Yeast genetics collaboration yields hope for improved diagnosis of urea cycle disorders

Fast and accurate diagnosis of urea cycle disorders (UCDs) may finally be within reach, thanks to the power of yeast genetics, an enterprising scientist, rapidly advancing technologies, and an ongoing research collaboration.

A team led by Aimée Dudley, Ph.D., senior investigator at the Pacific Northwest Research Institute, has developed a method using yeast genetics to rapidly measure the effects of thousands of variants of a gene that causes human disease. They are collaborating with experts from the Urea Cycle Disorders Consortium, which has been studying UCDs for more than 20 years, and other experts from Children’s National Hospital and Research Institute.

The group aims to shed light on “variants of uncertain significance”—genetic changes that may or may not cause disease—and help the medical community interpret often confusing and unhelpful genetic testing results.

Dr. Dudley’s team applied the method, called mutational scanning, to better understand the genetic underpinnings of the most common urea cycle disorder, ornithine transcarbamylase (OTC) deficiency, an X-linked condition. They measured the activity of 1,570 human OTC variants, ranked them by severity, and evaluated how well their experimental results agreed with the experiences of human patients.

Results were shared in a recent paper. A panel of experts is already considering the data they generated for clinical use in diagnosing OTC deficiency through a variant curation panel, a group of experts who weigh the evidence whether a variant causes disease.

The collaborators are now pursuing further research, including using the technique to better understand and diagnose all the urea cycle disorders by studying the other genes.?

Unraveling OTC

Urea cycle disorders are caused by small changes, called variants, in any one of eight genes. These variants can cause enzyme deficiencies severe enough to impair the process of metabolism, allowing ammonia to accumulate and cause brain damage, coma, and eventually death.

Thousands of possible genetic variants exist for the genes that cause each disorder. But the clinical significance—information on what level of disease (if any) those variants will cause in patients—is currently known for only a small portion of the variants.?

“A person’s genome can now be sequenced quickly, cheaply, and easily. However, geneticists need to know whether the variants they find are likely to cause disease or are completely harmless, and that information often isn’t available.”

“There is a problem in the field of human genetics,” says Dr. Dudley. “A person’s genome can now be sequenced quickly, cheaply, and easily. However, geneticists need to know whether the variants they find are likely to cause disease or are completely harmless, and that information often isn’t available.”

“Identifying a harmful variant can shorten the time to diagnosis and the time to treatment,” says coauthor Andrea Gropman, M.D., of Children’s National, who is also the principal investigator of the Urea Cycle Disorders Consortium. “For UCDs, the shorter the time to treatment, the better the patient outcome will be. This work is critically important for assessing the impact of variants found in genetic testing. Clarification of the variants of uncertain significance may impact prognosis and treatment, as well as inform participation in future clinical treatment trials.”

Stories of delayed or missed diagnoses are too familiar in the UCD community, and current diagnostic methods are far from perfect.

The dried blood spots (biochemical assays) used in newborn screening don’t work reliably for all urea cycle disorders, including OTC deficiency. The tests are administered by programs that are unevenly managed from state to state, and results can be too slow to prevent the worst outcomes. Diagnosing a patient based on high ammonia levels has obvious disadvantages: symptoms are confusing which can lead to delays, and changes in blood ammonia levels can cause permanent brain damage, coma and death. Ammonia testing can be difficult, as blood samples need special attention during collection, transport, and storage.

While genetic tests can now deliver results quickly and with great precision, they are only useful if doctors can interpret the results.

“If we had this technology 20 years ago when my daughter was diagnosed with OTC, we could have avoided so much pain.”

“We are incredibly excited about this research,” says Tresa Warner, president of the National Urea Cycle Disorders Foundation. Tresa’s daughter began showing symptoms of OTC at nine months, but it took five months of visiting hospital after hospital to reach a diagnosis. “If we had this technology 20 years ago when my daughter was diagnosed with OTC, we could have avoided so much pain.”

“In addition to speeding diagnosis, the information gained may help predict the course of a particular patient’s disorder,” she says. “It could allow doctors to personalize treatment, and even identify asymptomatic patients early enough that they can avoid the environmental triggers that cause late-onset disease. This research will change lives.”

Inside the technology

Because metabolism is such a basic process essential to life, it is similar—or highly “conserved”—between yeast and humans. Many of the same metabolic processes that happen in a human cell, also happen in a yeast cell. Studying yeast metabolism can help us better understand human metabolism.

Dr. Dudley calls herself both a yeast geneticist and a technologist. As a basic scientist, she uses yeast to better understand how cells function and to improve or develop new genetic techniques.

The new technique, or “assay,” her lab developed involves genetically engineering yeast cells to introduce a variant of a human disorder into their genetic code. For OTC, her team genetically modified 1,500-plus sets of yeast colonies to match each possible human variant. The colonies grew, were measured, and then ranked from largest to smallest.?

Colonies that grew bigger had healthier metabolisms, indicating that the variants they were given were benign or milder. Those that grow poorly or not at all had weaker metabolisms (with little or no enzyme activity), indicating that those variants were most severe or “pathogenic.” The work generates large data sets summarizing the results and ranking the variants from most to least severe.?

Time lapse video showing the growth of several yeast colonies. Video provided courtesy of the Pacific Northwest Research Institute.

The technique provides reliable data faster and more economically than other methods. Years of observation would be required to study even a handful of variants in human subjects. Creating a mouse model of one variant would require about a year before data collection could even begin. Computational methods to predict variant effects are too inaccurate to use for diagnosis.

Using yeast genetics, Dr. Dudley’s method can typically analyze all 1,000–3,000 variants of a gene in less than eight months.

This idea to apply yeast genetics to study rare diseases was sparked by a talk that Dr. Dudley attended at the University of Washington, where she first heard about the mutational scanning technology.

“To me, this was the biggest thing to happen in genomics in the last ten years. I realized that over time, our lab had built up several tools that could be powerfully applied in this method. I was so excited about it that I said, ‘We have to do it.’”

A chance decision led her to focus on UCD genes. Her team had just concluded work on another ultrarare disease when she realized that this work could inform other human diseases. She sought another viable pathway to study and chose the synthesis of arginine, which is what underlies the urea cycle.?

Her team was pursuing the UCD work independently until one of her colleagues sat on a scientific panel with Dr. Gropman. “My colleague made the connection, introduced us, and the rest is history,” says Dr. Dudley.

Teaming up with clinicians

That initial connection has blossomed into a three-year collaboration between basic and clinical scientists. In addition to Dr. Dudley and Dr. Gropman, the team now includes other experts from Children’s National including Nicholas Ah Mew, M.D., who is another clinician from the UCDC, along with Llubica Caldovic, Ph.D., an expert in human metabolism, and Hiroki Morizono, Ph.D., a biomedical informatics expert.

“Identifying a harmful variant can shorten the time to diagnosis and the time to treatment. For UCDs, the shorter the time to treatment, the better the patient outcome will be.”

“We were really fortunate to be able to interact with them,” says Dr. Dudley. “And they were so excited about the potential of having this kind of data that our groups, even as busy as everyone is, have been meeting every two weeks for over 3 years now.”

Through this collaboration, the team was able to use patient data from the scientific literature and the UCDC longitudinal study to verify the results of Dr. Dudley’s work.

“The really powerful thing about having these close interactions between clinicians, disease domain experts and technologists, is that our work informs their thinking, while their work and knowledge and understanding informs our thinking,” she says.

Some of the variant results uncovered in the yeast studies surprised clinicians, who are looking again at patient data to see what they can learn. The basic science team also realized their technique did not work as well as needed to give clear results for all disorders and are now refining their procedures to improve results?

The next step is to put the data to use in diagnosing patients. But, before that can happen, the findings must be reviewed by a panel of experts following guidelines set out by the American College of Medical Genetics.

Fortunately, the review is already underway. A Urea Cycle Disorders Variant Curation Expert Panel (VCEP) has been active since March 2021. Dr. Gropman, Dr. Caldovic, and Dr. Ah Mew are members of the volunteer panel, along with other experts from UCDC and beyond.

“Several different criteria are used and evaluated by these panelists. They now have our functional information, which is important for these variant curation efforts. It is not often available to them,” says Dr. Dudley.

She predicts that the process of using the data to inform practice will speed up as the panel gains experience with the types of data generated by yeast studies.

Like Tresa Warner, the UCDC’s Dr. Gropman is excited about where this research may lead.

“In the past, treatment was more of a one-size-fits-all,” says Dr. Gropman. “Now we know more about these patients.?We’ve studied them, through our work with UCDC and this yeast genetics work, to really understand how particular variants influence the enzyme capability and potentially the outcomes. Each patient is an individual, and the diagnosis will confer some individual differences in the therapeutic pathway.”

Looking ahead

Additional lines of research are being planned, including grouping patients for study according to the levels of enzyme activity revealed by their variants. These groupings may help doctors better understand symptoms and potentially personalize care. Carrier studies may also be in the future, which could reveal the health impacts on people who carry only one copy of a UCD gene, such as the mothers of sons born with OTC.

Dr. Dudley and her collaborators are committed to continuing this line of research. They are currently writing grants and seeking funding to support it.

The entire field of genetic testing is advancing rapidly. Dr. Dudley predicts that soon, every newborn—not just very sick babies—will have their whole genome sequenced at birth. Several large studies are already underway testing the concept at sites across the country. One site was able to go from blood draw to diagnosis within three hours.

“I’m enthusiastic,” says Dr. Dudley. “We have made a commitment to apply this approach to the full set of all eight urea cycle genes if humanly possible.” She hopes to eventually apply her method to study other rare diseases as well.

“I think this is going to be a really big deal for the UCD community,” she says. And it is a very big deal for her as well. As a basic scientist, she never expected to have this kind of direct interaction with clinicians and impact on patients.

“The fact that we get to do the technological things that we’re excited about, that we’re good at, and that we also get to have an impact, is huge. I can look in a database and literally count the number of people who might have a serious disease-causing variant,” she says. “That is very different from our usual work. Physicians and sometimes even families thank us for what we do. This is the most rewarding work that I and my lab have ever done.”

—Jill Schlabig Williams

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The research performed in the Dudley lab was funded by the National Institutes of Health (NIH) through grants from the National Institute of General Medical Sciences (R01GM134274) and the National Human Genome Research Institute (T32HG00035).

The Urea Cycle Disorders Consortium?(UCDC) is part of the Rare Diseases Clinical Research Network (RDCRN). It is funded by the NIH under grant number U54HD061221 as a collaboration between by the National Center for Advancing Translational Sciences (NCATS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)?

The National Urea Cycle Disorders Foundation (NUCDF) is the driving force behind efforts to speed diagnosis, improve treatments, and find a cure for urea cycle disorders. The nonprofit organization also serves as a lifeline to patients, families, and medical professionals worldwide seeking information, support, and hope. Contact NUCDF at [email protected].