Early morning on a Sunday, my grandfather went for a walk. He realized that his right leg was stiffer and less flexible when he wanted to move fast. He has been suffering from this condition for quite a long time. He became very tired and returned home worrying about his problem. He was confused about whom to approach for getting it screened and diagnosed. We consulted doctors, who suspected arthritic joint disease – a type of autoimmune disease. They based their diagnosis on a recent, advanced diagnostic method that uses cell-free circulating deoxyribonucleic acid (DNA) in the blood.

Diagnosis, using such cell-free molecules, is non-invasive. Neurodegenerative diseases and cancers can be detected using this technique even before the onset, primarily by using blood samples. An interesting study by Vishnu Swarup and team from the All India Institute of Medical Sciences, New Delhi discovered that in Friedreich’s ataxia, which is a type of neurodegenerative disease, there was a three-fold increase in the plasma DNA levels in suspected individuals compared to the normal category. This reinforces the fact that these cell-free DNA molecules are crucial biomarkers.

The novel discoveries of many years of effort by researchers have played a pivotal role in revolutionizing advanced diagnostic techniques. Multiple groups of scientists have demonstrated that the levels of nucleic acids such as DNA, ribonucleic acid (RNA), micro RNAs (miRNAs), mitochondrial RNAs and long non-coding RNAs are enhanced in the blood of affected individuals. These molecules, referred to as cell-free DNAs and RNAs, are not detected in healthy tissues.

Often, these nucleic acids are modified by the addition of specific molecules (called methyl marks) on their surfaces. These are like crowns on the king and queen that visually signify their importance. This is scientifically termed as epigenetic modification. Adding an extra methyl group to DNA on specific genes regulates the protein expression, resulting in altered levels of proteins in the tissues. The addition of methyl groups is performed by an enzyme known as DNA methyl transferase (DNMT) present inside the nucleus of the cell where DNA is located. Specifically, the cytosine (C) base of DNA gets modified into different forms such as 5‑methylcytosine (5mC), 5‑hydroxymethylcytosine (5hmC), 5‑formylcytosine (5fC) and 5‑carboxylcytosine (5caC). The type of modification varies based on the tissue of origin and the type of disease. Based on this, the severity of the disease is known and accordingly, the treatment regimen can be planned for patients.

The methyl marks on DNA surfaces are identified using laboratory-based assays such as methylLight, methyl-specific polymerase chain reaction (PCR), methylation-sensitive high-resolution melting and MIRA (methylated CpG island recovery assay). Here, DNA is isolated from the collected tissue sample and subjected to PCR following bisulfite treatment. This treatment can only convert unmethylated cytosine in DNA to uracil (one of the four nucleobases in the nucleic acid RNA). The regions that are methylated remain unchanged during PCR-based sequencing and can be easily detected. In methylLight, which is a fluorescence-based technique, methylation-specific fluorescent antibodies are used to detect modified DNA marks. Such techniques have resulted in ease of diagnosis for health care providers, which ultimately help in efficient disease management. 

This diagnostic method — using cell-free DNA — has recently shown promise for prenatal diagnosis as well. Such diagnosis is important in detecting early signs of abnormal pregnancy, including genetic diseases such as Down’s syndrome, Edward’s syndrome and Turner’s syndrome. Fetal-specific DNA marks can be easily distinguished, even though maternal blood is used for clinical testing.

In addition to autoimmune diseases and prenatal diagnosis, the method can be used to diagnose diabetes, inflammation, stroke, trauma, neurological disorders such as Alzheimer’s and Parkinson’s diseases, mitochondrial disorders and metabolic syndromes. Moreover, with the advancement in technologies such as artificial intelligence and machine learning, large-scale data-rich repositories such as The Cancer Genome Atlas (TCGA), BLUEPRINT, and the Encyclopedia of DNA Elements (ENCODE) provide the necessary computational platforms to support relevant diagnostic techniques. These tools and databases will help the future growth of precision medicine and personalized care for patients. The simultaneous developments in molecular diagnostics and disease-specific database repositories will benefit the healthcare system, ultimately creating patient awareness for early prevention and healthy living.