Systemic autoinflammatory diseases (SAIDs) primarily affect the innate immune response and are distinct disease entities, though they may overlap somewhat with autoimmune disorders (1). The spectrum of SAIDs has been expanded in recent years (2). Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a specialized group of intracellular proteins that play a critical role in the regulation of host immune responses (3, 4). Mutations in the NLR family members have been linked to a variety of autoinflammatory diseases (5).

Two examples of NLRs associated with SAIDs are NLRP3 and NLRP12. The NLRP3 gene encodes Cryopyrin, and mutations cause gain-of-function, leading to Cryopyrin-associated Periodic Syndrome (CAPS) (6), aka NLRP3-associated autoinflammatory disease (NLRP3-AID) (5). CAPS encompasses a spectrum of phenotypes ranging from mild Familial Cold Autoinflammatory Syndrome Type 1 (FCAS1), Muckle-Wells syndrome (MWS) to severe Chronic Infantile Neurological Cutaneous and Articular (CINCA), or Neonatal-onset Multisystem Inflammatory Disease (NOMID) (7, 8). NLRP3-AID is typically caused by NLRP3 gene missense mutations of high penetrance; however, an intermediate form has been reported to be associated with a low-penetrance variant, NLRP3 Q705K, aka Q703K (9, 10).

Genetic variations in NLRP12 cause NLRP12-AID (11), aka Familial Cold Autoinflammatory Syndrome Type 2 (FCAS2) due to its similarity to FCAS1 and MWS, and both NLRP12-AID and CAPS are phenotypically characterized by conjunctivitis, urticaria and arthralgias triggered by exposure to cold, as well as neurosensory hearing loss (11). Similarly, NLRP12-AID is associated with NLRP12 missense mutations of high penetrance resulting in gain-of-function; however, nearly 50% of reported cases to date harbor the NLRP12 variant, F402L, a low-penetrance variant (12). To date, molecular testing has been key for differentiating between these two diseases, as they are thought to be clinically indistinguishable. Like CAPS, NLRP12-AID is a rare autosomal dominant disease. There have been approximately 60 cases reported in the literature as of the year 2020 (13). Most reported cases were pediatric. In addition, we have not seen any reports of cases of concurrent NLRP3 and NLRP12 variants. Based on the Infevers database (https://infevers.umai-montpellier.fr/web/search.php?n=4) as of July 24, 2023, total numbers of sequence variants identified for NLRP3 and NLRP12 are 264 and 82, respectively. Both NLRP3-AID and NLRP12-AID are caused by a spectrum of a number of validated known and yet-to-be validated variants. Based on the current literature, both NLRP3-AID and NLRP12-AID are clinically indistinguishable. Herein, we investigated the largest monocentric cohort of adult-onset patients to compare these two diseases in order to identify clinical parameters for distinction. We also examined the frequency of disease-associated variants in two large control datasets to support their contributory roles in these diseases.

NLRP3-AID and NLRP12-AID are autosomal dominant disease and patients share similar phenotypes, including cold-induced urticaria/inflammatory disease and neurosensory hearing loss. Genotyping, therefore, has been key for their distinction (11, 12). In the present study, patients in both Group 1 and 2 had a family history of the disease in approximately 45% ( Table 3 ). We found statistically significant differences in certain clinical phenotypes among disease groups. In NLRP3-AID, complaints of headache and dizziness were significantly more common (P value=0.0176), consistent with a previous report of 79% of CAPS patients with known pathogenic NLRP3 variants and 73% of those with low-penetrance variants (9). We also found that GI symptoms (diarrhea) in patients with NLRP12-AID were significantly more common than among the NLRP3 group. In a previous report, only 25% of patients with pathogenic NLRP3 variants reported abdominal pain and none diarrhea (9). In support of our finding are two previous reports that a CAPS mouse transgenic model carrying human NLRP3 pathogenic mutations maintain homeostasis in the gut microbiota and are resistant to experimental colitis (19), whereas NLRP12 defect results in microbiome disturbance and colitis (20). In our study, hearing loss occurred infrequently across all groups with no significant difference (33% patients with NLRP3 variants). In contrast, sensorineural hearing loss was reported for 79% of patients with NLRP3 pathogenic variants and 18% with NLRP3 low-penetrance variants (9), indicating that mutations of high-penetrance may be more pathogenic for hearing loss. In addition, we noted that four patients had livedo reticularis and this occurred only among NLRP12 carriers. Taken together, our data suggest that these clinical features can be used for distinguishing these two diseases.

Over 60% of our patients in Group 1 carry the low-penetrance variant, NLRP3 Q705K, which is known to increase risk for CAPS (9). Compared with the large ARIC data, the variant frequency was significanly higher in our patient group ( Table 1 ). NLRP3 is primarily involved in inflammasome activation leading to production of proinflammatory cytokines like IL-1β, and its mutations cause gain-of-function leading to NLRP3-AID. In a multicenter study of 45 patients with low penetrance NLRP3 variants including 19 with Q705K vs 28 CAPS patients with known pathogenic variants, the former patients had autoinflammatory features, such as fever (76%), headaches (73%), rash (80%), sensorineural hearing loss (18%), arthralgia (84%), abdominal pain (56%), diarrhea (18%), and elevated ESR/CRP(26%-34%) (9); these clinical findings and their frequencies are quite similar to those in our patients with NLRP3 variants ( Table 3 ). In vitro studies confirmed that cells expressing NLRP3 Q705K exhibited mildly increased caspase 1 activity and cleavage, and also that these patients responded to IL-1 inhibition therapy (9). In another study, human monocytic cell lines transduced with Q705K produced a significant higher level of IL-1β and IL-18 than wild type, confirming the gain-of-function (21). In agreement with the literature, 67% of our patients with NLRP3 variants received IL-1 inhibitors with a good to excellent response. Taken together, both our and literature data strongly support that autoinflammatory features can be caused by the low-penetrance NLRP3 variants. Of note, compared with patients with low-penetrance variants, those with known pathogenic NLRP3 variants (A439V, E311K and T348M) had a lower frequency of fever (18%) and GI symptoms (0-25%), and a higher frequency of sensorineural hearing loss (79%) (9).

Over 50% of our patients in Group 2 carry the low-penetrance variant, NLRP12 F402L, which is linked to NLRP12-AID. In fact, 44% to 50% of reported cases carried the NLRP12 variant (12, 13). Symptomatic patients with F402L can be children and adults. In a review of 33 pediatric cases, females accounted for 65%, and major clinical manifestations included recurrent fever in 100% of patients, polyarthralgias in 55%, abdominal pain/diarrhea in 48%, rash in 45%, neurosensory hearing loss in 21%, headaches in 24%, and elevated CRP/ESR in 55% (13, 22). These manifestations and frequencies are similar to our findings in adult-onset patients with NLRP12-AID ( Table 3 ). To date, there have been few case-control studies to estimate the MAF of NLRP12 F402L. One study involved 5 patients vs 11 F402L carriers among 94 healthy subjects (MAF 0.05) (23) and the other involved 6 patients vs zero F402L carriers among 72 healthy subjects (MAF 0) (24). Apparently, these studies had sample sizes too small to more accurately estimate MAF or determine the effect size of this variant. To overcome the shortcomings of the small sample sizes, we compared our patients with the large ARIC data, and found that the frequency of the variant was significantly lower in the control as compared with patient’s group, supporting the previously reported role of NLRP12 F402L in the disease, NLRP12-AID. While we have not seen functional studies of NLRP12 F402L variant, bioinformatic analysis points to its pathogenicity (13). In addition, one of our patients carried two NLRP12 variants, A682T and F402L. Supporting the clinical significance of this finding is a recent report of a symptomatic adult female with carriage of two heterozygous NLRP12 variants, R352C/F402L inherited from both parents (13).

NLRP12 can negatively regulate pro-inflammatory signaling and inflammation (11, 25, 26). Wild type NLRP12 has a pro-inflammatory effect on caspase 1 signaling and speck formation, induces the release of the inflammasome-dependent cytokines IL-1β and IL-18, and has anti-inflammatory effect on NF-kB activation. It has been reported that the NLRP12 R352C missense mutation is associated with a gain-of-function with regard to caspase 1 processing (27, 28). In our current study, few cases of NLRP12-AID received IL-1 inhibitors with inconsistent effectiveness, by contrast to patients with NLRP3 variants. The mechanisms underlying the therapeutic differences between the two diseases are unclear. The negative regulation of proinflammatory cytokines by NLRP12 suggests that its function may be inflammasome-independent (26). This will need further translational study using larger cohorts of patients.

It is well known since the completion of the human genome project in 2003 (29) that many genetic variants that contribute to disease lie on a spectrum from rare alleles with large effect sizes to more common alleles with small effect sizes. There is a “gray zone” between these two extremes, such as NLRP3 Q705K and NLRP12 F402L in our study, that is ill-defined with regard to terminology, classification and clinical reportability (30). To supplement the traditional interpretation for monogenic autosomal dominant or recessive diseases, we recently proposed and defined the concept “Genetically Transitional Disease” (GTD). GTD refers to a disease status between monogenic and polygenic, where a mutation is necessary, but not sufficient, to cause disease (31). This concept has been used to certain rheumatic diseases (32). Autoinflammatory diseases with low frequency/penetrance variants as in our study are good examples. The GTD concept and related nosology may help with molecular laboratory reports of genetic testing results by inclusion of some “benign variants” or “variants of uncertain significance” (VUS) and inform genetic counselling (33).

Nine patients in our study carry concurrent NLRP3 and NLRP12 variants, including NLRP3 Q703K with NLRP12 F402L in 6/38 (15.8%) patients. This digenic combination was significantly more frequent than the ARIC control (1.25%) and was not identified in the DDC cohort control. We recently reported the contributions of variant combinations of NOD2 and other SAID genes to mixed NLR-associated SAID and their implications (34). To date, we have not seen any reports of the variant combination, NLRP3/NLRP12 only in the literature. Our current data suggest that the variant combination may contribute to digenic disease, where combinations of variants in two genes are required for disease expression (35). In addition, our study has provided new information on the frequency of this digenic combination in control populations as there have been no such data available from the general population to date. Our study indicates that GI involvement may be used to distinguish between NLRP3-AID and NLRP12-AID. Indeed, GI symptoms in CAPS with NLRP3 mutations are rare likely due to homeostasis maintenance from T regulatory cells induced by the remodeling of intestinal microbiota from hyperactive NLRP3 (19). By contrast, NLRP12 mutations can cause more GI symptoms as in our current study. Under normal conditions NLRP12 interacts with NOD2, blocking NOD2 signaling. NOD2 mutations can cause GI symptoms as in Crohn disease and Yao syndrome (36). NLRP12 deficiency induces tolerance of bacterial muramyl dipeptide (MDP), a specific stimulator to initiate NOD2 signaling pathway (37).

In our present study, GI symptoms appear more common in patients with NLRP3/NLRP12 variants than those with NLRP12 variants only, suggesting a synergistic role of both NLRP3 and NLRP12 variants in causing GI disturbance. Regarding the potentially differential impact of NLRP12 mutations and the digenic variants on disease phenotypes and expressivity, further study using a larger sample size is warranted. To our knowledge, there are no basic or translational studies of potential NLRP3-NLRP12 interaction in autoinflammatory diseases. However, in a human and murine study of infection using peripheral blood mononuclear cells, malaria induced the assembly of ASC, NLRP3 and NLRP12 inflammasomes jointly and promoted cleavage of procaspase-1, leading to IL-1β and hypersensitivity to bacterial superinfection. This study may favor a potential interaction between NLRP3 and NLRP12 under autoinflammatory state particularly with regard to infectious triggers (38). We also noted that compared with patients with NLRP3 variants, plasma cytokine profile was more frequently ordered or tested among patients with NLRP12 variants, with IL-10 levels frequently elevated. Interestingly, both wild type NLRP12 protein and IL-10 are anti-inflammatory (39), suggesting that there could be synergism of these two molecules in autoinflammatory regulation and control.

There are limitations in our study. This is a single center retrospective study with the benefit of uniformity and standardization of the study population seen in an Autoinflammatory Disease Referral Center. Relative to studies of common diseases, the sample size is small due to investigation of rare diseases or clinical scenarios. For the potential role of combined variants from different genes in SAIDs, we previously assumed that the composite effects could be synergistic, antagonistic, or both. This will need further functional study in the future.

In summary, our study has provided a new insight into the distinction of NLRP3-AID and NLRP12-AID. GI symptoms seem more common in NLRP12-AID and headaches/dizziness seem less common than NLRP3-AID. We also provide further evidence of the diagnostic utility of some genetic variants. For patients with suspected SAIDs, low penetrance variants should not be overlooked when molecular testing results are reported or interpreted. Unlike many common diseases for which there are readily available guidelines or consensus, SAIDs are rare diseases as in our study, and best evidence may come from case reports and case series (40).

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Stony Brook University Institutional Review Board (approval number IRB2021-00042, initial approval date February 3, 2021). The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because this is a retrospective study.

MY: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing. ZD: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing. BN-M: Investigation, Resources, Writing – review & editing. BX: Data curation, Investigation, Methodology, Resources, Validation, Writing – review & editing. JY: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing. HN: Investigation, Methodology, Resources, Writing – review & editing. OA: Resources, Writing – review & editing. PG: Methodology, Writing – review & editing. QY: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.