The Correlation between Gene Expression and DNA Methylation Levels of RAS p21 Protein Activator 3 (RASA3) Gene in Saudi Autistic Children

The potential role of DNA methylation pattern in autism has been provided by revealing the differences in methylation level of multiple genes which are significantly associated with their expression and implicated in ASD pathogenesis. RASA3 is a member of GTPase-activating proteins, RASA3 is highly expressed in brain tissues and can be deregulated by different epigenetic mechanisms. Many studies reported that differentially expressed RASA3 is correlated with its aberrant methylation. Accordingly, this has been suggested that deferentially expression of RASA3 may be correlated with its methylation levels which could play a role in ASD which brought our attention to identify differentially-expressed genes that could be associated with their methylation level of ASD in Saudi population, by performing comparative gene expression of RASA3 then investigate its relation to methylation level. This study was conducted on 18 Saudi autistic children as well as their healthy-control siblings. Relative expression of a candidate gene (RASA3) was measured using RT-qPCR. Furthermore, MethyLight assay was performed to estimate methylation level and evaluate its impact on RASA3 expression. Interestingly, RASA3 expression has found to be dysregulated in ASD cases. In contrast, MethyLight assay result showed no differences in the methylation patterns among ASD cases in the candidate region. However, it remains an open question whether these dysregulations of RASA3 expression could be a biomarker for early screening/detection of some cases which may also suggest a role for RasGAPs in autistic brain function.

Blood-Based Detection of Colorectal Cancer Using Cancer-Specific DNA Methylation Markers

Cancer tissues have characteristic DNA methylation profiles compared with their corresponding normal tissues that can be utilized for cancer diagnosis with liquid biopsy. Using a genome-scale DNA methylation approach, we sought to identify a panel of DNA methylation markers specific for cell-free DNA (cfDNA) from patients with colorectal cancer (CRC). By comparing DNA methylomes between CRC and normal mucosal tissues or blood leukocytes, we identified eight cancer-specific methylated loci (ADGRB1, ANKRD13, FAM123A, GLI3, PCDHG, PPP1R16B, SLIT3, and TMEM90B) and developed a five-marker panel (FAM123A, GLI3, PPP1R16B, SLIT3, and TMEM90B) that detected CRC in liquid biopsies with a high sensitivity and specificity with a droplet digital MethyLight assay. In a set of cfDNA samples from CRC patients (n = 117) and healthy volunteers (n = 60), a panel of five markers on the platform of the droplet digital MethyLight assay detected stages I–III and stage IV CRCs with sensitivities of 45.9% and 95.7%, respectively, and a specificity of 95.0%. The number of detected markers was correlated with the cancer stage, perineural invasion, lymphatic emboli, and venous invasion. Our five-marker panel with the droplet digital MethyLight assay showed a high sensitivity and specificity for the detection of CRC with cfDNA samples from patients with metastatic CRC.

Performance of a MethyLight assay for methylated SFRP2 DNA detection in colorectal cancer tissue and serum

Background: Colorectal cancer is one of the five most common cancers in China, and its incidence is steadily increasing. An accurate and non-invasive screening method is needed to increase the population uptake of colorectal cancer screening. Secreted frizzled-related protein 2 ( SFRP2) has been found to be hypermethylated in most colorectal cancer patients, and it may fulfill the role of a non-invasive biomarker for colorectal cancer screening. Methods: Methylation status of SFRP2 was examined in 17 cancer tissues and paired adjacent paracancer tissues by a new SFRP2 MethyLight assay, which was also used to test the serum of 62 patients with colorectal cancer and 55 normal individuals. Results: The limit of detection of the SFRP2 MethyLight assay was about 200 pg per reaction. The SFRP2 methylation level was higher in 94.1% colorectal cancer tissues than in paired adjacent paracancer tissues ( P<0.001). The sensitivity and specificity of SFRP2 for detecting colorectal cancer in serum were 69.4% (95% confidence interval (CI) 56.2, 80.1%) and 87.3% (95% CI 74.9, 94.3%), respectively. Conclusion: SFRP2 methylation in serum has the potential to be a non-invasive biomarker for colorectal cancer screening.

Establishment of the MethyLight Assay for Assessing Aging, Cigarette Smoking, and Alcohol Consumption

The environmental factors such as aging, smoking, and alcohol consumption have been reported to influence DNA methylation (DNAm). However, the versatility of DNAm measurement by DNAm array systems is low in clinical use. Thus, we developed the MethyLight assay as a simple method to measure DNAm. In the present study, we isolated peripheral blood DNA from 33 healthy volunteers and selected cg25809905, cg02228185, and cg17861230 as aging, cg23576855 as smoking, and cg02583484 as alcohol consumption biomarkers. The predicted age by methylation rates of cg25809905 and cg17861230 significantly correlated with chronological age. In immortalized B-cells, DNAm rates of two sites showed a younger status than the chronological age of donor. On the other hand, the predicted age of the patients with myocardial infarction (MI) was not accelerated. The methylation rate of cg23576855 was able to discriminate the groups based on the smoking status. The DNAm rate of cg02583484 was reduced in subjects with habitual alcohol consumption compared to that of subjects without habitual alcohol consumption. In conclusion, our MethyLight assay system reconfirms that aging, smoking, and alcohol consumption influenced DNAm in peripheral blood in the Japanese. This MethyLight system will facilitate DNAm measurement in epidemiological and clinical studies.

The CIMP Phenotype inBRAFMutant Serrated Polyps from a Prospective Colonoscopy Patient Cohort

Colorectal cancers arising via the serrated pathway are often associated withBRAFV600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association betweenBRAFV600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such asMLH1, p16,andIGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated withBRAFmutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%).MLH1methylation was mainly observed in the proximal colon and was significantly associated withBRAFmutation and CIMP-high. The number of samples methylated forp16andIGFBP7was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.

Aberrant CpG Island Hypermethylation in Pediatric Gastric Mucosa in Association With Helicobacter pylori Infection

Context.—Helicobacter pylori infection is primarily acquired during childhood and persists throughout life in the absence of eradication with antibiotics. Helicobacter pylori infection induces methylation in the promoter CpG island loci in gastric epithelial cells. Thus, aberrant CpG island hypermethylation in gastric epithelial cells likely occurs early in life, although there are no existing data supporting this notion. Objectives.—To identify whether aberrant CpG island hypermethylation occurs in pediatric stomach mucosa in association with H pylori infection and to compare methylation profiles of samples from pediatric and adult stomach tissues. Design.—We analyzed pediatric (n = 47) and adult (n = 38) gastric mucosa samples for their methylation status in 12 promoter CpG island loci using the MethyLight assay and compared the number of methylated genes and the methylation levels in individual genes between H pylori–positive and H pylori–negative sample results and between pediatric and adult samples. Results.—The average number of methylated genes was significantly higher in H pylori–infected pediatric samples than in H pylori–negative pediatric samples (3.4 versus 0.3, P &lt; .001) and in H pylori–infected adult samples than in H pylori–negative adult samples (7.6 versus 0.9, P &lt; .001). Seven genes showed significantly higher methylation levels in H pylori–infected pediatric samples than in H pylori–negative pediatric samples (all values were P &lt; .05). Conclusions.—These results indicate that CpG island hypermethylation occurs in pediatric gastric mucosa in association with H pylori infection and that the genes affected by H pylori–associated hypermethylation were similar in pediatric and adult samples.