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NGS in clinical diagnostics

Thirty-five years ago, Dr. Janet Davison Rowley sat at a microscope in her lab at the University of Chicago and made a remarkable discovery in cancer biology, that leukemia is caused by the translocation of a chromosome. In other words, it is a disease of the DNA. Today, thanks to next generation sequencing (NGS), we can zoom in on that chromosome and others to determine exactly which nucleotide bases are implicated in a plethora of human diseases.

Towards Personalized Medicine

Sequencing of the first human genome in 2003 cost billions of dollars and took close to a decade to complete. Today, the same project costs as little as $1000 and the cost per base is still dropping. Advances in data storage and the continuous accumulation of sequence data gives us better control over data management and enriches our analyses, respectively. With these advantages, NGS can now take us one step closer to personalized medicine. Here are a few examples that demonstrate the potential for NGS in personalized approaches to medicine.

1. The Role of Monogenic Diseases in Neonatal Death

Monogenic diseases, or rare single gene mutation diseases, are a common cause of neonatal death. Although more than 3,500 monogenic diseases have been identified to date, only a few of them have screening tests. In a study conducted at Mercy Children’s Hospital, Kansas City MO, whole genome sequencing was employed for the molecular diagnosis of diseases in affected babies. Two samples were sequenced. It took around 50 hours to complete the sample and library preparation, sequencing and analysis. These samples were sequenced on a rapid track Illumina Hiseq 2500, 2×100 read length sequenced to reach 50x coverage. RUNES (a variant-caller software) analyzed the samples within two and half hours. This resulted in the identification of two significant heterozygous mutations. This is an example of how NGS plays a role in situations where we don’t know exactly where to zoom in and look.

2. Story of A Cancer Survivor…and Researcher

Whilst studying medicine and cancer at Washington University at St. Louis, Dr. Lukas Wartman received an acute lymphoblastic leukemia (ALL) diagnosis. After chemotherapy and 2 years in remission, he experienced his first relapse. Since his relapse happened after a bone marrow transplant, NGS represented a valid option to understand exactly what was going on. He had his genome sequenced at the Genome Institute, Washington University. Transcriptome analysis revealed elevated expression of FLT3, a growth regulator gene. Fortunately, there was an FDA-approved FLT3 protein inhibitor available for the treatment of kidney cancer, and apart from insurance companies not paying for this drug, there were no major obstacles in trying it out for ALL. Lukas responded well to this treatment and recovered. You can read more about his story here.

3. Human Leukocyte Antigen Identification

The human leukocyte antigen (HLA) gene family contains the genes required for a group of closely related proteins known as the HLA complex. This complex is instrumental to the immune system’s ability to distinguish an individual’s own proteins from foreign proteins (e.g. microbes, parasites, organ transplants). The HLA is analogous to the major histocompatibility complex (MHC), present in many other species. The HLA complex contains more than 200 genes on chromosome 6, and understanding these genes is critical to successful organ transplantation and the avoidance of organ rejection by the recipient’s immune system (i.e. graft-versus-host disease). NGS makes it possible to perform HLA typing and HLA matching rapidly so that organ recipients have the best chance of a successful and safe organ transplant.

Challenges to NGS Becoming a Diagnostic Tool

Despite reductions in cost, there are still several challenges to NGS becoming a mainstay in all diagnostic labs. The instrumentation required for in-house NGS runs is expensive and setting up an NGS facility and analyzing data can take a lot of time, requiring specialized personnel. Whole genome/whole exome sequencing (WGS) can take several weeks to complete between sample preparation, sequencing and data analysis. The ideal time for a diagnostic test would be in the range of one to three days. Sequencing on multiple platforms with higher coverage may add to the cost and time. Technical challenges include under-representation of GC rich genes, the requirement for more in-depth sequencing and variation in analysis strategies. For more information on the technical issues surround NGS, check out some of our other NGS articles here.

Privacy and Protection Surrounding NGS

Another important aspect to consider is privacy and protection. Library construction, sequencing and analysis might actually be the most straightforward parts of NGS where diagnostics are concerned. On October 11th 2012, the presidential bioethical commission of the United States published an important report, entitled ‘Privacy and Progress in Whole Genome Sequencing‘. This report recommended that genome sequencing not be undertaken without the permission of the individual, that sequencing information be protected and that counseling be provided as a prerequisite for genetic testing by WGS. These recommendations help to ensure privacy protection e.g. by preventing unsolicited sequencing of human remains on discarded coffee cups or plates at restaurants.

NGS – Ethics and Education

The final and perhaps most important issue in WGS is the discovery of information that the patient might rather not receive! Analysis may hit upon pre-existing but undiagnosed conditions for which there may or may not be an available treatment. Alternatively, analysis might reveal a mutation that may or may not manifest into a syndrome. Personal genomics is fast becoming a widespread diagnostic tool, forcing us all to face some huge ethical questions, such as: should disease-specific counseling develop in parallel to NGS as a diagnostic tool?, is it the responsibility of the biological community to be the educators of personal genomics? and how do we help the general public understand the potential of the information, to be progressive about it and avoid misuse? For more discussion on these questions, check out the review article from Rigter et al., (2013)3.

Further Reading:

  1. Goldberg B, Sichtig H, Geyer C, Ledeboer N, Weinstock GM. 2015. Making the Leap from Research Laboratory to Clinic: Challenges and Opportunities for Next-Generation Sequencing in Infectious Disease Diagnostics. mBio. 6(6): e01888-15.
  2. Kamps R, Brandao RD, van den Bosch BJ, Paulussen ADC, Xanthoulea S, Blok MJ, Romano A. 2017. Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification. Int J Mol Sci. 18(2): 308.
  3. Rigter T, Henneman L, Kristoffersson U, Hall A, Yntema HG, Borry P, Tönnies H, Waisfisz Q, Elting MW, Dondorp WJ, Cornel MC. 2013. Reflecting on Earlier Experiences with Unsolicited Findings: Points to Consider for Next-Generation Sequencing and Informed Consent in Diagnostics. Hum Mutat. 34/10):1322-1328.

Originally published in 2012. Updated and republished in June 2017.

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