Following one unexplained case to a new rare bone marrow failure disorder causing high p53 levels

When only a handful of patients share an unexplained set of symptoms, how do physicians determine whether they have found a new disease?

That was the challenge that Marcin Wlodarski, MD, PhD, Bone Marrow Failure and Myelodysplastic Syndrome Clinic (BMF/MDS Clinic) director and Department of Hematology associate member, recently faced. Wlodarski treated a patient who came to the clinic with an unexplained bone marrow failure syndrome, a type of disorder marked by the body’s inability to make enough blood cells. Wlodarski suggested enrollment in the INSIGHT-HD clinical trial, which is designed to uncover unknown genetic causes of bone marrow failure and other hematologic disorders.

The patient’s choice to participate in the clinical trial was the key turning point in identifying a new disease. St. Jude researchers sequenced the patient’s DNA, finding that it did not have any of the trademark mutations known to cause these disorders. Instead, they identified a genetic change in a previously unknown to be associated with bone marrow failure gene called MDM4. Wlodarski’s team then searched for any prior unexplained cases across the world that matched his patient’s symptoms.

He found a single example.

Fortunately, the physicians who reported that case had documented that the patient and their family had a mutation in MDM4, a protein that interacts with p53, often called “the guardian of the genome.” While loss of p53 activity is common in cancer, p53 hyperactivity can create a different problem. In blood stem cells, too much p53 signaling can trigger these cells’ self-destruction, impairing the body’s ability to produce other blood cells. When researchers looked at the St. Jude patient’s DNA sequence, they found a mutation in the same MDM4 gene, suggesting that the two groups may have found the source of a novel rare disease.

“One case would not have been enough,” Wlodarski explained. “If we found only one patient, it wouldn’t establish a new bone marrow failure-causing gene. We needed to find multiple cases across multiple institutions to solidify that this was a robust finding.”

Building a global cohort

Once Wlodarski found the other case and identified the potential disease-causing mutation, he enlisted the help of many others to characterize the new disease. He began close to home, working with Senthil Bhoopalan, MBBS, PhD, Department of Bone Marrow Transplantation & Cellular Therapy. Bhoopalan is an expert in Diamond-Blackfan anemia syndrome, which has symptoms like, but distinct from, the novel disorder. Together, they reached out to the small but tight-knit rare disease research community across the world. Their search identified six patients across the United States, France and Germany. Whole-exome sequencing revealed MDM4 loss-of-function mutations in every case. The researchers published these findings recently in Blood.

The genetic pattern was compelling, but how the mutations caused the patients’ bone marrow failure remained unclear. The researchers needed to understand how those mutations impacted blood development. Therefore, the other lead author of the study, Richa Sharma, MD, formerly of the Department of Hematology (now at the Cleveland Clinic), with support from the Department of Hematology’s Human iPSC Core, led by Lei Han, PhD, reprogrammed patient cells into induced pluripotent stem cells, or iPSCs, which can develop into many different cell types. They then directed those cells back toward blood stem cells and observed how well they functioned.

“When we tried to differentiate blood cells from stem cells with any of these mutations, they all had problems,” Bhoopalan said. “They couldn’t differentiate well, and they had very high p53 activity.”

The result provided functional evidence that loss of MDM4 increases p53 levels and disrupts blood formation. Together with the genetic findings across multiple patients, the data supported recognition of a previously uncharacterized bone marrow failure disorder, though how MDM4 impacted p53 was unclear.

A broken bridge leaves p53 unchecked

When the scientists turned their investigation to understanding how MDM4 mutations cause this disease, they first set their sights on how it interacted with p53. They already knew that MDM4 interacts with both p53 and a related protein, MDM2. MDM2 helps regulate p53 by marking it for destruction, preventing the protein from accumulating unnecessarily. As MDM4 binds both proteins through separate regions of its structure, the researchers suspected that it might help connect them. 

To find out, Bhoopalan deleted one or the other of the two major parts (domains) of the MDM4 protein — the one that binds to MDM2 and the other that binds to p53 — and observed their effects in healthy stem cells. Removing either domain produced the same result of impaired blood cell formation and elevated p53 levels.

“It looks like we need both MDM4 domains,” Bhoopalan explained. “So, MDM4 may act like a bridge connecting MDM2 to p53 to help mark the protein for destruction, but in the case of this disease, part of that bridge is collapsed, preventing MDM2 from marking p53, resulting in the pro-cell death protein’s accumulation.”

If MDM4’s loss of function really was the problem leading to disease-causing levels of p53, then there should be ways to rescue blood formation. To test that idea, the scientists used CRISPR-Cas9 to recreate mutations in MDM4 and TP53 genes using blood stem cells from healthy donors. Blood stem cells lacking MDM4 disappeared over time, as expected if p53 accumulates and signals for self-destruction. However, cells lacking both MDM4 and TP53, the gene encoding p53, survived because p53 could not accumulate and signal for cell death.

“Regardless of the mutation, we think that too much p53 is building up and inhibiting blood production and promoting cell death,” Wlodarski said. “Now that we know what to look for, we have one more way to diagnose a patient’s bone marrow failure.”

A long path to improving patient care

Already, clinicians are incorporating the MDM4 mutations into gene panels they give to patients with a suspected bone marrow failure disorder, which will help identify more cases. In the future, a patient’s MDM4-mutation status could potentially be used to guide care, though there are still many unknowns.

“There is a lot we still want to learn about these diseases,” Bhoopalan said. “We don’t understand why these patients have different symptoms, and we don’t know if there is a mutation-specific clinical symptom and clinical course, so we cannot give a strong recommendation on the best treatment approach.”

Those differences are substantial. The patient treated at St. Jude experienced spontaneous improvement in symptoms. In contrast, other patients developed myelodysplastic syndrome, or MDS, a condition that can progress to leukemia, which may require very different approaches to care. However, now that clinicians are on the lookout for the gene and its mutations, the St. Jude investigators and their peers can collect the evidence needed to guide such treatment.

Scientists identify new rare diseases in partnership with patients

The journey to diagnose this new disease began with a single patient who agreed to be part of the INSIGHT-HD clinical trial at St. Jude. That one courageous individual set off a chain of events that became a worldwide collaborative search that found the source of this rare disease.

“These findings show how important it is for patients to take part in research studies, because their participation is what enables us to identify these new syndromes,” Wlodarski said. “They are the real key that helps us uncover novel genetic causes of bone marrow failures and begin improving care for the next generation of patients.”