our study reports lack of association of the splice variant with reduced risk of NAFLD, and mitigating the effect of PNPLA3 variant. Ethnicity-based validation must be carried out before including it in assessing protection against NAFLD.

The ubiquitin E3 ligase BFAR promotes degradation of PNPLA3

A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We previously showed that PNPLA3 is ubiquitylated and subsequently degraded by proteasomes and autophagosomes and that the PNPLA3(148M) variant interferes with this process. To define the machinery responsible for PNPLA3 turnover, we used small interfering (si)RNAs to inactivate components of the ubiquitin proteasome system. Inactivation of bifunctional apoptosis regulator (BFAR), a membrane-bound E3 ubiquitin ligase, reproducibly increased PNPLA3 levels in two lines of cultured hepatocytes. Conversely, overexpression of BFAR decreased levels of endogenous PNPLA3 in HuH7 cells. BFAR and PNPLA3 co-immunoprecipitated when co-expressed in cells. BFAR promoted ubiquitylation of PNPLA3 in vitro in a reconstitution assay using purified, epitope-tagged recombinant proteins. To confirm that BFAR targets PNPLA3, we inactivated Bfar in mice. Levels of PNPLA3 protein were increased twofold in hepatic lipid droplets of Bfar-/- mice with no associated increase in PNPLA3 mRNA levels. Taken together these data are consistent with a model in which BFAR plays a role in the post-translational degradation of PNPLA3. The identification of BFAR provides a potential target to enhance PNPLA3 turnover and prevent FLD.

The Intersection of Genetic Factors, Aberrant Nutrient Metabolism and Oxidative Stress in the Progression of Cardiometabolic Disease.

Cardiometabolic disease (CMD), which encompasses metabolic-associated fatty liver disease (MAFLD), chronic kidney disease (CKD) and cardiovascular disease (CVD), has been increasing considerably in the past 50 years. CMD is a complex disease that can be influenced by genetics and environmental factors such as diet. With the increased reliance on processed foods containing saturated fats, fructose and cholesterol, a mechanistic understanding of how these molecules cause metabolic disease is required. A major pathway by which excessive nutrients contribute to CMD is through oxidative stress. In this review, we discuss how oxidative stress can drive CMD and the role of aberrant nutrient metabolism and genetic risk factors and how they potentially interact to promote progression of MAFLD, CVD and CKD. This review will focus on genetic mutations that are known to alter nutrient metabolism. We discuss the major genetic risk factors for MAFLD, which include Patatin-like phospholipase domain-containing protein 3 (PNPLA3), Membrane Bound O-Acyltransferase Domain Containing 7 (MBOAT7) and Transmembrane 6 Superfamily Member 2 (TM6SF2). In addition, mutations that prevent nutrient uptake cause hypercholesterolemia that contributes to CVD. We also discuss the mechanisms by which MAFLD, CKD and CVD are mutually associated with one another. In addition, some of the genetic risk factors which are associated with MAFLD and CVD are also associated with CKD, while some genetic risk factors seem to dissociate one disease from the other. Through a better understanding of the causative effect of genetic mutations in CMD and how aberrant nutrient metabolism intersects with our genetics, novel therapies and precision approaches can be developed for treating CMD.

The pleiotropic approach to coronavirus disease-19 pathogenesis: The impact of liver diseases associated host genetic variants.

Coronavirus disease-2019 (COVID-19) is a novel multisystemic viral disease caused pandemic. The disease impact involves liver and associated systems. Undoubtedly, host genetic background influences the predisposition and prediction of infection. Variants among human populations might increase susceptibility or protect against severe outcomes. In this manner, rs738409 variant of patatin-like phospholipase domain-containing protein 3 gene appears to be protective in some populations in spite of its aggravating effect on non-alcoholic fatty liver diseases (NAFLDs) and steatohepatitis. DRB1*15:01 allele of human leukocyte antigen is associated with protective effect in European and Japanese populations. DRB1*03:01 contrarily increases the susceptibility of severe COVID-19 infection in European populations. rs1260326 in glucokinase regulatory protein gene, rs112875651 in tribbles homolog 1 gene, rs429358 in apolipoprotein 1, and rs58542926 in transmembrane 6 superfamily 2 alleles are found related with NAFLD and obesity; thus, hypercoagulability and severe COVID-19 outcomes. In chronic or acute liver diseases, comorbid syndromes are the key factors to explain increased severity. There might not be a direct association between the variant and severe COVID-19 infection. As it is concluded, there are genes and variants known and unknown yet to be studied to reveal the association with disease severity.

Altered profiles of circulating cytokines in chronic liver diseases (NAFLD/HCC): Impact of the PNPLA3I148M risk allele.

Individuals carrying the risk variant p.I148M of patatin-like phospholipase domain-containing protein 3 (PNPLA3) have a higher susceptibility to fatty liver diseases and associated complications, including HCC, a cancer closely linked to chronic inflammation. Here, we assessed circulating cytokine profiles for patients with chronic liver diseases genotyped for PNPLA3. Serum concentrations of 22 cytokines were measured by multiplex sandwich-ELISA. The cohort comprised 123 individuals: 67 patients with NAFLD without cirrhosis (57 steatosis, 10 NASH), 24 patients with NAFLD with cirrhosis, 21 patients with HCC (15 cirrhosis), and 11 healthy controls. Receiver operator characteristic analyses were performed to assess the suitability of the cytokine profiles for the prediction of steatosis, cirrhosis, and HCC. HGF, IL-6, and IL-8 levels were increased in patients, with ∼2-fold higher levels in patients with cirrhosis versus healthy, while platelet derived growth factor-BB (PDGF-BB) and regulated on activation, normal T cell expressed and secreted (RANTES) showed lower concentrations compared to controls. Migration inhibitory factor and monocyte chemoattractant protein-1 (MCP-1) were found at higher levels in NAFLD samples (maximum: NAFLD-cirrhosis) versus healthy controls and HCC samples. In receiver operator characteristic analyses, migration inhibitory factor, IL-8, IL-6, and monocyte chemoattractant protein-1 yielded high sensitivity scores for predicting noncirrhotic NAFLD (vs. healthy). The top combination to predict cirrhosis was HGF plus PDGF-BB. Migration inhibitory factor performed best to discriminate HCC from NAFLD; the addition of monokine induced gamma (MIG), RANTES, IL-4, macrophage colony-stimulating factor (M-CSF), or IL-17A as second parameters further increased the AUC values (> 0.9). No significant impact of the PNPLA3I148M allele on cytokine levels was observed in this cohort. Cytokines have biomarker potential in patients with fatty liver, possibly suited for early HCC detection in patients with fatty liver. Patients carrying the PNPLA3 risk allele did not present significantly different levels of circulating cytokines.

Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC.MethodsWe sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples.ResultsComprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3β and MAPK signaling.ConclusionsOur study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.

Abstract 13791: Non-Alcoholic Fatty Liver Disease Risk SNP Variants Affect Lipid Metabolism

Introduction: The impact of non-alcoholic fatty liver disease (NAFLD) risk SNP variants on changes in lipid metabolism in the general population has yet to be determined. We aimed to assess this association using a large prospective cohort study. Methods: A total of 1488 Japanese men and women (median age 62 years and median body mass index 24.7 kg/m2) free of cardiovascular disease were studied. Among NAFLD risk SNPs, variants of patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR), and neurocan (NCAN) were assessed. Plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (sdLDL-C), LDL-triglycerides (LDL-TG), high-density lipoprotein cholesterol (HDL-C), HDL3-C, triglycerides (TG), lipoprotein(a), and adiponectin were measured at both baseline and after a five-year follow-up. Univariate and multivariate analyses, Kruskal-Wallis, Wilcoxon’s rank sum, and paired t-tests were performed to examine the associations between NAFLD risk SNPs and lipoproteins. Results: All of the measured lipoproteins increased significantly over 5 years. The TM6SF2 variant did not affect lipoproteins at baseline. However, after 5 years, the nonCC group had significantly lower HDL-C and HDL3-C and significantly higher LDL-C, sdLDL-C, and LDL-TG than the CC group. For the GCKR variant, TG, LDL-C, sdLDL-C, and LDL-TG were significantly higher in the CT/TT group at baseline, and LDL-C, sdLDL-C, and LDL-TG were still significantly higher in the same group at 5 years versus the CC group. The PNPLA3 and NCAN variants did not affect lipoproteins in this study. Conclusions: NAFLD risk SNP variants associated with gene products regulating both glucose and lipid metabolism can affect atherogenic lipoprotein levels.

PNPLA3 rs738409 polymorphism and kidney dysfunction: an association beyond nonalcoholic fatty liver disease?

This commentary is primarily devoted to a recent observational study by Mantovani and colleagues (Aliment Pharmacol Ther. 2023; 57: 1093-102) examining the adverse effect of the patatin-like phospholipase domain-containing protein-3 (PNPLA3 ) rs738409 G allele on the kidney function in a cohort of 1,144 middle-aged Italian individuals with metabolic dysfunction. In this study, the authors found that the PNPLA3 rs738409 G allele was significantly associated with lower levels of estimated glomerular filtrate rate (eGFR), even after adjusting for not only common anthropometric and cardiometabolic risk factors but also ethnicity, serum liver enzymes, use of drugs against dyslipidemia and chronic kidney disease polygenic risk score. Additionally, in a subgroup of 144 patients followed for a median of 17 months, the authors also found that the PNPLA3 rs738409 G allele was independently associated with a faster eGFR decline. Commenting on the cohort study by Mantovani et al ., we also summarized the rapidly expanding evidence linking the PNPLA3 rs738409 variant with the risk of kidney disease. Furthermore, we discussed the potential research implications of these findings.

Gene Variants Implicated in Steatotic Liver Disease: Opportunities for Diagnostics and Therapeutics.

Non-alcoholic fatty liver disease (NAFLD) describes a steatotic (or fatty) liver occurring as a consequence of a combination of metabolic, environmental, and genetic factors, in the absence of significant alcohol consumption and other liver diseases. NAFLD is a spectrum of conditions. Steatosis in the absence of inflammation is relatively benign, but the disease can progress into more severe forms like non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. NAFLD onset and progression are complex, as it is affected by many risk factors. The interaction between genetic predisposition and other factors partially explains the large variability of NAFLD phenotype and natural history. Numerous genes and variants have been identified through large-scale genome-wide association studies (GWAS) that are associated with NAFLD and one or more subtypes of the disease. Among them, the largest effect size and most consistent association have been patatin-like phospholipase domain-containing protein 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), and membrane-bound O-acyltransferase domain containing 7 (MBOAT7) genes. Extensive in vitro and in vivo studies have been conducted on these variants to validate these associations. The focus of this review is to highlight the genetics underpinning the molecular mechanisms driving the onset and progression of NAFLD and how they could potentially be used to improve genetic-based diagnostic testing of the disease and develop personalized, targeted therapeutics.

Pathology and Pathogenesis of Metabolic Dysfunction-Associated Steatotic Liver Disease-Associated Hepatic Tumors.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive fat accumulation in the livers of patients without a history of alcohol abuse. It is classified as either simple steatosis (nonalcoholic fatty liver) or nonalcoholic steatohepatitis (NASH), which can progress to liver cirrhosis and hepatocellular carcinoma (HCC). Recently, it was suggested that the terms "metabolic dysfunction-associated steatotic liver disease (MASLD)" and "metabolic dysfunction-associated steatohepatitis (MASH)" should replace the terms "nonalcoholic fatty liver disease (NAFLD)" and "nonalcoholic steatohepatitis (NASH)", respectively, with small changes in the definitions. MASLD, a hepatic manifestation of metabolic syndrome, is rapidly increasing in incidence globally, and is becoming an increasingly important cause of HCC. Steatohepatitic HCC, a histological variant of HCC, is characterized by its morphological features resembling non-neoplastic steatohepatitis and is closely associated with underlying steatohepatitis and metabolic syndrome. Variations in genes including patatin-like phospholipase domain-containing protein 3 (PNPLA3), transmembrane 6 superfamily 2 (TM6SF2), and membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7) are associated with the natural history of MASLD, including HCC development. The mechanisms of HCC development in MASLD have not been fully elucidated; however, various factors, including lipotoxicity, inflammation, reactive oxygen species, insulin resistance, and alterations in the gut bacterial flora, are important in the pathogenesis of MASLD-associated HCC. Obesity and MASLD are also recognized as risk factors for hepatocellular adenomas, and recent meta-analyses have shown an association between MASLD and intrahepatic cholangiocarcinoma. In this review, we outline the pathology and pathogenesis of MASLD-associated liver tumors.

PNPLA3 rs738409 Genetic Variant Inversely Correlates with Platelet Count, Thereby Affecting the Performance of Noninvasive Scores of Hepatic Fibrosis.

Noninvasive tests (NITs) including platelets (PLTs) have been proposed to replace hepatic biopsy for the diagnosis of nonalcoholic fatty liver disease (NAFLD), or as more recently redefined, metabolic dysfunction-associated steatotic liver disease (MASLD). There has been reported an inverse correlation between PLTs and progressive MASLD, which is also affected by the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 C>G mutation. However, the correlation between low PLTs and PNPLA3 genotype has been poorly investigated. We stratified 1155 biopsy-proven MASLD patients according to PNPLA3 genotype. The hepatic expression of genes involved in megakaryopoiesis was investigated in n = 167 bariatric patients by RNAseq. PLT count progressively decreased according to the number of PNPLA3 at-risk alleles, irrespective of the presence of advanced fibrosis. The hepatic expression of genes involved in PLT biogenesis was associated with the PNPLA3 GG genotype. Finally, the presence of the PNPLA3 homozygosity flattened the accuracy of fibrosis-4 (FIB-4) in discriminating histological fibrosis stages. The PNPLA3 GG genotype may underpower the accuracy of NITs which include PLT count in identifying those patients with potentially reversible stages of fibrosis.

Interaction between estrogen receptor-α and PNPLA3 p.I148M variant drives fatty liver disease susceptibility in women

Fatty liver disease (FLD) caused by metabolic dysfunction is the leading cause of liver disease and the prevalence is rising, especially in women. Although during reproductive age women are protected against FLD, for still unknown and understudied reasons some develop rapidly progressive disease at the menopause. The patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant accounts for the largest fraction of inherited FLD variability. In the present study, we show that there is a specific multiplicative interaction between female sex and PNPLA3 p.I148M in determining FLD in at-risk individuals (steatosis and fibrosis, P < 10−10; advanced fibrosis/hepatocellular carcinoma, P = 0.034) and in the general population (P < 10−7 for alanine transaminase levels). In individuals with obesity, hepatic PNPLA3 expression was higher in women than in men (P = 0.007) and in mice correlated with estrogen levels. In human hepatocytes and liver organoids, PNPLA3 was induced by estrogen receptor-α (ER-α) agonists. By chromatin immunoprecipitation and luciferase assays, we identified and characterized an ER-α-binding site within a PNPLA3 enhancer and demonstrated via CRISPR–Cas9 genome editing that this sequence drives PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional multilineage spheroids with stellate cells. These data suggest that a functional interaction between ER-α and PNPLA3 p.I148M variant contributes to FLD in women.

A nutrigenetic precision approach for the management of non-alcoholic fatty liver disease

The Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 single nucleotide polymorphism (SNP) is one of the major genetic determinant of non-alcoholic fatty liver disease (NAFLD) and is strongly regulated by changes in energy balance and dietary factors. We aimed to investigate the association between the PNPLA3 rs738409 SNP, nutrient intake and NAFLD severity. PNPLA3-rs738409 SNP was genotyped in 181 patients with NAFLD who completed the EPIC Food Frequency Questionnaire. Liver steatosis was evaluated by Controlled Attenuation Parameter (CAP) (Fibroscan®530, Echosens). According to the established cut-off, a CAP value≥300dB/m was used to identify severe steatosis (S3). An independent group of 46 biopsy-proven NAFLD subjects was used as validation cohort. Overall, median age was 53 years (range 44; 62) and 60.2% of patients were male. Most subjects (56.3%) had S3 and showed increased liver stiffness (p<0.001), AST (p=0.003) and ALT levels (p<0.001) compared to those with CAP<300dB/m. At logistic regression analyses we found that the interaction between carbohydrates intake and the carriers of the PNPLA3 G risk allele was significantly associated with S3 (p=0.001). The same result was confirmed in the validation cohort, were the interaction between high carbohydrate intake (48%) and PNPLA3 SNP was significantly associated with steatosis ≥33% (p=0.038). The intake of greater than or equal to 48% carbohydrate in NAFLD patients carriers of the CG/GG allele of PNPLA3 rs738409 may increase the risk of significant steatosis.

Saturated Fat-Mediated Upregulation of IL-32 and CCL20 in Hepatocytes Contributes to Higher Expression of These Fibrosis-Driving Molecules in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases, ranging from liver steatosis to metabolic dysfunction-associated steatohepatitis (MASH), increasing the risk of developing cirrhosis and hepatocellular carcinoma (HCC). Fibrosis within MASLD is critical for disease development; therefore, the identification of fibrosis-driving factors is indispensable. We analyzed the expression of interleukin 32 (IL-32) and chemokine CC ligand 20 (CCL20), which are known to be linked with inflammation and fibrosis, and for their expression in MASLD and hepatoma cells. RT-PCR, ELISA and Western blotting analyses were performed in both human liver samples and an in vitro steatosis model. IL-32 and CCL20 mRNA expression was increased in tissues of patients with NASH compared to normal liver tissue. Stratification for patatin-like phospholipase domain-containing protein 3 (PNPLA3) status revealed significance for IL-32 only in patients with I148M (rs738409, CG/GG) carrier status. Furthermore, a positive correlation was observed between IL-32 expression and steatosis grade, and between IL-32 as well as CCL20 expression and fibrosis grade. Treatment with the saturated fatty acid palmitic acid (PA) induced mRNA and protein expression of IL-32 and CCL20 in hepatoma cells. This induction was mitigated by the substitution of PA with monounsaturated oleic acid (OA), suggesting the involvement of oxidative stress. Consequently, analysis of stress-induced signaling pathways showed the activation of Erk1/2 and p38 MAPK, which led to an enhanced expression of IL-32 and CCL20. In conclusion, cellular stress in liver epithelial cells induced by PA enhances the expression of IL-32 and CCL20, both known to trigger inflammation and fibrosis.