Global hypomethylation pattern in systemic sclerosis: An application for absolute quantification of epigenetic DNA modification products by 2D-UPLC-MS/MS.

BACKGROUND: Systemic sclerosis (SSc) is a rare autoimmune disease characterized by progressive fibrosis of the skin and internal organs. Besides genetics risk factors, understanding the epigenetic modifications in SSc has been gaining acceleration in recent years. Epigenetic modifications are reversible and defined as druggable targets. In this context, it is highly important to present a systemic perspective to epigenetic modifications of SSc in terms of both pathogenesis and clinical utility. MATERIAL AND METHODS: DNA samples from the whole blood specimens of the 41 SSc patients and 27 healthy controls (HCs) were obtained. Absolute quantification of 5-mC, 5-hmdC, 5-cadC, 5-fdC, and 5-hmdU as the DNA methylation and demethylation products were performed using 2D-UPLC-MS/MS. Demographic data and clinical scores were recorded in detail. RESULTS: 5-hmdU was significantly higher in SSc patients while 5-hmdC was lower compared to the HCs (p < 0.01, p = 0.012 respectively). 5-cadC and 5-fdC had upward trend in SSc (p = 0.064; p = 0.066). These results support that SSc patients tend to have a global hypomethylation pattern. Clinical analyzes revealed that lung, gastrointestinal, joint, and vascular involvement of SSc is also associated with increased demethylation or decreased methylation profile. CONCLUSION: We performed absolute quantification of epigenetic DNA modification products in SSc for the first time. We demonstrated an upward trend in global hypomethylation in SSc. Furthermore, as a result of detailed clinical analyzes, the relationship between lung, GIS, and vascular involvement with epigenetic changes was shown. We believe that absolute quantification of DNA methylation and demethylation products with novel technologies can provide a deep understanding of disease pathogenesis and has the potential to mark an era for developing new therapeutic strategies.

Urinary Measurement of Epigenetic DNA Modifications and 8-oxodG as Possible Noninvasive Markers of Colon Cancer Evolution.

The active DNA demethylation mechanism involves 5-methylcytosine (5-mCyt) enzymatic oxidation with the subsequent formation of 5-hydroxymethylcytosine (5-hmCyt), which can be further oxidized to 5-formylcytosine (5-fCyt) and 5-carboxylcytosine (5-caCyt). The products of active DNA demethylation are released into the bloodstream and eventually also appear in urine. We used online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) to compare DNA methylation marks and 8-oxo-2'-deoxyguanosine (8-oxodG) in colorectal cancer and pre-cancerous condition in urine. The study included four groups of subjects: healthy controls, patients with inflammatory bowel disease (IBD), persons with adenomatous polyps (AD), and individuals with colorectal cancer (CRC). We have found that the level of 5-fCyt in urine was significantly lower for CRC and polyp groups than in the control group. The level of 5-hmCyt was significantly higher only in the CRC group compared to the control (2.3 vs. 2.1 nmol/mmol creatinine). Interestingly, we have found highly statistically significant correlation of 5-hydroxymethyluracil with 5-hydroxymethylcytosine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-methyl-2'-deoxycytidine in the CRC patients' group.

Urinary Measurement of Epigenetic DNA Modifications: A Non-Invasive Assessment of the Whole-Body Epigenetic Status in Healthy Subjects and Colorectal Cancer Patients.

Active mechanism of DNA demethylation can be responsible for the activation of previously silenced genes. Products of 5-methylcytosine oxidation are released into the bloodstream and eventually excreted with urine. Therefore, whole-body epigenetic status can be assessed non-invasively on the basis of the urinary excretion of a broad spectrum of epigenetic modifications: 5-hydroxymethylcytosine (5-hmCyt), 5-formylcytosine (5-fCyt), 5-carboxycytosine (5-caCyt), and 5-hydroxymethyluracil (5-hmUra). We have developed a specific and sensitive, isotope-dilution, automated, online, two-dimensional ultra-performance liquid chromatography system with tandem mass spectrometry (2D UPLC-MS/MS) to measure 5-hmCyt, 5-fCyt, 5-caCyt, and their deoxynucleosides in the same urine sample. Human urine contains all of the modifications except from 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC). A highly significant difference in the urinary excretion of 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) was found between healthy subjects and colorectal cancer patients (3.5 vs. 7.8 nmol mmol-1 creatinine, respectively), as well as strong correlations between the majority of analyzed compounds.