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DOI: 10.1159/000492965 © 2018 S. Karger AG, Basel

Received: March 9, 2018 www.karger.com/orl
Accepted: August 16, 2018
Published online: October 11, 2018

Research Article

Nasal Symptom Questionnaire:

Our Proposed Scoring System and Prognostic
Factors in Chronic Rhinosinusitis
Takahiro Saito a, b Kenzo Tsuzuki a Hiroki Nishikawa c
      Ken Okazaki a  

Kengo Hashimoto a Masafumi Sakagami a
   

a Department of Otolaryngology-Head and Neck Surgery, Hyogo College of Medicine,

 

Nishinomiya, Japan; b Department of Otolaryngology, Nippon Telegraph and Telephone

 

West Corporation Osaka Hospital, Osaka, Japan; c Center for Clinical Research and
 

Education, Hyogo College of Medicine, Nishinomiya, Nishinomiya, Japan

Keywords
Chronic rhinosinusitis · Nasal symptom questionnaire · Visual analogue scale · Endoscopic
sinus surgery

Abstract
Objectives: We aimed to verify the usefulness of our proposed nasal symptom question-
naire (NSQ) scoring system and to determine predictors linked to the improvement in nasal
symptoms in chronic rhinosinusitis (CRS) patients undergoing endoscopic sinus surgery
(ESS). Patients and Methods: Ninety-one CRS patients undergoing ESS and 144 volunteers
with results of NSQ available were enrolled. The NSQ consists of 10 items. Receiver-operat-
ing-characteristic (ROC) curve, area under the ROC curve (AUC), internal consistency, and
test-retest reproducibility of the NSQ were analyzed. The relationship of the NSQ score with
the visual analogue scale (VAS) was examined. After classifying into two groups, i.e., (i) “im-
provement group” and (ii) “no-improvement group,” factors linked to the improvement in
NSQ after ESS were investigated. Results: ROC-AUC (0.9318), the Cronbach α coefficient
(0.8696), and the test-retest coefficient (0.8131) showed high reliability. NSQ score signifi-
cantly correlated with VAS in both pre- (p < 0.0001, rs = 0.6007) and postoperative stages
(p < 0.0001, rs = 0.5975). The postoperative NSQ scores significantly decreased compared
with baseline levels (p < 0.0001). In multivariate analysis, the computed tomography (CT)
score by the Lund-Mackay scoring system revealed to be significant (p = 0.0481). Conclu-
sion: Our proposed NSQ scoring system was well verified. The CT score can be helpful for
predicting the improvement in nasal symptoms after ESS in CRS patients.
© 2018 S. Karger AG, Basel

Kenzo Tsuzuki, MD, PhD

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Department of Otolaryngology-Head and Neck Surgery

Hyogo College of Medicine
1-1, Mukogawacho, Nishinomiyashi, Hyogo 663-8501 (Japan)
E-Mail kenzo @ hyo-med.ac.jp
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Saito et al.: Our Proposed Scoring System and Chronic Rhinosinusitis

Introduction

Sinonasal diseases can cause various symptoms such as sneezing, nasal obstruction,
nasal discharge, and olfactory loss, and thereby decrease the quality of life (QOL) [1–3]. It is,
therefore, essential to evaluate changes in subjective symptoms and QOL in patients with
sinonasal disease. To evaluate nasal symptoms and QOL, several questionnaires have been
preferably used: the 20-Item Sino-Nasal Outcome Test (SNOT-20) [4, 5], SNOT-22 [6–8] for
sinonasal diseases, the Japan Rhinitis Quality of Life Questionnaire (JRQLQ) [9] for allergic
rhinitis, and the self-administered odor questionnaire (SAOQ) [10] for olfactory dysfunction.
However, in daily clinical practice, there is concern about their complexity due to many
question items in these questionnaires, and a simple evaluation method of nasal symptoms
and QOL has been needed. Therefore, we created a novel simplified nasal symptom question-
naire (NSQ) consisting of 10 items, and we have used the NSQ for evaluating subjective
symptoms in patients with sinonasal diseases since June 2015.
Chronic rhinosinusitis (CRS) is one of the most common chronic diseases in adults and
severely affects QOL in patients [11–14]. CRS results in loss of daily work productivity, and
this is not a trivial amount. Productivity loss from CRS rivals that of other chronic disease
processes such as asthma, diabetes, and heart diseases [15, 16]. CRS is typically classified into
2 types: CRS with nasal polyps (NP) and CRS without NP [17–19]. Clinical features of CRS with
NP with eosinophil-dominant inflammation as “eosinophilic CRS (ECRS)” were first reported
in Japan in 2001 [20]. Clinically, ECRS is associated more closely with CRS with NP, clinical
complaints of nasal obstruction, and olfactory loss, and more frequently linked to comorbid-
ities such as asthma and aspirin hypersensitivity [21–24]. For the treatment of CRS, there is
currently an accepted treatment paradigm first with appropriate medical therapy such as
macrolide therapy and then with surgery if patients are refractory to medical therapy [2, 25,
26]. Endoscopic sinus surgery (ESS) can be indicated in such refractory cases. Postoperative
outpatient intranasal treatments under endoscopy and medication are also very important to
prevent recurring exacerbation [27, 28]. It is useful to estimate and evaluate a postoperative
sinonasal condition using an endoscopic scoring system [29, 30]. However, in daily clinical
practice, we certainly experience several cases whose subjective symptoms do not improve
even in CRS patients undergoing ESS. Thus, clarifying predictors associated with outcome in
CRS patients undergoing ESS is clinically of importance, although several prognostic factors
have already been reported [31–33].
The purpose of this study is to verify the usefulness of our proposed NSQ scoring system
and to determine predictors linked to clinical outcome, findings of nasal endoscopy, olfaction,
and sinonasal imaging in CRS patients treated with ESS.

Patients and Methods

Subjects
Between June 2015 and April 2018, 94 patients with CRS (60 men and 34 women; median age 51.0
years) who underwent ESS at the Department of Otolaryngology, Hyogo College of Medicine, and in whom
both pre- and postoperative NSQ results could be obtained, were enrolled in this study. ESS was performed
by 3 experienced rhinologists [34].
Patients were classified into ECRS (n = 45) and no-ECRS (n = 49) groups based on the Japanese Epide-
miological Survey of Refractory Eosinophilic Chronic Rhinosinusitis Study [20]. The criteria indicate ECRS
for a total score ≥11 from the following 4 items: (i) bilateral lesion (3 points); (ii) NP (2 points); (iii) ethmoid
sinus dominant or pansinusitis on computed tomography (CT) (2 points); and (iv) blood eosinophils > 2%
but ≤5%, 4 points; > 5% but ≤10%, 8 points; and > 10%, 10 points. Median duration of the postoperative
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follow-up was 11.0 months (range, 2.0–24.0 months). The baseline data and the latest postoperative data
were collected for analysis.
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Saito et al.: Our Proposed Scoring System and Chronic Rhinosinusitis

Nasal Symptoms Questionnaire (NSQ)

Below you will find a list of symptoms, difficulty of daily life, and overview. We would like to know
more about these problems. We would appreciate if you could answer the following questions about
your symptoms. Thank you for your participation.
Ⅰ. Symptoms
0. none 1. mild 2. moderate 3. severe
1. Sneezing, nasal itching □ □ □ □
2. Nasal discharge □ □ □ □
Characteristic of the nasal discharge? (watery・thick)
3. Nasal obstruction □ □ □ □
4. Postnasal drip, sputum □ □ □ □
5. Loss of smell □ □ □ □
6. Pain (headache, orofacial pain) □ □ □ □
7. Eye itching, watery eyes □ □ □ □
8. Cough, feeling of irritation in the throat □ □ □ □
Ⅱ. Difficulties in daily life due to the symptoms
1. Problems in daily life (study, work, outings, social life) □ □ □ □
2. Psychosomatic problems (sleep disorder, fatigue, depression) □ □ □ □
III. Overview
How do the nasal symptoms bother you? We would like to ask you to put a tick on the following line.

extremely easy extremely difficult

10cm length
We appreciate your participation
Department of Otolaryngology-Head and Neck Surgery, Hyogo College of Medicine, Hyogo, Japan.

Fig. 1. Our proposed nasal symptom questionnaire scoring system.

As a control group, a total of 144 volunteers (52 men and 92 women, median age 25 years), who were
staff members and students at the Hyogo College of Medicine, had been randomly selected. In the control
group, 121 healthy volunteers (48 men and 73 women, median age 24 years) without any sinonasal diseases
were analyzed to compare with the patients, and 61 volunteers (12 men and 49 women, median age 40 years)
could be retested on a > 5-day interval (median 7 days, ranging from 5 to 8 days) for test-retest reproduc-
ibility (including duplication).

Nasal Symptom Questionnaire

We proposed a novel NSQ scoring system to easily evaluate patients suffering from sinonasal diseases
(Fig. 1). The NSQ is a self-administered survey consisting of 10 items with 2 parts (I–II): (I) 8 items related
to nasal symptoms: (1) sneezing and/or itching of the nose, (2) nasal discharge, (3) nasal obstruction,
(4) postnasal drip and/or sputum, (5) olfactory loss, (6) pain (tooth, buccal, and/or facial pain, and/or
headache), (7) eye itching and/or epiphora, and (8) cough and/or feeling of irritation in the throat; and (II)
2 QOL-related items: (9) reduced productivity at school/work and limitation of outdoor life and/or social
functioning, and (10) sleep problems and general physical and/or emotional problems. Each item of the NSQ
was divided into 4 levels: no symptoms at all (0 points); mild (1 point); moderate (2 points), and severe (3
points). Total points (NSQ score) (ranging from 0 to 30) were analyzed. In addition, the visual analog scale
(VAS) regarding nasal symptoms, consisting of a 10-cm linear scale with the opposing ends of the scale
(extremely easy [0%] and extremely difficult [100%]), which is commonly used both in pre- and postoper-
ative stages, was also applied in the third NSQ part (III).
According to the postoperative change in the total NSQ score, patients were classified into 2 groups:
(i) “the improvement group” when the total NSQ score after ESS decreased 3 points or more compared with
the baseline level, and (ii) “the no-improvement group” when the total NSQ score after ESS decreased 2 points
or less compared with the baseline level. Three patients with preoperative total NSQ score 2 points or less
than 2 points were excluded as unevaluable cases. Consequently, a total of 91 patients were analyzed in this
study (Table 1). We also asked 144 volunteers in the control group whether the question items and numbers
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of the NSQ were appropriate or not, and the results were investigated.
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Saito et al.: Our Proposed Scoring System and Chronic Rhinosinusitis

Table 1. Baseline characteristics (n = 91)

Age, years 51 (13–87)

Gender, males/females 58/33
Bilateral disease, yes/no 65/26
Nasal symptom questionnaire, points 12 (3–25)
Visual analogue scale, points 77 (6–100)
ECRS/no-ECRS 43/48
Presence of bronchial asthma, yes/no 27/64
Presence of nasal septum deviation, yes/no 65/26
Mean recognition threshold 3.9 (0–5.8)
CT score by the Lund-MacKay scoring system, points 10 (0–24)
Polyp score, points 1 (0–4)
Total IgE level, IU/mL 115 (5–2,220)

Data are expressed as numbers or medians (ranges). ECRS, eosinophilic chronic rhinosinusitis; CT,
computed tomography.

Nasal Polyp Score

NP were endoscopically classified into 5 grades according to their size: no polyp (0 points); a single
small polyp in the middle nasal meatus (1 point); several polyps confined to the middle nasal meatus
(2 points); polyps in the common nasal meatus beyond the middle nasal meatus or olfactory cleft polyps
(3 points); and polyps filling the common nasal meatus (4 points) [35]. The NP score can easily evaluate the
change in size before and after ESS.

Olfactory Tests
Olfactory tests were performed using the T&T olfactometer and intravenous olfaction test, both of
which are covered by health insurance and are commonly used for olfactory examination in our country [36].
The T&T olfactory test consists of 5 odorants: (i) β-phenylethyl alcohol, which smells like a rose; (ii) methyl
cyclopentenolone, which smells like burning; (iii) isovaleric acid, which smells like sweat; (iv) γ-undecalactone,
which smells like fruit; and (v) skatole, which smells like garbage (Takasago Industry Ltd., Tokyo, Japan).
Recognition thresholds were determined in each odorant. The average value in these 5 recognition thresholds
was defined as olfactometry function.

CT Score
CT findings were scored in accordance with the Lund-MacKay scoring system [37, 38]. The maxillary,
frontal, anterior, and posterior ethmoid, and sphenoid sinuses on both sides were scored from 0 to 2 points
(partial opacification: 1 point and complete opacification: 2 points). The ostiomeatal complex was scored
from 0 (no opacification) to 2 points (opacification). The total score was considered as the total CT score
(possible range: 0–24 points).

Statistical Analysis
Categorical parameters were compared by the Fisher exact test. Continuous parameters were compared
by paired or unpaired t test, Mann-Whitney U test, or Spearman rank correlation coefficient rs, as applicable.
The pretreatment NSQ scores were analyzed using a conventional receiver-operating-characteristic (ROC)
curve in order to determine the optimal cutoff points that yielded the highest combined sensitivity and spec-
ificity (Youden Index) with respect to distinguishing subjects without any sinonasal diseases from those with
them at the pretreatment stage. The positive and negative predictive values and the area under the curve
(AUC) were also calculated. The Cronbach α coefficient indicating internal consistency, test-retest reproduc-
ibility, known-group difference, and responsiveness to surgery (ESS) were also analyzed in order to validate
the NSQ.
For predicting treatment outcomes (i.e., improvement in NSQ score >3 points or more), candidate vari-
ables were selected by univariate analysis; parameters with p < 0.10 were subjected to a multivariate logistic
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regression analysis. For the purpose of analyzing the significance of predictors in multivariate analysis, vari-
ables analyzed were divided by the median values for all cases (n = 91) and treated as dichotomous covariates.
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Saito et al.: Our Proposed Scoring System and Chronic Rhinosinusitis

1.0

0.8

0.6

Sensitivity
0.4 ROC-AUC: 0.9318
Cutoff value: 4 points
Sensitivity: 0.9341
0.2 Specificity: 0.7769
Positive predictive value: 0.7589
Negative predictive value: 0.9400
Fig. 2. ROC analysis of NSQ. The
ROC-AUC was 0.9318, suggesting 0
0 0.2 0.4 0.6 0.8 1.0
a high diagnostic ability. The opti-
1 – specificity
mal cutoff value was considered
to be reasonable at 4 points.

The following parameters potentially related to the ESS outcome were examined in univariate analyses: age,
gender, presence of bronchial asthma, ECRS or no-ECRS, presence of nasal septum deviation, preoperative
mean recognition threshold, total IgE level, NP score, and CT score by the Lund-Mackay scoring system.
Clinical data were demonstrated as median values (ranges) unless stated otherwise. Statistical significance
was set at p < 0.05. Statistical analysis was performed with JMP software (version 13.0; SAS Institute Inc.,
Cary, NC, USA).

Results

Validation of the NSQ

ROC analysis demonstrated that nasal symptoms can be predicted with a sensitivity of
0.9341 and a specificity of 0.7769 using a cutoff value of 4 points on the NSQ score (Fig. 2).
The ROC-AUC was 0.9318, suggesting an excellent diagnostic ability [39]. The Cronbach α
coefficient indicating internal consistency was of high reliability (0.8696) (n = 61) [40, 41].
The test-retest reliability coefficient was 0.8131 (n = 61). There was a strong significant
correlation between test and retest (n = 61, rs = 0.7706, p < 0.0001) [40, 41]. NSQ scores
showed significant differences between groups of healthy volunteers (1 [0–16]) and patients
(12 [3–25]) (p < 0.0001). Significant differences (p < 0.05) were also confirmed in all 10 items
of the NSQ. Of the 144 volunteers, all (100%) and 141 (97.9%) subjects answered that the
question items and numbers of the NSQ were appropriate, respectively.

Baseline Characteristics in the Patient Group

Baseline data in the patient group (n = 91) are shown in Table 1. There were 58 men and
33 women with a median age of 51.0 years. ERCS was found in 43 patients (47.3%), and no
ERCS in 48 patients (52.7%). The preoperative NSQ score ranged from 3 to 25 (median, 12),
while the preoperative VAS score ranged from 6 to 100 (median, 77). The preoperative
median NSQ score in the ERCS group was significantly higher than that in the no-ECRS group
(11 [0–25] vs. 9 [0–19], p = 0.0425). Particularly ECRS patients showed high scores regarding
olfaction loss. There were 70 of 91 patients (76.9%) in the improvement group and 21 (23.1%)
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in the no-improvement group.

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Color version available online

n = 91 n = 91
100 100

80 80

60
60
VAS

VAS
40
40
20
20 rs = 0.6007 rs = 0.5975
p < 0.0001 0 p < 0.0001
0
5 10 15 20 25 0 5 10 15 20
a NSQ b NSQ

Fig. 3. The relationship between NSQ and VAS in the pre- (a) and postoperative (b) stages.

Color version available online

25
n = 91
p < 0.0001
20

15
NSQ score

10

Fig. 4. Changes in NSQ scores af- 5

ter ESS. For all cases, the median

(range) postoperative NSQ scores 0
(4 [0–18]) significantly decreased
Pre-ESS Post-ESS
compared with baseline levels
(12 [3–25], p < 0.0001).

Relationship between NSQ Score and VAS Score

Total NSQ and VAS scores showed significant positive correlations in both pre- (n = 91,
p < 0.0001, rs = 0.6007; Fig. 3a) and postoperative stages (n = 91, p < 0.0001, rs = 0.5975; Fig. 3b).
In ECRS, total NSQ and VAS scores displayed significant positive correlations in the pre- (n = 43,
p = 0.0005, rs = 0.5069) and postoperative stages (n = 43, p < 0.0001, rs = 0.6306). In the no-ECRS
group, total NSQ and VAS scores also revealed significant positive correlations in the pre- (n =
48, p < 0.0001, rs = 0.6514) and postoperative stages (n = 48, p < 0.0001, rs = 0.5792).

Changes in NSQ Scores after ESS

For all cases, the median postoperative NSQ scores (4 [0–18]) significantly decreased
compared with baseline levels (p < 0.0001; Fig. 4). The median postoperative NSQ scores in
the ECRS group (6 [0–18]) significantly decreased compared with baseline levels (p < 0.0001).
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The median postoperative NSQ scores in the no-ECRS group (3 [0–18]) also significantly
decreased compared with baseline levels (p < 0.0001).
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Table 2. Univariate analysis for the improvement in nasal symptom questionnaire scores after endoscopic
sinus surgery

Improvement No-improvement p value

group (n = 70) group (n = 21)

Age, years 51 (13–73) 52 (25–87) 0.5237

Gender, males/females 47/23 11/10 0.3007
ECRS or no-ECRS 32/38 11/10 0.6261
Presence of bronchial asthma, yes/no 22/48 5/16 0.5939
CT score by the Lund-Mackay scoring system 11 (0–24) 8 (1–18) 0.0429
Polyp score 1 (0–4) 1 (0–3) 0.0478
Presence of nasal septum deviation, yes/no 51/19 14/7 0.5909
Total IgE level, IU/mL 125 (5–2,220) 72.2 (8.6–1,900) 0.4803
Average recognition threshold 4.4 (0–5.8) 2.1 (0.2–5.8) 0.0687

Data are expressed as numbers or medians (ranges). ECRS, eosinophilic chronic rhinosinusitis; CT,
computed tomography.

Table 3. Multivariate analysis of factors linked to the improvement in the nasal symptom questionnaire after
endoscopic sinus surgery

Hazard ratio 95% CI p value

Polyp score ≥1 point 3.369 0.510–7.406 0.2520

Average recognition threshold ≥3.9 4.201 0.299–5.912 0.7753
CT score by the Lund-Mackay scoring system ≥10 points 6.837 1.027–25.044 0.0481

CI, confidence interval; CT, computed tomography.

Uni- and Multivariate Analyses of Parameters Contributing to the Improvement in NSQ

Scores after ESS
Univariate analysis identified the following factors as those with statistical or marginal
significance associated with the improvement in NSQ scores after ESS: polyp score (p =
0.0478), CT score by the Lund-Mackay scoring system (p = 0.0429), and average recognition
threshold (p = 0.0687) (Table 2). The hazard ratios and 95% confidence intervals calculated
using multivariate analysis for the 3 parameters (p < 0.10) in univariate analysis are
presented in Table 3. Only CT score was significantly different in multivariate analysis of
parameters contributing to the improvement in NSQ scores after ESS (hazard ratio = 6.837,
p = 0.0481).

Proportion of Patients with Improvement in NSQ Scores after ESS Stratified by

Preoperative CT Score
The proportion of patients with improvement in NSQ scores after ESS in patients with
preoperative CT score >10 is significantly higher than that in patients with preoperative CT
score <9 (85.1% [40/47] vs. 68.2% [30/44], p = 0.0474; Fig. 5).
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% p = 0.0474
100
85.1%
40/47
80
68.2%
30/44
60

40

20
Fig. 5. Proportion of patients with
improvement in NSQ after ESS
stratified by preoperative CT 0
CT score 10 or more CT score 9 or less
score by the Lund-Mackay scor-
ing system.

Discussion

In this study, we aimed to elucidate the usefulness of our proposed NSQ scoring system
and to determine predictors associated with the improvement in nasal symptoms in CRS
patients undergoing ESS. As mentioned earlier, several questionnaires have been proposed
and validated [4–10]. However, due to their complexity, they may not be easily manageable,
and it may be difficult to completely perform all questionnaires in daily clinical practice. Thus,
in our proposed NSQ scoring system, questions about symptoms were narrowed down to 8
items characteristic of allergy- and sinusitis-related symptoms, and together with the 2 items
on QOL, the total was set to 10 items. In our data, almost all volunteers answered that the
question items and numbers of the NSQ were appropriate. The NSQ and VAS scores showed
significant positive correlations in both pre- and postoperative stages. These results denoted
that our proposed NSQ scoring system is simple and useful to evaluate nasal symptoms in CRS
patients.
According to a judgment criterion of the ROC analysis, the ROC-AUC of 0.9318 could be
evaluated as excellent accuracy [39]. The cutoff value was clinically considered to be
reasonable at 4 points on the NSQ scale with a sensitivity of 0.9341 and a specificity of 0.7769.
The Cronbach coefficient (α = 0.8696), indicating internal consistency, could be appropriately
considered as high reliability [40, 41]. The test-retest showed a high reproducibility with a
coefficient of 0.8131 and a strong significant correlation between test and retest. Both total
and all 10 NSQ items showed significant differences between groups of healthy volunteers
and CRS patients. These results suggest that the NSQ scoring system could be reliable.
NSQ significantly decreased after ESS. In our experience, VAS also significantly decreased
after ESS both in the ECRS group and no-ECRS group [36]. Thus, our ESS showed favorable
results for CRS patients refractory to medical therapies.
In our multivariate analysis of parameters contributing to the improvement in NSQ after
ESS, only the CT score revealed to be significant, and the proportion of patients with
improvement in NSQ after ESS in patients with preoperative CT score >10 was significantly
higher than that in patients with preoperative CT score <9. In patients with preoperative CT
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score <9, indication for ESS should be carefully reviewed because nasal symptoms may not
improve in such patients. Preoperative CT score significantly correlated with preoperative
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Saito et al.: Our Proposed Scoring System and Chronic Rhinosinusitis

NSQ in this study (n = 91, p = 0.0358, rs = 0.4829). Patients with high CT scores have severe
symptoms, and the range of the sinonasal area to be treated by ESS can be widened in such
patients. Regarding olfactory function, the severity of the olfactory disorder and CT opacifi-
cation correlated significantly with the preoperative stage [38]. A high improvement rate of
75% in anosmic patients could be obtained in olfactory function after ESS [42]. It is considered
that as the extent of the sinonasal area normalized by ESS is wide, symptoms can improve to
a greater extent. Smith et al. [31] reported that preoperative CT scores approached signifi-
cance as being predictive of outcome after ESS for CRS patients. Stewart et al. [43] also demon-
strated that disease severity assessed by a pretreatment CT scan is a strong predictor of
clinical outcome, and CRS patients with higher symptom severity based on CT scans showed
significantly greater improvement. These reports are in line with our current results.
Further multicenter studies will thus be required to confirm the usefulness of the NSQ
score. However, clinical utility of the NSQ as an easy method of estimating the condition of
patients suffering from nasal symptoms was suggested in this study.
In conclusion, ESS can be useful for the treatment of CRS patients refractory to medical
therapies, and the CT score can be a useful predictor of clinical outcome. ESS should be care-
fully reviewed in patients with mild nasal symptoms to improve the patients’ QOL. Finally,
our proposed NSQ scoring system may be a promising scoring system.

Acknowledgment

We would like to thank all volunteers who answered the NSQ. We also gratefully acknowledge the help
of our technical assistants, Ms. Yumi Kida and Mrs. Midori Tanide. This work was partly supported by grants-
in-aid for scientific research (Japan Society for the Promotion of Science, KAKENHI: Nos. JP25462671 and
JP16K11220) from the Japan Society for the Promotion of Science, and the Practical Research Project for
Rare/Intractable Diseases from the Japan Agency for Medical Research and Development (AMED).

Statement of Ethics

The ethical committee meeting in our institution acknowledged our current study protocol (approval
Nos. 1512 and 2298). This study strictly followed all regulations of the Declaration of Helsinki.

Disclosure Statement

The authors have no conflicts of interest to declare.

Author Contributions

Conception and design of the study were developed by K.T., T.S., and M.S. Data analysis and interpre-
tation were performed by T.S., K.O., K.H., and K.T. T.S., K.O., K.H., and K.T. were responsible for the acquisition
of data. T.S., K.T., and H.N. were involved in drafting of the article.

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