Multi-omics mendelian randomization identifies ferroptosis-related genetic variants linked to NAFLD risk

Abstract

Background: Ferroptosis, an iron-dependent cell death, contributes to non-alcoholic fatty liver disease (NAFLD), but causal genes remain unclear. This study identifies ferroptosis-related genes driving NAFLD to fill this gap.

Methods: Summary data-based Mendelian Randomization (SMR) analyzed 564 ferroptosis-related genes for causal links to NAFLD by integrating mQTL/eQTL/pQTL data with GWAS, validated in FinnGen and the GWAS Catalog. To further substantiate our findings, expression of lead candidate genes was validated in independent human liver tissue datasets from the Gene Expression Omnibus (GEO). HepG2 cells were used to assess candidate gene expression, oxidative stress, and ferroptosis under free fatty acids (FFA) treatment. We evaluated cell viability, lipid ROS levels, gene expression, protein levels, and oxidative stress markers.

Results: SMR analysis revealed an association between SLC2A6 methylation/expression and NAFLD risk. Hypermethylation at cg02257517 was associated with increased NAFLD risk (OR=1.032, 95 % CI=1.013-1.051), while higher SLC2A6 expression correlated with lower risk (OR=0.919, 95 % CI=0.87-0.97). Multi-omics analysis confirmed an inverse relationship between SLC2A6 expression and cg02257517 methylation (OR=0.741, 95 % CI=0.66-0.832), suggesting hypermethylation downregulates SLC2A6, increasing NAFLD susceptibility. Those results were further validated in HepG2 cells, where the candidate gene demonstrated a protective role against FFA-induced oxidative stress and ferroptosis. Overexpression of the gene significantly mitigated these pathological effects, supporting its potential as a therapeutic target.

Conclusion: Multi-omics Mendelian randomization identified SLC2A6 hypermethylation as a causal NAFLD risk factor by suppressing its expression. Functional validation in disease-relevant models revealed SLC2A6-mediated protection against oxidative stress and ferroptosis, highlighting its potential as a therapeutic target for NAFLD through epigenetic or gene-based interventions.

Keywords

Non-Alcoholic Fatty Liver Disease / Ferroptosis / Multi-Omics / Mendelian Randomization Analysis / Quantitative Trait Loci / DNA Methylation / Genome-Wide Association Study

Declarations

Not applicable.

Authors’ contributions

Weifeng Wu: Investigation, Formal analysis. Yizhong Bao: Conceptualization. Ying Yuan: Data curation. Jianjun Zhang: Writing - review & editing. Yue Zhang: Conceptualization. Xiaohu Yang: Writing - review & editing, Validation, Formal analysis. Zhen Wang: Writing - review & editing, Writing - original draft, Conceptualization. Xiao Liu: Writing - review & editing, Writing - original draft, Investigation, Formal analysis, Data curation, Conceptualization. Huadi Zhou: Methodology. Chenhan Shou: Investigation.

Ethics approval and consent to participate

As the study utilized publicly accessible databases, there was no requirement for ethics committee approval or informed consent from participants. All methods were performed in accordance with the relevant guidelines and regulations.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Funding

Not applicable.

Declarations of Competing Interests

The authors declare no competing interests.

Acknowledgements

Not applicable.

Authors' other information

Not applicable.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.prmedi.2025.100063.

References

[1] Mitra S, De A, Chowdhury A. Epidemiology of non-alcoholic and alcoholic fatty liver diseases. Transl Gastroenterol Hepatol. 2020; 5:16.

[2] Tacke F, Weiskirchen R. Non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH)-related liver fibrosis: mechanisms, treatment and prevention. Ann Transl Med. 2021; 9:729.

[3] Geh D, Anstee QM, Reeves HL. NAFLD-associated HCC: progress and opportunities. J Hepatocell Carcinoma. 2021; 8:223-239.

[4] Mehta KJ, Farnaud SJ, Sharp PA. Iron and liver fibrosis: mechanistic and clinical aspects. World J Gastroenterol. 2019; 25:521.

[5] Henry L, Paik J, Younossi Z. M. the epidemiologic burden of non-alcoholic fatty liver disease across the world. Aliment Pharmacol Ther. 2022; 56:942-956.

[6] Huang DQ, El-Serag HB, Loomba R. Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2021; 18:223-238.

[7] Muthiah MD, Cheng Han N, Sanyal AJ. A clinical overview of non-alcoholic fatty liver disease: a guide to diagnosis, the clinical features, and complications—what the non-specialist needs to know. Diabetes Obes Metab. 2022; 24:3-14.

[8] Raza S, Rajak S, Upadhyay A, Tewari A, Sinha RA. Current treatment paradigms and emerging therapies for NAFLD/NASH. Front Biosci. 2021; 26:206.

[9] Jonas W, Schürmann A. Genetic and epigenetic factors determining NAFLD risk. Mol Metab. 2021; 50:101111.

[10] Gensluckner S, Wernly B, Datz C, Aigner E. Iron, oxidative stress, and metabolic dysfunction—associated steatotic liver disease. Antioxidants. 2024; 13:208.

[11] González-Domínguez Á, et al. Iron metabolism in obesity and metabolic syndrome. Int J Mol Sci. 2020; 21:5529.

[12] Wu J, et al. Ferroptosis in liver disease: new insights into disease mechanisms. Cell Death Discov. 2021; 7:276. https://doi.org/10.1038/s41420-021-00660-4

[13] YH L. Study on the mechanism of apoptosis induced by environmental endocrine disruptor nickel sulfate in thyroid and pancreatic tissues and cells in rats. Lanzhou University; 2020.

[14] Porcu E, et al. Mendelian randomization integrating GWAS and eQTL data reveals genetic determinants of complex and clinical traits. Nat Commun. 2019; 10:3300.

[15] Zhou N, et al. FerrDb V2:update of the manually curated database of ferroptosis regulators and ferroptosis-disease associations. Nucleic Acids Res. 2023;51:D571-D582.

[16] Wu Y, et al. Integrative analysis of omics summary data reveals putative mechanisms underlying complex traits. Nat Commun. 2018; 9:918.

[17] Võsa U, et al. Large-scale cis-and trans-eQTL analyses identify thousands of genetic loci and polygenic scores that regulate blood gene expression. Nat Genet. 2021; 53:1300-1310.

[18] Pietzner M, et al. Mapping the proteo-genomic convergence of human diseases. Science. 2021;374:eabj1541.

[19] Consortium G. The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science. 2020; 369:1318-1330.

[20] Sun Z, et al. Genetic variants in HFE are associated with non-alcoholic fatty liver disease in lean individuals. JHEP Rep. 2023; 5:100744.

[21] Namjou B, et al. GWAS and enrichment analyses of non-alcoholic fatty liver disease identify new trait-associated genes and pathways across eMERGE Network. BMC Med. 2019; 17:1-19.

[22] Zhu Z, et al. Integration of summary data from GWAS and eQTL studies predicts complex trait gene targets. Nat Genet. 2016; 48:481-487. https://doi.org/10.1038/ng.3538

[23] Wu Y, et al. Integrative analysis of omics summary data reveals putative mechanisms underlying complex traits. Nat Commun. 2018; 9:918. https://doi.org/10.1038/s41467-018-03371-0

[24] Giambartolomei C, et al. Bayesian test for colocalisation between pairs of genetic association studies using summary statistics. PLoS Genet. 2014; 10:e1004383. https://doi.org/10.1371/journal.pgen.1004383

[25] Rasooly D, Peloso GM, Giambartolomei C. Bayesian genetic colocalization test of two traits using coloc. Curr Protoc. 2022; 2:e627.

[26] Zuber V, et al. Combining evidence from Mendelian randomization and colocalisation: review and comparison of approaches. Am J Hum Genet. 2022; 109:767-782.

[27] Zhu Z, et al. Integration of summary data from GWAS and eQTL studies predicts complex trait gene targets. Nat Genet. 2016; 48:481-487.

[28] Chu B, et al. ALOX12 is required for p53-mediated tumour suppression through a distinct ferroptosis pathway. Nat Cell Biol. 2019; 21:579-591.

[29] Shi J-F, et al. Targeting ferroptosis, a novel programmed cell death, for the potential of alcohol-related liver disease therapy. Front Pharmacol. 2023; 14:1194343.

[30] Zheng Y-D, Zhang Y, Ma J-Y, Sang C-Y, Yang J-L. A Carabrane-type sesquiterpenolide carabrone from carpesium cernuum inhibits SW 1990 pancreatic cancer cells by inducing ferroptosis. Molecules. 2022; 27:5841.

[31] Huang B, Yu Z, Cui D, Du F. MAPKAP1 orchestrates macrophage polarization and lipid metabolism in fatty liver-enhanced colorectal cancer. Transl Oncol. 2024; 45:101941.

[32] Huby T, Gautier EL. Immune cell-mediated features of non-alcoholic steatohepatitis. Nat Rev Immunol. 2022; 22:429-443. https://doi.org/10.1038/s41577-021-00639-3

[33] Zhang Y, Qin H, Bian J, Ma Z, Yi H. SLC2As as diagnostic markers and therapeutic targets in LUAD patients through bioinformatic analysis. Front Pharmacol. 2022; 13:1045179.

[34] Chen S-Y, et al. Investigating the expression and function of the glucose transporter GLUT 6 in obesity. Int J Mol Sci. 2022; 23:9798.

[35] Song W, Li D, Tao L, Luo Q, Chen L. Solute carrier transporters: the metabolic gatekeepers of immune cells. Acta Pharm Sin B. 2020; 10:61-78.

[36] Byrne FL, et al. Metabolic vulnerabilities in endometrial cancer. Cancer Res. 2014; 74:5832-5845.

[37] Basaranoglu M, Basaranoglu G, Bugianesi E. Carbohydrate intake and nonalcoholic fatty liver disease: fructose as a weapon of mass destruction. Hepatobiliary Surg Nutr. 2015; 4:109.

[38] Mueckler M, Thorens B. The SLC 2 (GLUT) family of membrane transporters. Mol Asp Med. 2013; 34:121-138.

[39] De Vito F, et al. Association between higher duodenal levels of the fructose carrier glucose transporter-5 and nonalcoholic fatty liver disease and liver fibrosis. J Intern Med. 2024; 295:171-180.

[40] DeBosch BJ, Chen Z, Saben JL, Finck BN, Moley KH. Glucose transporter 8 (GLUT8) mediates fructose-induced de novo lipogenesis and macrosteatosis. J Biol Chem. 2014; 289:10989-10998.

[41] Zhang W, et al. Exosome GLUT1 derived from hepatocyte identifies the risk of non-alcoholic steatohepatitis and fibrosis. Hepatol Int. 2023; 17:1170-1181. https://doi.org/10.1007/s12072-023-10520-1

[42] Maedera S, et al. GLUT6 is a lysosomal transporter that is regulated by inflammatory stimuli and modulates glycolysis in macrophages. FEBS Lett. 2019; 593:195-208.

[43] Du J, Ji Y, Qiao L, Liu Y, Lin J. Cellular endo-lysosomal dysfunction in the pathogenesis of non-alcoholic fatty liver disease. Liver Int. 2020; 40:271-280.

[44] Do Van B, et al. Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC. Neurobiol Dis. 2016; 94:169-178.

[45] Monteleone L, et al. PKCα inhibition as a strategy to sensitize neuroblastoma stem cells to etoposide by stimulating ferroptosis. Antioxidants. 2021; 10:691.

[46] Shu Y, et al. Hepatocyte-specific PKCβ deficiency protects against high-fat diet-induced nonalcoholic hepatic steatosis. Mol Metab. 2021; 44:101133.

[47] Zhang J, et al. PKCδ regulates hepatic triglyceride accumulation and insulin signaling in Leprdb/db mice. Biochem Biophys Res Commun. 2014; 450:1619-1625.

[48] Yang J, et al. Adipose transplantation improves olfactory function and neurogenesis via PKCα-involved lipid metabolism in Seipin Knockout mice. Stem Cell Res Ther. 2023; 14:239.

[49] Leppänen T, Tuominen RK, Moilanen E. Protein kinase C and its inhibitors in the regulation of inflammation: inducible nitric oxide synthase as an example. Basic Clin Pharmacol Toxicol. 2014; 114:37-43.

[50] Mishra D, Reddy I, Dey CS. PKCα Isoform inhibits insulin signaling and aggravates neuronal insulin resistance. Mol Neurobiol. 2023; 60:6642-6659.