Epigenetic marks do not follow the Mendelian laws of inheritance. The environment can alter the epigenotype of an individual when exposed to different external stressors. In lactating cows, the first stages of gestation overlap with the lactation peak, creating a negative energy balance that is difficult to overcome with diet. This negative energy balance could affect early embryo development that must compete with the mammary tissue for nutrients. We hypothesize that the methylation profiles of calves born to nonlactating heifers are different from those of calves born to lactating cows. We found 50,277 differentially methylated cytosines and 2,281 differentially methylated regions between these two groups of animals. A comethylation network was constructed to study the correlation between the phenotypes of the mothers and the epigenome of the calves, revealing 265 regions associated with the phenotypes. Our study revealed the presence of DMCs and DMRs in calves gestated by heifers and lactating cows, which were linked to the dam\'s lactation and the calves\' ICAP and milk EBV. Gene-specific analysis highlighted associations with vasculature and organ morphogenesis and cell communication and signalling. These finding support the hypothesis that calves gestated by nonlactating mothers have a different methylation profile than those gestated by lactating cows.

Aims: Early detection of colorectal cancer (CRC) provides substantially better survival rates. This study aimed to develop a blood-based screening assay named SPOT-MAS (\'screen for the presence of tumor by DNA methylation and size\') for early CRC detection with high accuracy. Methods: Plasma cell-free DNA samples from 159 patients with nonmetastatic CRC and 158 healthy controls were simultaneously analyzed for fragment length and methylation profiles. We then employed a deep neural network with fragment length and methylation signatures to build a classification model. Results: The model achieved an area under the curve of 0.989 and a sensitivity of 96.8% at 97% specificity in detecting CRC. External validation of our model showed comparable performance, with an area under the curve of 0.96. Conclusion: SPOT-MAS based on integration of cancer-specific methylation and fragmentomic signatures could provide high accuracy for early-stage CRC detection.
A novel blood test for early detection of colorectal cancer. Colorectal cancer is a cancer of the colon or rectum, located at the lower end of the digestive tract. The early detection of colorectal cancer can help people with the disease have a higher chance of survival and a better quality of life. Current screening methods can be invasive, cause discomfort or have low accuracy; therefore newer screening methods are needed. In this study we developed a new screening method, called SPOT-MAS, which works by measuring the signals of cancer DNA in the blood. By combining different characteristics of cancer DNA, SPOT-MAS could distinguish blood samples of people with colorectal cancer from those of healthy individuals with high accuracy.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

Pyrosequencing, a real-time sequencing technology, is considered a \"gold standard\" for quantitative allele quantification at single base resolution. Quantitative bisulfite Pyrosequencing determines DNA methylation level by analyzing artificial \"C/T\" SNPs at CpG sites within a specific Pyrosequencing assay. The bisulfite Pyrosequencing methylation assay design is DNA strand specific and the primer design should not contain any CpG sites and should be free of high-frequency mutations. Additionally Pyrosequencing assays must be tested for preferential amplification during bisulfite PCR to ensure the sequencing quantification accuracy and reproducibility. Pyrosequencing analysis gives a reproducible measurement of average methylation at several CpG sites within the Pyrosequencing assay directly from a PCR product, rapidly and accurately for many samples at a time. It is therefore well suited for clinical research, validation of whole-genome methylation screening results, and global methylation analysis using repetitive elements including LINE-1, Alu, and Sat2. Pyrosequencing reproducibility and accuracy result in low measurement variance, thereby increasing the likelihood of early detection of small changes in methylation levels that may become apparent in response to treatment. For example, the high reproducibility of the LINE-1 assay is important for detecting the relatively small daily changes in methylation levels associated with hypomethylation. This enables detection of differences in patterns between normal and disease tissue such as in tumor suppresser genes, and to determine global methylation changes in response drug treatments. Relatively low cost and easy automation allows the researcher to increase the experiment\'s sample population to detect trends that would otherwise not have a sufficient sampling basis for statistical significance.