Tumor necrosis factor receptor–associated periodic syndrome characterized by a mutation affecting the cleavage site of the receptor: Implications for pathogenesis


Tumor necrosis factor receptor (TNFR)–associated periodic syndrome (TRAPS; MIM no. 142680) is a potentially lethal, autosomal-dominantly inherited autoinflammatory syndrome characterized by recurrent attacks of fever, skin lesions, and abdominal, joint, or muscle pain (1–3). To date ∼20 mutations in the membrane-distal domains of TNFR superfamily 1A (TNFRSF1A) in patients with TRAPS have been reported (4–6). Defective shedding and reduced serum levels have been demonstrated in vitro (1, 5). It has been hypothesized that some mutations would indirectly interfere with activation-induced cleavage (1, 5), although this process occurs at Asn201–Val202 close to the transmembrane region (7). The aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the receptor at the same region and facilitates cleavage (8). Herein we describe a novel TNFRSF1A mutation (I199N) in a German family with dominantly inherited recurrent fever and arthritis. This amino acid substitution is predicted to cause hydrogen bond formation within the cleavage site of TNFRSF1A. We also demonstrate that this mutation causes defective shedding of the receptor.

All participants tested gave their informed consent. The index patient (case III-2, Figure 1A) is a 36-year-old woman of southern German extraction with recurrent (occurring roughly monthly to quarterly) attacks of fever, pharyngitis, arthritis/arthralgia, and low back pain. The first attack was noted when she was 19 years old; there were no obvious triggers. Episodes usually started in the evening with a maximum intensity close to midnight and often lasted several days, sometimes more than a week. Remarkably, during pregnancy and breast-feeding, the attacks vanished. Correlations of the symptoms with levels of acute-phase reactants are shown in Table 1. The 66-year-old mother of the index patient (case II-2) reported an almost identical history, including maximum intensity of attacks in the late evening and lack of episodes during pregnancy and lactation. Onset of her disease was at 20 years of age. After menopause the attacks became milder with pharyngitis and fever only, and truly periodic (every 4 weeks). The maternal grandfather (case I-1) was reported to have had recurrent fever of unknown etiology. The children of the index patient (case IV-1, 6 years old and case IV-2, 3 years old) are healthy to date. No DNA tests have been performed. The unaffected 42-year-old brother of the index patient (case III-1) never experienced periodic fever attacks and is otherwise healthy.

Figure 1.

Pedigree of and mutation in the study family with tumor necrosis factor receptor–associated periodic syndrome (TRAPS). A, Pedigree of 4 generations. Open symbols represent healthy individuals; solid symbols represent affected individuals. The mutation was confirmed in cases II-2 and III-2, and TRAPS in case I-1 (deceased) was suspected upon history. Question marks indicate that individuals have not been studied (case IV-1, 6 years old and case IV-2, 3 years old). B, DNA sequence of wild type (upper panel) and mutation (lower panel). Arrow indicates the nucleotide 596 T→A substitution. C, Three-dimensional structure of the wild-type (upper panel) and mutated (lower panel) protein, created with the Swiss-Model server (http://www.expasy.ch/spdbv). Residue 199 is shown in blue. The hydrogen bonds formed by the mutated N199 are shown as green broken lines.

Table 1. Correlation between symptoms and levels of serum TNFα and acute-phase reactants in the index patient*
February 2, 2002/no symptomsFebruary 11, 2002/TRAPS attack
  • *

    The TRAPS (tumor necrosis factor receptor–associated periodic syndrome) attack included arthralgia, myalgia, and fever (39.2°C). TNFα = tumor necrosis factor α; IL-6 = interleukin 6; ESR = erythrocyte sedimentation rate; CRP = C-reactive protein.

TNFα, pg/ml (normal ≤8.1)4.227.9
IL-6, pg/ml (normal 0.1–5.4)<59.7
ESR, mm/hour (normal 6–20)2045
CRP, mg/dl (normal ≤0.8)0.32.5

Polymerase chain reaction (PCR) was performed with intron-based primers (primer sequences available upon request). The PCR products were purified with the PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosciences, Foster City, CA). The products were purified with the Dye-Ex Purification Kit (Qiagen) and analyzed in an ABI Prism model 3100 sequencer (Applied Biosciences). Mutation screening of controls was performed using TaqMan allelic discrimination. Peripheral blood from patients (III-2 and II-2) and several normal donors was analyzed for phorbol myristate acetate (PMA)–induced TNFRSF1A (CD120a) shedding by flow cytometry using an EPICS XL flow cytometer (Coulter, Hialeah, FL). Gates were set on the granulocyte population. Sera from the index patient collected during an asymptomatic period, from the healthy, unaffected brother of this patient, and from 13 normal donors were screened for soluble TNFRSF1A levels by enzyme-linked immunosorbent assay (R&D Systems, Wiesbaden, Germany).

In both patients, but not in the healthy brother of the index patient, a missense mutation (nucleotide 596 T→A, exon 6) was detected (Figure 1B). This T-to-A transition results in an amino acid substitution of hydrophobic isoleucine to polar asparagine (I199N). One hundred ninety-two ethnically matched control chromosomes (from 96 controls) were screened for this mutation. None of the controls carried the mutation (data not shown). Moreover, a BLAST search revealed that a hydrophobic residue is present at this amino acid position in the rat, cat, and mouse, indicating an evolutionary conservation of this residue. The 3-dimensional structure visualized with an automated modeling server demonstrated that asparagine in place of isoleucine causes the formation of 2 new hydrogen bonds (Figure 1C): one from N199 to Q198, and the other from N199 to N201, an essential amino acid of the cleavage site of TNFRSF1A (7). Furthermore, granulocytes from both patients lost the capacity to cleave the receptor upon activation (Figure 2), whereas PMA-induced shedding of CD16 was unaffected (data not shown). Measurement of soluble TNFRSF1A from the sera of the index patient, her healthy brother, and 13 controls indicated that the patient's level was below the lowest of the values in normal donors, whereas her brother's was within the normal distribution of the 13 controls tested (Figure 3).

Figure 2.

Activation-induced tumor necrosis factor receptor superfamily 1A shedding. Peripheral blood leukocytes from 3 normal donors (ND) and both patients with tumor necrosis factor receptor–associated periodic syndrome (cases III-2 and II-2) were stimulated with 10 ng/ml phorbol myristate acetate (PMA) for 20–30 minutes, followed by staining with phycoerythrin-conjugated anti-CD120a. Mean fluorescence intensity (MFI) was determined by flow cytometric analysis with gating on granulocytes.

Figure 3.

Serum levels of soluble tumor necrosis factor receptor superfamily 1A (sTNFRSF1A). Sera from 13 normal donors (ND), the index patient carrying the tumor necrosis factor receptor–associated periodic syndrome mutation (case III-2), and her mutation-negative brother (case III-1) were examined for sTNFRSF1A concentrations by sandwich enzyme-linked immunosorbent assay. Values shown represent the mean from 2 identical aliquots measured.

In conclusion, we have identified a novel missense mutation in the TNFRSF1A gene causing TRAPS in a German family. This mutation is the first one to be discovered in close proximity to the transmembrane region within a highly conserved stretch. The I199N exchange induces 2 new hydrogen bonds, one of them affecting Asn201, the core of the cleavage site. The structural findings were supported by the demonstration of defective receptor shedding, reduced levels of soluble TNFRSF1A, and attack-associated elevations of soluble TNFα. We therefore hypothesize that the I199N mutation might not only hinder ARTS-1–assisted cleavage of the receptor, but might also directly interfere with the core of the cleavage site. This novel mutation is further evidence for the role of an intact shedding process in the homeostatis of the TNF/TNFR system in humans and provides a structural basis for defective shedding in the pathogenesis of TRAPS.


Supported by grants from the Interdisciplinary Center for Clinical Research (IZKF C13) and from the ELAN Fonds fuer Forschung und Lehre (grant of the University of Erlangen-Nuremberg.