LC-MS/MS Quantification of the Methylation Status of Clinical Samples of NASH Liver Tissues

Nonalcoholic fatty liver disease (NAFLD) is a common hepatic disorder in individuals who drink little or no alcohol. A key marker associated with NAFLD is the accumulation of fats in the liver (> 5% of liver weight). NAFLD is becoming a global health problem, and currently, almost one in four adults in the United States has NAFLD. NAFLD can progress to a more severe stage called nonalcoholic steatohepatitis (NASH), characterized by inflammation, cell injury, and fibrosis. Progression of NASH can lead to end-stage liver diseases such as liver cancer. It has been reported that aberrant epigenetic modification patterns are associated with inappropriate gene expression and the pathogenesis of NAFLD and NASH. It has been reported that histone modifications initiated the development of insulin resistance, a NAFLD-associated clinical trigger.[i] Another key epigenetic modification that controls the NAFLD and NASH progression is DNA methylation. DNA methyltransferases (DNMT) catalyze the transfer of the methyl group from S-adenosylmethionine (SAM) into DNA. Interestingly, hepatic DNMT expression levels in patients with steatohepatitis were increased relative to normal individuals.[ii] Moreover, relevant differences in the methylation patterns can distinguish patients with advanced versus mild NAFLD stages. Genes that regulate steatohepatitis, fibrosis, and carcinogenesis have the most dysregulation in their methylation status.[iii]

Recently, Visikol studied the epigenetics of four liver biopsies, two NASH patients, and two normal individuals, to establish the baseline levels of global DNA methylation and N6Me RNA methylation. DNA and RNA were extracted from the tissues using kits (Qiagen, Cat. Nos. 69504 and 74104), enzymatically hydrolyzed utilizing our optimized protocol, and finally, the targeted nucleosides were separated and quantified by our LC-MS/MS instrument. Figure 1 summarizes our findings on the baseline of the global DNA and RNA methylation from the clinical samples. Interestingly, one of the NASH livers showed a significant global DNA methylation percentage increase relative to the healthy baseline (Figure 1A). However, the global N6Me RAN methylation percentage was maintained among all the clinal samples (Figure 1B). These results suggest that in NASH liver2, post-translational mechanisms might be operating.

Figure 1. The top panel shows normal liver and NASH liver. The bottom panel shows Baseline global DNA methylation percentage (A), and global N6Me global RNA methylation (B) of liver tissues from healthy individuals and NASH patients quantified by LC-MS/MS. 

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[i] Ling C., Groop L. Epigenetics: A molecular link between environmental factors and type 2 diabetes. Diabetes2009, 58, 2718–2725.

[ii] Pirola CJ, Gianotti TF, Burgueño AL, Rey-Funes M, Loidl CF, Mallardi P, Martino JS, Castaño GO, Sookoian S. Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease. Gut., 2013, 62, 1356-1363.

[iii] Murphy SK, Yang H, Moylan CA, Pang H, Dellinger A, Abdelmalek MF, Garrett ME, Ashley-Koch A, Suzuki A, Tillmann HL, Hauser MA, Diehl AM. Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease. Gastroenterology. 2013, 145, 1076-1087.

2022-09-09T07:41:23-05:00Tags: , , , , |

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