INTRODUCTION
The pathogenesis of hereditary systemic autoinflammatory diseases (SAIDs) along with their genetic basis are increasingly investigated which led to new entities being discovered and described. Familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), and tumor necrosis factor type 1 A receptor-associated periodic syndrome (TRAPS) are among the most common and well-known diseases of this type, monogenic in nature, and they often occur in the Middle East. (1–3) The hallmark of these diseases is innate immune system dysfunction presenting with symptoms such as recurrent fever, arthritis, rash, and serositis, with amyloidosis as a possible complication. (1–3)
Familial cold-induced autoinflammatory syndrome (FCAS), chronic infantile neurologic, cutaneous, articular syndrome (CINCA), and Muckle-Wells syndrome (MWS) fall within the family of CAPS. (1–3) They are associated with NLRP3 gene mutations, which lead to interleukin (IL)-1β secretion disruption. (1–3) In a subset of patients with CAPS without NLRP3 gene mutations, it has been found that mutations in the NLRP12 gene are responsible for these syndromes. (2, 3) The NLRP12 protein is known to have both anti and proinflammatory roles, downregulating NF-κB and stimulating IL-1β and IL-18, respectively, thus in theory, variants could cause SAID. (2–4) Common symptoms of NLRP12-associated SAID, or FCAS 2, include fever in 93% of cases, followed by musculoskeletal symptoms in 61%, urticaria in 52%, abdominal symptoms in 38%, headache in 22%, and elevated acute phase reactants in 30% of cases, which is triggered by exposure to the cold in 36% of cases; and other than that symptoms such as fatigue, other types of rashes, and conjunctivitis have also been reported. (2, 4) Antihistamines, non-steroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids are used in treatment as needed. (2, 4) There has been positive results with anakinra, and research suggests the possibility of using tumor necrosis factor (TNF) as well as IL-6 inhibitors, although the exact behaviour of cytokines in this disease is unclear. (2, 4)
A number of variants of NLRP12 have been reported to date, including c.1206C>G, most commonly, and R284X, D294E, R352C, c.2072+3insT, c.1352G>A, among others (2, 4). Herein we present the case of a patient with symptoms suggestive of a hereditary SAID, in particular FCAS 2, with a variant in the NLRP12 gene which has not been reported to date.
CASE REPORT
A 40-year-old male patient was referred to our rheumatology clinic in spring 2021 by an infectious disease specialist after extensive workup for recurrent febrile episodes. The patient had several febrile episodes the previous winter, generally with elevated C-reactive protein (CRP) and was treated repeatedly with antibiotics. Upon the use of antibiotics, the CRP level normalised. The bouts of fever were usually followed by unspecific chest pain and dry cough, and occasionally the patient experienced morning stiffness in his right hip. The patient also experienced bouts of fever two winters ago, but to a milder extent. Workup included infectious disease testing, anti-nuclear antibodies (ANA), computerised tomography (CT) of the thorax, echocardiography, and leukocyte scintigraphy, which were normal or insignificant. At this point, SAID was considered.
Approximately a month later, during follow-up, the patient reported another bout of fever with an elevated CRP level of 25 mg/L and a leukocyte range of 20x10^9/L. As recommended, echocardiography was done during an attack, and its results were insignificant. Genetic testing was arranged at this time.
Through genetic testing, sequencing of clinical exome LG235, heterozygous variant in the NLRP12 gene, NM_001277126.1, position 19:54314063, c.850C>G was detected, which causes the substitution of arginine for glycine in codon 284, p.Arg284Gly. This variant is absent from databases. Predictions from in silico analysis showed that the variant does not damage the structure or the function of the protein, although no functional studies are available to confirm this.
Illumina TruSight One reagent set was used to prepare libraries for DNA sequencing. NextSeq 550 analyser was used for sequencing, and Variant Interpreter for data analysis. Online Mendelian Inheritance in Man (OMIM), Clinical Variant (ClinVar) Database, Human Gene Mutation Database (HGMD), Single Nucleotide Polymorphism Database (dbSNP), Genome Aggregation Database (GnomAD), Clinical Genome (ClinGen) Resource, 1000 Genomes Project, Database of Genomic Variants (DGV) databases were searched. The analysis included 4800 genes known to cause disease, as annotated in OMIM, ClinVar and HGMD, for the following phenotypes: recurrent fever, fever of unknown origin, elevated CRP, leukocytosis.
Based on the clinical presentation and considering the genetic testing results, despite the lack of functional studies, the patient was diagnosed with NLRP12-associated hereditary SAID or FCAS 2. Colchicine was recommended as a treatment method. At follow-up, a significant reduction in the number of febrile attacks with colchicine was noted, which may additionally confirm the diagnosis.
DISCUSSION
Our patient presented with a clinical presentation highly suggestive of SAID, more specifically CAPS, considering the recurrent fever and elevated CRP primarily during winter, and with the NLRP12 gene variant, we narrowed this down to the possibility of FCAS 2 or NLRP12-associated SAID. The patient presented with cough and chest pain, which may be indicative of serositis, although this was not objectively shown. Although the pathogenesis of NLRP12-associated SAIDs and effect of NLRP12 variants is unclear, such variants have been associated with FCAS 2, and shown to be causative. (2–4) The c.850C>G gene variant in our patient was shown to be benign with in silico analysis, but in the absence of another clear cause of the patient’s clinical presentation, it is possible that this variant is the one that caused the disease. Therefore, the patient was diagnosed with FCAS 2 or NLRP12-associated SAID, despite the lack of functional studies.
The method for whole exome sequencing (WES) in recent years has become readily available, reliable, and cost effective, (5) and it has even been implemented and routinely performed in a national centre in Zagreb. WES can confirm diagnosis, especially in the case in which a pathogenic disease variant is detected in a patient with a matching clinical phenotype for the assumed disease. (5) However, in other detected variants, also known as variants of unknown clinical significance, as in our case, a definite diagnosis cannot be made. (5) Chances increase based on the location and type of mutation, and computational studies, or in silico analysis. (5) However, these tools are not designed for clinical practice, and results should be interpreted with caution. (5) At this time, functional studies remain as the only option for validating such variants, but current methods such as mRNA or protein expression, rescue experiments, and animal or cell culture model systems, are not easily or readily attainable. (5) In our case, the mutation location and in silico analysis currently do not suggest a pathogenic variant, but a functional study would be needed for confirmation. Unfortunately, such a study is not available at our centre due to personnel, equipment, and funding limitations.
Although results using colchicine for treatment of NLRP12-associated SAIDs are weak, considering the relatively mild clinical presentation of the patient, it was decided to try the treatment method with colchicine first before opting for other, more uncomfortable (subcutaneous) or costly (novel biologics) treatment method which has been suggested in literature. (2, 4) With significant reduction in the number of attacks during follow-up, colchicine proved to be a successful treatment in the case of our patient.
Late disease onset is questionable in this case, but given the clinical presentation and associated genetic variant, an acquired SAID, such as adult-onset Still’s disease or Schnitzler’s syndrome, is less likely to occur. Considering that the region that the patient resides in has mild winters, and the fact that temperatures in that region rarely go below 0 °C, it is possible that this, in combination with an aging immune system which may have lost the ability to compensate for disbalances, may explain why the symptoms did not manifest earlier in this patient.
In conclusion, although there is a lack of functional studies in this case, given the clinical presentation indicative of CAPS, with the exclusion of other causes, and a favourable response to colchicine as a treatment method, we consider that the diagnosis of CAPS is the final one in the case of our patient (more specifically FCAS 2 or NLRP12-associated SAID). Furthermore, the discovered novel c.850C>G NLRP12 gene variant could possibly be the one that causes this disease. With whole exome sequencing becoming a routine method, it is likely that an increasing number of novel gene variants of unknown clinical significance will be discovered. This will lead to an increase in the need for validating these variants through functional studies, which will pose an issue until such studies become more readily available and cost effective.
Acknowledgments: Josipa Mateševac, Fran Borovečki
Conflict of interest statement: The authors declare no conflict of interest.