By Paul Nicolaus
March 3, 2020 | An international group of scientists has discovered a previously unknown autoinflammatory disease and identified its underlying biological origin. The disease, dubbed cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome, is caused by mutations in the receptor-interacting protein kinase 1 (RIPK1) gene.
“The presenting symptoms are basically those of lifelong recurrent fevers,” Daniel Kastner, National Human Genome Research Institute (NHGRI) scientific director told Diagnostics World News, as well as swelling of the lymph nodes. Other symptoms can include abdominal pain, gastrointestinal issues, headaches, and an enlarged spleen or liver. While not life-threatening, the disorder can lead to lifelong pain and disability.
In a study published in Nature (doi: 10.1038/s41586-019-1828-5), he and colleagues describe patients with a history of inflammatory symptoms who were diagnosed with CRIA syndrome.
The paper defines a new mutation responsible for auto-inflammatory syndromes, explained Anca Askanase, a rheumatologist, associate professor of medicine, and director of Rheumatology Clinical Trials at Columbia University Medical Center who was not involved in the research.
“Elegant genetics work is followed by confirmation and characterization of the mutation in an animal model,” she told Diagnostics World News in an email.
We have known for some time that while there is a genetic predisposition to developing an autoimmune or autoinflammatory disease, there are often outside or environmental factors provoking the genes to cause expression, explained Tiffany Caplan, co-founder of Central Coast Center for Integrative Health, who was not involved in the study.
“What this new research is highlighting is the direct link of causation to a very specific genetic mutation that appears to not only be the key to identifying this specific autoinflammatory disease but is also the key to treatment,” she told Diagnostics World News in an email.
Discovery Spans Two Decades, Three Families
When Kastner’s group encountered the condition in a young girl from Texas roughly a decade ago, exome sequencing technology wasn’t available just yet. “This was before the advent of serious genomic sequencing,” he said, “in which one could sequence all of the genes in someone’s genome.”
At the time when he and colleagues first saw this patient, the technology was at the stage where, if a researcher had a candidate gene in mind, it was possible to sequence that gene to search for a mutation. Or if there were multiple members of a family, it was possible to perform linkage analysis to figure out what chromosome the gene would be on and then use that information to make informed guesses before sequencing candidate genes. “But in that family,” he said, “we only had one person and so we didn’t have any clues.”
Several years later, however, the technology had progressed, and Steven Boyden, a postdoctoral fellow in Kastner’s lab, was handling a sequencing project that led to a new development.
“I have a large clinic in which we see patients with periodic fever syndromes,” explained Kastner. In total, he and colleagues had a group of several dozen patients with undiagnosed periodic fever syndromes, and Boyden hypothesized that some of those patients could have a de novo mutation—one that has arisen in the child but was not present in either parent.
The idea was that he would perform whole-exome sequencing in the patient—the young girl from Texas—in addition to sequencing the two parents to search for genetic variants present in the patient but not in her parents. From there, he could analyze any variants and determine whether or not the findings made sense in terms of this particular clinical picture.
When he looked at the panel of patients and their parents using whole-exome sequencing, he found that this particular patient did have a mutation in RIPK1, which was already known to be an important molecule associated with inflammation.
When Boyden analyzed the mutation, he found that it is located at the cleavage site of RIPK1, Kastner explained. It is cleaved by caspase-8, a particular enzyme inside the white blood cell that essentially turns off its inflammatory effect. “And if you lacked the ability to cleave RIP kinase, it was at least plausible to think that you might have continuous inflammation,” he said.
From there, Boyden conducted studies revealing that the mutation did inhibit the cleavage of RIPK1. He also found some in vitro evidence to suggest that it would lead to prolonged inflammation, according to Kastner, at least based on some white blood cell assays.
“But still, in order to be confident that the mutation actually leads to the disease, you’d like to see another patient unrelated to the first patient who would have a similar mutation,” Kastner added. “In this field, until you have the second case you don’t have that sense of certainty that you’ve really found it.”
For some time, the researchers searched for another case of a patient with fever and swelling of the lymph nodes as well as a mutation in RIPK1, but to no avail. While revisiting materials from families seen in the past, however, a surprising finding emerged.
“We started looking at a family that we had seen back in the late 1990s,” he said, roughly a decade before finding the mutation in the girl from Texas. This three-generation family from California has five affected individuals, and exome sequencing revealed a mutation in RIPK1 at the same amino acid as in the patient from Texas.
Whereas the patient from Texas had an asparagine instead of an aspartate, it was a histidine rather than an aspartate in the second family. “So it was just a different amino acid substitution, but that substitution—the histidine for aspartate—would also be predicted to render the RIP kinase protein uncleavable.”
About a year ago, the researchers came across a third family from New York with a mutation in RIPK1 associated with the same clinical picture, he said, also at position 324. In that family, the mutation was a substitution of tyrosine for aspartate.
The phenomenon, Kastner pointed out, resembled lightning striking the same place three times.
“It’s really remarkable, when you think about it, that here you have in three different families, three different mutations in RIP kinase,” he said. All of them are at the same amino acid, and all of them render RIPK1 virtually indestructible.
Understanding the Molecular Mechanisms
Although Kastner and his team made the connection between CRIA syndrome and RIPK1 mutations, they still wanted to better understand how these mutations led to chronic inflammation. To learn more, they turned to a group led by Najoua Laloui and John Silke at the Walter and Eliza Hall Institute in Australia.
Their research team has investigated the role of the same RIPK1 protein, which is one of the key players of the Tumor necrosis factor (TNF) signaling pathway, explained Che Stafford, a postdoctoral researcher at the Ludwig Maximilian University of Munich in Germany who worked in Silke’s lab while pursuing his PhD.
This pathway is activated by TNF, an inflammatory messenger, that either leads to the production of more inflammatory messengers or cell death, Stafford told Diagnostics World News in an email. In a healthy system, TNF is tightly regulated and is needed to fight infection. If regulation is lost, however, excessive inflammation can occur.
Using mouse models and cellular biology, Lalaoui and colleagues explored how cleavage of RIPK1 is required for proper TNF signaling. They engineered mice with a mutation in RIPK1 that prevents its cleavage, which usually occurs upon TNF stimulation mediated by the caspase-8 enzyme. The mice displayed increased levels of cytokines and cell death, he explained, which are both hallmarks of inflammation.
“Intriguingly, when the collaboration was formed between the research groups, they realized that the patient mutations also resulted in an un-cleavable form of RIPK1,” Stafford noted. “Since the mice displayed similar inflammatory symptoms to the patients, it provided a good model to study the disease.”
Another Data Point, New Treatment Potential
When Kastner and colleagues first saw the patient from Texas—before they had completed the sequencing and found the clue about RIPK1—they were aware that she did not have a known periodic fever syndrome because they had tested her for Familial Mediterranean fever and some of the other known genetic periodic fever syndromes.
“On the other hand, we did know that patients—even with undiagnosed periodic fever syndromes—do respond to certain types of drugs,” Kastner explained.
After trying an inhibitor of Interleukin-1 (IL-1) and an inhibitor of TNF, which she did not respond to, they turned to another option. “The third major category of biologic that one can use in patients with undiagnosed recurrent fever syndromes is an IL-6 inhibitor, tocilizumab, and that was actually the medication to which she responded,” he said.
When they later tried tocilizumab on the affected individuals from the other two families, there were “fairly beneficial” effects. It varies from patient to patient, he said, but in general patients respond reasonably well to IL-6 inhibition.
“But this is an empiric observation, and we don’t have at this point a full accounting of why it is that inhibiting IL-6 as opposed to IL-1 or TNF would have a beneficial effect for these patients,” Kastner added. “So this is in the to-be-discovered category for future work is to figure out how inhibiting IL-6 would have a salutary effect on these patients.”
One upside to the discovery of CRIA is that it provides another data point in terms of genes that can be looked at for patients that have undiagnosed recurrent fevers.
“We see a lot of children that have undiagnosed recurrent fevers where infections have been ruled out and they don’t have cancer, fortunately, but where there’s not an explanation,” he added. “Nowadays, with the advent of DNA sequencing, they can be tested for mutations in known genes that cause recurrent fevers, and so this adds another gene that can be tested for.”
In some cases, these patients have their exomes sequenced. For someone who’s interpreting that data, it gives them the precedent that if they find a child with a mutation in RIPK1 that it is probably positive of their syndrome. “And of course, given our experience with IL-6 inhibition, it suggests a treatment for that patient,” he said.
The discovery of CRIA syndrome could also be more far-reaching. The findings suggest that unchecked cell death could play a critical role in other inflammatory conditions and that inhibitors of RIPK1 may be a useful tool in a broad spectrum of inflammatory illnesses. Some, however, caution against reading too much into the newly published research.
“While there is a lot to be learned from these findings,” noted Caplan, the functional medicine practitioner, “there is still more to be learned in the fields of genetics and epigenetics before applying this information or ‘blaming genetics’ for the other 100+ known autoimmune and autoinflammatory conditions plaguing society today.”
Paul Nicolaus is a freelance writer specializing in science, nature, and health. Learn more at www.nicolauswriting.com.