Methylation in cell-free DNA

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 Hi readers! Welcome back to another post on cell-free DNA! This time we will look at another type of change that can occur in DNA – methylation. 




Not all genes are expressed in a cell at the same amount at a given time. Some genes are expressed in a lower amount and some are expressed in a higher amount. By gene expression, I mean production of a protein from the gene encoding it. Changes in gene expression which do not affect the DNA sequence are called epigenetic changes. 

Methylation is a type of epigenetic change used by the cells to turn off gene expression. It changes a component of the DNA molecule called the nucleotide base. In this event, a methyl group is added to the carbon at the fifth position of a cytosine ring, resulting in the formation of a 5-methylcytosine. Methylation of a gene can block its expression compared to genes that are not methylated. In a cell, addition or removal of methyl groups are used to regulate gene expression. 

In cancer, genes that could stop a cancer cell from growing and dividing are often hyper-methylated or excessively methylated. This blocks the expression of these genes. Scientists have widely studied the role of DNA methylation in cancer. They have examined the methylation status of many genes which function in important cellular processes such as growth, survival and death. 

For example, P16 is a gene which blocks cell division. Many studies have reported P16 to be inactivated in various cancers. It is known to have abnormal methylation patterns in breast cancer, liver cancer, lung cancer, esophageal cancer and colorectal cancer. 

As we saw before in the last post about mutations in cell-free DNA, methylation patterns of DNA in the tumor can also be detected in cell-free DNA. So, methylation is another attribute of cell-free DNA that is being widely studied for non-invasive testing in cancer. In terms of non-invasive testing, methylation can be tested in two ways:

1. Whether a selected sequence is methylated or not, or

2. How many of the cfDNA gene sequences are methylated

The first one is qualitative, and we link the presence or absence of methylation to disease occurrence or treatment response. The second way is quantitative: we measure the number of DNA fragments containing the methylation and link that to treatment response.

Let’s look at some studies in which researchers have evaluated methylated cfDNA sequences in disease diagnosis and treatment response!

P16 in Liver Cancer

Wong et al. studied, in 2003, the methylation profile of p16 in plasma cell-free DNA of liver cancer patients. In this study, the researchers measured the number of p16 sequences which were methylated. They measured and compared the levels before and after surgery. They found that the amounts of methylated sequences reduced after surgery by 12-fold! Thus, they concluded that detection of methylated sequences in plasma can be useful in detecting and monitoring liver cancer.

RASSF1A in breast cancer and melanoma

Avraham et al. evaluated methylation status of the RASSF1A gene in breast cancer patients. They selected RASSF1A from five genes (RASSF1, RARB, GSTPI, DAPK1 and HIN1) evaluated in tumor biopsies. RASSF1A was methylated in 80% of breast tumors. In this study, the researchers tested the presence or absence of methylated sequences and correlated it with response to treatment. So, they collected serum samples and checked the methylation status before and after neoadjuvant chemotherapy. Patient who showed complete response to treatment did not have detectable levels of methylated RASSF1A sequences. But patients who showed partial or minimal response continued to have methylated RASSF1A sequences in their serum after treatment. The researchers then concluded that the clearance of RASSF1A sequences after treatment shows that the tumor is responding to the treatment. Therefore, testing for presence of methylated RASSF1A sequences can help to monitor treatment response in breast cancer patients. 

RASSF1A in liver cancer 

RASSF1A methylation was also studied in liver cancer by Chan et al. They evaluated the use of RASSF1A methylation in diagnosing liver cancer as well as in its prognosis. They performed the study in two parts. In the first part, they compared the methylation levels in liver cancer patients, chronic hepatitis-B (HBV) carriers and healthy people. They found that the liver cancer patients and HBV carriers had higher levels of RASSF1A methylation than healthy individuals. When they checked the levels in liver cancer patients after surgery, they observed that the levels had reduced. This indicates that the RASSF1A levels could be useful in monitoring response after surgery.  In the second part, they compared methylation levels in two groups of hepatitis-B virus carriers – those who developed liver cancer and those who didn’t. The HBV carriers who developed liver cancer had higher levels of RASSF1A methylation than those who didn’t develop liver cancer. Therefore, this study shows that screening HBV carriers using RASSF1A methylation status may help diagnose liver cancer at an early stage and help in early treatment.

So, this was about methylation in cell-free DNA and its applications in disease. I hope you enjoyed this post. Watch out for my next post!




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