NOD2 versus MEFV: Differential diagnosis of Yao syndrome and familial Mediterranean fever

Introduction

Systemic autoinflammatory disease (SAID) is a group of periodic inflammatory diseases without detectable auto-antibodies or antigen-specific T lymphocytes.^[1] Familial Mediterranean fever (FMF) is the most prototypic of SAIDs,^[2] clinically marked by recurrent short episodes of fever and serositis. FMF has been a century-old disease, and its gene MEditerranean FeVer (MEFV) is localized to chromosome 16p13 and encodes for a protein called pyrin or marenastrin,^[3] and MEFV mutations cause the disease.

Nucleotide-binding oligomerization domain-containing protein-2 (NOD2) is a cytosolic NOD-like receptor (NLR).^[4] Since its discovery in 2001,^[4] NOD2 gene mutations have been associated with Crohn's disease (CD),^[5] Blau syndrome,^[6] and Yao syndrome (YAOS), formerly named as NOD2-associated autoinflammatory disease (NAID).^[7] Since our initial report in 2011, YAOS is characterized by periodic fever, dermatitis, polyarthritis, gastrointestinal (GI), and sicca symptoms with eyelid swelling and is linked to certain NOD2 variants.^[7,8,9,10] Both YAOS and FMF share similar clinical phenotypes or resemble each other in some cases, thus posing diagnostic challenges clinically. Moreover, both NOD2 and MEFV genes are mapped at chromosome 16, and their gene products belong to the superfamily of death domain proteins.^[11] Both NOD2 and pyrin proteins play important roles in the regulation of apoptosis, cytokine processing, and inflammation.^[12] However, a potential relationship between these two disorders has yet to be studied. Herein, we report a case series in conjunction with the literature to illustrate the similarities and differences between YAOS and FMF on clinical and molecular levels.

Patients and Methods

In this retrospective study, electronic medical records of a series of patients with YAOS were reviewed, who fulfilled the diagnostic criteria consisting of characteristic phenotype and the presence of NOD2 variants with the exclusion of other related diseases.^[7] The study was approved by the Institutional Review Board of Stony Brook University Hospital with a waiver of informed consent. These patients underwent extensive workup, including genetic testing for periodic fever syndrome 6-gene panel (MEFV, TNFRSF1A, NLRP3, MVK, NLRP12, and NOD2). All patients initially carried the diagnosis of MEFV-negative FMF and received treatment with colchicine. An abstract on this case series was previously presented at the EULAR annual meeting in 2020.^[13]

Our prior presentation of a published abstract summarizing 3 patients at the American College of Rheumatology Annual Meeting was also used in this article. In this case series, examination of the NOD2 and MEFV genes for mutations was performed by DNA polymerase chain reaction and DNA sequencing. The phenotypes and genotypes of these patients were individually described.^[14]

Results

Mutations of Both MEFV and NOD2 May Contribute to SAIDs in an Individual or Family

Discussion

Our case analysis has indicated that YAOS can partially resemble FMF in clinical phenotypes, thus YAOS should be contemplated as an FMF differential diagnosis, particularly in atypical cases. Molecular analysis of periodic fever syndromes should cover NOD2 whole gene sequencing to help distinguish these two diseases. YAOS and FMF are distinct diseases. Based on the literature data including our prior publications, the demographics, clinical phenotypes, genotypes, and therapies of these two diseases are summarized (Table 1). In addition to different gene mutations, there are distinguishing clinical features between these diseases. For instance, in FMF, the febrile episode is short (<3 d), the erysipelas-like rash tends to occur on the distal lower extremities, and acute abdomen with more constipation than diarrhea is common. A majority of FMF patients have a sustainable response to colchicine. In YAOS, the duration of each disease flare can be several days or longer, erythematous patches often occur on the upper body (face and chest), and mild abdominal cramping and bloating with non-bloody loose stools or diarrhea are common. Intermittent eyelid swelling and distal lower extremity swelling can occur. Most patients do not respond to colchicine or a minority of patients may have a temporary response.

Our study also suggests that both MEFV and NOD2 mutations together may contribute to mixed and heterogeneous autoinflammatory phenotypes for YAOS and FMF in individual patients. The clinical phenotype of patient 4 shared similar phenotypes with her aunt who had FMF with carriage of a heterozygous MEFV mutation. Given her longer duration of disease flares, rash locality, poor response to colchicine, and the absence of MEFV mutations, further genetic testing identified the presence of the NOD2 variants. Thus, YAOS was diagnosed and sulfasalazine was initiated with symptomatic resolution. This case suggests that both MEFV and NOD2 variants may run in a family. Both patients 5 and 6 carried MEFV and concurrent NOD2 variants and had overlapping and mixed phenotypes for both FMF and YAOS. FMF genotype is usually composed of either homozygous or compound heterozygous MEFV mutations. However, a single copy of MEFV gene mutations can be seen in approximately 25% of patients with FMF.^[15] YAOS is considered to be a genetically complex disease and is generally sporadic, though 10–15% of cases may have a positive family history. YAOS is associated with the NOD2 variants, IVS8+158 in nearly all patients, IVS8+158/R702W in up to 30%, and IVS8+158/1007fs, G908R, or other rarer NOD2 variants in some other patients.^{[9, 16]}

CD is another NOD2-associated disease. Approximately 30% of patients with CD harbor NOD2 variants.^[17] There have been studies investigating the association between FMF and CD. Some reported a higher prevalence of CD among patients with FMF, and others found an increased rate of FMF among CD patients.^{[18, 19]} Nevertheless, these studies demonstrated no association of the MEFV mutations with CD susceptibility. Instead, the presence of these mutations appears to be associated with a stricturing disease pattern and significantly more extra-intestinal manifestations of CD, suggesting that the MEFV gene may be a modifier of CD disease severity.^{[18, 20]} A recent study has demonstrated that the three common NOD2 variants (1007fs, G908R, and R702W) are not associated with increased susceptibility to develop FMF, and the coexistence of these NOD2 variants and MEFV mutations might contribute to higher rates of erysipelas-like erythema, acute scrotum attacks, colchicine resistance, and a more severe disease as compared with FMF patients without mutations.^[21] In our study, both patient 5 and 6 carried combined heterozygous NOD2 variant IVS8⁺¹⁵⁸ and MEFV mutations, but their clinical phenotypes were atypical for FMF with poor treatment response to colchicine. These data indicate that the NOD2 variant might serve as a modifier gene for FMF or form compound heterozygotes with the MEFV mutation to cause variable phenotypes. With more advanced molecular technology, such as next-generation sequencing, two or more genetic mutations from two or more different genes are occasionally identified in patients with SAIDs.^[11] Based on our prior study, a combination of MEFV and NOD2 mutations may be relatively common^[11] and they could be synergistic.

Both NOD2 and MEFV genes are located at Chromosome 16, and they are structurally and functionally similar (Figures 1 and 2).^{[22,23,24,25]} NOD2 contains 2 N-terminal caspase activation recruitment domains (CARDs), a central NLR (NACHT or nucleotide-binding domain [NBD]), and 6 C-terminal leucine-rich repeats (LRRs) (Figure 1A). The NOD2 proteins are primarily present in the cytosols of monocytes, macrophages, dendritic cells, and intestinal endothelial cells. NOD2 functions through its recognition of bacterial peptidoglycan via RICK or RIP2, thus activating p38 MAP kinases and NF-kB, resulting in inflammation^[22] (Figure 2). Pyrin is encoded by MEFV and has five domains, including a PYRIN domain (PYD), a bZIP transcription factor basic domain, a B-box zinc finger domain, an alpha-helical domain, and a B30.2 (PRYSPRY) domain (Figure 1B). Pyrin is expressed in granulocytes, monocytes, dendritic cells, and synovial fibroblasts. Pyrin regulates caspase-1 activation and consequently IL-1b production via the interactions of its N-terminal PYRIN and C-terminal B30.2 domains. Pyrin is cleaved by caspase-1 and the cleaved N-terminal fragment is translocated to the nucleus and enhances NF-kB activation (Figure 2).^[23] As both MEFV and NOD2 genes are located on the same chromosome, we hypothesize that these two genes might affect each other and modify the phenotypes of FMF and YAOS. Further study would be needed to understand their relationship and combined influence on disease expression.

Figure 1

Schematic representation of the gene and protein structures of NOD2 and MEFV. (A) NOD2 gene and protein. (B) MEFV gene and protein. CARD, caspase activation recruitment domain; CC, coiled-coil; LRR, leucine-rich repeat; MEFV, MEditerranean FeVer; NBD, nucleotide-binding domain; NOD2, nucleotide-binding oligomerization domain-containing protein-2; PYD, pyrin domain; CC, coiled-coil; SPRY, Dictyostelium discoideum dual-specificity kinase SpIA and ryanodine receptor.

Figure 2

Signaling pathways and molecular interactions of NOD2 and pyrin. (1) Bacterial product, MDP, binds cytosolic NOD2 and, in turn, activates the Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RIP2). The phosphorylated RIP2 subsequently activates NF-kB and p38/ERK/JNK, leading to the inflammatory response. (2) By binding apoptosis-ASC, pyrin disrupts the cryopyrin-ASC interaction and specifically inhibits activation of caspase 1. Mutation in pyrin leads to apoptosis and NF-kB activation by reducing the inhibitory pathway. (3) NOD2 also interacts with other NLRs, leading to activation of caspase 1 with adaptor protein ASC. ASC, associated speck-like protein; CARD, caspase activation recruitment domain; CC, coiled-coil; ERK, extracellular-signal-regulated kinase; JNK, JUN aminoterminal kinase; LRR, leucine-rich repeat; MDP, muramyl dipeptide; NOD2, nucleotide-binding oligomerization domain-containing protein-2; NLRs, NOD-like receptors; NBD, nucleotide-binding domain.

Limitations of the Study

This study is retrospective and its sample size is small. No familial segregation analysis was performed. Further study of larger case series is needed in future research in this regard.

Conclusion

YAOS and FMF share similar clinical phenotypes and can masquerade each other, notably in the scenario of atypical cases of FMF, but they are phenotypically and genotypically distinct. Molecular analysis should cover NOD2 whole gene sequencing to help distinguish these diseases. Both MEFV and NOD2 mutations can be simultaneously detected in an individual or family, and the coexistence of both gene mutations may contribute to mixed and heterogeneous phenotypes. Clinical interpretation should base on the correlation between phenotype and genotype data.