BACKGROUND: Although metabolic associate fatty liver disease (MAFLD) is associated with obesity, it can also occur in lean patients. MAFLD is more aggressive in lean patients compared to obese patients, with a higher risk of mortality. Specific biomarkers to diagnose differentially lean or overweight MAFLD are missing. Histones and nucleosomes are released in the bloodstream upon cell death. Here, we propose a new, fast, imaging and epigenetics based approach to investigate the severity of steatosis in lean MAFLD patients. RESULTS: A total of 53 non-obese patients with histologically confirmed diagnosis of MAFLD were recruited. Twenty patients displayed steatosis grade 1 (0-33%), 24 patients with steatosis grade 2 (34-66%) and 9 patients with steatosis grade 3 (67-100%). The levels of circulating nucleosomes were assayed using enzyme-linked immunosorbent assay, while individual histones or histone dimers were assayed in serum samples by means of a new advanced flow cytometry ImageStream(X)-adapted method. Circulating nucleosome levels associated poorly with MAFLD in the absence of obesity. We implemented successfully a multi-channel flow methodology on ImageStream(X), to image single histone staining (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2). We report here a significant depletion of the levels of histone variants macroH2A1.1 and macroH2A1.2 in the serum of lean MAFLD patients, either individually or in complex with H2B. CONCLUSIONS: In summary, we identified a new circulating histone signature able to discriminate the severity of steatosis in individuals with lean MAFLD, using a rapid and non-invasive ImageStream(X)-based imaging technology.

Department of Adaptive Biotechnologies Global Change Research Institute CAS Brno Czech Republic

Department of Gastroenterological Endocrine Metabolic and Nephro Urological Sciences Fondazione Policlinico Universitario A Gemelli IRCCS Rome Italy

Department of Medical and Surgical Sciences and Advanced Technologies GF Ingrassia University of Catania Catania Italy

Institute for Liver and Digestive Health Division of Medicine University College London London UK

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Research Unit of Molecular Genetics of Complex Phenotypes Bambino Gesù Children's Hospital IRCCS Rome Italy

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Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018;15(1):11–20. PubMed

Eslam M, Sanyal AJ, George J, International Consensus P MAFLD: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease. Gastroenterology. 2020;158(7):1999–2014.e1. PubMed

Anstee QM, Reeves HL, Kotsiliti E, Govaere O, Heikenwalder M. From NASH to HCC: current concepts and future challenges. Nat Rev Gastroenterol Hepatol. 2019;16(7):411–428. PubMed

Mazzoccoli G, Miele L, Marrone G, Mazza T, Vinciguerra M, Grieco A. A role for the biological clock in liver cancer. Cancers. 2019;11(11):1778. PubMed PMC

Mazzoccoli G, Miele L, Oben J, Grieco A, Vinciguerra M. Biology, epidemiology, clinical aspects of hepatocellular carcinoma and the role of sorafenib. Curr Drug Targets. 2016;17(7):783–799. PubMed

Lazarus JV, Ekstedt M, Marchesini G, Mullen J, Novak K, Pericas JM, et al. A cross-sectional study of the public health response to non-alcoholic fatty liver disease in Europe. J Hepatol. 2020;72(1):14–24. PubMed

Deurenberg-Yap M, Schmidt G, van Staveren WA, Deurenberg P. The paradox of low body mass index and high body fat percentage among Chinese, Malays and Indians in Singapore. Int J Obes Relat Metab Disord. 2000;24(8):1011–1017. PubMed

Duarte SMB, Stefano JT, Miele L, Ponziani FR, Souza-Basqueira M, Okada L, et al. Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study. Nutr Metab Cardiovasc Dis. 2018;28(4):369–384. PubMed

Denkmayr L, Feldman A, Stechemesser L, Eder SK, Zandanell S, Schranz M, et al. Lean patients with non-alcoholic fatty liver disease have a severe histological phenotype similar to obese patients. J Clin Med. 2018;7(12):562. PubMed PMC

Hagstrom H, Nasr P, Ekstedt M, Hammar U, Stal P, Hultcrantz R, et al. Risk for development of severe liver disease in lean patients with nonalcoholic fatty liver disease: a long-term follow-up study. Hepatol Commun. 2018;2(1):48–57. PubMed PMC

Golabi P, Paik J, Fukui N, Locklear CT, de Avilla L, Younossi ZM. Patients with lean nonalcoholic fatty liver disease are metabolically abnormal and have a higher risk for mortality. Clin Diabetes. 2019;37(1):65–72. PubMed PMC

Kountouras J, Polyzos SA, Katsinelos P, Doulberis M, Zavos C, Kazakos E, et al. Letter: helicobacter pylori in lean and obese patients with non-alcoholic fatty liver disease. Aliment Pharmacol Ther. 2017;46(6):637–638. PubMed

Sookoian S, Pirola CJ. Systematic review with meta-analysis: the significance of histological disease severity in lean patients with nonalcoholic fatty liver disease. Aliment Pharmacol Ther. 2018;47(1):16–25. PubMed

Bedogni G, Bellentani S, Miglioli L, Masutti F, Passalacqua M, Castiglione A, et al. The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol. 2006;6:33. PubMed PMC

Lo Re O, Maugeri A, Hruskova J, Jakubik J, Kucera J, Bienertova-Vasku J, et al. Obesity-induced nucleosome release predicts poor cardio-metabolic health. Clin Epigenetics. 2019;12(1):2. PubMed PMC

Ramachandran S, Henikoff S. Nucleosome dynamics during chromatin remodeling in vivo. Nucleus. 2016;7(1):20–26. PubMed PMC

Holdenrieder S, Stieber P. Clinical use of circulating nucleosomes. Crit Rev Clin Lab Sci. 2009;46(1):1–24. PubMed

McAnena P, Brown JA, Kerin MJ. Circulating nucleosomes and nucleosome modifications as biomarkers in cancer. Cancers. 2017;9(1):5. PubMed PMC

Bauden M, Pamart D, Ansari D, Herzog M, Eccleston M, Micallef J, et al. Circulating nucleosomes as epigenetic biomarkers in pancreatic cancer. Clin Epigenetics. 2015;7:106. PubMed PMC

Holdenrieder S, Stieber P, von Pawel J, Raith H, Nagel D, Feldmann K, et al. Circulating nucleosomes predict the response to chemotherapy in patients with advanced non-small cell lung cancer. Clin Cancer Res. 2004;10(18 Pt 1):5981–5987. PubMed

Rahier JF, Druez A, Faugeras L, Martinet JP, Gehenot M, Josseaux E, et al. Circulating nucleosomes as new blood-based biomarkers for detection of colorectal cancer. Clin Epigenetics. 2017;9:53. PubMed PMC

Roth C, Pantel K, Muller V, Rack B, Kasimir-Bauer S, Janni W, et al. Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression. BMC Cancer. 2011;11:4. PubMed PMC

Xu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, et al. Extracellular histones are major mediators of death in sepsis. Nat Med. 2009;15(11):1318–1321. PubMed PMC

Huang H, Evankovich J, Yan W, Nace G, Zhang L, Ross M, et al. Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice. Hepatology. 2011;54(3):999–1008. PubMed PMC

Buschbeck M, Hake SB. Variants of core histones and their roles in cell fate decisions, development and cancer. Nat Rev Mol Cell Biol. 2017;18(5):299–314. PubMed

Bereshchenko O, Lo Re O, Nikulenkov F, Flamini S, Kotaskova J, Mazza T, et al. Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome. Clin Epigenetics. 2019;11(1):121. PubMed PMC

Giallongo S, Lo Re O, Vinciguerra M. Macro histone variants: emerging rheostats of gastrointestinal cancers. Cancers. 2019;11(5):676. PubMed PMC

Lo Re O, Douet J, Buschbeck M, Fusilli C, Pazienza V, Panebianco C, et al. Histone variant macroH2A1 rewires carbohydrate and lipid metabolism of hepatocellular carcinoma cells towards cancer stem cells. Epigenetics. 2018;13(8):829–845. PubMed PMC

Lo Re O, Fusilli C, Rappa F, Van Haele M, Douet J, Pindjakova J, Rocha SW, Pata I, Valčíková B, Uldrijan S, Yeung RS, Peixoto CA, Roskams T,Buschbeck M, Mazza T, Vinciguerra M. Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma. Hepatology. 2018;67(2):636–50. PubMed

Lo Re O, Vinciguerra M. Histone MacroH2A1: a chromatin point of intersection between fasting, senescence and cellular regeneration. Genes (Basel) 2017;8(12):367. PubMed PMC

Lo Re O, Mazza T, Giallongo S, Sanna P, Rappa F, Vinh Luong T, et al. Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4(+)CD25(+)FoxP3(+) regulatory T cells activation. Theranostics. 2020;10(2):910–924. PubMed PMC

Ogle LF, Orr JG, Willoughby CE, Hutton C, McPherson S, Plummer R, et al. Imagestream detection and characterisation of circulating tumour cells - a liquid biopsy for hepatocellular carcinoma? J Hepatol. 2016;65(2):305–313. PubMed

Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313–1321. PubMed

Sperling R, Bustin M. Histone dimers: a fundamental unit in histone assembly. Nucleic Acids Res. 1976;3(5):1263–1275. PubMed PMC

Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature. 1997;389(6648):251–260. PubMed

Rappa F, Greco A, Podrini C, Cappello F, Foti M, Bourgoin L, et al. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma. PLoS One. 2013;8(1):e54458. PubMed PMC

Podrini C, Koffas A, Chokshi S, Vinciguerra M, Lelliott CJ, White JK, et al. MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis. FASEB J. 2015;29(5):1676–1687. PubMed

Pazienza V, Panebianco C, Rappa F, Memoli D, Borghesan M, Cannito S, et al. Histone macroH2A1.2 promotes metabolic health and leanness by inhibiting adipogenesis. Epigenetics Chromatin. 2016;9:45. PubMed PMC

Wan D, Liu C, Sun Y, Wang W, Huang K, Zheng L. MacroH2A1.1 cooperates with EZH2 to promote adipogenesis by regulating Wnt signaling. J Mol Cell Biol. 2017;9(4):325–337. PubMed

Sun K, Jiang P, Cheng SH, Cheng THT, Wong J, Wong VWS, et al. Orientation-aware plasma cell-free DNA fragmentation analysis in open chromatin regions informs tissue of origin. Genome Res. 2019;29(3):418–427. PubMed PMC

Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell. 2016;164(1-2):57–68. PubMed PMC

Hurtado-Bages S, Guberovic I, Buschbeck M. The macroH2A1.1 - PARP1 axis at the intersection between stress response and metabolism. Front Genet. 2018;9:417. PubMed PMC

Szanto M, Bai P. The role of ADP-ribose metabolism in metabolic regulation, adipose tissue differentiation, and metabolism. Genes Dev. 2020;34(5-6):321–340. PubMed PMC

Changolkar LN, Singh G, Cui K, Berletch JB, Zhao K, Disteche CM, et al. Genome-wide distribution of macroH2A1 histone variants in mouse liver chromatin. Mol Cell Biol. 2010;30(23):5473–5483. PubMed PMC

Borghesan M, Mazzoccoli G, Sheedfar F, Oben J, Pazienza V, Vinciguerra M. Histone variants and lipid metabolism. Biochem Soc Trans. 2014;42(5):1409–1413. PubMed

Fracanzani AL, Petta S, Lombardi R, Pisano G, Russello M, Consonni D, et al. Liver and cardiovascular damage in patients with lean nonalcoholic fatty liver disease, and association with visceral obesity. Clin Gastroenterol Hepatol. 2017;15(10):1604–1611. PubMed

Ginley BG, Emmons T, Lutnick B, Urban CF, Segal BH, Sarder P. Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy. Sci Rep. 2017;7(1):17755. PubMed PMC

Feng RN, Du SS, Wang C, Li YC, Liu LY, Guo FC, et al. Lean-non-alcoholic fatty liver disease increases risk for metabolic disorders in a normal weight Chinese population. World J Gastroenterol. 2014;20(47):17932–17940. PubMed PMC

Furuncuoglu Y, Tulgar S, Dogan AN, Cakar S, Tulgar YK, Cakiroglu B. How obesity affects the neutrophil/lymphocyte and platelet/lymphocyte ratio, systemic immune-inflammatory index and platelet indices: a retrospective study. Eur Rev Med Pharmacol Sci. 2016;20(7):1300–1306. PubMed

Gao B, Tsukamoto H. Inflammation in alcoholic and nonalcoholic fatty liver disease: friend or foe? Gastroenterology. 2016;150(8):1704–1709. PubMed PMC

Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28(7):412–419. PubMed

Movsisyan NK, Vinciguerra M, Lopez-Jimenez F, Kunzova S, Homolka M, Jaresova J, et al. Kardiovize Brno 2030, a prospective cardiovascular health study in Central Europe: Methods, baseline findings and future directions. Eur J Prev Cardiol. 2018;25(1):54–64. PubMed

Medina-Inojosa JR, Vinciguerra M, Maugeri A, Kunzova S, Sochor O, Movsisyan N, Geda YE, Stokin GB, Lopez-Jimenez F. Prevalence of ideal cardiovascular health in a Central European community: results from the Kardiovize Brno 2030 Project. Eur J Prev Cardiol. 2020;27(4):441–3. PubMed

Kozakova M, Palombo C, Eng MP, Dekker J, Flyvbjerg A, Mitrakou A, et al. Fatty liver index, gamma-glutamyltransferase, and early carotid plaques. Hepatology. 2012;55(5):1406–1415. PubMed

Huang X, Xu M, Chen Y, Peng K, Huang Y, Wang P, et al. Validation of the fatty liver index for nonalcoholic fatty liver disease in middle-aged and elderly Chinese. Medicine. 2015;94(40):e1682. PubMed PMC

Elliott P, Peakman TC, Biobank UK. The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine. Int J Epidemiol. 2008;37(2):234–244. PubMed

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