The Value of DNA Methylation Detection in Aging Assessment and Disease Diagnosis

Abstract: During aging and disease occurrence, the methylation level of DNA undergoes tissue and organ specific changes. These changes can be used to evaluate the aging and disease of organs and systems. The cell free DNA (cfDNA) in the blood comes from various organs and tissues of the body. Research has found that tissue tracing of cfDNA can be achieved by detecting the methylation of cfDNA. This article will introduce the use of DNA methylation for detection to assess aging in individuals, tissues and organs. And through methylation of DNA (especially cfDNA in blood samples), cancer detection and evaluation of cancer treatment efficacy. And introduce relevant research on DNA methylation in other diseases.

Keywords: Methylation, blood cell free DNA, organ and tissue tracing, cancer, aging

1. Introduction to DNA methylation

DNA methylation is a chemical modification of DNA in mammals, which refers to the covalent bonding of the cytosine 5 'carbon bond of CpG dinucleotide to a methyl group (as shown in Figure 1a) under the action of DNA methyltransferase, which affects gene expression and phenotype. Methylation can cause changes in chromatin structure and DNA conformation, leading to changes in protein-protein interactions, particularly in the interaction between the transcription promoter region and the transcription initiation complex, resulting in changes in gene expression levels (methylation near mammalian genes is shown in Figure 1b).

During the generation of mammalian offspring, DNA methylation undergoes a process of erasure and reconstruction, which is called reprogramming of DNA methylation. In the early stages of embryo implantation and embryo formation, demethylation occurs in the fertilized egg and the initially replicated morula and blastocyst stages, respectively. During the embryo implantation stage, there is a wave of methylation process, which, due to the protection of methylation, suppresses most genes and only allows the expression of housekeeping genes. In the late stage of embryo implantation, the pattern of methylation varies for different stages and tissues and organs. This methylation defines different cell types, and this change usually lasts for a relatively long period of time.

In the process of aging, as age increases, methylation at different sites will change, and a common trend is that the level of methylation at different sites in tissues tends to 50%, which is a random state. For different organs and cell types, the methylation levels of a set of sites can be used to accurately predict the corresponding age. The predicted biological age is a measure of the aging level of organs and tissues.

In many diseases, the methylation levels of multiple loci, especially the promoter regions of genes, can change. For example, abnormal high methylation levels can lead to inhibition of gene expression. The changes in DNA methylation are considered important factors in the development of cancer. Low methylation can lead to genomic instability, while hypermethylation can inhibit gene expression, but it can serve as a site for methylation editing.

It is not realistic to directly collect samples from different organs and tissues for monitoring the health of different organs. In recent years, the development of cell free DNA (cfDNA) detection technology in blood has provided an opportunity to understand the health and aging status of organs and tissues through blood samples. cfDNA comes from different organs and tissues, and the extraction of this part of cfDNA information can reveal the health information of its source organs and tissues. This information can be used for aging assessment of organs and tissues, as well as for the detection of related diseases. This article mainly introduces the research and detection of aging assessment and age-related diseases through methylation of cell free DNA.

2. DNA Methylation Detection Technology

The detection of DNA methylation can be divided into overall methylation level detection and gene site specific methylation level detection. Here, we only focus on gene site specific methylation level detection. For gene site-specific methylation level detection, there are currently mainly chip based methylation detection and whole genome methylation detection based on high-throughput sequencing. Here, we will briefly introduce them. 

1). Chip-based methylation detection technology: This type of detection can only obtain detection of methylation levels at certain loci on the genome, and the cost of detection is relatively lower than that of whole genome methylation detection. This type of detection first obtains DNA fragments, which are then hybridized onto a chip for methylation level detection. At present, there are three main strategies for DNA processing and capture. The first is based on the conversion of bisulfite, enrichment methods based on methylation antibodies or MBD (Methyl-CpG-Binding Domain) affinity, and the other is restriction endonucleases for digestion.

2). Whole genome methylation detection technology based on high-throughput sequencing: This detection obtains the methylation levels of various points on the whole genome through high-throughput sequencing, and the current price of this detection is relatively high. At present, there are two detection methods based on second-generation sequencing, WGBS (Whole Genome Bisulfite Sequencing) and RRBS (Reduced Representation Bisulfite Sequencing). The former is the gold standard for whole genome methylation detection, while the latter detection method can detect approximately 85% of CpG islands in the human genome.

The number of studies is still small due to the fact that the price of methylation testing is still relatively high, but methylation has great value as an important part of epigenetics. As the price of methylation testing can be lowered in the future, more research will be conducted.

3. Cell Free DNA Tissue and Organ Tracing

The tissue and organ tracing of cfDNA provides a foundation for organ and tissue aging and disease assessment through DNA methylation. Cell free DNA refers to the DNA that is free in the blood and comes from dead cells in various parts of the body. Recent studies have found that the tissue origin of cfDNA can be traced through tissue and organ specific cfDNA information. Through the encapsulation mode of cfDNA nucleosomes, organ specific methylation site information, and the association between adjacent methylation sites, the tissue and organ traceability of cfDNA can be achieved.

cfDNA can be used for accurate tissue and organ tracing through methylation, providing valuable localization information for obtaining aging and disease status of tissues from blood samples. This enables the detection of cfDNA methylation to accurately detect aging and disease status in different parts of the body. In recent years, related research has continuously emerged, providing a foundation for clinical applications.

4. DNA Methylation for Aging Assessment

Biological age is the age that reflects the actual aging degree of an individual, organ or tissue, which is different from the actual age of the individual. Currently, biological age is an important comprehensive indicator for aging assessment. If the biological age is greater than the actual age, it indicates an accelerated aging rate compared to peers. If it is less than the biological age, it indicates a slower aging rate compared to peers. Biological age is evaluated through aging markers. At present, the recognized standards for aging markers come from the American Federation for Aging Research (AFAR): 1. It must be able to predict the rate of aging, in other words, accurately identify an individual's position in the life process. It must predict an individual's lifespan better than its actual age; 2. It must be able to monitor the basic process of aging, rather than the symptoms or outcomes of diseases; 3. It must be able to be easily detected without causing harm to the human body. For example, blood tests or imaging tests; 4. It must work on both humans and laboratory organisms, such as mice. In this way, testing can be conducted on laboratory animals before testing on humans.

By using different types of aging markers, researchers have developed different types of biological age, telomere length, blood biochemical indicators, DNA methylation, transcriptome, proteome, small RNA, wearable device data, and other indicators that can be used to evaluate biological age. Comparing different biological ages, a prominent feature of methylation age is that the predicted actual age is relatively accurate, and the correlation coefficient with actual age can usually reach 0.95 or above.

Epigenetic changes are an important part of the molecular mechanism of aging, with three levels of epigenetics: DNA methylation, histone modification, and chromatin reprogramming. DNA methylation affects gene expression by affecting gene transcription. A site on a DNA strand has two states: methylated and unmethylated. If multiple pieces of DNA at the same site are detected simultaneously, the proportion of site methylation will be obtained, which will change with age. This age dependent ratio provides a method for evaluating age. From the methylation sites, identify a group of age related and relatively independent sites, and predict age as the biological age of methylation based on this group of sites (refer to review literature).

1). Methylation age has organ resolution: The methylation of different organs is not entirely the same, depending on different gene expression requirements. In this way, organ and tissue specific sites can be used to evaluate the aging of organs and tissues separately. Currently, most studies assess biological age based on DNA methylation in whole blood or white blood cells. The cancer treatment process can obtain cancer tissues from different organs and healthy controls, providing samples for the methylation age of different organs.

2). The manifestation of methylation age in disease populations: The biological age evaluated using different types of aging markers may reflect different aspects of aging in the body. Different researchers have developed methylation age using different sites and algorithms. The effectiveness of biological age testing is to examine its role in disease prediction, life expectancy prediction, and anti-aging interventions. The methylation changes in tissues and organs may reflect relatively long-term and stable changes, and the assessed methylation age has a clear signal in chronic diseases or chronic aging symptoms. The acceleration of methylation age is closely related to cancer (as shown in Figure 2), neurological disorders, Down syndrome, Parkinson's disease, Werner's syndrome, physiological and cognitive health, as well as the condition of elderly individuals with long lifespans.

In summary, methylation age can serve as an accurate method for predicting age, while also having a good indicative effect on age-related symptoms and diseases.

5. Application and Research of DNA Methylation in Age-Related Diseases

Due to the fact that methylation of cfDNA can be traced to organs and tissues, and the amount of cfDNA released into the bloodstream increases when various organs undergo lesions, methylation in some chronic diseases also has special signals. Based on this information, methylation of cfDNA plays an important role in disease diagnosis, and some research results have shown promising prospects. The methylation level on cfDNA, somatic mutations in genes on cfDNA, their content in the blood, and the distribution of cfDNA fragments contain different information and have different values for disease detection. Here, we will only introduce research on detection through cfDNA methylation signals. At present, research and application in this area are the most common in cancer, and there are relatively few in other neurological and metabolic diseases, as well as other diseases.

1). cfDNA methylation is used for cancer detection: Because methylation signals can be traced to tissues, and when organs and tissues undergo lesions, the amount of cfDNA originating from that tissue increases. The application of methylation in cancer diagnosis and treatment is reflected in cancer diagnosis and evaluation of cancer treatment efficacy. In addition to the biological age of methylation mentioned above, which can accelerate aging in cancer patients, recent studies have also shown that methylation detection of cfDNA can provide more information.

At present, research on methylation in cancer diagnosis and treatment is still rapidly developing, and there will be more and better research in the future. High sensitivity detection will gradually target early cancer, and even in the stage where imaging has not yet been detected, cancer can be detected, enabling patients to receive treatment earlier.

2). DNA methylation for the study of Alzheimer's disease (AD): Alzheimer's disease (AD) is a neurodegenerative disease that undergoes degenerative changes in the brain before diagnosis. Early diagnosis and diagnostic markers have high value for treatment. Currently, some studies have been conducted on the methylation of brain tissue in Alzheimer's patients. The appearance of different disease symptoms in individuals with similar or even identical genetic backgrounds suggests that epigenetics may differ in the occurrence and development of Alzheimer's disease, and DNA methylation in different brain regions may change. Some of the current conclusions are still controversial. The value of detecting methylation changes in the blood is higher. At present, there is not much research on the methylation of DNA in the blood for the diagnosis of Alzheimer's disease, and further strengthening is needed.

3). Methylation research in other diseases: At present, there are also many studies on DNA methylation in metabolic diseases. Although some diseases can be explained by gene mutations, gWAS (gene wide association study) research has found that the associated gene loci can only explain a very small portion (<5-10%) of cases. The influence of environment and lifestyle habits may occur through epigenetics. By detecting DNA methylation in the blood, a range of metabolic diseases, such as obesity related loci, were identified. A study found that the methylation of adipose tissue in mice changed after feeding a high-fat diet, resulting in symptoms similar to obesity and other diabetes. As a validation, the author also found that methylation sites of these changes were repeatedly found in adipose tissue of obese individuals. It is worth noting that the changes in this type of methylation are relatively mild, less than 5-10%, and the physiological and psychological effects of these changes are still uncertain. At present, no definite correlation has been found between such changes and the occurrence of diseases. Although it is not yet possible to explain the impact of methylation changes on diseases from a mechanistic perspective, DNA methylation sites on certain specific genes in blood samples can now serve as biomarkers for certain autoimmune diseases, such as systemic lupus erythematosus.

6. Prospect

In summary, DNA methylation detection can provide information that is different from DNA sequences, mining the information in DNA methylation, especially the cfDNA methylation information of easily obtained samples, providing valuable tools for anti-aging practices, as well as the diagnosis and treatment of geriatric diseases, especially cancer. cfDNA methylation can be used for tissue tracing, providing enormous potential for further research and development of diagnostic and therapeutic technologies. There will be more practical research and technologies worth looking forward to in the future.

Reference: Clinical Laboratory, Issue 7, 2020, Special Topic on Geriatrics and Laboratory Testing