Beyond NLRP3: expanding the spectrum of NLR-associated autoinflammatory diseases

Abstract

Objectives

Gain-of-function mutations in NLRP3 represent the prototypical cause of inflammasome-driven systemic autoinflammatory diseases (SAIDs). Other members of the nucleotide-binding domain and leucine-rich repeat–containing (NLR) family, including NLRP1, NLRP7, NLRP12, and NLRC4, may also contribute to systemic inflammatory phenotypes. We aimed to characterize patients carrying NLR-variants.

Methods

Patients referred for SAIDs and harbouring NLR-variants were retrospectively analysed. All patients underwent targeted next-generation sequencing and variants were interpreted according to ACMG criteria. Demographics, clinical manifestations, laboratory, and treatment data were recorded.

Results

Thirty-three patients (70% female; age 19–62 years) were included. Recurrent fever was the predominant manifestation and frequently accompanied by musculoskeletal symptoms and prominent mucocutaneous findings, including erythematous and urticarial rashes, erythema nodosum–like lesions, oral aphthae, and conjunctivitis. Distinct phenotypic patterns were observed across variant groups. NLRP1 variants were often associated with FMF-like features, including recurrent fever and serositis, occasionally with autoimmune features. NLRP7 variants presented with recurrent febrile episodes with mucocutaneous involvement. NLRP12 variants typically presented with periodic fever syndromes and maculopapular or erythema nodosum–like lesions. NLRC4 variants were observed with episodic systemic inflammation with gastrointestinal involvement and atypical cutaneous manifestations including erysipelas-like erythema and angioedema. Coexisting MEFV variants were identified in a subset. Approximately one-third responded to colchicine, whereas another third required IL-1 blockade.

Conclusion

Patients carrying rare non-NLRP3 NLR variants demonstrated a broad and overlapping spectrum of systemic autoinflammatory phenotypes, frequently marked by mucocutaneous involvement. These descriptive observations highlight the need for further functional studies to clarify the pathogenic significance of these variants and importance of expanded genetic evaluation beyond MEFV in patients with atypical inflammatory presentations and describe clinical responses to colchicine and IL-1 inhibition in a subset of patients.

Graphical abstract

Graphical Abstract.

Rheumatology key messages.

• Non-NLRP3 NLR variants expand the genetic spectrum of systemic autoinflammatory diseases.

• NLR variants show overlapping phenotypes with prominent mucocutaneous involvement.

• Not only IL-1 inhibition but also colchicine may be effective in patients with non-classical NLR-associated SAIDs.

Introduction

Systemic autoinflammatory diseases (SAIDs) are characterized by recurrent episodes of sterile systemic inflammation driven mainly by dysregulation of innate immune pathways [1]. Many monogenic SAIDs converge on inflammasomes, cytosolic multiprotein complexes that activate caspase-1 and promote maturation of IL-1β and IL-18 [2]. Gain-of-function variants in NLRP3, responsible for cryopyrin-associated periodic syndromes (CAPSs), represent the prototypical inflammasome-mediated SAID and have established IL-1–targeted therapy as a paradigm of precision-medicine in autoinflammation [3].

While NLRP3 is the best-characterized inflammasome sensor, other members of the nucleotide-binding domain and leucine-rich repeat–containing (NLR-) family may also drive SAIDs. NLRP1, NLRP7, NLRP12, and NLRC4 are NOD-like receptors (NLR) capable of assembling inflammasomes and regulating inflammatory responses. Variants in these genes have increasingly been associated with SAID phenotypes, often exhibiting variable penetrance and heterogeneous organ involvement [3].

The widespread use of next-generation sequencing (NGS) panels has expanded the detection of rare variants, including variants of uncertain significance and combinations across multiple autoinflammatory genes. This has complicated genotype–phenotype correlations and supports the concept of oligogenic or mixed autoinflammation [4]. Curated databases, such as INFEVERS, together with standardized frameworks for variant interpretation from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP), are essential for integrating genetic findings with clinical phenotype [5, 6].

In this study, we aimed to characterize adult patients carrying variants in NLRP1, NLRP7, NLRP12, and NLRC4, focusing on clinical, laboratory, and treatment response in line with the SAID concept.

Material and methods

Study design and patients

This single-centre, retrospective observational study included adult patients referred to a tertiary rheumatology centre for evaluation of suspected SAIDs between October 2020 and January 2026. Patients harbouring variants in NLRP1, NLRP7, NLRP12, or NLRC4 identified during genetic screening were included. Demographic characteristics, clinical manifestations, laboratory findings, and treatment data were retrieved from medical records. Clinical phenotypes were analysed in relation to the identified variants.

The study protocol was approved by the Institutional Research Ethics Committee (approval number: 28.06.2024/09.2024.737), and all patients provided written informed consent for genetic testing and the use of anonymized clinical data.

Genetic analysis

Peripheral blood samples were collected in EDTA tubes, and genomic DNA was extracted using standard procedures. Targeted NGS was performed using the AmpliSeq Library Kit Plus Autoinflammatory Diseases Panel (Thermo Fisher Scientific) on the Ion GeneStudio S5 Plus System (Ion Torrent platform). This panel includes genes associated with monogenic autoinflammatory syndromes, including ADAM17, CARD14, CDC42, CEBPE, CECR1, ELANE, F12, IFIH1, IL10, IL10RA, IL10RB, IL1RN, IL36RN, ISG15, LPIN2, MEFV, MVK, NCSTN, NLRC4, NLRP1, NLRP3, NLRP7, NLRP12, NOD2, PLCG2, PSMA3, PSMB8, PSMB9, PSTPIP1, RBCK1, RELA, SAMD9L, SH3BP2, SLC29A3, TNFRSF1A, TNFRS11A, TNFAIP3, TRAP1, TRNT1, UBA1, and WDR1.

Sequencing data were analysed using Ion Reporter software (version 5.20). Variants were annotated using publicly available databases, including ClinVar, OMIM, dbSNP, gnomAD, and RefSeq, and classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines and the INFEVERS database as pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign [5, 6]. Large genomic rearrangements and deep intronic variants outside the targeted regions were not assessed.

Results

A total of 33 patients with variants in non-NLRP3 NLR-inflammasome genes were included. The cohort comprised 10 patients with NLRP1, 4 with NLRP7, 11 with NLRP12, and 8 with NLRC4 variants. The median age was 28 years (range 18–62), and 23 patients (70%) were female. Clinical manifestations were heterogeneous but demonstrated overlapping inflammatory phenotypes. Recurrent fever was the most common presenting feature and was frequently accompanied by musculoskeletal symptoms (arthralgia or episodic arthritis) and prominent mucocutaneous involvement, including erythematous and urticarial rashes, erythema nodosum–like lesions, recurrent oral aphthae, and conjunctivitis (Table 1, Fig. 1).

Table 1.

Detailed description of each patients with any of NLRP1, NLRP7, NLRP 12 or NLRC4 variants.

Patient	Gender	Age	NLR mutation	Pathogenicity	Other mutation	Symptoms	Laboratory	Previous treatment	Final treatment
1	Male	28	NLRP1 c.164C > T HT	VUS	–	Fever, arthralgia, ROA, diarrhoea, macular rash	Normal APR	COLC	COLC
2	Male	27	NLRP1 c.790delT HT	VUS	–	Fever, ROA, arthralgia, conjunctivitis, urticaria, tonsillopharyngitis, erythema nodosum	Elevated APR	COLC	COLC
3	Male	52	NLRP1 c.790delT HT	VUS	–	ROA, genital ulcer, multiple cerebral aneurysms, hearing loss,	Elevated APR	CS, MMF	CS, MMF
4	Female	19	NLRP1 c.1877T > C HT	VUS	MEFV c.2177T > C HT PAT	Fever, abdominal pain, peritonitis, macular rash on neck and leg, nausea	Elevated APR	COLC	COLC
5	Female	19	NLRP1 c.1877T > C HT	VUS	–	Fever, peritonitis, abdominal pain, chronic renal failure, tubulointerstitial nephritis, arthralgia, nausea, vomiting, pericarditis, macular rash, ROA, periorbital oedema	Elevated APR, ANA 1/160 homogeneous,	COLC, CS	CS, ANA
6	Female	42	NLRP1 c.1877T > C HT	VUS	–	Pericarditis	Elevated APR	COLC	None
7a	Female	43	NLRP1 c.2712T > A HT	VUS	MEFV c.2282G > A HT LP/MEFV c.2177T > C HT LP	Fever, peritonitis, urticaria, arthralgia (Dx of FMF for 14 years but persistent urticaria)	Elevated APR	COLC; ANA	COLC; CAN
8	Female	40	NLRP1 c.3577C > A HT	VUS	–	Erythematous lesions on legs	APR normal, ANA 1/100 homogeneous	None	None
9	Female	19	NLRP1 c.3645G > C HT	VUS	–	Fever, generalized rash, erythema nodosum, myalgia	Elevated APR, Bilateral wall thickness on CCA, right renal artery, SMA (Takayasu arteritis)	CS, AZA, TOCI	CS, COLC, TOCI
10	Male	25	NLRP1 c.4157G > A HT	VUS	–	Fever, Abdominal pain (Dx of FMF for 18 years but atypical attacks)	Elevated APR	COLC	COLC
11	Female	32	NLRP7 c.1196G > A HT VUS	VUS	MEFV c.2084A > G HT LP/TRAP1 c.2107C > T HT VUS	Fever, lymphadenopathy, arthralgia, arthritis, sacroiliitis, folliculitis, urticaria, abdominal pain, peritonitis, pleuritis, periorbital oedema, conjunctivitis	Elevated APR	CS, SSZ, COLC, SECU, ADA,	COLC
12	Male	29	NLRP7 c.1614T > A heterozygote	VUS	MVK c.155G > A HT LB	Fever, ROA, generalized folliculitis, erythematous rash, lymphadenopathy, arthritis, inflammatory back pain	Elevated APR, negative but erythematous pathergy	COLC	COLC
13	Female	19	NLRP7 c.2471 + 1G > A HT	PAT	MEFV c.1223G > A HT VS	Fever, tonsillopharyngitis, peritonitis, abdominal pain, arthralgia, arthritis, urticaria	Elevated APR	COLC	COLC
14	Female	21	NLRP7 c.2471 + 1G > A HT	PAT	MEFV c.2282G > A HT PAT/MEFV c.1223G > A HT VUS	Fever, arthralgia, arthritis, urticaria, rash	Elevated APR	COLC	COLC
15	Female	26	NLRP12 c.79_81delAAG HT	VUS	–	Myalgia, arthralgia, arthritis, ROA	Elevated APR	COLC	COLC
16	Female	62	NLRP12 c.632G > A HT	VUS	–	Fever, EN, macular rash, (Dx of RA for 19 years and Crohn’s disease for 20 years)	Elevated APR	CS, MTX, SSZ, IFX, ADA, CERT	CS, SSZ
17	Female	29	NLRP12 c.779 C > T HT	VUS	–	Generalized erythematous macular rash, nausea, vomiting, diarrhoea, leukocytoclastic vasculitis	Elevated APR	COLC, ANA	COLC, ANA
18	Male	28	NLRP12 c.883_884delGG HT VUS	VUS	–	Fever, abdominal pain, ROA, genital ulcer, conjunctivitis, arthralgia, arthritis, lymphadenopathy, generalized macular rash	Elevated APR; negative pathergy	COLC, ANA	COLC, ANA
19	Female	18	NLRP12 c.1027C > T HT VUS	VUS	–	Fever, abdominal pain, peritonitis, arthralgia	Elevated APR	CS, COLC	COLC
20	Female	19	NLRP12 c.1054C > T HM	VUS/LP	MEFV c.2040G > C HT PAT	Fever, abdominal pain, peritonitis, pleuritis, arthralgia, arthritis	Elevated APR	COLC	None
21	Female	23	NLRP12 c.1054C > T HT	VUS/LP	–	Fever, nausea, myalgia, tonsillopharyngitis, arthralgia, arthritis	Elevated APR, RF, ACPA, ANA negative, (dx of JRA for 17 years)	CS, MTX, ADA, IFX, TOCI	CS, ANA
22	Male	30	NLRP12 c.1343G > C HT	VUS	–	Abdominal pain, pericardial affusion	Elevated APR; ACPA positive	COLC	COLC
23	Female	39	NLRP12 c.1572G > A HT	VUS	MEFV c.2282G > A HT PAT	Erythematous macular rash, arthralgia, arthritis, EN	Elevated APR	CS, COLC	COLC
24	Male	19	NLRP12 c.2502C > A HT	VUS	NCSTN c.968G > A HT VUS	EN, panniculitis, EBL, ROA, conjunctivitis, myocarditis, abdominal pain, diarrhoea, folliculitis	Elevated APR	CS, COLC	COLC
25	Female	43	NLRP12 c.2984T > C HT	VUS	MEFV c.2080A > G HM PAT	Fever, arthralgia	Persistently elevated APR	COLC	COLC
26	Male	43	NLRC4 c.283G > T HT	LP	–	Itchy, non-painful swelling attacks on extremities	Elevated APR	COLC	COLC
27	Female	24	NLRC4 c.541C > T HT	VUS	MEFV c.2177T > C HT PAT	Fever, peritonitis, pleuritis, lymphadenopathy, tonsillopharyngitis, arthralgia, arthritis (Dx of FMF for 2 years)	Elevated APR	COCL	COLC
28	Female	22	NLRC4 c.1024G > A HT	VUS	SAMD9L c.1964delC HT LP	Fever, EN, arthralgia, arthritis, macular ash, tonsillopharyngitis	Elevated APR	COLC	COLC
29	Female	38	NLRC4 c.1099T > C HT	VUS	–	Fever, peritonitis, abdominal pain, arthralgia, arthritis (Dx of FMF for 15 years but atypical attacks)	Elevated APR	COLC, SSZ	COLC, SSZ, ANA
30	Female	44	NLRC4 c.1679A > G HT	VUS	–	Fever, arthralgia, chronic renal failure,	Elevated APR, ANA 1/320 homogeneous, Anti-SSA positive	CS, COLC	COLC, ANA
31a	Female	22	NLRC4 c.2567C > A HT	LP	MEFV c.2080A > G HT PAT	Fever, peritonitis, pleuritis, severe EBL, arthritis, ROA, urticaria, angioedema on face (Dx of FMF for 16 years but atypical skin sx)	Elevated APR	COLC, CS, ANA	COLC, CAN
32	Male	42	NLRC4 c.2614 + 6A > G HT	VUS	IL36RN c.244-5G > A HT VUS	Fever, myalgia, abdominal pain, renal microaneurysm, lower extremity decreased blood flow (PAN?)	Elevated APR, ANA negative, Anti-Scl70 positive	CS, AZA, MMF	CS, MMF
33a	Female	39	NLRC4 c.2828A > T HT	VUS	MEFV c.442G > C HT VUS	Fever, abdominal pain, subileus, pleuritis, folliculitis, conjunctivitis, angioedema, periorbital oedema, urticaria, nausea, proteinuria, ROA, genital ulcer, Raynaud, myalgia, arthralgia, arthritis	Elevated APR, pathergy negative, renal biopsy excluded amyloidosis	CS, COLC, ANA	CS, CAN

Abbreviations: ADA: adalimumab, ANA: anakinra, ANA: anti-nuclear antibody, APR: acute phase reactants, AZA: azathioprine, CAN: canakinumab, CERT: certolizumab, COLC: colchicine, CS: corticosteroid, Dx: diagnosis, HM: homozygous, HT: heterozygous, IFX: infliximab, LB: likely benign, LP: likely pathogenic, MMF: mycophenolate mofetil, MTX: methotrexate, PAN: polyarteritis nodosa, PAT: pathogenic, ROA: recurrent oral aphthae, SSZ: sulphasalazine, TOCI: tocilizumab, VUS: variant of unknown significance.

^aThese cases were also represented in the referenced article of our group.

Figure 1.

Cutaneous manifestations associated with NLR-related gene variants. (a) Patient carrying the NLRP12 c.779C > T variant presenting with erythematous, maculopapular lesions involving the distal lower extremity, compatible with inflammatory skin involvement. (b) Patient with NLRP12 c.2502C > A mutation showing bilateral erythema and non-pitting oedema of the feet and ankles, suggesting an inflammatory oedematous phenotype. (c) Patient with combined NLRP7 c.1614T > A and MVK c.155G > A variants, displaying widespread erythematous reaction after pneumococcal induced skin pathergy test without induration, consistent with aggravated systemic inflammatory activation. (d) Patient with NLRP12 c.883_884delGG mutation exhibiting diffuse, symmetric maculopapular eruptions across the trunk, reflecting chronic inflammatory skin involvement. (e) Patient carrying NLRC4 c.2567C > A together with MEFV c.2080A > G, presenting with different attacks of marked erythema, swelling, and tenderness of the both foot and ankle representing an aggressive form of erysipelas like erythema, compatible with acute inflammatory flares, and attacks of angioedema. (f) Patient harbouring NLRC4 c.2828A > T and MEFV c.442G > C variants, showing prominent erythema involving the extremities and attacks of urticaria, consistent with systemic autoinflammatory cutaneous involvement

Despite this overlap, variant-specific patterns emerged (Table 1). Ten patients harboured NLRP1 variants; two carried the same c.790delT and three shared the c.1877T > C variant. Patients carrying NLRP1 variants often had recurrent fever and serositis reminiscent of FMF, and a subset showed atypical findings such as persistent skin lesions or coexisting autoimmune manifestations (e.g. large-vessel arteritis in one patient, and chronic arthritis with positive ANA in another), indicating a mixed autoinflammatory-autoimmune phenotype.

Patients carrying NLRP7 variants presented with recurrent febrile episodes accompanied by systemic inflammation and mucocutaneous findings. Among four patients, two sharing the same splice-site mutation c.2471 + 1G > A. Interestingly, one patient with an NLRP7 variant (c.1614T > A) also carried a low-activity MVK variant and developed recurrent fever, rash, lymphadenopathy, and a non-indurated erythematous pathergy reaction (Fig. 1c), suggesting a potential contribution of NLRP7 variants to a mucocutaneous-dominant autoinflammatory phenotype.

Eleven patients carried NLRP12 variants, two carried the same variant c.1054C > T. In patients carrying NLRP12 variants, periodic fever with cutaneous involvement were prominent. These patients frequently exhibited recurrent fever accompanied by maculopapular eruptions or erythema nodosum–like lesions, often involving the lower extremities, together with arthralgia or arthritis (Fig. 1a and b).

All of eight NLRC4 variants were distinct. Patients carrying NLRC4 variants typically presented with episodic systemic inflammation characterized by high fever and gastrointestinal symptoms. In addition, recurrent erysipelas-like erythema and episodes of angioedema affecting the face or extremities were observed (Fig. 1e and f). These cutaneous manifestations are not characteristic of classical NLRC4-associated hemophagocytic phenotypes and suggest a broader inflammasome-driven clinical spectrum.

Coexisting heterozygous MEFV variants were identified in a substantial proportion of patients. In some cases, atypical features, such as persistent urticaria, severe erythematous flares, or prominent mucosal involvement, extended beyond classical FMF presentations, supporting a potential synergistic effect between MEFV and NLR variants in shaping complex phenotypes.

Nearly all patients received colchicine as first-line therapy, particularly those with FMF-like features. Treatment response was assessed during routine clinical follow-up based on both clinical and laboratory outcomes, including reduction or complete resolution of inflammatory attacks (frequency, duration, and severity), improvement of associated inflammatory manifestations, and normalization or marked improvement of acute-phase reactants. Approximately one-third of patients achieved satisfactory clinical and laboratory disease control with colchicine alone or in combination with low-dose corticosteroids, including some patients without MEFV variants. Patients with persistent disease activity despite colchicine were subsequently escalated to IL-1 blockade (anakinra or canakinumab), after which satisfactory clinical and laboratory control was achieved. Corticosteroids were frequently used for musculoskeletal or autoimmune manifestations. A small number of patients with overlapping autoimmune features received additional biologic agents, including infliximab or tocilizumab, according to their clinical phenotype.

Discussion

Our cohort provides a descriptive clinical characterization of patients carrying rare non-NLRP3 NLR variants and expands the spectrum of phenotypes observed in these patients. We observed overlapping SAID phenotypes among patients carrying variants in NLRP1, NLRP7, NLRP12, and NLRC4 in which mucocutaneous inflammation emerges as a prominent and clinically informative feature. Although recurrent fever was the leading manifestation, the accompanying mucocutaneous spectrum, including erythematous and urticarial rashes, erythema nodosum–like lesions, oral aphthae, conjunctivitis, erysipelas-like erythema, and angioedema, was striking and often exceeded expectations for classical FMF. These observations are consistent with previous studies suggesting that different inflammasome sensors may contribute to overlapping IL-1β/IL-18-mediated inflammatory phenotypes with variable organ involvement, particularly affecting the skin and mucosa [7].

Gene-specific patterns were identifiable despite substantial overlap. Patients with NLRP1 variants frequently exhibited FMF-like attacks characterized by recurrent fever and serositis, but a subset showed persistent cutaneous disease and/or autoimmune overlap, suggesting an autoinflammatory–autoimmune continuum rather than discrete syndromic entities [8]. This is consistent with NLRP1-associated autoinflammation with arthritis and dyskeratosis (NAIAD), where constitutive inflammasome activation and high IL-18 were emphasized, and with genetic associations linking NLRP1 to autoimmune susceptibility [9]. Reports of germline NLRP1 gain-of-function causing inflammatory skin syndromes and cancer susceptibility further support the plausibility of prominent cutaneous disease in this subgroup and fit with the strong keratinocyte biology of NLRP1 [10].

NLRP12-variant patients often resembled a periodic fever syndrome with maculopapular eruptions or erythema nodosum–like lesions and frequent musculoskeletal involvement [11]. Since NLRP12 mutations were first linked to NLRP12-associated autoinflammatory disease with disrupted NF-κB signalling, historically FCAS2, going with hereditary periodic fever, subsequent work has showed broad expressivity beyond cold-induced attacks, commonly involving rash and musculoskeletal symptoms and sometimes gastrointestinal complaints [12]. Clinically, NLRP12 should be considered in ‘undifferentiated’ recurrent fever or SAID presentations when skin involvement is prominent.

NLRC4-variant patients were distinguished by episodic systemic inflammation with high fever and gastrointestinal symptoms accompanied by recurrent erysipelas-like erythema and angioedema. Activating NLRC4 activation have classically been linked to IL-18–dominant inflammation, enterocolitis, and recurrent macrophage activation syndrome and our findings suggest that cutaneous manifestations may represent an under-recognized component of NLRC4-associated disease, even in the absence of overt hyperinflammatory syndromes [13, 14].

The NLRP7 subgroup is of particular interest, as NLRP7 is best known for recurrent hydatidiform mole and reproductive wastage, but mechanistic studies demonstrate an NLRP7 inflammasome in human macrophages capable of driving caspase-1–dependent IL-1β/IL-18 maturation [15, 16]. In our cohort, heterozygous NLRP7 variants were associated with recurrent febrile mucocutaneous inflammatory episodes, supporting a potential role as a modifier gene within an oligogenic autoinflammatory framework.

A key observation was the frequent coexistence of MEFV variants with NLRP1, NLRP7, NLRP12, or NLRC4 variants. Several of these patients also exhibited clinical manifestations that are not typically associated with classical FMF, including persistent urticaria, severe erythematous flares, oral aphthae, conjunctivitis, lymphadenopathy, and other prominent mucocutaneous inflammation, which prompted extended genetic evaluation. In addition to MEFV, several patients also carried variants in other autoinflammatory disease-associated genes, including MVK, IL36RN, SAMD9L, TRAP1, and NCSTN, further highlighting the genetic complexity observed in this cohort. While the present study cannot determine the independent contribution of each genetic variant, these observations are consistent with the growing recognition of mixed or oligogenic autoinflammatory diseases (MAID), in which variants affecting multiple innate immune pathways may collectively modify disease expression and contribute to phenotypic heterogeneity [17]. Future functional, segregation, and larger multicentre studies will be required to clarify the biological significance of these interactions.

Therapeutically, our findings suggest a pragmatic, stepwise treatment approach that largely mirrors the management of FMF-like autoinflammatory disease. Colchicine was used as first-line therapy in nearly all patients. Approximately one-third achieved satisfactory clinical and laboratory disease control, defined by reduction or complete resolution of inflammatory attacks, improvement of associated inflammatory manifestations, and normalization or marked improvement of acute-phase reactants, including some patients without MEFV variants. Although colchicine is classically recommended as first-line therapy in FMF for attack control and prevention of AA amyloidosis, its role in other SAIDs (e.g. CAPS, TRAPS, and MKD) is generally considered limited because IL-1-driven inflammasome activation is predominant, continuous and strongly associated with gasdermin D–mediated pyroptosis [18, 19]. However, FMF is characterized by more regulated and episodic pyrin inflammasome activation, in which cytokine release predominates over overt pyroptotic cell death. Notably, the meaningful colchicine responsiveness observed in our NLR-variant cohort suggests that inflammasome activity in these conditions may retain partial colchicine sensitivity, resembling a pyrin-like regulatory pattern rather than a fully NLRP3-driven phenotype. Mechanistic studies support this observation, demonstrating that colchicine can modulate inflammasome-related pathways, including inhibition of caspase-1 activation and downstream cytokines such as IL-18. The clinical response observed in a subset of our patients may be hypothesized that colchicine may remain a reasonable initial therapeutic option in selected patients with non-NLRP3 NLR variants; however, these observations should be interpreted as descriptive clinical outcomes rather than evidence of a specific pathogenic mechanism.

Approximately one-third of patients with persistent disease activity despite colchicine required escalation to IL-1 blockade (anakinra or canakinumab), after which satisfactory clinical and laboratory disease control was achieved in line with current ACR/EULAR recommendations for IL-1–mediated SAIDs [19]. Therefore, this escalation reflected routine clinical management of refractory inflammation and should not be interpreted as direct evidence supporting the pathogenicity of the identified variants. Corticosteroids were primarily used as short-term bridging therapy during severe flares or in patients with prominent articular or autoimmune manifestations and were generally tapered following clinical improvement. A small subset of patients with clear autoimmune overlap required non–IL-1 biologics, including TNF inhibitors or tocilizumab, further reflecting the clinical heterogeneity of this cohort [20].

These findings have practical implications for diagnostic evaluation, particularly in FMF-endemic settings. In patients with recurrent fever and FMF-like features accompanied by prominent mucocutaneous involvement, namely urticaria, angioedema, erysipelas-like erythema, diffuse maculopapular eruptions, erythema nodosum–like plaques, recurrent aphthae or conjunctivitis, extended genetic screening beyond MEFV should be considered, including NLRP1, NLRP12, and NLRC4, and potentially NLRP7 in selected cases. Because expanded panels with NGS increase detection of VUSs, interpretation should follow ACMG/AMP standards and leverage curated resources such as INFEVERS, together with careful phenotyping [4–6].

This study has several limitations. Its retrospective, single-centre design introduces potential referral bias towards complex cases, and subgroup sizes limit robust genotype–phenotype correlations. Most detected variants were classified as VUS according to ACMG criteria. Therefore, our findings should not be interpreted as evidence of pathogenicity or causality. Rather, this study provides descriptive clinical observations that may help generate hypotheses for future segregation analyses, functional studies, cytokine profiling, and inflammasome activation assays to clarify the biological significance of these variants. Finally, targeted NGS panels do not capture deep intronic variants, regulatory regions, or large genomic rearrangements, and therefore may underestimate the full genetic complexity.

In conclusion, patients carrying rare or pathogenic variants in NLRP1, NLRP7, NLRP12, and NLRC4 demonstrated a broad spectrum of overlapping systemic autoinflammatory phenotypes, frequently accompanied by prominent mucocutaneous manifestations and coexisting autoinflammatory gene variants, particularly MEFV. Gene-specific clinical tendencies were observed, although considerable phenotypic overlap remained the dominant pattern. These descriptive findings broaden the recognized clinical spectrum associated with non-NLRP3 NLR variants and support further investigation into the potential clinical relevance of these variants in SAIDs. In clinical practice, extended genetic evaluation may be considered in selected patients with atypical inflammatory presentations and colchicine may represent a reasonable initial therapeutic option, followed by IL-1 blockade in refractory cases. Future prospective, multicentre studies incorporating segregation analyses, functional assays, and cytokine profiling will be essential to clarify the pathogenic significance of these variants, refine genotype–phenotype correlations, and guide mechanism-based therapeutic strategies.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Funding

No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Disclosure statement: The authors have declared no conflicts of interest.