The phenotype and genotype of Chinese adult patients with NLRP3-associated autoinflammatory disease

NLRP3-associated autoinflammatory disease (NLRP3-AID) is a spectrum of autosomal dominant inherited diseases associated with NLRP3 gene mutations. Reports of Chinese NLRP3-AID cases are limited to date. In the present study, we aim to describe the phenotype and genotype of a cohort of Chinese adult NLRP3-AID patients This single-center study included sixteen adult patients diagnosed with NLRP3-AID at Department of Rheumatology, Peking Union Medical College Hospital from April 2015 to September 2021. Whole-exome sequencing using next-generation sequencing was performed in each patient. Clinical data and mutational information were compared with a European cohort. The median age of disease onset was 16 (0–46) years old, and adult-onset was observed in 4 patients (25%). The median time of diagnosis delay was 20 (0–39) years. Five patients (31.3%) had family history of similar symptoms. The most common clinical manifestations were recurrent fever (93.8%), arthralgia/arthritis (81.3%), skin rash (75%), myalgia (62.5%), and central nervous system manifestations (50%). Heterozygous NLRP3 variants detected in these patients were p.T348M (n = 4, 25%), Q703K, V70M, K129R, M116I, P38S, V442I, D303G, G326E, A439V, K829T, L632F and V198M (n = 1, separately). All the variants were missense mutations. We reported the largest case series of Chinese adult NLRP3-AID patients. The distinct symptoms of NLRP3-AID patients suggest the heterogeneity of disease. P38S, M116I, K129R, V442I and K829T were identified as novel NLRP3 variants. These data expand the clinical phenotypic and genotypic profiles of NLRP3-AID. Key Points • We characterized the clinical and genetic features of sixteen Chinese adult NLRP3-AID patients. • Thirteen NLRP3 gene variants were confirmed in this cohort, and P38S, M116I, K129R, V442I and K829T were identified as novel variants. • Clinical data and mutation information were compared with a European cohort. • We hope these data would expand the phenotypic and genotypic profile of NLRP3-AID and raise the awareness of early diagnosis and accurate treatment among rheumatologists. Key Points • We characterized the clinical and genetic features of sixteen Chinese adult NLRP3-AID patients. • Thirteen NLRP3 gene variants were confirmed in this cohort, and P38S, M116I, K129R, V442I and K829T were identified as novel variants. • Clinical data and mutation information were compared with a European cohort. • We hope these data would expand the phenotypic and genotypic profile of NLRP3-AID and raise the awareness of early diagnosis and accurate treatment among rheumatologists.


Introduction
Systemic autoinflammatory diseases (SAIDs) are a group of genetically heterogeneous disorders characterized by febrile attacks and unspecific systemic inflammation resulting from dysregulation of the innate immune system [1,2]. Since the initial research in the late 1990s, an increasing number of SAIDs have been classified. At present, more than 40 monogenic SAIDs were defined [3]. NLRP3-associated autoinflammatory disease (NLRP3-AID, OMIM 606416), previously called cryopyrin-associated periodic syndrome (CAPS), is a continuum of monogenetic autoinflammatory diseases caused by gain-of-function NLRP3 mutations [4]. NLRP3 gene is located on chromosome 1q44, encoding NLRP3 protein, which consists of three regions: the pyrin domain (PYD) in the N-terminal, the NACHT central domain and the leucine-rich repeats (LRR) containing domain in the C-terminal. NLRP3 can recruit the apoptosisassociated speck-like protein containing a CARD (ASC). Then, pro-caspase-1 is connected to ASC to assemble the NLRP3 inflammasome, a crucial immune sensor in the innate immunity. Gain-of-function mutations in NLRP3 gene result in the aberrant activation of NLRP3 inflammasome, triggering the activation of pro-caspase-1, the overproduction of proinflammatory cytokines interleukin-1β (IL-1β) and IL-18, and gasdermin-D-mediated pyroptosis, consequently leading to systemic inflammation [5]. Of note, an international collaborative study revealed that the majority of mutation-negative NLRP3-AID cases are due to low-level NLRP3 mosaicism [6].
Hal Hoffman et al. reported the first NLRP3-AID case in 2001 [7]. NLRP3-AID encompasses three conditions of increasing severity: familial cold-induced autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and chronic infantile neurological cutaneous articular syndrome (CINCA). FCAS is characterized by cold-induced fever, arthralgia and urticaria-like rashes. The main clinical features of are recurrent febrile attacks, urticaria-like rash, arthritis, conjunctivitis, sensorineural hearing loss and amyloidosis. CINCA is the most severe phenotype of NLRP3-AID, manifesting as childhood onset of fever, rash, conjunctivitis, osteoarthropathy and central nervous system (CNS) inflammation including developmental delay and seizures [8]. To prevent long-term organ damages, the early diagnosis and swift initiation of effective treatments are mandatory [9]. Anti-IL-1 biologics (anakinra, canakinumab and rilonacept) are recommended to treat NLRP3-AID. They have sustained efficacy in controlling symptoms and improving quality of life [10].
Due to the rarity, the real-world awareness and practice of NLRP3-AID in China remain limited. Through literature review, we searched only two publications describing cohorts of Chinese pediatric patients with NLRP3-AID [11,12], and some case reports from our team [13][14][15]. In this study, we aimed to summarize the clinical and genetic characteristics of Chinese adult NLRP3-AID patients.

Methods
This single-center prospective study included sixteen adult patients diagnosed with NLRP3-AID at Department of Rheumatology, Peking Union Medical College Hospital from April 2015 to September 2021. The diagnosis was based on clinical criteria proposed by Kuemmerle-Deschner et al., which requires elevation of inflammatory markers and at least two of the following six typical signs or symptoms: urticarial rash, cold-triggered flares, chronic aseptic meningitis, neurosensorial hearing loss, muskoloskeletal symptoms, and skeletal abnormalities [16]. Exclusion criteria included tumor and infectious diseases, et al. Demographic information and detailed clinical records including white blood cell (WBC) counts, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum tumor necrosis factor (TNF)-α and IL-6 level were carefully documented and analyzed. Whole-exome sequencing based on the NovaSeq 6000 next-generation sequencing platform was performed in each patient. Clinical data and mutation information were compared with a European cohort. This study was approved by the Institutional Review Board of Peking Union Medical College Hospital. All the participants have given their informed consents.

Clinical characteristics
The demographic information and clinical features of adult NLRP3-AID patients in this study were summarized in Table 1. According to the clinical manifestations described in the introduction, all the sixteen patients were diagnosed as MWS. Of them, the gender ratio of male to female was 9:7. All patients were of Chinese Han ethnicity. The median age of disease onset was 16 (0-46) years old, and adult-onset was observed in 4 patients (25%). The median time of diagnosis delay was 20 (0-39) years. Five patients (31.3%) had family history of similar symptoms.
Glucocorticoids were given to 7 patients with partial symptom relief in 6 patients (85.7%). Due to the unavailability of IL-1 inhibitors in China, TNF-α inhibitors (etanercept and adalimumab) were used in 7 patients with improvement of fever, rashes and arthritis (100%). However, severe complications such as brain atrophy and hearing loss were not obviously relieved.

Genotype-phenotype relationship
In four patients with T348M mutation, the median age of disease onset was 2 (0-10) years old. All of the four patients experienced severe ocular symptoms (100%), referring to papilledema and optic neuritis. In addition, chronic aseptic meningitis and sensorineural hearing loss were found in 3 patients (75%), respectively. Whereas in those with other variants (n = 12), the incidences of abovementioned complications were 6.3%, 33.3% and 33.3% respectively, proving the relationship between the severity of NLRP3-AID and NLRP3 T348M mutation.
Interestingly, two patients in our cohort had symptoms of Behçet's syndrome. Patient 12 was a 45-year-old woman presenting with recurrent fever, cold-induced urticaria, oral and genital ulcers, conjunctivitis, uveitis, headache and hearing loss. Whole-exome sequencing identified a heterozygous NLRP3 A439V mutation in the proband and her daughter. This is the firstly recorded Chinese pedigree of NLRP3-AID presented with Behçet's syndrome [15]. Patient 14 was a 33-year-old man who suffered from recurrent oral and genital ulcers, skin erythema, arthritis and sensorineural hearing loss. Genetic testing identified a heterozygous L632F mutation in the NLRP3 gene. In addition, gene sequencing did not identify histocompatibility leukocyte antigen (HLA)-B * 51 mutation in these two patients, which is associated with Behçet's syndrome susceptibility. Based on these cases, there may be a potential association between A439V/L632F mutation and the coexistence of NLRP3-AID and Behçet's syndrome. D303N were reportedly associated with severe phenotypes in NLRP3-AID [19]. Here, we observed serious sequela such as bilateral sensorineural hearing loss and vision impairment in patient 8 bearing D303G variant. In contrast, patient 11 carrying G326E variant had relatively mild clinical manifestations without obvious organ damage.

Comparison between Chinese cohort and European cohort
To date, the largest cohort of NLRP3-AID was a study from Eurofever Registry [20], which enrolled 136 patients, including 128 Caucasian and 8 Asian. In the light of this research, R260W was the most frequent mutation (26.5%, n = 36). Other common variants included T348M (n = 20), A349V (n = 14), V198M (n = 13), E311K (n = 9), Q703K (n = 9), and D303N (n = 5). 19.9% (n = 27) patients carried other rare variants and 2.0% (n = 3) did not carry NLRP3 variants (Fig. 2). The gender ratio of male to female in Chinese and European series were similar (9:7 vs. 1:1). The median age at disease onset of our patients was older than the European cohort (16 vs. 0.8 years old), and the average time of diagnosis delay was longer (20 vs. 15 years). In terms of symptoms, recurrent fever (93.8 vs. 84.0%), myalgia (62.5 vs. 44%), and hearing impairment (62.5 vs. 42%) were more commonly seen in our case series than the European cohort. While the incidences of rashes (75 vs. 97%) and eye involvement (43.7 vs. 71%) were relatively lower. The frequencies of CNS and joint involvement of the two cohorts were alike. Four patients (25%) in our cohort reported abdominal pain or diarrhea, which was seldom recorded in the European patients. About 4% patients (5/136) from the European Registry cohort developed amyloidosis, yet no amyloidosis was seen in our patients ( Table 2).

Discussion
NLRP3-AID is a continuum of autosomal dominant inherited autoinflammatory disorders. This study confirms the clinical features of patients with NLRP3-AID described in previous reports. Sixteen Chinese Han patients were all diagnosed as MWS, the moderate type of NLRP3-AID. The mean time of diagnosis delay was 20 years, implying the lack of awareness of NLRP3-AID among Chinese population. In our study, characteristic symptoms of MWS were recurrent fever, urticaria-like rashes and late-onset organ involvement such as neurological symptoms, hearing and vision impairment. The distinct symptoms in each individual suggested the heterogeneity of disease. The disease severity and complications may vary with the mutation type and treatment process.
In this cohort, 75% MWS patients were child-onset while 25% patients were adult-onset, implying NLRP3-AID should be considered in both pediatric and adult patients. Most patients had their initial visits at departments of dermatology or infection diseases for the unexplained fever and rashes. A small proportion of patients initially attended departments of ophthalmology or otology for the vision or hearing loss. Clinically, misdiagnosis and delayed diagnosis are common to see due to the rarity and phenotypic variability of this autoinflammatory disorder [21]. Awareness of NLRP3-AID among patients presenting with unknown reason of recurrent fever, rashes and musculoskeletal manifestations, as well as accurate diagnosis and appropriate treatments would avoid organ damage and prevent disease progression, especially the irreversible sequelae such as chronic aseptic meningitis, hydranencephaly, brain atrophy, optic papilledema, optic neuritis, vision impairment and hearing loss.
Compared with the European cohort, fever, myalgia and hearing impairment were more common, yet rash and eye involvement were less common in Chinese patients. In our cohort, eight patients (50%) presented with the intermittent urticarial-like rashes on the trunk, limbs or face. Four patients had erythema nodosa on the lower limbs, which was seldom seen in the Europe cohort or other researches [22]. Notably, 25% patients in our cohort had abdominal pain/ diarrhea, however, it was not mentioned in European patients [20]. In untreated NLRP3-AID patients, amyloidosis represents a serious long-term complication, and according to previous research, up to a quarter of MWS patients may develop amyloidosis [23,24]. Meanwhile, in the European Registry cohort, about 4% patients developed amyloidosis. Consistently, in Chinese pediatric patients with NLRP3-AID, only one patient (6.7%) got amyloidosis [11], whereas none of our adult patients had amyloidosis. We suggest that ethnic difference and different genotypes may partially account for the diversity of clinical phenotype.
To date, a total of 262 NLRP3 variants are listed in the Infevers database. Most variants leading to NLRP3-AID are found in exon 3 of the NLRP3 gene, encoding the NACHT domain [18]. Few non-exon 3 variants have been reported before [25][26][27]. In the present study, thirteen NLRP3 variants were identified, and five of them (P38S, M116I, K129R, V442I and K829T) were unreported. The minor allele frequencies (MAF) of these five missense variants are < 0.00 5, < 0.00013, < 0.00013, < 0.0002, < 0.0002 respectively in the Asian population. Based on American College of Medical Genetics and Genomics (ACMG) guidelines, V442I was identified as a likely pathogenic variant, while the ratings of the other four unreported variants were uncertain significance due to the limited evidence by now. Further functional experiments are required to confirm the diseases-causing roles of these variants. Importantly, from the perspective of phenotype, patients carrying abovementioned variants fulfill clinical criteria for NLRP3-AID. Taken together, we preliminarily identify them as novel variants. Intriguingly, four of the novel variants found in our cohort are non-exon 3 variants: K829T in exon 6, K129R, M116I in exon 2, and P38S in exon 1. This illustrates non-exon 3 variants may also lead to clinical symptoms and should be considered as a unique feature of Chinese NLRP3-AID patients. Based on previous studies, T348M was the most common mutation in NLRP3-AID patient [28][29][30]. This variant is associated with early disease onset, chronic course sensorineural hearing loss and neurological phenotype [20,30,31]. Early disease onset was also predictive of a more severe outcome [20]. Consistent with these findings, in Chinese patients, T348M is related to serious CNS manifestations including sensorineural hearing loss, chronic aseptic meningitis, hydrocephalus and brain atrophy [32]. In addition, we have also reported that T348M was associated with a variety of ocular manifestations, such as conjunctivitis, uveitis, papilledema, optic neuritis and optic atrophy, which led to impaired vision in some cases [33]. In the present research, up to a quarter of Chinese patients carried T348M variant, and they presented with earlier disease onset and more severe sequelae when compared with patients carrying other mutations. These data verified the positive correlation between T348M variant and the severity of clinical genotype.
Autoinflammatory diseases used to be defined in opposition to autoimmune diseases due to the lack of involvement of the adaptive immune system and circulating autoantibodies [34]. Nevertheless, further investigations in this field suggest that most rheumatic and musculoskeletal diseases can be placed along a spectrum of disorders, with autoinflammatory diseases and autoimmune diseases representing the two ends of this spectrum, setting no obvious boundaries [35]. In line with this growing viewpoint, we observed a rare patient with positive ANA and antiphospholipid antibodies, as we have reported before [14]. This patient presented with lupus-like presentations as well as positive auto-antibodies, hence systemic lupus erythematosus and antiphospholipid syndrome should be taken into account. Nevertheless, the abovementioned diseases could not explain all aspects of her manifestations. She had clinical features of recurrent inflammation and carried an explicit NLRP3 T348M variant. Therefore, we speculate this is a special case, who had characteristics of both NLRP3-AID and lupus-like disease. Moreover, in this study, we also found two NLRP3-AID patients complicated with Behçet's Syndrome, and one of them has been reported by our team [15]. These cases provide clinical evidence of overlapping features between autoinflammatory diseases and autoimmune diseases, in addition, between monogenic and polygenic autoinflammatory diseases.
Recently, pediatricians Zhou et al. from PUMCH have reported a Chinese NLRP3-AID cohort [12]. They recruited 14 child-onset NLRP3-AID patients, and 8 of them were diagnosed with NOMID, the severe type of NLRP3-AID. They found 12 NLRP3 gene variants in these patients and L361W (NM_004895) was identified as novel, which has been added in the Infevers database thereafter. In comparison, the present study has reported 16 NLRP3-AID patients and 4 of them had disease-onset during adulthood, indicating the presence of adult-onset in NLRP3-AID. All patients were diagnosed with MWS according to the moderate manifestations in our study. Similarly, it is common to observe recurrent fever, musculoskeletal manifestations, leukocytosis and increased CRP/ESR in both cohorts. While the incidence of rashes (75% vs. 100%) and neurological symptoms (50% vs. 86%) in our patients were relatively lower than their cases. Besides, both studies reported a patient with positive ANA antibodies, which may be related to the race difference. With respect to the genotype, T348M and G326E were found in both cohorts, while the remaining 10 variants in Zhou's research and 11 variants our study were different, and we recorded 5 novel variants. In short, Zhou et al. and we have reported two groups of Chinese NLRP3-AID patients, aiming to offer more information about clinical and genetic features of Chinese NLRP3-AID patients.

Conclusions
Herein, we described the largest case series of Chinese adult NLRP3-AID patients. Due to the rarity and complexity of NLRP3-AID, diagnosis delay remains an unsolved problem, resulting in severe complications such as hearing and vision loss. We confirmed thirteen variants in the NLRP3 gene, among which, P38S, M116I, K129R, V442I and K829T were identified as novel variants. We hope these data would expand the phenotypic and genotypic profile of NLRP3-AID.