Discovery of new PI shows value of grants for exploratory research

When Dr. Megan Cooper, director of the clinical immunology program and The Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies at St. Louis Children’s Hospital, applied for the Immune Deficiency Foundation’s research grant program in 2018, she didn’t know exactly what she was looking for. She wanted to use the grant to find a genetic explanation for the severe infections and autoimmune symptoms of two young boys who were medical mysteries. Her resulting work implicated variants in the toll-like receptor 8 (TLR8) gene for their condition. The research not only added a new disorder, TLR8 gain-of-function (GOF), to the list of primary immunodeficiencies (PIs) but also put a whole category of overlooked gene variants, called somatic variants, in the spotlight. 

Cooper is a pediatric rheumatologist by training and studied cancer immunology during her PhD at The Ohio State University. Describing the evolution of her interests, Cooper said, “During my PhD work I was attending a large meeting focused on cancer, and I heard a talk about gene therapy for SCID [severe combined immunodeficiency]...it was amazing to me that you could fix this disease in children. …It just kind of opened my eyes to these inborn errors of immunity.”

Cooper moved to St. Louis Children’s Hospital for her residency in pediatrics, then Washington University in St. Louis for a fellowship in pediatric rheumatology. Her experience working on another disorder that causes both severe infections and autoimmunity, STAT3 GOF, helped prime her for the TLR8 GOF discovery. At St. Louis Children’s Hospital, she came across children with early-onset immune dysregulation similar to STAT3 GOF who could not be definitively diagnosed. 

“What the IDF grant gave me—I’ll be honest—was the freedom to perform some of these exploratory sequencing studies on patients that we didn’t know what was wrong with them,” Cooper said about her grant application. “As a clinical immunologist, I knew there [was] certainly something wrong with this child’s immune system. And if we can better understand it, then we can provide them with a specific diagnosis and, potentially, better therapy.”

“There’re very few grants that allow you to do that kind of discovery work,” Cooper pointed out.

Whole exome sequencing (WES), the type of genetic testing Cooper used, determines the DNA sequence for all of the protein-coding regions of all of a person’s genes. Its strength is that researchers don’t have to know beforehand which genes to look at because they get data back on all of a person’s genes. 

That flood of data can also be overwhelming, though. One study of almost 50,000 people in the United Kingdom found an average of 80 coding variants in WES data per individual. For a patient with clinical symptoms, each of those variants could be responsible for their condition—or not. Researchers have to sift through them and decide which, if any, are worth pursuing.

What Cooper got back were WES results that weren’t easy to interpret. The two boys with immune dysregulation had variants in the TLR8 gene on the X chromosome. The TLR8 protein recognizes certain germs once they invade a cell, triggering downstream immune responses, such as inflammation. However, variants in TLR8 had not been associated with any form of PI before. 

There was another oddity in the data. The variants were only there in a fraction of the sequencing data that covered the TLR8 gene for each boy. Since boys have only one X chromosome, the only possible explanation was that the boys had cells coexisting in their bodies with different TLR8 sequences. That is, they had somatic, rather than germline, variants.

In high school biology, students learn that a person’s DNA is the same in every cell in their body, but that is an oversimplification. Sometimes, during a baby’s development in the womb, an error in copying DNA to create new cells leads to what’s called a somatic variant. In these cases, the baby is born with cells that contain two different DNA sequences—cells without the variant and cells with the variant that grew from the original miscopied cell. Before the era of deep sequencing techniques like WES, scientists simply did not have the tools to ‘see’ somatic variants. 

When asked how she zeroed in on the TLR8 variants given the puzzling WES results, Cooper replied, “It was a little bit serendipity and a little bit just looking at one patient at a time.” 

Cooper and her colleagues at Washington University registered as clinicians interested in TLR8 on GeneMatcher, a nonprofit website that connects scientists interested in the same human gene. Through the site, she collaborated with other clinicians and identified additional boys with TLR8 variants, all with symptoms of immune dysregulation. Cooper also reached out to the National Institutes of Health (NIH), where colleagues there were able to identify additional patients with variants in TLR8 and immune dysregulation. 

“It’s like a dating service for genes,” Cooper said, describing GeneMatcher. “So there were other people out there that had found some patients with changes in toll-like receptor 8 but weren't really sure what it meant.”

The larger pool of patients with similar symptoms, including low numbers of neutrophils, enlarged spleen and/or liver due to increased generation of B and T cells, and infections, gave Cooper more evidence that she was on the right track. She and her collaborators then showed that immune cells from these patients were more sensitive to molecules that activate TLR8 and that once activated, the protein was more difficult to turn off, resulting in uncontrolled inflammation. Cooper concluded that the variants in the TLR8 gene result in a protein that is more active than it should be (has gained function), leading to both immune deficiency and autoimmunity.

On the wider impact of her discovery of TLR8 GOF, Cooper explained, “I think that previously people had not been very interested in studying toll-like receptor 8 because there had not been an important role defined for our immune system, as it has some overlapping functions with other TLR receptors. But I think that this has shown that toll-like receptor 8 function is obviously very tightly regulated because when you lose that regulation, it leads to human disease.”

“I think the other thing that it's done, for the field and for me personally, in combination with some other recent studies, is to ignite an interest in somatic mosaicism as causing inborn errors of immunity.”

Since publishing the discovery of TLR8 GOF in 2021, Cooper and the network of researchers she works with have found other patients with the disorder, including a girl with a TLR8 variant present in all of her cells (known as a germline variant). Despite having another copy of the TLR8 gene that functions properly, she has the same severe symptoms as boys with somatic TLR8 GOF. This individual demonstrates that TLR8 GOF can be inherited in an X-linked dominant manner. It is the first PI documented to be X-linked dominant, and there are only a handful of other human disorders with X-linked dominant inheritance. 

Cooper continues to focus on discovering unknown gene variants that explain immune dysregulation, particularly somatic variants. At a talk this past spring at the Clinical Immunology Society, she encouraged clinicians to look for somatic variants when they can’t find a genetic explanation for a patient’s PI symptoms. 

“I never would have predicted five years ago that [somatic mosiacism] would be a major theme of my lab,” Cooper said. “And as a physician, it's rewarding to try to think about new ways that we can diagnose our patients.”