NGS – A Powerful Tool For Disease Diagnostics

Key takeaways:

• NGS is used for disease biomarker discovery and biomarker validation.
• Whole exome sequencing has been used for diagnostics of poorly characterised diseases by identifying novel mutations and biomarkers.
• Whole genome sequencing is well-suited for diagnostics of rare diseases by identifying complex mutations such as chromosomal translocations.
• NGS is the pivotal system for the detection of diseases and the generation of personalised pharmacogenomics profiles.

NGS diagnostics: Range of diseases

For the diagnosis of complex diseases that involve multiple genes, NGS is the most suitable option. These diseases include autism, connective tissue disorders, cardiomyopathies (disease of the heart muscle that impedes blood pumping), and disorders of sex development (Di Resta et al., 2018). For instance, in 2009 Richard Lifton and colleagues at Yale University diagnosed the clinical condition of a patient using whole exome sequencing (WES) (exome = protein coding regions). The patient, a five months old infant, was suspected to have Bartter’s syndrome. However, the results of WES revealed the presence of a mutation that resulted in congenital chloride diarrhoea (Choi et al., 2009). The symptoms of both diseases are similar: intestinal problems and dehydration. In this and other cases, conventional genotyping methods such as karyotyping, fluorescent in-situ hybridisation (FISH) and restriction fragment length polymorphism analysis (RFLP) failed to properly diagnose the disease. The WES strategy opted for by Lifton was sheer brilliance and the decisive factor in finding the correct diagnosis, as around 85% of the disease-causing mutations are present in the exome. It also cut the costs for the diagnosis to a fraction of the other option of whole genome sequencing (WGS).

Image 2: Depiction of the personalised therapeutic approach.

Currently, “big NGS data” is utilised by some clinical laboratories for the development of personalised therapeutic approaches (image 2). This will help in understanding disease mechanisms, along with strategies to prevent and treat diseases. Soon clinicians will be able to utilise the patient’s genetic information to choose specific medical care that has the highest chance of success. Before this can be realised, legal, ethical and technological challenges need to be clarified by the respective authorities that, at present, are working on developing guidelines.
Nevertheless, there are three main NGS strategies for diagnostics purposes: NGS panels, WES and WGS. The choice of strategy depends on the nature of the individual disease and available funds.

NGS diagnostics by Whole Genome Sequencing: For rare diseases and beyond

As the name indicates, WGS includes sequencing the coding (exons), non-coding (introns) and regulatory regions. WGS enables the identification of novel gene mutations including complex mutations such as chromosomal translocations and copy number variations (CNVs). It enables the diagnosis of rare disease that, when combined, affect more than 300 million people worldwide (Liu et al., 2019; Marshall et al., 2020).

As additional routine procedure in research laboratories, WGS is used to analyse infectious agents such as bacteria and viruses (Ilyas, 2017). Thus far, PCR-based genetic tests have usually been performed for viral infections but not for bacteria. Isolation, correct species identification and susceptibility testing of bacteria may take 1 to 2 days. But for slow growing strains, like Mycobacterium tuberculosis, phenotypic testing could take weeks. Additionally, less than 2% of bacteria are cultivable in laboratory conditions, which could complicate testing even more (Wade, 2002). In those cases, WGS is the preferred detection method. NGS also helps in analysing of the genetic changes that result in antibiotic resistances and, therefore, enable clinicians to prescribe effective antibiotics. For example, the genome of M. tuberculosis is homogenous and antibiotic resistances can arise through deletion, insertion or nucleotide substitution (Achtman, 2008; Ilyas, 2017).

Personalised medicine: NGS takes medicine to the next level

Despite some challenges, NGS is paving the way towards personalised medicine! NGS has the potential to accelerate the detection of disorders and generate personalised pharmacogenomics (effect of genes on the individual’s response to drugs) profiles. After all, the genetic makeup of patients is not completely identical and different genetic mutations can cause the same disease and symptoms. Therefore, the correct diagnosis of a disease and taking into account the individual’s response to a therapeutic agent, which is genetically determined, is crucial. NGS can help in comprehending the biology of the disease and can assist in making clinical decisions based on an individual’s genetic profile. Ashley and colleagues at Stanford University School of Medicine, for example, sequenced the whole genome of a 40 years old healthy male patient with a family history of osteoarthritis, premature coronary artery disease and sudden death. The team focused on three types of variants: rare mutations, novel mutations and mutations that modulate drug response. Interestingly, the investigation found 63 known pharmacogenetic variants. The presence of these mutations suggested that this individual will respond well to statin while being resistant to clopidogrel. As both drugs are prescribed to prevent atherosclerotic cardiovascular disease, the findings of the study will help clinicians to plan individual prevention and treatment strategies for the patient if needed. Additionally, the investigation identified three rare mutations that are associated with sudden death, and another gene variant associated with coronary artery disease. These findings could also contribute to creating a personalised treatment plan for the individual in future (Ashley et al., 2010; Samani et al. 2010).

With the incorporation of NGS in clinical settings, the availability of genomic information will not be a limiting factor to guide and optimise therapies anymore. The dream of applying genetics to clinical medicine can finally become reality.