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New Diagnostic Test for Familial Mediterranean Fever Emerges from Inflammation Study


NEW YORK (360Dx) – Based on an unexpected finding from a study of inflammation, researchers at VIB and Ghent University in Belgium have developed an immunoassay that may improve the diagnosis of familial Mediterranean fever (FMF) and lead to earlier therapy.

The scientists, led by Mohamed Lamkanfi at the Center for Inflammation Research at VIB and Ghent University, are currently validating the assay and are transferring it to other clinical laboratories. In addition, they are in discussion with a potential licensing partner who might commercialize the test as a diagnostic kit.

FMF is a genetic autoinflammatory disease that belongs to a set of disorders known as periodic fever syndromes and affects about 150,000 patients worldwide, in particular individuals originating from the Mediterranean region and the Middle East.

Most cases are caused by mutations in both copies of the MEFV gene, which encodes the protein pyrin, and more than 310 such mutations are known to date. Pyrin plays a role in the activation of a type of inflammasome, a multiprotein complex that regulates inflammation and results in the release of cytokines interleukin 1beta and interleukin 18. However, it is not known how exactly MEFV mutations affect pyrin's actions.

"That's why we got interested in this," said Lamkanfi, whose lab has a longstanding interest in inflammation. "It's [about the] inflammasome, and the disease is not very well understood so far. There had been some hypotheses, and we wanted to dig deeper and understand exactly what's going on."

In their initial studies, the researchers showed that both the bacterium Clostridium difficile and its enterotoxin TcdA can activate pyrin and the inflammasome in mouse macrophages as well as in human peripheral blood mononuclear cells (PBMCs).

They also found that colchicine, a drug that inhibits the polymerization of microtubules in the cell, can prevent the toxin-induced inflammasome activation in PBMCs, leading to reduced release of IL-1beta and IL-18. However, somewhat surprisingly, this was not the case for PBMCs from FMF patients — they continued to secrete IL-1beta and IL-18 after being treated with colchicine or other microtubule assembly inhibitors.

To further study whether this reaction was specific to FMF, the researchers, in collaboration with a group in Italy, repeated the experiment with PBMCs from five healthy donors and nine FMF patients with a variety of mutations, as well as four patients with CAPS disease and seven patients with juvenile idiopathic arthritis. Again, only the cells from FMF patients continued to release the cytokines when treated with microtubule inhibitors, suggesting that mutated pyrin can activate the inflammasome without the presence of intact microtubules.

In addition to shedding new light on the function of pyrin in the cell, the researchers, who published their findings in the Proceedings of the National Academy of Sciences last year, reckoned that their discovery could also be exploited as a diagnostic test for FMF. "We thought, given the diagnostic issue that's still out there in the clinic, we can use this unique feature … that is associated with the disease to screen patients suspected of inflammation" for FMF, Lamkanfi explained. "This will hopefully help to bring down the time to a correct diagnosis."

FMF is normally diagnosed based on clinical symptoms, taking the patient's ethnic background into account. However, it is often mistaken for related diseases with similar symptoms, he said, and on average, it takes FMF patients five years to obtain a diagnosis. Patients are frequently misdiagnosed with appendicitis, for example, which can lead to unnecessary surgery.

FMF patients usually suffer from periodic fevers and often have other symptoms, such as abdominal pain, joint pain, or pleuritis. A major long-term complication of untreated FMF is amyloidosis, where protein clumps are deposited in organs like the kidney, which can lead to kidney failure.

The disease can be treated with colchicine, which suppresses the inflammatory attacks and is successful in the majority of FMF patients but has risks associated with it and does not work in related autoinflammatory diseases. Thus, a timely diagnosis is important, and given the possible kidney complications, "the sooner you start treatment, the better," Lamkanfi said.

A genetic test for FMF that looks for mutations in the MEFV gene exists, he said, but its costs are quite high and it is not always conclusive because some FMF patients only have a mutation in one copy of the gene, while patients with other inflammatory diseases may be carriers of FMF.

The ELISA test that Lamkanfi and his team developed, on the other hand, is low-cost, fast and easy to perform, and would be the first immunoassay for the disease, he said.

To perform the assay, a patient's blood is drawn, PBMCs are isolated, and bacterial toxin is added to trigger the inflammatory response. Then a microtubule inhibitor is added to half the sample but not the other half. Both samples are incubated for several hours, and an ELISA for inflammatory cytokines, in particular IL-1beta, is performed. If the assay is positive in both samples, this indicates they came from an FMF patient.

The next step is to validate the assay in a cohort of 100 to 200 FMF patients, as well as individuals with other inflammatory disorders, in order to get an idea of its sensitivity and specificity. Lamkanfi and his colleagues are currently looking for additional collaborations to obtain blood samples from patients and are hoping to complete the trial in about a year. One of the aims of the study is to cover some of the rarer FMF mutations that were not part of the original work. Initially, the trial will only include patients with validated genetic mutations, he said, but going forward, the researchers would like to "go blind" and test samples from patients who don't have a diagnosis yet.

In the meantime, his team has started to transfer the assay to other academic clinical centers, including labs in the Netherlands and in Southern Italy.  

The VIB-Ghent team has also filed a patent application on immunological FMF diagnosis, with the aim to license the assay to a company to commercialize it as a diagnostic kit. This could be a firm that already offers genetic testing for FMF, or one that specializes in diagnostic immunoassays and would like to add an FMF assay to its portfolio, Lamkanfi said. The team recently started discussions with a currently undisclosed potential licensing partner.

The price of a commercial FMF immunoassay would likely be lower than that of a genetic test, he said, noting that some ELISA-based tests costs between $25 and $50.