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A year ago, the Kunzman family in Oregon had some accidents — Matthew Kunzman, 13, suddenly coughed and was accompanied by a high fever. During the pandemic, any COVID-19-like symptom can cause a high degree of alertness, and doctors' diagnoses have been mixed: the good news is that Matthew didn't get COVID-19, and the bad news is that Matthew's symptoms may have originated from the heart.
Matthew Kunzman is one of 12 patients who benefited from the technology | References[2]; Courtesy of Jenny Kunzman
Local doctors told Matthew's parents that he had signs of myocarditis. This is an inflammation of the heart that makes it difficult for this vital organ to maintain its normal function and not be able to send enough blood to the whole body. Subsequent test results confirmed the hypothesis: Matthew's heart was failing, which was beyond the treatment capacity of the local clinic. Doctors recommended that Matthew go to Stanford Hospital immediately, where matthew's life could be saved.
Matthew briefly dodged death's surprise attack. A few hours later, his father flew him to Stanford for a visit. The next day, his condition deteriorated rapidly. When his mother arrived the next day, he was already in use of a life support system. If you wait a few more days, the consequences can be unimaginable.
Euan Ashley is a professor at Stanford University School of Medicine and an expert in genetics and biomedical data. After hearing about Matthew's case, he had a different view in his mind - overall, Matthew was a very healthy young man. Sudden heart failure, there may be two main reasons: one is the Diagnosis of Oregon Doctor, that is, myocarditis induced by the virus or other physical discomfort; the other is genetic reasons, the effects of a genetic mutation, which finally crushed Matthew's heart over time.
Professor Euan Ashley, head of this study (Stanford School of Medicine)
These two causes have their own possibilities, but they have completely different treatment options – myocarditis is often reversible. With treatment, heart function can return to normal levels. But this is not the case with heart disease caused by genetic mutations. If the cause is really a genetic defect, the only treatment option is a heart transplant.
Dr. John Gorzynski of Professor Ashley's research group contacted Matthew's parents about an ongoing study in their lab for rapid genome sequencing and asked permission to include Matthew in the study. For this request, Matthew's parents immediately agreed. They couldn't wait to know what exactly was wrong with Matthew.
With their consent, the researchers drew a few milliliters of Matthew's blood and sent them for rapid genome sequencing. A few hours later, the sequencing results returned, revealing that Matthew's genome did indeed have a mutation in the gene that caused the disease. After obtaining this information, the doctors immediately put Matthew on the waiting list for a heart transplant. After three weeks, he waited for a whole new heart. A year later, Matthew's mother says he's still alive and healthy.
In addition to their joy, they may not have thought that Matthew was involved in a landmark study. The technique that made the key diagnosis for Matthew completed the sequencing of the whole genome in just a few hours. "Usually, if you can sequence a patient's genome in a few weeks and return the results, most doctors will say it quickly." Professor Ashley said.
At the turn of the century, when humans first completed the sketching of the genome, few could have predicted that in just over two decades, genome sequencing would become a tool for diagnosing individual diseases — at a time when scientists in many countries spent more than a decade and nearly $3 billion to complete genome sketches. Put on any one individual, this is unimaginably astronomical.
Today, with the development of molecular biology technology, the threshold of genome sequencing technology is constantly declining. Commercial sequencing of individual genomes became possible a few years ago. In a few thousand dollars, in a matter of weeks, a blueprint for life of your own will be sequenced. In the eyes of experienced doctors, this blueprint also contains clues to genetic diseases, and there are also signs leading to the direction of treating diseases.
The cost of sequencing the human genome has fallen rapidly over the past two decades | Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) Available at: www.genome.gov/sequencingcostsdata. Accessed 01/13/2021.）
But as Professor Ashley says, existing genome sequencing techniques have gone a lot faster than the turn of the century, but for critically ill patients like Matthew, it's still too slow. The traditional sequencing time of a few weeks, the average healthy person can afford to wait, Matthew can not afford to wait. In order to make timely diagnoses of patients like Matthew, researchers also need to develop faster sequencing techniques. Fast sequencing means faster diagnosis, faster treatment, and patients may spend less time in intensive care units or recover faster. More critically, this speed increase cannot be sacrificed to accuracy.
To meet this need, the researchers came up with the idea of "nanoporous" technology. It's a technique that's already widely used in gene sequencing, and its principle is easy to understand — when long-stranded DNA molecules pass through nano-sized voids, they produce corresponding current changes depending on the base. By identifying this current change, the corresponding base can be reversed, thereby reducing the sequence of DNA.
Professor Ashley contacted Oxford Nanopore Technologies and used the PromethION 48 sequencing system developed by the company. There are a total of 48 flow cells in the system, each of which can complete the work of genome sequencing alone. To save time, the researchers decided to have 48 flow pools working at the same time, dramatically speeding up the pace.
Schematic of DNA sequencing by nanoporous technology | DataBase Center for Life Science (DBCLS), CC BY 4.0, via Wikimedia Commons
Parallel sequencing, while increasing speed, also produces massive amounts of data that exceed the analytical capabilities of the lab. To this end, the research team further transferred the data to the cloud to expand computing power. At the same time, the team has also developed algorithms to scan and sequence the genetic information obtained while looking for mutations that may lead to genetic diseases. Finally, scientists will also compare the found genetic variants with published papers to confirm.
Between December 2020 and May 2021, the researchers recruited 12 patients at two Stanford-based hospitals and performed rapid genome sequencing using the techniques described above for assisted diagnosis. Yesterday, scientists presented the results of their phased work in the New England Journal of Medicine. According to the introduction, the minimum time from collecting a patient's blood sample to obtaining the initial diagnosis result is only 7 hours and 18 minutes.
Speed is certainly a very compelling point of the job. According to a press release from the Stanford School of Medicine, in one case, if the diagnostic link is removed and only the sequencing part of the genome is counted, it takes only 5 hours and 2 minutes, which also sets a Guinness World Record.
But speed is by no means the only bright spot in this study. In fact, a major feature of nanopore technology is the sequencing of long sequences. Traditional genome sequencing methods first break the genome into small pieces for sequencing, and then use the standard human genome as a reference to reassemble the sequencing results into a genome. But such an approach sometimes misses some genomic fragments or misses some genetic variation. Long sequence sequencing can sequence DNA fragments up to tens of thousands of bases, providing more detail.
The New England Journal of Medicine article mentions that more than half of the sequencing fragments exceed 25 kb. Overall, each genome can produce 173 to 236 Gb of data, with 94 percent alignment identity and autosomal coverage of 46 to 64x.
Schematic diagram of this study | References[1]
"With long sequence sequencing, it is easier to find mutations on large fragments of the genome," Professor Ashley commented, "and it is almost impossible to find some of these variants without the use of some long sequence sequencing methods." ”
The technology can also answer medical questions that are difficult to answer with traditional diagnostic techniques. The research team presented another case - a 3-month-old baby developed epilepsy of unknown etiology, so the hospital took a two-pronged approach, using standard clinical genetic diagnosis on the one hand, and using this new whole genome rapid sequencing technology on the other hand. It took just over 8 hours for the latter to give the answer — the child had a mutation in the CSNK2B gene, which was known to be linked to neurodevelopmental disorders that led to early-onset epilepsy. In contrast, the standard clinical genetic diagnosis does not include the CSNK2B test, so the information returned by the test is of limited help to patients.
This research result has attracted the attention of many doctors and scientists. Stanford doctors are excited about the prospect of a genetic diagnosis being completed in a matter of hours, and renowned scientist Professor Eric Topol said on his social media that "the future is coming."
Professor Eric Topol social media screenshots
Of course, this work has only been used on a small scale at present, and there is still a lot of room for optimization in terms of cost and accuracy. But this pioneering attempt has shown us that rapid sequencing of the genome promises to be an important part of the diagnosis, bringing timely and important diagnostic information to families of patients suspected of having a rare genetic disorder. Fast, accurate, individualized... Perhaps, this is what the future of medical diagnosis looks like.
bibliography
[1] John E. Gorzynski et al., (2022), Ultrarapid Nanopore Genome Sequencing in a Critical Care Setting, NEJM, DOI: 10.1056/NEJMc2112090
[2] Fastest DNA sequencing technique helps undiagnosed patients find answers in mere hours, Retrieved January 13, 2022, from https://med.stanford.edu/news/all-news/2022/01/dna-sequencing-technique.html
[3] Scientists describe new approach in NEJM, using Oxford Nanopore DNA sequencing technology to improve prognosis in critically ill patients, in less than 8 hours, Retrieved January 13, 2022, from https://nanoporetech.com/about-us/news/scientists-describe-new-approach-nejm-using-oxford-nanopore-dna-sequencing-technology
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