Generalized Cytokine Increase in the Setting of a Multisystem Clinical Disorder and Carcinoid Syndrome Associated with a Novel NLRP12 Variant

A 63-year-old female patient of northern and central European descent presented to the gastrointestinal clinic for symptoms of abdominal pain and non-bloody diarrhea. She also described a history of chest pressure, tachycardia, shortness of breath, flushing, and hypoglycemia. At the time of presentation, the patient’s symptoms were partially improved with subcutaneous octreotide; however, she continued to have episodic abdominal pain and intermittent elevations in hepatic transaminases. In addition, she reported a long-standing history of symptoms including fevers, oral ulcers, Raynaud’s phenomenon, polyarthritis, and a mild malar rash. At one point, she was diagnosed with undifferentiated connective tissue disorder with a positive antinuclear antibody at 1:160 and low titer IgM anti-cardiolipin antibody. She was previously treated with prednisone, methotrexate, and azathioprine and eventually transitioned to leflunomide with some improvement in symptoms. None of these symptoms were affected by exposure to environmental temperature changes. In addition, the patient’s history was significant for reported numbness and tingling in the base of the neck and right upper extremity, and she was diagnosed with thoracic outlet syndrome (TOS) with a positive nerve conduction study. She underwent two surgeries with subtotal resection of multiple muscles, and biopsy of the right scalene muscle revealed fibrosis and chronic inflammation. The past medical history is also significant for hypothyroidism, asthma, bilateral avascular necrosis of the shoulders requiring decompression surgeries, endometriosis, IgG deficiency requiring intravenous immunoglobulin, and gastroesophageal reflux. Her social history is negative for smoking or alcohol, and additional family history is significant for colon cancer in her mother. Her physical exam was largely normal except for hepatomegaly. Laboratory studies demonstrated a normal white blood cell count, but with a differential significant for an eosinophil count as high as 240 cells per microliter of blood. Additionally, she had intermittently abnormal liver tests with elevated transaminases and alkaline phosphatase and elevated lipase levels ranging from 66 to 132 units per liter. On subsequent evaluation when the patient was receiving immunosuppressive treatment, autoantibodies, including repeat antinuclear antibody, were negative. Although her 24-h urine 5-hydroxyindoleacetic acid (5-HIAA) was elevated, serum insulin, C-peptide, gastrin, serotonin, and chromogranin-A levels were all within normal range. Transglutaminase IgA, endomysial IgA, and gliadin antibodies were negative. Hepatitis serologies and EBV IgG antibody were non-reactive. Liver biopsy showed periportal inflammation. Given her elevated urine 5-HIAA and symptoms concerning for carcinoid syndrome, the patient underwent extensive gastrointestinal workup including octreotide and 68 Ga-DOTATATE PET/CT scans, small bowel enteroscopy, and endoscopic ultrasound which showed elevated DOTATATE activity localized to a small focus in the left hepatic lobe, along the lesser curvature of the stomach, tail of the pancreas, and small bowel. Her abdominal symptoms improved after treatment with octreotide. MRCP revealed mild prominence of the central biliary tree and hepatomegaly, but no pancreatic tumor. Genome-level sequencing was requested due to concern for one of the multiple endocrine neoplasias or autoinflammatory disorders, given her constellation of symptoms. Exome sequencing was performed using the Agilent SureSelect Clinical Research Exome XT kit on an Illumina HiSeq 2500 and identified a heterozygous c.536C > T (p.Thr179Ile) variant in the NLRP12 gene ( Fig. 1 , 2 ); no mutation in any of the familial cancer syndrome genes or in any of the other known periodic fever genes was identified. Analysis of serum cytokines revealed elevated levels of pro-inflammatory (interleukin (IL)-1β, IL-6, IL-12, and TNF-α), Th1 (IL-2, IFN-γ), and Th2 (IL-4, IL-5, IL-13) cytokines, but not of Th17 (IL-17) and IL-10 ( Table 1 ). No mutations in any cytokine genes were detected. Levels of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6, were persistently elevated over several months ( Fig. 3 ). Treatment with a single dose of canakinumab, a human monoclonal antibody targeting IL-1β, resulted in some improvement in the diarrhea and abdominal pain. The patient’s daughter was seen at UCLA in consultation shortly after we identified the novel genomic variant. The daughter also expressed similar symptoms of diarrhea, abdominal pain, and flushing, with the notable addition of urticaria. Her medical history was also significant for hypothyroidism and TOS, and her laboratory studies revealed an elevated 24-h urine 5-HIAA. Whole exome sequencing was positive for the identical NLRP12 variant.

HPFs or systemic autoinflammatory diseases are a group of disorders characterized by recurrent fevers and systemic inflammation and are categorized into at least six disorders based on clinical criteria. Among the NLRP subfamily, the NLRP3 autosomal-dominant mutations are known to be associated with three of these disorders, including familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), as it encodes the dysfunctional protein, cryopyrin, which is involved in the processing and secretion of IL-1β, eventually leading to systemic inflammation [ 3 ]. FCAS is usually mild with generalized inflammatory attacks including skin urticaria, fevers, or joint pains triggered by cold, while MWS is more severe, causing progressive hearing loss and renal amyloidosis [ 4 ]. New autoinflammatory disorders with their associated genes and mutations are still being identified and reported regularly in the literature. Over the last 10 years, other mutations in the NLRP12 gene have been shown to be associated with HPFs, as its nucleotide-binding site, sequence, and effects on IL-1β are similar to that of NLRP3 . In the first reported study of disease-causing mutations in NLRP12, Jeru et al. sought to determine the molecular basis of a disorder in twin brothers who presented with symptoms overlapping FCAS and MWS—namely sensorineural hearing loss and the triggering effect of cold—without mutations in the known HPF genes. Sequencing of NLRP12 revealed a heterozygous nonsense mutation (c.850C > T, p.Arg284X) in both brothers, as well as their father, who displayed a milder phenotype. A second NRLP12 mutation (c.2072 + 3insT) was identified in a young girl in a different family presenting with periodic fevers, abdominal pain, and arthralgia associated with exposure to cold, similar to FCAS. Her father was found to have the same mutation and had similar symptoms as a child. This study elucidated the autosomal-dominant mode of inheritance of NLRP12 -associated syndromes and designated these syndromes as FCAS-2 or NLRP12 -AD [ 2 ]. At least 46 cases have been reported thus far of NLRP12 variants in patients [ 3 – 6 ]. Among the reported NRLP12 variants, p. F402L (c.1206 C > G) is the most frequent NLRP12 mutation (16/30, 53%) [ 3 ]. The other reported NLRP12 gene variants are p. R284X (c.850 C > T) and c.2072 + 3insT (as above), p. D294E (c.882 C > G), p.R352C (c.1054 C > T), p. G448A (c.1343 G > C), and p.H304Y (c.910 C > T). Although the disorder is more prominent among Caucasians, there are now multiple reported cases of Chinese patients with NLRP12 -AD [ 3 ]. About 70% of reported patients have had a childhood onset with overall male-to-female ratio of 7:5 [ 3 ]. Our patient does not have features typical of FCAS, other than abdominal pain, diarrhea, arthralgia, and arthritis, which are relatively nonspecific symptoms, and she has a previously unreported novel NLRP12 variant (heterozygous g.54314377G > A, c.536C > T, p.Thr179Ile). NLRP12 -AD patients typically present with periodic fever (28/30, 93%), cold exposure as a trigger (20/30, 66%), urticaria-like rash (19/30, 63%), arthralgia/arthritis (18/30, 60%), myalgia (18/30, 60%), abdominal pain/diarrhea (13/30, 43%), headache (11/30, 36%), and lymphadenopathy (9/30, 30%), and elevated acute phase reactants (9/30, 30%) [ 2 , 7 – 11 ]. Splenomegaly, sensorineural deafness, and thoracic pain have been reported in less than 10% patients. Our patient and her daughter lack most of these typical features and instead present with a multisystem illness and autoimmune diathesis, including positive ANA, polyarthritis, muscle inflammation and fibrosis, and hypothyroidism. Their particular NLRP12 variant, not previously reported in the literature or databases, may therefore suggest a new hereditary syndrome. Although no biological model (either cellular or animal) exists for this specific variant, Functional Analysis through Hidden Markov Models (FATHMM) predicts that this mutation may be damaging with a score of − 2.26 (VarSome database: NM_001277126.1 :c.536C > T) [ 12 , 13 ]. The clinical spectrum associated with NLRP12 gene variants in 48 cases, including 2 cases in this report, is summarized in Fig. 4 . A similar case report recently described a 20-year-old female postmortem who expressed two heterozygous non-synonymous amino acid substitutions in the NLRP12 gene, encoding the NALP12 protein (p.H304Y and p.A629D) [ 14 ]. Her phenotype consisted of progressive B cell lymphopenia/antibody deficiency diagnosed as common variable immunodeficiency, juvenile idiopathic arthritis, and development of intestinal inflammatory amyloidosis. No cold-induced symptoms, skin rashes, urticaria, or periodic fever could be observed or elucidated in the index patient or related family members. Transcriptome analyses revealed steady-state normal IL-1β expression, but significantly increased baseline transcription of XCL2, CXCL9, CXCL10 , and IFN-γ. Our patient presents with features that resemble the above reported case, specifically in relation to low IgG levels, polyarthritis, diarrhea, and lack of cold-induced symptoms, though obviously she has survived much longer. Furthermore, our patient’s diarrhea, atopic symptoms, and eosinophilia could be secondary to an increase in various cytokines associated with NLRP12 mutation. In experimental studies of mice receiving Nlrp12 −/− T cells, excessive levels of Th2 cytokines (such as IL-4, IL-5, and IL-13) were detected, promoting atopic dermatitis of the skin and ears as well as colitis, colitis-associated colon cancer, and atypical autoimmune encephalomyelitis [ 15 ]. Consistently, our patient had increased circulating IL-4, IL-5, and IL-13 and gave a history of bronchial asthma, eosinophilia, allergies, and recurrent skin rash. Her daughter with the same NLRP12 variant has had recurrent urticaria. Similarly, high amounts of Th2 cytokines have been implicated in the development of fibrosis and could contribute to the presence of muscle fibrosis in our patient [ 16 ]. Recently, NLRP12 has been shown to have a protective role on intestinal inflammation by inhibiting the NF-kB pathway ( Fig. 5 ) and through alterations of the intestinal microbiome [ 17 ]. Nlrp12 deficiency in mice predisposes to tumorigenesis [ 18 , 19 ]. In humans, the expression levels of NLRP12 gene and protein are dysregulated in malignant prostate tumor tissues [ 20 ]. Consistently, our patient and her daughter had features suggestive of carcinoid syndrome (though without a tissue diagnosis). Further studies are needed to establish association between our newly discovered NLRP12 variant and the development of carcinoid syndrome and other malignancies. In terms of treatment, only one-third of patients respond to glucocorticoids and anti-histamine drugs, and thus, IL-1 inhibitors, such as anakinra, may be beneficial given the role of IL-1β in the pathogenesis of NLRP12 -AD [ 3 ]. However, another study reported recurrence of symptoms on anakinra within a few months of treatment, which was concerning for an acquired resistance to the medication [ 9 ]. Canakinumab, a human monoclonal antibody targeting IL-1β, could be efficacious in patients who are resistant to anakinra, as has been shown in other autoinflammatory disorders; however, there have been no reported cases of treatment with canakinumab in NLRP12 -AD. After a single administration of canakinumab, our patient reported some improvement in diarrhea and abdominal pain. Encouraged by this observation, we have initiated long-term treatment with canakinumab in this patient. In summary, we present a previously unreported variant in NLRP12 in a patient and her daughter with a multisystem illness without classic symptoms of NLRP12 - AD , but with persistently elevated levels of IL1β and several other cytokines. They presented with chronic nonspecific symptoms including flushing, diarrhea, abdominal pain, atopy, and arthralgia, which were severe enough in our patient to require treatment with several immunosuppressive drugs. Our observations warrant the search for mutations in NLRP12 and related genes in patients with such nonspecific and severe symptoms, within the context of the ever-expanding constellation of autoinflammatory disorders.

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a group of cytoplasmic sensors that survey danger signals released by invading pathogens or damaged tissue [ 1 ]. Mutations in the NLRP subfamily affect pro-inflammatory mediators and are associated with autoinflammatory disorders known as hereditary periodic fevers (HPFs) [ 2 ]. These disorders cause nonspecific systemic symptoms, which make them a diagnostic challenge and often overlooked. We describe a previously unreported variant c.536C > T (p.Thr179Ile) in NLRP12 in association with an atypical multisystem illness with carcinoid syndrome as well as atopic and autoimmune features, the latter potentially related to the role of NLRP12 in regulating inflammatory cytokine production.