Methylation Test

General Description

DNA methylation is a chemical reaction in which a methyl group (composed of three hydrogen atoms and one carbon atom) attaches to a cytosine base in the DNA nucleotide chain. This binding occurs at cytosine-guanine dinucleotides (CpG), which are especially abundant in gene promoter and regulatory regions. Methylation within a gene’s DNA sequence generally deactivates the gene, meaning it suppresses its protein coding, making it a crucial mechanism in gene expression regulation. It is a type of epigenetic modification, meaning it affects gene expression without altering the DNA sequence. Although all the cells in an organism contain the same DNA sequence, their methylation patterns are different. Unlike the DNA sequence, methylation patterns are not inherited from gametes but rather form during embryonic development and are maintained through cell reproduction. Furthermore, throughout an individual’s life, changes in the methylation pattern of their genome also occur.

Genetic methylation tests analyze variations in the DNA sequence that affect methylation capacity to identify specific genetic variants in genes related to this process. In other words, these tests evaluate the methylation status of a gene. Methylation influences numerous bodily processes, so the presence of certain genetic variants can impact methylation efficiency and the body’s ability to carry out these processes.

When Are They Indicated?

Methylation tests have various applications in clinical medicine, including the following:

• Cancer diagnosis: Both hypermethylation of tumor suppressor genes and hypomethylation of oncogenes (mutated cancer-causing genes) promote disease development. Thus, certain cancers such as prostate, liver, stomach, colon, breast, or cervical cancer can be detected early.
• Cardiovascular disease prevention: Certain genetic variants in the methylation pathway can cause an increase in homocysteine, an amino acid whose overproduction is closely linked to the risk of atherosclerosis, hypertension, thrombosis, or heart failure, among others. Detecting these variants allows for planning dietary changes to correct homocysteine levels.
• Congenital disease diagnosis: Methylation alterations in specific DNA regions can cause genetic disorders such as Prader-Willi syndrome, muscular dystrophies, or Angelman syndrome.
• Autoimmune disease diagnosis: Diseases such as lupus, rheumatoid arthritis, or systemic sclerosis are associated with methylation alterations in the promoter regions of specific genes.
• Prenatal diagnosis: The analysis of fetal free DNA methylation can identify chromosomal abnormalities or epigenetic alterations that affect fetal health.
• Identifying genetic predisposition to mental disorders: Methylation alterations can affect neurotransmitter synthesis and, therefore, influence the development of mental illnesses.

How Is It Performed?

To perform a methylation test, a DNA sample must be extracted from the patient. Typically, the sample is obtained from blood or saliva.

Once the sample is collected, it is sent to the laboratory, where various procedures can be carried out to assess the methylation status of the DNA segment of interest. One of the most commonly used methods, due to its speed and sensitivity, is methylation-specific polymerase chain reaction (MSP). The procedure begins by treating the DNA section with sodium bisulfite, which causes unmethylated cytosines in CpG dinucleotides to convert into uracils (one of the four nitrogenous bases in RNA), while methylated cytosines remain unchanged.

To observe the presence or absence of cytosines and determine the DNA segment’s methylation status, amplification is required, meaning billions of copies are created. This is done using polymerase chain reaction (PCR), which involves reacting the sample with a polymerase enzyme and primers—small DNA sequences specifically designed to complement the segment in the sample. The copies are made through multiple cycles of heating and cooling. During each cycle, the DNA double helix separates into two single strands, to which the primers attach. These are extended by the polymerase, which can add nucleotides to the ends of each primer. This synthesizes two new DNA strands, which will be duplicated in the same manner in subsequent cycles. During synthesis, uracils convert into thymine, allowing for the distinction between methylated and unmethylated CpG dinucleotides.

Risks

Collecting DNA samples for methylation tests poses no risks or complications.

However, during bisulfite treatment, cytosine-to-uracil conversion may be incomplete, potentially leading to the misinterpretation of unmethylated cytosines as methylated ones. Additionally, bisulfite causes extensive degradation of DNA sequences and higher losses during processing, which can affect test success.

What to Expect from a Methylation Test

The procedures for extracting a patient’s DNA sample are generally quick and simple. To collect a blood sample, a needle puncture is performed on a vein in the forearm. Before doing so, the area is disinfected, and an elastic band is placed to facilitate vein filling and make the puncture easier. Once the sample is drawn, the band is removed, and a sterile dressing is applied to stop the bleeding.

Saliva sample collection is performed using a swab, gently rubbing it along the inner cheek lining and under the tongue. The swab must be placed in the corresponding container to prevent contamination. In some cases, the patient may collect the sample at home using a specific kit.

Specialties That Request Methylation Tests

Methylation tests are diagnostic tools developed by genetics specialists and are primarily used in oncology. However, they may also be requested in other specialties such as cardiology, neurology, gynecology, or rheumatology.