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Ambiguous alteration patterns of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) involved in Alzheimer's disease (AD) obstructed the mechanism investigation of this neurological disorder from epigenetic view. Here, we applied a fully quantitative and validated LC-MS/MS method to determine genomic 5mC and 5hmC in the brain cortex of 3 month-aged (12, 15, and 18 month) AD model mouse and found significant increases of 5mC and 5hmC levels in different months of AD mouse when compared with age-matched wild-type control and exhibited rising trend from 12-month to 18-month AD mouse, thereby supporting genomic DNA methylation and hydroxymethylation were positively correlated with developing AD. [ABSTRACT FROM AUTHOR]

Tumor suppressor genes (TSGs) with DNA methylation has been suggested as effective biomarkers for cancer identification and promoter methylation in TSGs has been widely discussed. However, the feasibility of TSG exon methylation used for discriminating cancers was not fully clarified yet. In this study, genomic DNA was isolated from colorectal tissues and responding cancer-adjacent counterparts, and then bisulfite-converted. The exon 1 region of four popular TSGs including ALX4, FBN1, MLH1 and BCL2 was amplified from bisulfite-treated DNA, using nest- polymerase chain reaction (PCR) technique. The purified amplicon was hydrolyzed and then used to detect the methylation level using a robust and convenient LC-MS/MS method. The methodological validation revealed the favorable sensitivity and accuracy of established LC-MS/MS approach. The LC-MS/MS result showed that the methylation level of ALX4 and FBN1 exon in colorectal cancerous tissues (23.70 ± 10.85 and 33.23 ± 6.64) was significant higher than that in cancer-adjacent tissues (12.15 ± 7.08 and 22.08 ± 4.46) with statistic difference, and their value of area under curve (AUC) in receiver operator characteristic curve (ROC) analysis were higher than 0.8. On the other hand, the methylation level of MLH1 and BCL2 was 13.95 ± 6.93 and 15.46 ± 2.41 in CRC tissues and 13.87 ± 3.47 and 12.84 ± 1.91 in cancer-adjacent tissues, respectively. It is demonstrated that the methylation alteration of MLH1 and BCL2 exhibited a non-differential association between two groups. The finding indicated exon methylation of ALX4 and FBN1 could effectively distinguish CRC from non-CRC tissue and exon-based methylation should be used as potential biomarkers for cancer identification. [ABSTRACT FROM AUTHOR]

The identification of biomarkers would be benefit for the diagnosis and treatment of colorectal cancer in clinic. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was effective indicators to reveal the occurrence and development of cancers. To screen out the methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer-adjacent tissues collected from 30 CRC patients and then bisulfite-converted. The exon regions of 5 targeted genes (CNRIP1, HIC1, RUNX3, p15, and SFRP2) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and evaluated by using the formula. The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC-MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer-adjacent individuals, while that of HIC1, p15, and SFRP2 had no difference between 2 subjects. These finding supported that CNRIP1 and RUNX3 can be used as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC-MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation is associated with various diseases. Quantitative analysis of regional DNA methylation levels of some specific genes would aid in diseases diagnosis and risk stratification. In this study, we developed a robust method for detecting DNA methylation level over targeted genomic regions using nucleobases quantification in bisulfite amplicons by isotope dilution liquid chromatography tandem mass spectrometry coupled with a simple equation. This method had wide detection range (from 0% to 100% methylation) and high accuracy while more time-saving compared to clonal bisulfite sequencing method. The application for clinical tissue samples showed good applicability and cost effectiveness. This analytical method is suitable for quantifying average DNA methylation level over targeted genomic regions and expected to be a useful tool for detecting DNA methylation biomarkers. [ABSTRACT FROM AUTHOR]