The association between mutations in the MEFV gene and FMF was reported for the first time in 1997. It has been shown that variants in exon 10 account for almost 80% of typical FMF cases. Genetic analysis may be performed to confirm FMF diagnosis since it increases the sensitivity of the criteria recently proposed by Eurofever/PRINTO [23]; however, the previous diagnostic criteria did not include genetic testing as part of the normal work-up [24, 25]. Indeed, there is a lack of conclusive genetic evidence for around 30% of patients diagnosed with clinical FMF [26].

This study focused on the p.R202Q gene variation, that is commonly considered as a polymorphism according to the Genome Aggregation Database (gnomAD); currently, there are limited data about the significance of this alteration, however, recent studies have shown that it may be a disease-causing mutation [1, 15]27,28,29,– [30].

Therefore, this study aimed to investigate the role of MEFV p.R202Q alteration in a cohort of patients who received a diagnosis of probable FMF based on Tel Hashomer criteria. In these patients, we evaluated several aspects linked to inflammasome activation and inflammation: the patients’ clinical profile, the alteration of plasma cytokines, the response of monocytes / whole blood to PKN inhibition and finally, the cytological evaluation of leukocytes.

Our p.R202Q carriers indeed shared clinical similarities with canonical FMF, including a positive response to colchicine, with no differences between p.R202Q homozygous or heterozygous patients.

We observed a marked increase in IL-1β levels both in FMF and FMF-like patients, whereas p.R202Q patients exhibited lower levels; IL-18 levels were higher in FMF than in HDs, but no differences were found between p.R202Q patients and controls.

Proinflammatory IL-1β and IL-18 released by monocytes from HDs, FMF, FMF-like and p.R202Q patients were measured upon LPS + UCN0-1 treatment. Monocytes from FMF patients secreted significantly more IL-1β and IL-18 than HDs and p.R202Q monocytes after LPS + UCN-01 treatment. Three patients with the p.R202Q variant exhibited an increase in IL-1β that exceeded 600 pg/ml. These three patients underwent a repeat genetic analysis via Next-Generation Sequencing (NGS) panel, showing the presence of other gene variations, all recognized as VUS, on IFIH1, TNFRSF1A and MVK genes, respectively. We cannot exclude that these variants may have influenced both the clinical phenotype and the response to the LPS + UCN-01 functional assay. Statistical analyses performed after excluding these individuals from the p.R202Q patient group showed that the functional assay response in the remaining p.R202Q patients was comparable to that of HD (Figure S3 - S6). These findings may suggest that patients with p.R202Q, as well as other subjects who are likely to have other unrecognized or non-classically pathogenic variants, should be carefully evaluated through functional assays and genetic sequencing through panels with Next-generation Sequencing (NGS) approach or whole exome sequencing (WES) should be considered to avoid the risk of undertreatment.

The functional assay may also be performed using a small volume of human whole blood, supporting a more convenient and straightforward approach for FMF diagnosis. The preliminary data obtained with this assay are comparable to those from the monocyte-based assay; we did not observe differences in IL-1β and IL-18 release between p.R202Q patients and HDs. However, this assay requires further validation with a larger sample size as it may be affected by donor variability (leukocyte composition, disease activity at the time of sampling) and ongoing pharmacological treatments.

Inflammasome activation in FMF patients, mediated by PKC inhibition, was blocked by colchicine in line with the efficacy of this drug in patients. In p.R202Q patients, colchicine reduced the release of IL-18 and IL-1β only in the three individuals who exhibited inflammasome activation following LPS + UCN-01 treatment and who also carried mutations in the IFIH1, MVK, or TNFRSF1A genes (Figure S2). Notably, all three patients also showed a clinical response to colchicine in vivo. However, it is important to note that the in vitro response to colchicine observed in this functional assay does not fully reflect the patients’ clinical response to treatment. This assay specifically evaluates the effect of colchicine in the context of UCN-01–mediated pyrin inflammasome activation. In contrast, the clinical efficacy of colchicine in vivo likely results from its multiple pharmacological effects, including inhibition of neutrophil activation, modulation of chemokines and prostanoid production, as well as reduction of neutrophil and endothelial cell adhesion [31].

Another factor that suggests the activation of pyrin is the induction of pyroptosis. During inflammasome activation, GSDMD forms plasma membrane pores that facilitate Ca2+ influx, resulting in the calpain-dependent maturation of IL-1α and its release from pores [22]. In p.R202Q patients the levels of IL-1α released from UCN-01 treated monocytes were comparable to those found in HDs, suggesting that this variant does not alter pyrin function.

Several studies reported that the frequency of p.R202Q homozygosity is very low, and it has been associated with autoinflammatory manifestations, suggesting a potent dosage-dependent pro-inflammatory effect of this variant [29]. However, our data showed no differences between homozygous and heterozygous p.R202Q patients in the release of IL-1β, IL-18 and IL-1α from LPS + UCN-01 treated monocytes.

Taken together, these data (release of IL-1β, IL-18, IL-1α, and differences between homozygous and heterozygous patients) showed that the p.R202Q variant responded similarly to wild-type pyrin.

The impact of LPS, widely recognized as a potent activator of monocytes, in this study was useful to investigate the difference in the responsiveness of p.R202Q monocytes compared to HDs. An unexpected result was the response of p.R202Q monocytes to LPS treatment. We found that IL-1β release was higher in p.R202Q monocytes than in HDs, a mechanism probably not related to pyrin activation. Further studies will be necessary to assess whether the release of IL-1β after LPS treatment may depend on the activation of an “alternative inflammasome” pathway in p.R202Q patients.

Finally, we performed a cytologic evaluation of leukocytes from these patients to evaluate the presence of genomic instability, cell death rate, and abnormal neutrophilic subsets. Neutrophils are the most abundant circulating leukocytes during attacks in FMF patients and they rapidly change their characteristics as they get activated [32,33,34,35]. We found a high percentage of hypersegmented N and band N in FMF, FMF-like and p.R202Q patients. A heterogeneous population of these cells (hypersegmented and band N) might reflect their activated state even in the absence of acute attack. These data might suggest that patients experience chronic low-grade inflammation even between flares. Hypersegmentation of N was found in many microenvironments and disease states (cancer and acute - chronic inflammation) [36]. However, the functional consequences of this hypersegmented nuclear morphology are unknown and future studies are required to characterize this population and to evaluate their function. Finally, we found higher levels of cell death rate and NA in patients than HDs. This could be related to inflammation-induced cell death and nuclear damage. The study conducted by Varga et al. reported indeed that cell death mechanisms (e.g. NETosis and pyroptosis) can occur in neutrophils from inactive FMF patients [37]. Future studies will be needed to assess the role of pyroptosis in the pathogenesis of FMF.

In conclusion, our findings suggest that the p.R202Q variant does not interfere with the second regulatory mechanism of pyrin activation. Nevertheless, we acknowledge that this assay does not rule out the possibility that p.R202Q may impact pyrin function through alternative, as yet unidentified, mechanisms. Therefore, while our data do not support a direct functional consequence of p.R202Q in this context, the presence of this variant may still contribute to the clinical phenotype observed in some patients. From a diagnostic standpoint, the inclusion of p.R202Q in routine MEFV genetic screening may remain relevant, particularly in complex cases (potential involvement of additional genes responsible for autoinflammatory conditions) or when present in combination with other MEFV variants.

We would be remiss not to mention some of the limitations of our study. Firstly, the p.R202Q patients’ recruitment: we did not evaluate the possible carriage of MEFV p.R202Q mutation in the control group; rather, we requested the genetic test after choosing individuals who displayed symptoms typically of FMF, even though it is a common variation in the population. However, despite selecting patients with autoinflammatory symptoms, p.R202Q patients showed an almost absent response to UCN-01 stimulation compared to FMF patients. Given the potential classification of p.R202Q as a low-penetrance variant, a distinct functional response would be expected at least in homozygous individuals. However, in our study, we did not observe any significant differences in inflammasome activation or cytokine release between homozygous and heterozygous p.R202Q carriers. Moreover, previous data from a cohort of 218 healthy donors recruited in our geographical area showed that the percentage of individuals homozygous for the p.R202Q variant was very low (0.07%) [38]. Based on this, we performed an additional analysis (Figure S7), comparing the response to the LPS + UCN-01 treatment in homozygous p.R202Q patients with HDs. No significant differences were observed in the functional assay response, suggesting that the presence of the p.R202Q variant in the homozygous state does not result in abnormal pyrin activation. Although sequencing HDs would have provided another layer of certainty, our overall data indicate that the p.R202Q variant does not appear to impair the secondary regulatory mechanism of pyrin.

Secondly, our sample size was too small to draw any definitive conclusions. A comparison involving a larger cohort of FMF patients, stratified by genotype and including other VUS and benign variants, should be performed to better define the functional relevance of these findings.

Thirdly, the inclusion of patients who were receiving colchicine treatment at the time of sample collection. While we acknowledge that colchicine could potentially influence certain cellular responses, we believe that it does not significantly impact the final results of our functional assays. In particular, for the functional assay performed on isolated monocytes, the cells were cultured in colchicine-free conditions for 24 h, allowing for a washout of the drug. This approach minimizes the influence of colchicine treatment on the assay outcome. Furthermore, previous studies that used this type of assay have shown that colchicine did not affect the final outcome of the assay [6]. Colchicine treatment could potentially influence neutrophil counts, however, in our cohort, we found no significant alterations in this parameter. Studies in the literature suggest that colchicine primarily affects neutrophil function rather than morphology, and the usual colchicine doses do not result in significant cytological abnormalities [32, 39, 40].