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Clinical Laboratories and Pathology Groups

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Stanford Researchers Discover Junk DNA Affects Gene Expression

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.

Scientists at Stowers Institute for Medical Research and Duke University School of Medicine contributed to the study.

The researchers published their findings in the journal Science titled, “Short Tandem Repeats Bind Transcription Factors to Tune Eukaryotic Gene Expression.”

Polly Fordyce, PhD

“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 London EDIT Lab blog 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, kMITOMI, 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.”

Junk DNA Affects Blood Pressure

In another study, researchers at The Hospital for Sick Children (SickKids), which is affiliated with the University of Toronto, Ontario, examined the possible effect of non-coding DNA on genes related to blood pressure.

“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.

In “Systematic Characterization of Regulatory Variants of Blood Pressure Genes,” published in the journal Cell Genomics, the SickKids scientists noted that high throughput technology identified “regulatory variants at blood pressure loci.”

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.   

—Donna Marie Pocius

Related Information:

Stanford Medicine-led Study Clarifies How “Junk DNA” Influences Gene Expression

Short Tandem Repeats Bind Transcription Factors to Tune Eukaryotic Gene Expression

J for Junk DNA Does Not Exist!

Repetitive DNA Regulates Gene Expression

Illuminating Genetic Dark Matter: How “Junk DNA” Influences Blood Pressure

Systematic Characterization of Regulatory Variants of Blood Pressure Genes

The 99 Percent of the Human Genome

Canadian Scientists and Medical Researchers Urge Health Canada to Regulate Laboratory-developed Tests

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

Health Canada is the latest government healthcare organization under pressure to enact legislation that regulates laboratory-developed tests (LDTs). In a public commentary, several members of the Institute of Health Policy, Management and Evaluation (IHPME) at the University of Toronto in Ontario, urged Canadian lawmakers to follow the European Union’s lead and find ways to monitor LDTs in Canada.

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:

  • Kelly Holloway, PhD, Research scientist at University of Toronto;
  • 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
  • Falsification of data.

However, in “FDA Looks to Clamp Down on Laboratory-Developed Tests and Put an End to ‘Wild West of Medicine’: Might CLIA Problems at Theranos Support FDA’s Position?Dark Daily, May 4, 2016, Roger D. Klein, MD, JD, Chair of the Association for Molecular Pathology (AMP) Public Relations Committee, and Medical Director, Molecular Oncology at Cleveland Clinic, called a report released by the FDA in 2015 “mostly a hodgepodge of outlier assays.”

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.

—JP Schlingman

Related Information:

Health Canada Needs to Act on Laboratory-developed Diagnostics

Laboratory Developed Tests

The Public Health Evidence for FDA Oversight of Laboratory Developed Tests: 20 Case Studies

Framework for Regulatory Oversight of Laboratory Developed Tests (LDTs)

FDA Discussion Paper on Laboratory Developed Tests

FDA Announces Intention to Regulate LDTs as Devices

FDA Official Makes Case in Favor of LDT Guidance

Regulation of Laboratory Developed Tests by FDA: Time for the Agency to Cease and Desist Until Congress Enacts Legislation

Johns Hopkins University Study Finds Laboratory-Developed Liquid Biopsy Tests Can Give Different Results; Call for ‘Improved Certification’ of Medical Laboratories That Develop These LDTs

FDA Looks to Clamp Down on Laboratory-Developed Tests and Put an End to ‘Wild West of Medicine’: Might CLIA Problems at Theranos Support FDA’s Position?

British Medical Laboratory Test for Early Screening of Lung Cancer Shows Promising Interim Results in Large Trial; Could Lead to Other Simple Blood Tests for Cancer Detection

EarlyCDT-Lung test followed by X-rays and CT scans proves up to five times more likely to detect cancer than current standard of care

Encouraging preliminary results from a 12,000-person clinical trial into the effectiveness of a non-invasive medical laboratory blood test for the early detection of lung cancer could signal an advance that leads to creation of similar anatomic pathology screening tests for the early detection of other cancers.

Interim results of a study using the potentially life-saving blood test EarlyCDT-Lung were presented on December 6, 2016, at the International Association for the Study of Lung Cancer’s 17th World Conference on Lung Cancer in Vienna, Austria. Initial results from the trial indicated the blood test can detect lung cancer potentially up to five years before symptoms develop. (more…)

Dental Plaque Could Lead to Clinical Laboratory Testing for Biomarkers to Identify Health Risks

Researchers have found that isolating a particular gene within the oral microbiome can reveal a huge amount of useful diagnostic information about a person’s health

Most people don’t think of dental plaque when they think about clinical laboratories. For the vast majority of people, the only diseases that dental plaque bring to mind are those of the mouth:

periodontitis; and,
dental caries.

Samples that are sent to medical labs and pathology laboratories are more often blood or tissue. However, that could be changing, thanks in part to the work being done at the Oral Microbiome and Metagenomics Research Lab (OMMR) at the University of Toronto. (more…)

Internationally-respected Experts in Clinical Pathology and Laboratory Medicine Ask: Why Don’t We Know More about Theranos’ Technology?

This secretive start-up medical laboratory testing company has not disclosed how its diagnostic test technology works, nor has it given laboratorians an opportunity to examine the technology

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.

One question these clinical pathologists and laboratory directors ask is why Theranos has so far been unwilling to provide more information about the lab testing technology it uses to deliver medical laboratory test results to patients and their referring physicians. Even as the company has declined to speak to the medical laboratory profession, Theranos has mounted a major public relations campaign designed to make a big impression on investors, business partners, and most recently on health insurers.

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…)