Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study

Abstract

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-β (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

FIGURE 1

Global DNA methylation and histone signatures in plasma of pediatric patients with nonalcoholic fatty liver disease (NAFLD). (A) Percentage of cell‐free DNA (cfDNA) methylation in NAFLD (red) and control (green) patients. ***p < 0.001 vs. controls. (B) Box plots of the standardized individual histones expression in NAFLD (red) and controls (green); outliers were removed; the horizontal bars represent the medians and the whiskers. *p < 0.05 vs. controls. (C) Percentage of cfDNA methylation in patients with nonalcoholic steatohepatitis (NASH) NAFLD (red) and no NASH (yellow). ***p < 0.001 vs. controls. (D) Box plots of the standardized histones expression in patients with NASH (red) and no NASH (yellow); outliers were removed; the horizontal bars represent the medians and the whiskers.

FIGURE 2

Correlation between circulating cfDNA methylation and histological traits in patients with NAFLD. Correlation between percentages of cfDNA methylation in patients with NAFLD (n = 67) stratified by steatosis (A), lobular inflammation (B), fibrosis (C), ballooning (D), and NAFLD activity score (NAS) (E).

FIGURE 3

Correlation between circulating histones and histological traits in patients with NAFLD. Correlation between macroH2A1.2 histone expression and steatosis (A) or NAS (B) in patients with NAFLD (n = 60); macroH2A1.2 histone expressions were quantile‐normalized to produce these plots.

FIGURE 4

Performance of cfDNA methylation, patatin‐like phospholipase domain containing 3 (PNPLA3) rs738409, and macroH2A1.2 in predicting children with NASH. K‐nearest‐neighbors (KNN) receiver operating characteristic (ROC) curves achieved combining cfDNA methylation, PNPLA3 rs738409, and macroH2A1.2 features. True positive rate (TPR) and false positive rate (FPR) are indicated in the graph. Abbreviation: AUC, area under the curve.

FIGURE 5

Identification of the best model for predicting NASH. (A) Right bars represent the AUC achieved using the single feature values to predict the NASH status; red and green bars indicate, respectively, the positive and negative associations between the feature and the NASH status; left bars represent the log p value from the Mann–Whitney test for the same features performed on the NASH and no‐NASH NAFLD groups. (B) KNN ROC curves achieved by combining additional features to the cfDNA methylation and PNPLA3 rs738409 features. TPR and FPR are indicated in the graph.