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Tumor necrosis factor receptorassociated periodic syndrome characterized by a mutation affecting the cleavage site of the receptorImplications for pathogenesis.

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Vol. 48, No. 8, August 2003, pp 2386–2388
© 2003, American College of Rheumatology
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 T3 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).
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
DOI 10.1002/art.11169
Tumor necrosis factor receptor–associated periodic
syndrome characterized by a mutation affecting the
cleavage site of the receptor: implications for
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
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)
Table 1. Correlation between symptoms and levels of serum TNF␣
and acute-phase reactants in the index patient*
February 2, 2002/ February 11, 2002/
no symptoms
TRAPS attack
TNF␣, pg/ml (normal ⱕ8.1)
IL-6, pg/ml (normal 0.1–5.4)
ESR, mm/hour (normal 6–20)
CRP, mg/dl (normal ⱕ0.8)
* The TRAPS (tumor necrosis factor receptor–associated periodic
syndrome) attack included arthralgia, myalgia, and fever (39.2°C).
TNF␣ ⫽ tumor necrosis factor ␣; IL-6 ⫽ interleukin 6; ESR ⫽
erythrocyte sedimentation rate; CRP ⫽ C-reactive protein.
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 T3 A
substitution. C, Three-dimensional structure of the wild-type (upper panel) and mutated (lower panel) protein, created with the Swiss-Model server
( Residue 199 is shown in blue. The hydrogen bonds formed by the mutated N199 are shown as green broken lines.
of defective receptor shedding, reduced levels of soluble
TNFRSF1A, and attack-associated elevations of soluble
TNF␣. 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
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.
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.
Martin A. Kriegel, MD
Ulrike Hüffmeier, MD
Elisabeth Scherb
Christina Scheidig
Thomas Geiler, MD
Joachim R. Kalden, MD
André Reis, MD
Hanns-Martin Lorenz, MD
University of Erlangen-Nuremberg
Erlangen, Germany
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Figure 3. Serum levels of soluble tumor necrosis factor receptor
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site, cleavage, syndrome, receptorimplications, pathogenesis, receptorassociated, necrosis, factors, mutation, characterized, periodid, affecting, tumors
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