How an MD-PhD Student With Hemophilia Made a Genetic Research Breakthrough

Nick Popp was an unhappy baby. He was colicky and constantly getting little bruises out of nowhere.

“My parents took me to the pediatrician and were told to put me on a table,” he says. “Where my dad picked me up, every single one of his fingertips left a bruise on my body.”

About a month later, Popp was diagnosed with hemophilia A, a rare genetic disorder that prevents his blood from clotting properly.

Popp, 34, is now a graduate of the University of Washington School of Medicine’s MD-PhD degree program and a pathology resident at Mass General Brigham in Boston. While at UW, he helped develop a new technology with Doug Fowler, PhD, a researcher in the UW School of Medicine’s Department of Genome Sciences, and Jill Johnsen, MD, a researcher in the Division of Hematology and Oncology and a hematologist at UW Medicine, to decode one of the genes that causes hemophilia and find all the possible variants in it.

A gene variant (aka mutation) is a change in the DNA sequence of a gene, which provides instructions for a protein to do its job. In most cases, variants have no effect on health. In some cases, though, they result in proteins that do not work well — or at all — or aren’t even produced. Depending on the gene where the variant occurs, this can lead to genetic diseases like hemophilia or cystic fibrosis or dramatically increase the risk for certain cancers.

Popp’s work on this gene is part of a broader global effort started by Fowler and his colleagues at UW Medicine to create an atlas of every variant of all 4,000 human-disease-related genes in order to provide diagnoses and improve therapies for anyone with a genetic disease, no matter how rare. It’s an ambitious goal, but one that would represent a huge advance in genetic medicine and change the lives of millions of people.

The evolution of hemophilia treatment

Hemophilia is dangerous because it can cause uncontrolled bleeding — before modern treatments, most people with severe hemophilia didn’t survive to adulthood.

People with hemophilia have a variant in the genes that make proteins called coagulation factors. People with hemophilia B, for example, can’t make enough functional coagulation factor IX, and people with hemophilia A have problems making functional factor VIII.

“When you get an injury, your blood vessels leak; that’s where the blood is coming from, and it’s the job of our coagulation system to patch that leak,” says Popp. “Factor VIII is part of that process. If you’re missing factor VIII or it doesn’t work in some way, then you can’t stop the leak, and you continue to bleed.”

This used to mean that a fall could be fatal for someone with hemophilia, especially if it led to bleeding in the brain. Repeated bleeding in the joints often caused severe physical disabilities.

In the middle of the last century, doctors used treatments that contained coagulation factors derived from blood donors. When someone with hemophilia was bleeding, it could be infused into the patient to help their blood clot.

Tragically, in the ’70s and ’80s, the blood supply for these treatments was contaminated with HIV and hepatitis. The tainted treatments infected over 9,000 people living with severe hemophilia in the U.S. at the time.

By the time Popp was born in the 1990s, researchers had figured out how to make recombinant coagulation factors in a lab without using human blood. This new approach eliminated the risk of contamination, but it still involved giving people with hemophilia frequent IV treatments.

Popp grew up in a small town in southern Illinois, an hour from the nearest hospital that offered the treatment. He would have three or four bleeding events per month, so the distance was a challenge.

“Until I was 5 years old, every time I had an injury, I was going to the emergency room, and I was spending hours there being worked up and getting treated,” says Popp. “I  started learning how to give myself IVs, and by the time I was 12, I was teaching new nurses how to do it.”

Because hemophilia occurs in only 1 in 5,000 male births in the U.S., many doctors and nurses, especially in rural areas, haven’t encountered it before and don’t know how to diagnose or treat it.

“A lot of my childhood was defined by being the weird kid who had this thing wrong with him that no one had ever heard of,” says Popp.

The pain of being excluded because of having hemophilia made him want to help other people with rare genetic diseases. This led him to pursue a career in medicine, becoming the first person in his family to go to college.

Nick Popp on the UW campus on Match Day                                    © Courtesy Nick Popp

Mapping the variants of a disease-causing gene

By the time Popp graduated from college, treatment for hemophilia had improved, including the introduction of newer therapies and regimens that better prevent bleeds.

This progress was incredibly impactful — Popp’s trips to the hospital decreased to less than once per year. But much remained unknown about how genetic variants affected the coagulation factors of people with the disease. Popp became interested in researching rare genetic diseases, as well as patient care, and was drawn to the UW School of Medicine because of its strong genetics program.

He moved to Seattle and began an MD-PhD degree program, where he found himself in the labs of Fowler and Johnsen.

Fowler and his team had been working on a way to look at all the variants of all disease-causing genes in the human genome to help figure out which mutations are innocuous and which ones cause disease.

“It turns out that knowing whether a variant is pathogenic or benign is a really hard problem,” says Fowler. “Just detecting that a variant is there is only the beginning.”

Just because a variant is in or near a disease-causing gene doesn’t mean it’s harmful — in fact, most of them probably aren’t. When a clinician doesn’t have enough information to say whether a gene variant is harmful or not, they call it a “variant of uncertain significance” or VUS. If you’ve ever gotten a genetic test, you might have received this result.

A genetic test with a VUS cannot help with diagnosis or treatment. For example, if you and your doctor decide to test for inherited breast cancer risk and your results come back with a VUS in the BRCA1 gene, it might mean you have an elevated risk for breast cancer, or it might not, so the clinician won’t be able to recommend next steps, like a preventive mastectomy.

In fact, approximately 90% of known genetic variants are VUS, and that’s just a fraction of the more than 6 billion possible variants in the human genome.

“So said another way, we mostly don’t know what variants do; it’s a huge problem,” says Fowler. “We need to know about all the variants that could ever exist in the genome because all of them are likely out there in the world already, and we’ll encounter them as we do more genetic tests.”

What do researchers do to solve a problem of this scale? Looking at variants one at a time would be a fool’s errand. So, Fowler and his team set out to create a technology that could sequence thousands of variants simultaneously rather than one by one. 

“What we figured out how to do was edit the genomes of the cells so that they each expressed a different variant,” says Fowler. “So now, instead of having one million cells in a dish all expressing the same variant, we have one million cells in a dish each expressing a different variant.”

They then measured the function of each variant and used the data to create a table that allowed you to look up the effect of any variant in that gene. This process is called multiplex assays of variant effects, or MAVE, and it is game-changing for clinical geneticists.

“When we see a variant in the clinic in a patient, we can go to our lookup table and say, ‘Aha, that’s a loss-of-function variant,’” says Fowler. “And that gives us evidence that we can then hand to the clinical geneticist who can incorporate it into their clinical variant interpretation, and now say that’s a pathogenic variant.”

Fowler’s team has found that MAVEs can reduce the number of VUS in a gene by 50% to 75%. 

A need for more data

One of those experts looking for more data on VUS was Johnsen, whose lab studies the genetics of bleeding disorders, including hemophilia.

“We were really feeling the weight of not having enough evidence,” says Johnsen. “We very often would find a DNA change, and we were pretty sure it caused hemophilia, but we didn’t have enough evidence to say that confidently.”

So Johnsen teamed up with Fowler to create MAVEs for the genes that cause hemophilia. And they had the perfect graduate student to work on it in Popp.

There was a problem, though: the MAVEs that had been created so far only worked for proteins that stayed in the cell so that you could track the variant’s effect. But coagulation factors are proteins that are secreted from the cell into the blood, where they’re impossible to track.

“I had to figure out a way that we could simultaneously identify the DNA variant and what the protein was doing, despite the fact that they were separated in space,” says Popp. “The easiest way to do this is just to tether it to the cell — it gets secreted, but it gets stuck on the way out.”

His plan worked, allowing him to build a MAVE for factor IX, the coagulation factor that doesn’t function properly in people with hemophilia B. The result is a comprehensive map of nearly every variant in the gene that makes factor IX. It allows researchers and clinicians to see not only whether the coagulation factor is being made but also whether it leaves the cell and if it has the right chemical additions needed to make it work in clotting. This development was so significant that Popp’s paper about it was published in the prestigious publication Nature Structural and Molecular Biology. 

Popp presenting his PhD research                                © Courtesy Nick Popp

“The tool that we developed doesn’t just allow us to say, ‘This variant doesn’t work correctly, and so therefore is clinically impactful,’ but also can let us say, ‘Well, this variant can be secreted, but doesn’t get modified correctly, and so it can’t function,’” says Popp. “Or this one can’t get secreted at all, so none of the rest of the things it is supposed to do will work.”

This more granular picture of the genetic variant’s function can help clinicians understand if someone might have a milder version of hemophilia or a severe version that could cause them to spontaneously start bleeding. It can also lead to personalized treatments tailored to what the coagulation factor is doing — a far cry from spending hours in the hospital waiting for an IV.

Meanwhile, Johnsen was able to use the data generated by the MAVE almost immediately in a high-stakes clinical situation. A premature baby shared a variant in the gene that creates factor IX with a sibling who had hemophilia B. If the baby also had hemophilia B, they needed to give him factor IX prophylactically. If he didn’t, giving this treatment could be extremely dangerous. Unfortunately, it was unclear whether the shared variant caused hemophilia B — it was a VUS. This meant that the clinical team was hesitant to use this variant to decide clinical care. Popp’s map of the factor IX gene provided the evidence they needed to use the variant to diagnose the baby with hemophilia B and provide the necessary treatment.

A monumental effort threatened by funding uncertainty

In addition to the MAVE that Popp made for the gene that produces factor IX, researchers around the world have used MAVEs to create maps for 40 of the 4,000 genes related to human disease. In order to tackle those other 3,960 genes, Fowler and his team have created the Atlas of Variant Effects Alliance, a global collaboration that aims to pool genome scientists’ efforts to create more MAVEs for more genes. The goal is an atlas of the variant effect maps of all 4,000 genes. 

But this monumental effort has been threatened by federal funding uncertainty in the past year. Without funding from the National Institutes of Health, it might not be possible to keep making MAVEs. And researchers like Fowler and Johnsen might also be unable to continue training graduate students like Popp.

“It’s very disruptive; you can’t start and stop the scientific complex,” says Johnsen. “There was a period of time when only Nick knew how to do this. If Nick had to leave and no one else had gotten a chance to learn it, then we wouldn’t have been able to keep going.”

And graduate students like Popp may not even enter the field in the first place when the career path looks so unstable.

“If a lab doesn’t have the money to fund the research and fund you as a graduate student, you can’t join that lab, and you can’t do the work that you may want to do,” says Popp. “It is greatly impacting how people in my position are considering their careers, not just in terms of what they want to do but also in terms of whether they can do it.”

And if Fowler and his collaborators aren’t able to continue making more MAVEs, clinicians and researchers won’t have maps for other genes that cause disease. This lack of evidence makes it harder to diagnose people and to develop better treatments. This has a cascading effect: learning about one rare disease, like hemophilia, often leads to discoveries that can be applied to many others. While only about 30,000 people in the U.S. have hemophilia, around 17 million people in the U.S. (about 1 in 20 people) have a rare genetic disease when looked at collectively.

“Nature is going to keep delivering inherited diseases because there are new variants all the time,” says Johnsen. “We need to be better at diagnosis, better at treatment, better at understanding the mechanisms; all those things come together to improve human health.”