Research findings could lead to new biomarkers for genetic tests and give clinical laboratories new capabilities to diagnose different health conditions
New insights continue to emerge about “junk DNA” (aka, non-coding DNA). For pathologists and clinical laboratories, these discoveries may have value and eventually lead to new biomarkers for genetic testing.
One recent example comes from researchers at Stanford Medicine in California who recently learned how non-coding DNA—which makes up 98% of the human genome—affects gene expression, the function that leads to observable characteristics in an organism (phenotypes).
The research also could lead to a better understanding of how short tandem repeats (STRs)—the number of times a gene is copied into RNA for protein use—affect gene expression as well, according to Stanford.
“We’ve known for a while that short tandem repeats or STRs, aren’t junk because their presence or absence correlates with changes in gene expression. But we haven’t known how they exert these effects,” said study lead Polly Fordyce, PhD (above), Associate Professor of Bioengineering and Genetics at Stanford University, in a news release. The research could lead to new clinical laboratory biomarkers for genetic testing. (Photo copyright: Stanford University.)
To Bind or Not to Bind
In their Science paper, the Stanford researchers described an opportunity to explore a new angle to transcription factors binding to some sequences, also known as sequence motifs.
“Researchers have spent a lot of time characterizing these transcription factors and figuring out which sequences—called motifs—they like to bind to the most,” said the study lead Polly Fordyce, PhD, Associate Professor of Bioengineering and Genetics at Stanford University, in a Stanford Medicine news release.
“But current models don’t adequately explain where and when transcription factors bind to non-coding DNA to regulate gene expression. Sometimes, no transcription factor is attached to something that looks like a perfect motif. Other times, transcription factors bind to stretches of DNA that aren’t motifs,” the news release explains.
Transcription factors are “like light switches that can turn genes on or off depending on what cells need,” notes a King’s College LondonEDIT Labblog post.
But why do transcription factors target some places in the genome and not others?
“To solve the puzzle of why transcription factors go to some places in the genome and not to others, we needed to look beyond the highly preferred motifs,” Fordyce added. “In this study, we’re showing that the STR sequence around the motif can have a really big effect on transcription factor binding, providing clues as to what these repeated sequences might be doing.”
Such information could aid in understanding certain hereditary conditions and diseases.
“Variations in STR length have been associated with changes in gene expression and implicated in several complex phenotypes such as schizophrenia, cancer, autism, and Crohn’s disease. However, the mechanism by which STRs affect transcription remains unknown,” the researchers wrote in Science.
Special Assays Explore Binding
According to their paper, the research team turned to the Fordyce Lab’s previously developed microfluidic binding assays (MITOMI, k–MITOMI, and STAMMP) to analyze the impact of different DNA sequences on transcription factor binding.
“In the experiment we asked, ‘How do these changes impact the strength of transcription factor binding?’ We saw a surprisingly large effect. Varying the STR sequence around a motif can have a 70-fold impact on the binding,” Fordyce wrote.
In an accompanying Science article titled, “Repetitive DNA Regulates Gene Expression,” Thomas Kuhlman, PhD, Assistant Professor, Physics and Astronomy, University of California, Riverside, wrote that the study “demonstrates that STRs exert their effects by directly binding transcription factor proteins, thus explaining how STRs might influence gene expression in both normal and diseased states.”
“This research unveils, for the first time, the intricate connection between how variants in the non-coding genome affect genes that are associated with blood pressure and with hypertension. What we’ve created is a kind of functional map of the regulators of blood pressure genes, “said Philipp Maass, PhD, Lead Researcher and Assistant Professor Molecular Genetics, University of Toronto, in a news release.
The research team used massively parallel reporter assay (MPRA) technology to analyze 4,608 genetic variants associated with blood pressure.
The findings could aid precision medicine for cardiovascular health and may possibly be adopted to other conditions, according to The Hospital for Sick Children.
“The variants we have characterized in the non-coding genome could be used as genomic markers for hypertension, laying the groundwork for future genetic research and potential therapeutic targets for cardiovascular disease,” Maass noted.
Why All the ‘Junk’ DNA?
Clinical laboratory scientists may wonder why genetic research has primarily focused on 20,000 genes within the genome, leaving the “junk” DNA for later investigation. So did researchers at Harvard University.
“After the Human Genome Project, scientists found that there were around 20,000 genes within the genome, a number that some researchers had already predicted. Remarkably, these genes comprise only about 1-2% of the three billion base pairs of DNA. This means that anywhere from 98-99% of our entire genome must be doing something other than coding for proteins,” they wrote in a blog post.
“Imagine being given multiple volumes of encyclopedias that contained a coherent sentence in English every 100 pages, where the rest of the space contained a smattering of uninterpretable random letters and characters. You would probably start to wonder why all those random letters and characters were there in the first place, which is the exact problem that has plagued scientists for decades,” they added.
Not only is junk DNA an interesting study subject, but ongoing research may also produce useful new biomarkers for genetic diagnostics and other clinical laboratory testing. Thus, medical lab professionals may want to keep an eye on new developments involving non-coding DNA.
Lack of regulations and quality management jeopardizes the quality and safety of LDTs, claim experts in clinical laboratory medicine in a commentary to Canadian policymakers
The IHPME members published their comments in the Canadian Medical Association Journal (CMAJ), a peer-reviewed journal owned by Joule Inc., a subsidiary of the Canadian Medical Association. In it, they claim “recent expansion of the molecular diagnostics industry has revealed weaknesses in Canada’s regulatory system for laboratory-developed tests, which are not subject to statutory regulations on medical devices.”
For pathologists and clinical laboratory professionals in both Canada and the United States, these recent actions show the concerns many experts have as they watch the explosive growth in the use of laboratory-developed tests in both countries. In many ways, the swift advances in molecular and genetic diagnostics is outrunning the ability of government regulators to keep pace with use of LDTs in clinical care settings.
In their commentary in CMAJ, the IHPME members also
claim the review and evaluation of LDTs in Canada is inconsistent. Some LDTs they
say, may endure stringent assessments and have endorsements by clinical
guidelines or findings that are published in scientific journals. Other LDTs,
however, may have no analysis at all.
In addition, the IHPME members point out that there is no
national registry kept of LDTs. They theorize that a lack of proper regulation,
controls, and quality management “has potentially jeopardized the delivery of
quality, safe, timely, and appropriate care.”
The researchers calling on Health Canada to address these
issues include:
Fiona A. Miller, PhD, Professor of Health Policy and IHPME Chair in Health Management Strategies;
François Rousseau, PhD, Professor, Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Quebec;
Alberto Gutierrez, PhD, Partner, NDA Partners LLC, former Director, Office of In Vitro Diagnostics and Radiological Health at the FDA’s Center for Devices and Radiological Health (CDRH);
Stuart Hogarth, PhD, Lecturer in Sociology of Science and Technology, University of Cambridge, Cambridge, UK.
During an exclusive presentation offered by The Dark Report (Dark Daily’s sister publication) in 2015, Alberto Gutierrez, PhD (above), who at that time was Director, Office of In Vitro Diagnostics and Radiological Health at the FDA, said, “LDTs are an area that will be difficult to regulate. There is a broad set of tests. Some of the LDTs are very good. Some of them require a lot of expertise from the pathologists and some of them don’t. Regulating LDTs in a way that makes sense and that does not disrupt what’s going on [in clinical laboratories] is going to be difficult.” (Photo copyright: FDA.)
Canadian Scientists Call on Health Canada to Take the
Lead on Regulating LDTs
In the US, the FDA has been making moves to regulate LDTs since 2010, with much opposition from clinical laboratories and In Vitro Diagnostic (IVD) manufacturers. The FDA describes LDTs as internally designed clinical laboratory tests that are developed, manufactured, and used within a single laboratory. They have not undergone government regulatory review, can be simple or complex, and can be utilized to detect a variety of analytes.
Health Canada is the name of a department that falls under
the purview of the Minister of
Health and is part of Canada’s Health
Portfolio. It is responsible for helping Canadians maintain and improve
their health. Other agencies included in the Health Portfolio are:
According to the IHPME paper, however, Health Canada
currently does not have a way to regulate LDTs, and no government agency in
that country is responsible for the oversight of laboratory-developed tests.
Only LDTs that are marketed as test kits are evaluated and reviewed by Health
Canada.
“The current laboratory regulatory system in Canada involves a mixture of public and private entities and operates with oversight from provincial governments, nongovernmental organizations, and professional societies,” the IHPME paper states, adding, “most provinces and territories rely on voluntary standards that are unevenly applied, with little auditing and systematic testing to ensure quality.”
The authors also note that the current lab regulations in
Canada apply only to the operations of the medical laboratories themselves,
encompassing such things as lab environments, personnel, accreditation, and
quality control. They believe the loophole regarding LDTs needs to be addressed,
and they urged Health Canada to “demonstrate leadership” by subjecting these
tests to regulations that are currently applied to medical devices and
pharmaceuticals.
Other Countries Regulate LDTs, though Not Without
Controversy
In support of their call to action, IHPME researchers noted
that Australia, the EU, and the US all have taken steps to regulate LDTs.
The Australian government began oversight of LDTs in 2010 by
subjecting high-risk LDTs to external evaluation and then tracking them in a
public registry.
An EU regulation, which was passed in 2017, will administer
regulatory review of LDTs manufactured on an industrial scale, which targets
commercial laboratories. The law exempts LDTs utilized within individual
hospital laboratories and should be fully implemented by 2022.
Though on its radar since the 1990s, in 2010, the FDA officially
announced its intent to regulate LDTs in the US. The agency released an initial
draft approach for doing so starting in 2014, held a public workshop on the
topic in 2015, and released a
discussion paper in 2017. At this time, however, the FDA is not regulating
LDTs, though the agency remains open to the possibility.
Dark Daily
has reported extensively over the years on the development of LDTs and the
controversy surrounding the FDA’s moves to regulate them.
According to the FDA
website, problems with several high-risk LDTs have been identified,
including:
Claims that are not adequately supported with
evidence;
Lack of appropriate controls which may yield
erroneous results; and
The FDA’s report, titled, “The
Public Health Evidence for FDA Oversight of Laboratory Developed Tests,” reviewed
20 case studies of LDTs for Lyme disease, ovarian cancer, whooping cough,
fibromyalgia, prostate cancer, autism, breast cancer, melanoma, Vitamin D, and
other conditions. The agency concluded that in many instances “patients have
been demonstrably harmed or may have been harmed by tests that did not meet FDA
requirements.”
Klein noted, however, that “The 20 tests described by FDA are mostly a hodgepodge of outlier assays including tests that were never offered, tests for which comparable FDA assays perform poorly, tests for poorly defined disorders with psychologic components, and use of an FDA-approved test off-label.” He continued, “That FDA could find only these dubious examples out of the many thousands of laboratory-developed procedures (LDPs) that benefit patients each day, calls into question the agency’s rationale for expanding its regulatory scope to include LDPs.”
Perhaps this is why the FDA has yet to implement regulations
for LDTs. The controversy continues.
Whether Health Canada will accept the advice of the IHPME
scientists and take steps to regulate laboratory-developed tests in Canada remains
to be seen. As more LDTs are created and manufactured, however, it is probable
that governments will continue to evaluate the administration and oversight of laboratory-developed
tests.
In both Canada and the United States, pathologists, clinical
laboratory managers, and executives at in vitro diagnostic manufacturers
can expect an ongoing tug-of-war between government regulators and the lab
industry over the most appropriate ways to regulate LDTs.
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Several internationally-respected clinical laboratory experts are asking serious questions about Theranos and its diagnostic testing technology, and they’ve gotten few answers to date. Though the number of experts is small, their credentials in the clinical laboratory profession are impressive. In addition, some have published their critiques of the start-up medical laboratory company in well-respected medical journals.
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The clinical laboratory company in Palo Alto, Calif., gets plenty of attention because it claims to have disruptive technology that will allow it to perform medical laboratory tests equivalent to the current standard of care. Theranos says it can do this using a capillary specimen and return results in four hours, while charging a price that is just 50% of Medicare Part B lab test fees. Given these assertions, it is natural that pathologists and laboratory scientists who perform tests for patients, are curious about the scientific basis of Theranos’ proprietary diagnostic technology and what evidence Theranos has developed to support its claims of comparable accuracy and reproducibility. (more…)
