Clinical significance of serum soluble
TNF receptor I/II ratio for thedifferential diagnosis of tumor necrosis
factor receptor-associated periodic
syndrome from other autoinflammatory
diseases

w
e
i
v
re

Junko Yasumura1*, Masaki Shimizu2, Tomoko Toma2, Akihiro Yachie2, Satoshi Okada1
1

Hiroshima University, Japan, 2Kanazawa
nazawa University, Japan

Submitted to Journal:
Frontiers in Immunology
munology

In

Specialty
cialty Section:
Primary
Immunodeficiencies
ry Immunodeficie
Article typ
type:
Original
Research Article
al ReseManuscript ID:
576152
Received on:
25 Jun 2020
Revised on:
05 Sep 2020

Frontiers website link:www.frontiersin.org

Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest

Author contribution statement
All authors were involved in the conception, design of the study and revising it critically for important intellectual content. JY andMS and TT were involved in the acquisition of data, analysis and interpretation of data. JY and MS wrote the manuscript. All
authors read and approved the final manuscript.

Keywords

w
e
i
v
re

Familial meditarranean fever, Kawasaki disease (KD), Soluble tumor necrosis factor receptor, Systemic juvenile idiopathic arthritis(sJIA), Tumor necrosis factor receptor associated periodic syndrome (TRAPS)

Abstract

In

Word count:

199

tives
Objectives
Genetic analysis of TNFRSF1A can confirm the diagnosis of tum
tumor necrosis factor receptor-associated periodic syndrome (TRAPS),
but interpretation
pretation of the pathogenesis of variants of unknown significance is sometimes required. The aim of this study was toevaluate thehe clinical significance of serum soluble tumor necrosis factor receptor type I (sTNFR-I)/II ratio to differentiate TRAPS
from other autoinflammatory diseases.
Methods
Serum sTNFR-I
FR and sTNFR-II levels were measured using an enzyme-linked immunosorbent assay in patients with TRAPS (n=5),
familial Mediterranean fever (FMF) (n=14), systemic juvenile idiopathic arthritis (s-JIA) (n=90), and Kawasaki disease (KD) (n=37) in
the active and inactive phase, along with healthy controls (HCs) (n=18).
Results
In the active phase, the serum sTNFR-I/II ratio in patients with s-JIA, KD, and FMF was significantly elevated compared with that inHCs, whereas it was not elevated in patients with TRAPS. In the inactive phase, the serum sTNFR-I/II ratio in patients with s-JIA
and FMF was significantly higher compared with that in HCs, and the ratio was lower in TRAPS patients than in patients with s-JIA
and FMF.
Conclusions
Low serum sTNFR-I/II ratio in the active and inactive phase might be useful for the differential diagnosis of TRAPS and otherautoinflammatory diseases.

Contribution to the field
There is considerable overlap in clinical manifestations and laboratory findings in autoinflammatory disorders. The absence ofdefinitive biomarkers makes the diagnosis difficult in patients with autoinflammatory disorders. Patients with TNF receptor
associated syndrome (TRAPS) develop recurrent fever, abdominal pain, myalgia, exanthema, arthralgia/arthritis, and ocular
involvement. However, based on the similarity in the clinical manifestations, TRAPS is often misdiagnosed as the otherautoinflammatory disorders, such as systemic juvenile idiopathic arthritis (s-JIA). Genetic analysis of TNFRSF1A can confirm thediagnosis of TRAPS, but interpretation of the pathogenesis of variants of unknown significance (VUS) is sometimes required. The
current study revealed that the serum soluble tumor necrosis factor receptor type I (sTNFR-I)/II ratio in patients with s-JIA,
Kawasaki disease, and familial Mediterranean fever (FMF) is significantly elevated, whereas it was not elevated in patients with
TRAPS in the active phase. In addition, the serum sTNFR-I/II ratio was lower in patients with TRAPS than in patients with s-JIA and
FMF in the inactive phase. These results clearly showed that the serum sTNFR-I/II ratio can be a useful indicator in the diagnosis of
TRAPS. This discovery contributes for the precise interpretation of the pathogenesis of VUS in TNFSF1A, facilitating accurate
diagnosis of TRAPS.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethics statements
Studies involving animal subjects
Generated Statement: No animal studies are presented in this manuscript.

Studies involving human subjects
Generated Statement: The studies involving human participants were reviewed and approved by the Institutional Review Board of
Kanazawa University. Written informed consent to participate in this study was provided by the participants' legal guardian/nextof kin.

Inclusion of identifiable human data
Generated Statement: No potentially identifiable human images or data is presented in this study.

In

w
e
i
v
re

Data availability statement
Generated Statement: The authors acknowledge that the data presented in this study must be deposited and made publicly
available in an acceptable repository, prior to publication. Frontiers cannot accept a manuscript that does not adhere to our open
data policies.

In

w
e
i
v
re

3

Clinical significance of serum soluble TNF receptor I/II ratio for thedifferential diagnosis of tumor necrosis factor receptor-associatedperiodic syndrome from other autoinflammatory diseases

4
5

Junko Yasumura1*, Masaki Shimizu2͊, Tomoko Toma2, Masato Yashiro3, Akihiro Yachie2͋
and Satoshi Okada1͋

671

Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences,
Hiroshima, Japan

8
9

2

1
2

Department of Pediatrics, Graduate School of Medical Sciences, Kanazawa University, Kanazawa,
Japan

w
e
i
v
re

10

3

Department of Pediatrics, Okayama University Hospital, Okayama,ayama, Japan

11†

Present address

12
13
14

Department of Child Health and Development, Graduate
aduate School of Medical anda Dental Sciences,
Tokyo Medical and Dental
ntal University
1-5-45 Yushima,ma, Bunkyo-ku, Tokyo 113 - 8510, JAPAN

15

‡

16
17
18

pondence:ndence:* Correspondence:
umura
mur
Junko Yasumura
2@2@hjunko-ma562@hiroshima-u.ac.jp

19
20
21
22

In

These authors contributed equally to this w
work

Keywords: familial Mediterranean fever1, Kawasaki disease2, soluble tumor necrosis factor
receptor3, systemic juvenile idiopathic arthritis4, tumor necrosis factor receptor-associatedperiodic syndrome5.

23
24

Abstract

25

Objectives

26
27
28
29
30

Genetic analysis of TNFRSF1A can confirm the diagnosis of tumor necrosis factor receptorassociated periodic syndrome (TRAPS), but interpretation of the pathogenesis of variants of
unknown significance is sometimes required. The aim of this study was to evaluate the clinicalsignificance of serum soluble tumor necrosis factor receptor type I (sTNFR-I)/II ratio to differentiate
TRAPS from other autoinflammatory diseases.

31

Methods

32
33

Serum sTNFR-I and sTNFR-II levels were measured using an enzyme-linked immunosorbent assay
in patients with TRAPS (n=5), familial Mediterranean fever (FMF) (n=14), systemic juvenile

sTNFR-I/II ratio for diagnosis
34
35

idiopathic arthritis (s-JIA) (n=90), and Kawasaki disease (KD) (n=37) in the active and inactive
phase, along with healthy controls (HCs) (n=18).

36

Results

37
38
39
40
41

In the active phase, the serum sTNFR-I/II ratio in patients with s-JIA, KD, and FMF was
significantly elevated compared with that in HCs, whereas it was not elevated in patients with
TRAPS. In the inactive phase, the serum sTNFR-I/II ratio in patients with s-JIA and FMF was
significantly higher compared with that in HCs, and the ratio was lower in TRAPS patients than inpatients with s-JIA and FMF.

42

Conclusions

43
44

Low serum sTNFR-I/II ratio in the active and inactive phase might be useful for the differential
diagnosis of TRAPS and other autoinflammatory diseases.

45
Introduction

w
e
i
v
re

46

1

47
48
49
50
51
52
53
54
55
56
57
58

ctor receptor-associated
iated periodic syndrome (TRAPS) is aan autosomal dominantly
Tumor necrosis factor
d autoinflammatory
utoinflammatory disease caused by mutations in TNFR
inheritedTNFRSF1A [1]. Symptoms of TRAPS
ecurrent fever, abdominal
abdomina pain,
ain, myalgia, exant
eexanthema, arthralgia/arthritis, and ocularinclude recurrent
ent.
nt. Clinical features and laboratorylaborato parameters in patients with TRAPS and other
involvement.
mmatorymatory diseases, includi
includautoinflammatory
including systemic juvenile idiopathic arthritis (s-JIA), Kawasaki disease
milial Mediterranean
Medi
Medit
(KD), and familial
fever (FMF), tend to overlap. These diseases share clinicalons such as fever, rash, and arthritis, as well as laboratory findings such as elevatedmanifestations
inflammatory markers. Furthermore, there are no definitive biomarkers for these diseases, making thediagnosis difficult. Genetic analysis of TNFRSF1A can confirm the diagnosis of TRAPS, butinterpretation of the pathogenesis of variants of unknown significance is sometimes required.
Although the pathogenesis of TRAPS remains unknown, low levels of serum soluble tumor necrosis
factor receptor type I (sTNFR-I) in TRAPS patients have been reported [1].

59
60
61
62

In this study, we aimed to demonstrate that the serum sTNFR-I/II ratio may be useful fordifferentiating TRAPS from other autoinflammatory diseases including FMF, s-JIA, and KD. We
measured serum sTNFR-I and sTNFR-II levels in patients with these autoinflammatory diseases andcompared them between each disease.

63

2

64

Original research

65

3Materials and methods

66

3.1

Participants

67
68
69
70

Five TRAPS patients from three families, fourteen FMF patients, 90 s-JIA patients, 37 KD patients,
and 18 healthy controls (HCs) were enrolled in this study. Two patients in one family, who wereported previously [2], had a T50M (p.Thr79Met) heterozygous mutation in TNFRSF1A and two in
another family had a C43R (p.Cys72Arg) heterozygous mutation and one in another family,

In

Article type2

sTNFR-I/II ratio for diagnosis
71
72
73
74
75
76
77
78
79
80
81
82

previously reported [3], had a C30Y (p.Cys59Tyr) heterozygous mutation in the same gene. The
initial diagnosis of two of the patients with T50M or C30Y mutation were s-JIA, while that in one ofthe patients with a C43R mutation was FMF. All FMF patients had a mutation in exon 10 of MEFV
(thirteen patients with M694I, one with M694V). The diagnosis of s-JIA was based on theInternational League of Associations for Rheumatology criteria [4]. The diagnosis of KD was basedon the classic clinical criteria as follows: fever persisting for at least 5 days, changes in extremities(acute phase: erythema of palms and soles, and edema of hands and feet; subacute phase: periungual
peeling of fingers and toes in weeks 2 and 3), polymorphous exanthem, bilateral bulbar conjunctival
injection without exudate, changes in lips and oral cavity (erythema, cracked lips, strawberry tongue,
diffuse injection of oral and pharyngeal mucosae), and cervical lymphadenopathy (≥1.5-cm diameter)[5]. The classic diagnosis of KD was based on the presence of ≥5 days of fever and ≥4 of the fiveprincipal clinical features [5].

83
84
85
86
87
88
89
90
91
92
93
94

The criteria for the active phase of TRAPS, FMF, and s-JIA are defined as follows: fever, rash,
CRP) learthritis, and serositis along with increased serum C-reactive protein (CRP)
levels. The criteria for the
hat can be seen in the active phase as
inactive phase on medication include no clinical symptoms thatollected
lected from three patients w
well as normal CRP levels. Serum samples were collectedwith TRAPS, 8 withhe active phase.
hase. Serum samples wer
a collected from
FMF, 90 with s-JIA, and 33 with KD in thewere alsos-JIA
JIA and 6 pa
five patients with TRAPS, 10 patients with FMF, 33 patients with s-JIA
patients with KD in
he clinical characteristics
cteristics of these patients in the active phasepthe inactive phase. The
are shown in Table
ents with s-JIA and KD had fever, but one
on patient
tient with TRAPS
TR
1. All patientsand one patient with FMF
ever in the active phase. M
ost
st patients with s-JIA
s-JIA
IA and KD had rash, and most patients with
had no fever
Most
se
pa
TRAPS andd FMF had serositis.
Only one patien
patient
with TRAPS was treated with a low dose ofe. The patients with FMF,
FMF KDK and s-JIA in the active phase received no treatments
prednisone.
ednisone, colchcolc
including prednisone,colchicine, immunosuppressants, and biologics.

95
96
97

This study was approved by the Institutional Review Board of Kanazawa University. All
participants provided written informed consent. The study was performed in accordance with the
ethical standards laid down in an appropriate version of the 1964 Declaration of Helsinki.

98

3.2

99
100
101
102

Sera were extracted from blood samples, divided into aliquots, frozen, and stored at −80°C until
analysis. Serum levels of sTNFR-I and sTNFR-II were measured using a commercial enzyme-linked
immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, Inc,
Minneapolis, MN, USA).

103

3.3

104
105
106
107
108

Statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA,
USA). Serum sTNFR-I and sTNFR-II levels and sTNFR-I/II ratio were presented as the median andinterquartile range (IQR). Comparisons between several groups were performed using one-way
analysis of variance with Tukey's multiple comparisons test. A P-value of <0.05 was considered
statistically significant.

109

4

Results

110
111

4.1

Comparison of serum sTNFR-I and sTNFR-II levels and sTNFR-I/II ratio in TRAPS andother autoinflammatory diseases in the active phase

In

w
e
i
v
re

Quantification of serum cytokines

Statistical analysis

3

sTNFR-I/II ratio for diagnosis
112
113
114
115
116
117
118
119
120
121
122

We measured serum sTNFR-I and sTNFR-II levels in patients with TRAPS in the active phase andcompared our findings with those observed in FMF, s-JIA, and KD patients and HCs. As shown in
Figure 1A and Table 2, serum sTNFR-I levels were significantly elevated in the active phase inpatients with s-JIA (median, 2,900 pg/mL; IQR 2,240–3,563) (p<0.0001) and KD (median, 2,400
pg/mL; IQR 1,860–3,160) (p<0.0001) compared with HCs (median, 835 pg/mL; IQR 795–1,083).
Serum sTNFR-I levels were significantly elevated in the active phase in patients with s-JIA
compared with FMF (median, 1,260 pg/mL; IQR 1,113–1,635) (p<0.01) and TRAPS (median, 920
pg/mL; IQR 890–1,000) (p<0.01). However, serum sTNFR-I levels in patients with TRAPS were notelevated compared with those in HCs and were significantly lower compared with those in patientswith s-JIA. Serum sTNFR-I levels in patients with TRAPS were also lower compared with those inpatients with FMF, although this was not statistically significant.

123
124
125
126
127
128
129
130

As shown in Figure 1B and Table 2, serum sTNFR-II levels were significantly elevated in theactive phase in patients with s-JIA (median, 6,250 pg/mL; IQR 4,550–7,963) (p<0.0001) and KD
hose in HCs (median, 3,125(median, 7,250 pg/mL; IQR 4,410–9,390) (p<0.0001) compared with thosenificantly elevated in the active phase in
pg/mL; IQR 2,730–3,775). Serum sTNFR-II levels were significantly
pared
ared with those in FMF patpapatients with s-JIA (p<0.05) and KD (p<0.05) comparedpatients (median, 3,340
II levels inn patients with KD were also
als significantly
pg/mL; IQR 2,375–3,858). Serum sTNFR-II
mpared with those in patients with TRAPS (media(melevated in the active phase compared(median, 3,100 pg/mL;
5).
2,330–3,550) (p<0.05).

131
132
133
134
135

hown in Figure 1C and Table 2, the serum sTNFRAs shownsTNFR-I/II ratio was significantly elevated in theasee in patients with FMF (median, 0.40
active phase0.401; IQR 0.349–0.497) (p<0.05), s-JIA (median, 0.440;
2–0.597)
0.597) (p<0.0001), and K
KD (median, 0.327; IQR 0.240̽0.469) (p<0.05) compared with
IQR 0.342–0.597)
median, 0.275;
0.275 IQR 0.250–0.303), whereas this ratio was not elevated in patients withthat in HCss (median,edian 0.297; IQR 0.282–0.382) compared with HCs.
TRAPS (median,

136
137

4.2

138
139
140
141
142
143
144

We also measured serum sTNFR-I and sTNFR-II levels in patients with TRAPS in the inactive phaseand compared these values with those obtained for patients with s-JIA, FMF, KD and HCs. As shownin Figure 1D and Table 2, serum sTNFR-I levels were significantly lower in the inactive phase inpatients with TRAPS (median, 444 pg/mL; IQR 350–495) compared with those in s-JIA patients(median, 1,040; 685–1,380) (p<0.01) and KD (median, 1,450; IQR 1,238–1,698) (p<0.001). SerumsTNFR-I levels in patients with TRAPS were also significantly lower compared with those in HCs(median, 835; IQR 795–1,083) (p<0.05).

145
146
147
148
149

As shown in Figure 1E and Table 2, serum sTNFR-II levels showed no differences in the inactive
phase among patients with TRAPS (median, 2,580; IQR 1,835–3,350) and FMF (median, 2,130; IQR
1,475–2,638), s-JIA (median, 2,750; IQR 2,090–3,925) , KD (median, 4,475; IQR 3,775–5,250) andin HCs (median, 3,125; IQR 2,730–3,775). Serum sTNFR-II levels in patients with FMF weresignificantly lower compared with those in patients with KD (p<0.01) and HCs (p<0.05).

150
151
152
153

As shown in Figure 1F and Table 2, the serum sTNFR-I/II ratio was significantly lower in theinactive phase in patients with TRAPS (median, 0.156; IQR 0.136–0.224) compared with FMF
patients (median, 0.451; IQR 0.334–0.541) (p<0.01), s-JIA patients (median, 0.379; IQR 0.262–
0.505) (p<0.01). In contrast, serum sTNFR-I/II ratio in patients with FMF and s-JIA was significantly

In

w
e
i
v
re

Comparison of serum sTNFR-I and sTNFR-II levels and sTNFR-I/II ratio in TRAPS andother autoinflammatory diseases in the inactive phase4

sTNFR-I/II ratio for diagnosis
154
155

elevated compared with those in HCs (median, 0.275; IQR 0.250–0.303) (FMF vs HCs, p<0.01; sJIA vs HCs, p<0.05).

156

4.3

157
158
159

As shown in Figure 2A, in the active phase, serum sTNFR-I levels and sTNFR-I/II ratio in patientswith s-JIA and KD were high. In contrast, both values in TRAPS patients were similar to those in
HCs. In patients with FMF, they were mildly elevated and higher than those in patients with TRAPS.

160
161

As shown in Figure 2B, in the inactive phase, serum sTNFR-I levels and sTNFR-I/II ratio inpatients with TRAPS were lower than those in HCs.

162

5

163
164
165
166
167
168
169

In this study, we demonstrated that in the active phase, the serum sTNFR-I/II ratio in patients with sJIA, KD, and FMF was significantly elevated compared with that in HCs, whe
whereas it was not/II rratio in patients withelevated in patients with TRAPS. In the inactive phase, the serum sTNFR-I/II
ared with that in HCs, but was lower
llow in patients with
FMF and s-JIA was significantly higher compared
JIA. From these findings,
ndings, low serum sTNFR
sTN
TRAPS compared with FMF and s-JIA.
sTNFR-I/II
ratio in theor the differential diagnosis of TR
active and inactive phase might be useful for
TRAPS and otherry diseases prior to genetic analysis for TRAPS.
autoinflammatory

170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188

PS
S is an autosomal dominantly inherited autoinflammatory
auto
a
TRAPS
disease caused by mutations in
1A [1]. The pathogenesis of TRAPTNFRSF1A
TRAPS remains unknown and is under investigation. One
xplanation
lanation is the shedding
shedd
sheddi hypothesis [1]. In normal conditions, after activation of thepossible explanation
extracellul
receptor, thee extracellu
extracellular region of TNFR1 is shed from the cell surface by metalloproteases.
fsTNFR-I effectively
neutralizes TNF-α. However, increased cell surface expression of TNFR-I and
decreased plasma levels of sTNFR-I were observed in patients with TRAPS, which induced
increased and prolonged TNFα signaling and decreased inhibition of circulating TNFα. However,further studies demonstrated that cleavage defects were not always observed in association with
TRAPS mutations. Recent studies demonstrated additional signaling defects in patients with TRAPS.
Rebelo et al. showed that mutant TNFR-I may aggregate and be retained in the cytoplasm, resulting
in defective cell surface expression and cell signaling [6]. Furthermore, Lobito et al. revealed thatmutant TNFR-I showed reduced surface expression, which was correlated with downregulated
apoptosis induction and NF-κB signaling [7]. The structurally altered mutant of TNFR-I failed tointeract with the wild-type receptor and formed abnormal self-aggregates that were retained in theendoplasmic reticulum. Misfolding of TNFR-I in the ER induces an inflammatory response throughthe unfolded protein reticulum [8], ligand-independent NFκB activation [9-11], and generation of
mitochondrial reactive oxygen species [12]. This misfolding hypothesis might explain how the
inflammatory phenotype of TRAPS may be associated with the induction of cytokines, such as IL-1β,
due to an unfolded protein response.

189
190
191
192
193
194
195
196

Clinical features and laboratory parameters in patients with TRAPS often overlap with those ofother autoinflammatory diseases, particularly FMF, s-JIA, and KD. Furthermore, there are no
definitive biomarkers for these diseases. This situation makes the clinical diagnosis of these patientsdifficult. Our patients with TRAPS were initially diagnosed with s-JIA or FMF. Furthermore, thepatient with TRAPS diagnosed as FMF had undetermined mutations outside of exon 10 of MEFV.
Thus, genetic analysis is not always the best approach for diagnosing these diseases, particularly inpatients with ambiguous genetic mutations. McDermott et al. reported serum s TNFR-I levels were
not elevated even in the active phase in patients with TRAPS [1]. However, we previously reported

Distribution map of serum sTNFR-II and sTNFR-I/II ratio

Discussion

In

w
e
i
v
re

5

sTNFR-I/II ratio for diagnosis
197
198
199

that serum sTNFR-I levels were significantly elevated in KD and s-JIA [13]. From these findings, wehypothesized serum sTNFR-I levels might be useful for differentiating TRAPS from otherautoinflammatory diseases whose clinical features are similar to TRAPS.

200
201
202
203
204
205
206
207
208
209
210
211
212
213
214

In this study, serum sTNFR-I levels in patients with TRAPS were not elevated even in the active
phase. Furthermore, serum sTNFR-I levels were lower than those in HCs in the inactive phase.
Nonetheless, serum sTNFR-II levels in patients with TRAPS did not differ from those in HCs in boththe active and inactive phase. Although we examined only patients with TRAPS with T50M, C43R
and C30Y mutations of TNFRSF1A, McDermott et al. also reported that serum sTNFR-I levels of
TRAPS patients with C33Y, T50M, C88Y, and C52F mutations of TNFRSF1A in the inactive phasewere lower than those in HCs, and in the active phase they were more elevated than those in theinactive phase, but were not as high as levels in rheumatoid arthritis and systemic lupus
erythematosus. Serum sTNFR-II levels of TRAPS patients with C33Y and C52F mutations in
TNFRSF1A were similar between the active phase and the inactive phase [1]. These findings indicatee, is a ccharacteristic of TRAPS.
that low serum levels of sTNFR-I, both in the active and inactive phase,
uld be considered in suspected TRAPS
Diagnosis of TRAPS is conducted via a genetic test and should
phase compared with other
patients with insufficient elevation of serum sTNFR-II levels in the active phash a significant
nt decrease in these levelautoinflammatory diseases, and also withlevels in the inactive phasecompared with HCs.

215
216
217
218
219
220
221
222
223
224
225
226
227

tudy, serum sTNFR-I
sTNFR
R-I levels in patients with s-JIA
s A werewer significantlys
In this study,
elevated in thehase compared with those in FMF patients. Serum sTNFR-II levels in patients with s-JIA andactive phaseignificantly elevated in the active phase
phas compared with those in FMF patients. The serum
KD were significantly
II ratio in patientspa
p tients
ients with FMF
FM was significantly elevated compared with that in HCs, andsTNFR-I/II
there were noo differences in the ratio between FMF and s-JIA, and KD. These findings indicate bothndd sTNF
sTN
sTNFR-I andsTNFR-II are increased in patients with s-JIA and KD, whereas sTNFR-I is
predominantly increased and sTNFR-II is not increased in patients with FMF. Furthermore, serumsTNFR-II levels in patients with FMF were significantly lower in the inactive phase compared withthose in s-JIA and KD patients. During cell-mediated immune responses, sTNFR-II is mainly shedfrom stimulated monocytic cells and lymphocytes whereas other cells responding to IFN-γ
preferentially shed sTNFR-I [14], but it is unclear why. However, monocytes/lymphocytes might
contribute more to the pathogenesis of s-JIA and KD compared with that of FMF, to which
neutrophils mainly contribute.

228
229
230
231
232
233
234

This study had some limitations. First, the sample number of TRAPS patients was very small. We
measured serum levels of sTNFR-I and sTNFR-II only in TRAPS patients with T50M, C43R and
C30Y mutations in TNFRSF1A. Second, we did not perform a cost-benefit analysis. Third, ingeneral, cytokine measurement by ELISA is limited to the laboratory level. Further studies toevaluate these levels in TRAPS patients with other mutations in TNFRSF1A are necessary. Larger
studies may help to define the true diagnostic value of sTNFR-I and the sTNFR-I/II ratio as clinicalmarkers.

235
236
237
238
239

In conclusion, the serum sTNFR-I/II ratio in TRAPS patients may be a useful indicator for thedifferentiation of TRAPS from FMF, s-JIA, and KD. Particularly, decreased serum sTNFR-I levels
and sTNFR-I/II ratio in the inactive phase and not increased those in the active phase may be usefulin cases of suspected TRAPS. Hence, genetic tests for TRAPS should be considered in patients withthese abnormal findings.

240

6

Conflict of Interest
6

sTNFR-I/II ratio for diagnosis
241

All authors declare that they have no conflict of interest.

242

7

243
244
245
246

All authors were involved in the conception, design of the study and revising it critically forimportant intellectual content. JY and MS and TT were involved in the acquisition of data, analysis
and interpretation of data. JY and MS wrote the manuscript. All authors read and approved the final
manuscript.

247

8

248
249
250

This study was approved by the Institutional Review Board of Kanazawa University and allparticipants provided informed consent, in accordance with an appropriate version of the 1964
Declaration of Helsinki.

251

9

252
253

m funding agencies in the public,
pu
This research did not receive any specific grant fromcommercial, or
not-for-profit sectors

254

10

255
256

We thank
k Harumi Matsukawa for technical assistance. We
W also thank H. Nikki March, PhD, from
Edanz Groupup (www.edanzediting.com/ac) ffor editing a draft of this manuscript.

257

11

258
259
260

1.
McDermott MF, Aksentijevich I, Galon J, McDermott EM, Ogunkolade BW, Centola M, etal. (1999). Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1,
define a family of dominantly inherited autoinflammatory syndromes. Cell. 97, 133–44.

261
262
263

2.
Junko Yasumura, Masakuni Wago, Satoshi Okada, Ryuta Nishikomori, Syuji Takei, Masao
Kobayashi. (2016). A 2-year-old Japanese girl with TNF receptor-associated periodic syndrome: Acase report of the youngest diagnosed proband in Japan. Mod Rheumatol. 26, 798-801.

264
265
266

3.
Akira Manki, Ryuta Nishikomori, Mami Nakata-Hizume, Taiji Kunitomi, Syuji Takei,
Tomoko Urakami, et al. (2006). Tumor necrosis factor receptor-associated periodic syndrome
mimicking systemic juvenile idiopathic arthritis. Allergol Int. 55, 337-41.

267
268
269

4.
Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al. (2004).
International League of Associations for Rheumatology classification of juvenile idiopathic arthritis:
second revision, Edmonton, 2001. J Rheumatol. 31, 390–2.

270
271
272
273

5.
Newburger JW, Takahashi M, Gerber MA, Gewitz MH, Tani LY, Burns JC, et al. (2004).
Diagnosis, treatment, and long-term management of Kawasaki disease: a statement for healthprofessionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council
on Cardiovascular Disease in the Young, American Heart Association. Pediatrics. 114, 1708–33.

274
275

6.
Rebelo SL, Bainbridge SE, Amel-Kashipaz MR, Radford PM, Powell RJ, Todd I, et al.
(2006). Modeling of tumor necrosis factor receptor superfamily 1A mutants associated with tumor

Author Contributions

Ethics statement

FundingIngments
Acknowledgments

erenceenReference

w
e
i
v
re

7

sTNFR-I/II ratio for diagnosis
276
277

necrosis factor receptor-associated periodic syndrome indicates misfolding consistent with abnormal
function. Arthritis Rheum. 54, 2674–87.

278
279
280

7.
Lobito AA, Kimberley FC, Muppidi JR, Komarow H, Jackson AJ, Hull KM, et al. (2006).
Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1
mutants in TNFR1-associated periodic fever syndrome (TRAPS). Blood. 108, 1320–7.

281
282
283

8.
Zhang K, Shen X, Wu J, Sakaki K, Saunders T, Rutkowski DT, et al. (2006). Endoplasmic
reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response. Cell. 124,
587–99.

284
285
286
287

9.
Yousaf N, Gould DJ, Aganna E, Hammond L, Mirakian RM, Turner MD, et al. (2005).
Tumor necrosis factor receptor I from patients with tumor necrosis factor receptor-associatedperiodic syndrome interacts with wild-type tumor necrosis factor receptor I and induces ligandindependent NF-kappaB activation. Arthritis Rheum. 52, 2906–16.

288
289

is facto
10.
Turner MD, Chaudhry A, and Nedjai B. (2012).. Tumour necrosis
factor receptor trafficking
flammatory
tory cytokine secretion. B
Bio Rep. 32, 105–12.
dysfunction opens the TRAPS door to pro-inflammatory
Biosci

290
291
292

bito AA, Bulua AC, Jackson AJ, ete al. (2010). Concerted
11.
Simon A, Park H, Maddipati R, Lobito
ype and mutant TNF
NF receptors enhances inflammation in TNF receptor 1-associatedaction of wild-type
99801–6.
8
periodic fever syndrome. Proc Natl Acad Sci USA. 107, 9801

293
294
295

ickie
kie LJ, Aziz AM, Savic S, Lucherini
Luch
Luche
12.
DickieOM, Cantarini L, Geiler J, et al. (2012). Involvementnding protein 1 and reactiverreof X-box binding
oxygen species pathways in the pathogenesis of tumour
ctor
or recepto
receptor
necrosis factor
receptor-associated periodic syndrome. Ann Rheum Dis. 71, 2035–43.

296
297
298
299

13.
Shimizu M, Yokoyama T, Yamada K, Kaneda H, Wada H, Wada T, et al. (2010). Distinct
cytokine profiles of systemic-onset juvenile idiopathic arthritis-associated macrophage activation
syndrome with particular emphasis on the role of interleukin-18 in its pathogenesis. Rheumatology(Oxford). 49, 1645–53.

300
301

14.
Diez-Ruiz A, Tilz GP, Zangerle R, Baier-Bitterlich G, Wachter H, and Fuchs D. (1995).
Soluble receptors for tumour necrosis factor in clinical laboratory diagnosis. Eur J Haematol. 54, 1–8.

302

12

303
304

12.1 Figure 1. Serum sTNFR-I and sTNFR-II levels and sTNFR-I/II ratio in different patientgroups in the active phase and inactive phase.

305
306
307
308
309
310
311

(A) sTNFR-I and (B) sTNFR-II serum levels and (C) sTNFR-I/II ratio in different patient groups in
the active phase. Serum levels of (D) sTNFR-I and (E) sTNFR-II and (F) sTNFR-I/II ratio in theinactive phase of different patient groups. Bars represent median values. Statistically significant
differences between each patient group are shown as *P<0.05, **P<0.01, ***P<0.001, and****P<0.0001. TRAPS, tumor necrosis factor receptor-associated periodic syndrome; FMF, familial
Mediterranean fever; s-JIA, systemic juvenile idiopathic arthritis; KD, Kawasaki disease; HC,
healthy control.

312

12.2 Figure 2. Distribution map of serum sTNFR-I and sTNFR-I/II ratio.

In

w
e
i
v
re

Figure legend

8

sTNFR-I/II ratio for diagnosis
313
314
315
316

Correlation between serum sTNFR-I levels and sTNFR-I/II ratio in each group in the active (A) andinactive phase (B). TRAPS, tumor necrosis factor receptor-associated periodic syndrome; FMF,
familial Mediterranean fever; s-JIA, systemic juvenile idiopathic arthritis; KD, Kawasaki disease;
HCs, healthy controls.

317

13

318

Table 1. Clinical characteristics and treatment of enrolled patients in the active phase

Table

TRAPS

FMF

s-JIA

KD

Number of patients (n)

3

8

90

33

Median age (IQR)

8 (3–33)

34 (19–45)

1 (0–3.5)

2 (0–3)

w
e
i
v
e
r
In

Sex (M, F)

1, 2

2, 6

47, 4318, 15

Clinical symptoms

Fever

2 (67%)%)

7 (87.5%)(87.5%90
0 (100%)33 (100%)

Ras
Rash

0 (0%)

0 (0%)

67 (74.4%)

28 (84.8%)

Arthralgia/Arthritis
gia/Arthritis
a/Arthritis

1 (33%)

0 (0%)

61 (67.8%)

0 (0%)

Conjunctivitis

1 (33%)

0 (0%)

0 (0%)

29 (87.9%)

Serositis

2 (66%)

8 (100%)8 (8.9%)0 (0%)

10.1 (5.7–15.2)

8.7 (4.2–12.2)

00Laboratory findings
CRP (mg/dL), mean (IQR)

14.1 (6.0–16.1)

3.6 (0.8–13.0)
Treatments

PSL (n) (mg/kg/day)

319
320

1 (0.15)0

IQR, interquartile range; TRAPS, tumor necrosis factor receptor-associated periodic syndrome; FMF, familial Mediterranean
fever; s-JIA, systemic juvenile idiopathic arthritis; KD, Kawasaki disease; CRP, C-reactive protein; PSL, prednisolone
Table 2. Serum sTNFR-I and sTNFR-II levels and sTNFR-I/II ratio in each autoinflammatory diseasesTNFR-I (pg/ml)

sTNFR-II (pg/ml)

sTNFR-I/II ratio

median (IQR)

median (IQR)

median (IQR)N

9sTNFR-I/II ratio for diagnosis

Active phase

TRAPS

3

920 (890–1000)

3100 (2330–3550)

0.297 (0.282–0.382)

FMF

81260 (1113–1635)

3340 (2375–3858)

0. 401 (0.349–0.497)

s-JIA

90

2900 (2240–3563)

6250 (4550–7963)

0.440 (0.342–0.597)

KD

332400 (1860–3160)

7250 (4410–9390)

0.327 (0.240–0.469)

Inactive phase

TRAPS

5

In

FMF

10

s-JIA
JIA
A

33
3

KD

6

w
e
i
v
re
444 (350–495))

2580 (1835(1835–3350)
835–
–335
50)

0.156 (0.136–0.224)

930
0 (557–1185)(557
557–1185)
185)2130 ((1475–2638)
147 –2638)
1475
38

0.451 (0.334–0.541)

1040
40 (685(685–1380)(685–138

2750 (2090–3925)

0.379 (0.262–0.505)

1450 (1238–1698)

4475 (3775–5250)

0.325 (0.322–0.328)

3125 (2730–3775)

0.275 (0.250–0.303)

Healthy controls

HCs

321
322
323

18

835 (795–1083)

IQR, interquartile range; TRAPS, tumor necrosis factor receptor-associated periodic syndrome; FMF, familial Mediterranean
fever; s-JIA, systemic juvenile idiopathic arthritis; KD, Kawasaki disease; HCs, healthy controls; sTNFR, soluble tumor
necrosis factor receptor

10Figure 1.TIF

Figure 2.TIFwi