Serum endotrophin, a type VI collagen cleavage product, is associated with increased mortality in chronic kidney disease
Serum endotrophin, a type VI collagen cleavage product, is associated with increased mortality in chronic kidney disease
Anthony Fenton 0 1 2 3
Mark D. Jesky 0 1 2 3
Charles J. Ferro 0 1 2 3
Jacob Sørensen 0 1 3
Morten A. Karsdal 0 1 3
Paul Cockwell 0 1 2 3
Federica Genovese 0 1 3
0 Funding: AF would like to acknowledge The Binding Site Ltd (Birmingham, UK) for the funding of his research fellowship. The Binding Site Ltd had
1 Data Availability Statement: Data are available upon request. The data restriction is enforced by the NRES Committee West MidlandsÐSouth Birmingham (contact person: Penelope Gregory , REC Manager, NRESCommittee.westmidlands-
2 Department of Renal Medicine, Queen Elizabeth Hospital , Birmingham , United Kingdom , 2 College of Medical and Dental Sciences, University of Birmingham , Birmingham , United Kingdom , 3 Nordic Bioscience A/S, Herlev Hovedgade, Herlev , Denmark
3 Editor: Abelardo I Aguilera, Hospital Universitario de la Princesa , SPAIN
Patients with chronic kidney disease (CKD) are at increased risk of end-stage renal disease (ESRD) and early mortality. The underlying pathophysiological processes are not entirely understood but may include dysregulation of extracellular matrix formation with accelerated systemic and renal fibrosis. We assessed the relationship between endotrophin (ETP), a marker of collagen type VI formation, and adverse outcomes in a cohort of patients with CKD.
We measured serum ETP levels in 500 patients from the Renal Impairment in Secondary
Care (RIISC) study, a prospective observational study of patients with high-risk CKD.
Patients were followed up until death or progression to ESRD. Cox regression analysis was used to assess the relationship between ETP and risk of adverse outcomes.
During a median follow-up time of 37 months, 104 participants progressed to ESRD and 66
died. ETP level was significantly associated with progression to ESRD (HR 1.79 [95% CI
1.59±2.02] per 10 ng/mL increase; HR 11.05 [4.98±24.52] for highest vs lowest quartile;
both P<0.0001). ETP level was also significantly associated with mortality (HR 1.60 [1.35±
1.89] per 10 ng/mL increase; HR 12.14 [4.26±34.54] for highest vs lowest quartile; both
P<0.0001). After adjustment for confounding variables, ETP was no longer significantly
associated with progression to ESRD but remained independently associated with mortality
(HR 1.51 [1.07±2.12] per 10 ng/mL increase, P = 0.019).
Serum ETP level is independently associated with mortality in CKD. This study provides the basis for further exploratory work to establish whether collagen type VI formation is mechanistically involved in the increased mortality risk associated with CKD.
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. JS, MAK and FG are full-time
employees at Nordic Bioscience A/S, and MAK
holds stocks in Nordic Bioscience A/S. Nordic
Bioscience is a privately-owned, smallÐmedium
size enterprise (SME) partly focused on the
development of biomarkers, including the
endotrophin ELISA used in this study. None of the
authors received fees, bonuses or other benefits
for the work described in the manuscript. The
specific roles of these authors are articulated in the
`author contributions' section.
Competing interests: The Binding Site Ltd
(Birmingham, UK) provided funding for the
research fellowship of AF. JS, MAK and FG are
fulltime employees at Nordic Bioscience A/S, and MAK
holds stocks in Nordic Bioscience A/S. These
associations do not alter our adherence to PLOS
ONE policies on sharing data and materials.
Patients with chronic kidney disease (CKD) are at increased risk of end-stage renal disease
(ESRD) and early mortality [
]. There is growing evidence that dysregulation of extracellular
matrix (ECM) formation is associated with accelerated systemic and renal fibrosis and
modulation of inflammation [
]. However, there is limited information on the events that drive this
process in human CKD.
Collagen type VI (Col6) forms a network of beaded microfilaments in the ECM of most
connective tissues, where it interacts with other ECM molecules and provides structural
support for cells. In addition to a mechanical role, Col6 has cytoprotective functions such as the
inhibition of apoptosis and oxidative damage, and the regulation of cell differentiation and
autophagy [3±5]. Following secretion of Col6 from its producing cell, the C-terminus of its α3
chain is cleaved offÐthis cleaved fragment is known as endotrophin (ETP, also termed
C6), and represents a dynamic read-out of Col6 expression . Col6, as one of the most abun
dant proteins in the glomerular ECM, is upregulated in renal disease [7±12] and we have
recently found that high levels of serum ETP are associated with adverse outcomes in patients
with kidney transplants, including allograft failure (unpublished data). However, it is not
known whether Col6 is dysregulated in CKD and if dysregulation is associated with adverse
To address this, we measured serum ETP as a marker of Col6 expression in a well-characterised prospective CKD cohort. We assessed the relationship between ETP level and clinical outcomes of progression to ESRD and mortality, including adjustment for known risk factors for adverse outcomes in patients with CKD.
Subjects and methods
Five-hundred participants from the Renal Impairment in Secondary Care (RIISC) Study (NCT01722383) were included in the study. The RIISC study is a prospective observational cohort study designed to identify determinants of adverse outcomes in CKD, and detailed methodology has previously been described .
In brief, patients in nephrology clinics in a single renal centre in Birmingham, UK, with
non-dialysis high-risk CKD were invited to participate in the study. High risk CKD was as
defined by the UK National Institute for Health and Care Excellence 2008 CKD guideline [
and comprised one or more of: estimated glomerular filtration rate (eGFR) < 30 mL/min/
1.73m2, or eGFR 30±59 mL/min/1.73m2 with a decline of 5 mL/min/1.73m2/year or 10
mL/min/1.73m2/5years, or a urinary albumin:creatinine ratio (ACR) 70 mg/mmol on three
occasions. Patients were excluded if they had received immunosuppression for
immune-mediated renal disease or if they were on renal replacement therapy (RRT). Patients consented to
follow-up for 10 years, or until death or the initiation of RRT. Ethical approval was granted by
South Birmingham Local Research Ethics Committee (reference: 10/H1207/6). All patients
provided written consent, and the study was conducted in accordance with the Declaration of
All participants had demographic, clinical, and laboratory data collected at recruitment and
during follow-up to produce detailed bio-clinical phenotyping. Comorbidity was recorded by
specific comorbidity and by calculating each individual's age-adjusted Charlson Comorbidity
Index (CCI) . Socioeconomic status (SES) was assessed using the Index of Multiple Deprivation (IMD 2010) Ðan individual was assigned a score and rank according to their postcode; lower scores and ranks indicate greater deprivation. Body mass index (BMI) was
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calculated as body weight (kg) height (cm) squared. Blood pressure (BP) was measured
using the BpTRU fully automated sphygmomanometer (BpTRU Medical Devices, Coquitlam,
BC, Canada), which obtains a series of six BP readings at one-minute intervals following a
five-minute rest period. Mean BP was derived from the average of the second to sixth BP
reading, which has been reported to be comparable to mean daytime BP from 24-hour ambulatory
BP monitoring . Mean arterial pressure (MAP) was calculated using the average systolic
(SBP) and diastolic blood pressure (DBP) and the following equation: DBP + (SBP − DBP]
3). Pulse pressure (PP) was calculated as SBP − DBP.
Data and samples from the six-month study follow-up visit were used for this analysis. No
prior formal sample size calculation was performed; the first 500 patients recruited into the
study who had both serum and urine available from their six-month visit were included; these
visits occurred between April 2011 and September 2014. Participants were followed up from
their six-month visit until death or the initiation of RRT. The last outcome data collection took
place on 3rd March 2016 and patients who had not reached a study end point were censored
on this date.
Serum and urine were processed immediately after collection according to pre-defined standard operating procedures and stored at -80ÊC until analysis. Biochemistry results from the local clinical laboratory were obtained from tests performed in accordance with the current standard of care.
Serum ETP level was measured using a competitive enzyme-linked immunosorbent assay
(ELISA) developed by Nordic Bioscience, Denmark. The monoclonal antibody employed in
the ELISA detects the last 10 amino acids of the α3 chain of Col6 (3168'KPGVISVMGT'3177),
but not the elongated peptide KPGVISVMGTA or truncated peptide KPGVISVMG; the
technical details and characterization of the assay have previously been described [
Serum creatinine measurements were performed on a Roche Modular Analyser using a
blank rated compensated Jaffe reaction, and eGFR was estimated using the creatinine-based
CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation . Urine ACR was measured using a Roche Hitachi 702 analyser.
C-reactive protein (CRP) was measured using the Full Range C-Reactive Protein Kit on a
SPA™ automated PLUS turbidimeter (The Binding Site Group Ltd, Birmingham, UK). The
normal range for CRP is between 0.1 and 9 mg/L, with 90 percent below 3 mg/L [
kappa (κ) and lambda (λ) free light chain (FLC) concentrations were measured by
nephelometry on a Dade-Behring BNTMII Analyser (Siemens AG, Erlangen, Germany) using particle
enhanced high-specificity homogenous immunoassays (Freelite™; The Binding Site Group Ltd,
Birmingham, UK). The normal reference ranges for serum FLC concentrations have been pre
viously described as κ: 3.3±19.4 mg/L and λ: 5.7±26.3 mg/L, with the assay sensitivity being
demonstrated as <1 mg/L. κ and λ FLC concentrations were combined to calculate the
combined serum FLC (cFLC) concentrations.
Statistical analyses were performed using IBM SPSS Statistics for Macintosh, Version 22
(Armonk, NY: IBM Corp, 2013). Baseline characteristics are described as a frequency and
percentage for categorical variables, and as a mean and standard deviation or median and
interquartile range (IQR) for normally and non-normally-distributed continuous variables
respectively. As there were little missing data, complete case analysis was performed.
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Serum ETP was analysed as both a continuous and a categorical variable by dividing the
cohort into quartiles (Q1-4). Differences between ETP quartiles in baseline characteristics
were assessed using Pearson's chi-square for categorical variables, and ANOVA (parametric)
or Kruskal-Wallis test (non-parametric) for continuous variables. ETP, eGFR, CRP, and cFLC
were log transformed before scatter plots and Pearson's correlation coefficients were used to
assess for linear associations.
Kaplan-Meier survival analysis was performed for quartiles of ETP, and mean times to
ESRD and death were estimated from the area under the survival curve. Cox proportional haz
ards regression was used to generate time-to-event data for progression to ESRD and death,
and perform both univariable and multivariable analyses of the association between baseline
characteristics and risk of ESRD and death. Known important confounding factors (eGFR and
ACR) and any other variables with a P<0.1 on univariable analysis were included as candidate
variables in the multivariable analysis, and a forward inclusion strategy was used to build final
multivariable models. When variables were highly correlated, only the most clinically relevant
was included as a candidate variable in the multivariable models: CKD-EPI eGFR was used
and not Cystatin C; of the four blood pressure parameters (SBP, DBP, MAP, and PP), only the
parameter most strongly correlated with the outcome of interest was used as a candidate
variable, viz. MAP for progression to ESRD, and PP for mortality. Results are presented as a
hazard ratio and 95% confidence interval (CI). The effect of adding ETP to a multivariable model
was assessed by the change in chi-square with its associated P value (an increase in chi-square
with a P<0.05 being taken as a significant improvement in model fit).
The primary outcomes of interest were 1) progression to ESRD and 2) death. ESRD was defined as the initiation of RRT (dialysis or renal transplantation) and was captured through the use of a local database of all patients who have commenced RRT. Patient mortality was captured through electronic patient records linked to the Office of National Statistics.
Of the 500 participants, two had an insufficient amount of serum available for analysis, so ETP
levels were measured and analyses performed for 498 participants. No other participants were
excluded from analysis. Baseline demographic, social, clinical, and laboratory data for the
study participants, divided by serum ETP quartile, are presented in Table 1. The cohort was
61.4% male, median age was 64 years (IQR 50±76), and ethnicity was 72.3% white, 18.1%
South Asian, 8.6% black, and 1.0% other. The primary renal diagnosis was
ischaemic/hypertensive nephropathy in 28.4%, glomerulonephritis in 18.5%, diabetic kidney disease in 10.5%,
polycystic kidney disease in 6.4%, and other/uncertain in 36.3%. Median eGFR was 26.5 mL/
min/1.73 m2 (IQR 19.4±34.6), and median ACR was 32.1 mg/mmol (IQR 6.2±128.8). Median
follow-up time, calculated using the reverse Kaplan-Meier method, was 37 months (range
0±58), and the shortest duration of event-free follow-up was 18 months; that is, any
participants followed up for less than 18 months had reached a study end-point.
Median ETP level was 23.1 ng/mL (IQR 16.8±30.1). Participants in each ETP quartile were
similar in terms of gender, ethnicity, and socioeconomic status, as shown in Table 1. However,
patients in higher quartiles were older, more likely to have diabetes mellitus (DM), and had
more comorbidity (as assessed by an age-adjusted CCI of 5). Patients in higher quartiles also
had a higher BMI, higher SBP, and lower DBP. Patients in higher quartiles were more likely to
have ischaemic/hypertensive nephropathy or diabetic kidney disease as their primary renal
disease and less likely to have glomerulonephritis, and also had worse renal function (lower
eGFR and higher cystatin C), higher CRP, and higher serum cFLC. There was no significant
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difference between quartiles in MAP and urinary ACR. ETP levels by primary renal disease are
shown in Fig 1.
After log transformation of both variables, there was a strong linear association between
serum ETP level and eGFR (Pearson's correlation coefficient r = -0.74, P<0.0001), indicating
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Fig 1. Boxplots showing serum ETP levels by primary renal diagnosis. The interrupted line represents
median ETP level for the whole cohort. ETP = endotrophin.
that ETP level increases with decreasing eGFR as shown in Fig 2. ETP level also showed a
strong positive correlation with cFLC (r = 0.67, P<0.0001) and a weak correlation with CRP
(r = 0.25, P<0.0001), as illustrated in Fig 3. There was no significant correlation between ETP
and ACR (r = 0.131, P = 0.07).
Progression to ESRD
One hundred and four (20.9%) participants progressed to ESRD, and the estimated mean time
to ESRD was 48.7 (95% CI 47.0±50.3) months. Univariable Cox regression analysis of baseline
characteristics and their hazard ratio for progression to ESRD is shown in Table 2. ETP level
when analysed as both a continuous variable and by quartiles, was significantly associated with
progression to ESRD. Hazard ratios for an increase in ETP of 5, 10, and 20 ng/mL were 1.34
(1.26±1.42), 1.79 (1.59±2.02), and 3.20 (2.51±4.07), respectively (all P<0.0001), and
participants in the highest quartile had a hazard ratio for progression to ESRD 11-fold that of the
lowest quartile. The number and percentage of patients reaching ESRD in each ETP quartile were
7 (5.6%), 21 (16.9%), 29 (23.2%), and 47 (37.9%) in Q1-Q4, respectively.
Other baseline characteristics significantly associated with increased risk of progression to
ESRD by univariable analysis included South Asian ethnicity, a primary renal diagnosis of diabetic nephropathy or polycystic kidney disease, an age-adjusted CCI score <5, absence of malignancy, higher SBP, higher DBP, higher MAP, higher cystatin C, lower CKD-EPI eGFR, increasing urine ACR, and higher serum cFLC.
After adjusting for eGFR and ACR, ETP was no longer independently associated with pro
gression to ESRD (HR 1.19 [0.98±1.45], P = 0.08). Cox p4 follows:-, owsÐQuartile ing ESRD
in each quartile was as follows: quartile 1 5.6%, Q2riable and by quartiles, is
signifiMultivariable Cox regression using a forward selection procedure produced a final model containing
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Fig 2. Association between renal function and serum ETP level. Scatter plot of ETP by CKD-EPI eGFR
(logarithmic scales), R2 = 0.547.
eGFR, ACR, age, MAP, cFLC, and a renal diagnosis of polycystic kidney disease, as shown in
Sixty-six (13.3%) participants died, and the estimated mean survival time was 52.0 (95% CI
50.6±53.5) months. Univariable analysis of baseline characteristics and their hazard ratio for
risk of death is shown in Table 2. ETP level was significantly associated with mortality when
analysed as both a continuous variable and by quartiles. Hazard ratios for a 5, 10, and 20 ng/
mL increase in ETP were 1.26 (1.16±1.37), 1.60 (1.35±1.89), and 2.55 (1.83±3.57), respectively
(all P<0.0001), and patients in the highest quartile had a 12-fold increased risk of death relative
to the lowest quartile. The number and percentage of participants in each ETP quartile who
died were 4 (3.2%), 12 (9.7%), 20 (16.0%), and 30 (24.2%), and estimated mean survival times
were 56 (54±57), 54 (51±56), 50 (47±53), and 43 (39±47) months, in Q1-4 respectively. A
Kaplan-Meier plot showing survival by ETP quartile is shown in Fig 4.
Other baseline characteristics significantly associated with an increased risk of mortality
were increasing age, DM, cardiovascular disease (ischaemic heart disease [IHD]),
cerebrovascular disease [CVD], peripheral vascular disease [PVD]), an age-adjusted CCI 5, higher SBP,
increased PP, increasing cystatin C, lower eGFR, and higher serum cFLC.
On multivariable analysis, after adjusting for age, eGFR, ACR, co-morbidities, PP and
cFLC, ETP significantly improved model fit (P = 0.030) and remained significantly associated
with mortality (HR 1.51 [1.07±2.12], P = 0.019). A forward selection procedure produced a
final multivariable model for mortality containing age, IHD, ACR, and ETP, as shown in
Table 4. These analyses indicate that ETP remained independently significantly associated
with mortality after adjustment for confounding factors.
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Fig 3. Scatter plots showing the association between ETP and markers of systemic inflammation. a)
ETP by cFLC, R2 = 0.445; b) ETP by CRP, R2 = 0.057. All scales are logarithmic. ETP = endotrophin;
cFLC = combined serum free light chains; CRP = C-reactive protein.
Of the 66 participants who died, the cause of death was available for 58 and was CVD in 17
(29%), malignancy in 16 (28%), infection in 9 (15%), and other in 16 (28%). Median (IQR)
serum ETP was 30.4 (20.5±35.8) for CVD, 24.5 (19.3±29.6) for malignancy, 28.3 (24.9±31.4)
for infection, and 31.4 (25.2±37.0) for other, and was not significantly different when analysed
by cause of death (P = 0.12).
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ESRD = end-stage renal disease; CI = con®dence interval; COPD = chronic obstructive pulmonary disease; CCI = Charlson's comorbidity index;
CKD-EPI = Chronic Kidney Disease Epidemiology Collaboration; eGFR = estimated glomerular ®ltration rate; cFLC = combined free light chains;
ETP = endotrophin; Q = quartile.
aAnalysed in 10-unit increases.
bAnalysed in 5-unit increases.
cAnalysed in 20-unit increases.
PLOS ONE | https://doi.org/10.1371/journal.pone.0175200
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Identifying the dominant biological processes associated with worse outcomes in patients with
CKD will facilitate our understanding of pathophysiology and identify targets that may lead to
improved outcomes, both through stratification and intervention. Tissue fibrosis and
remodelling have been implicated in both the progression of CKD and the increased mortality risk
associated with CKD. However, little is known about the associations between systemic
fibrosis and increased clinical risk in human kidney disease. We analysed serum ETP, a marker of
Col6 formation, in a cohort study of high-risk CKD and found an independent relationship between ETP level and mortality.
There was a significant inverse correlation between eGFR and serum ETP levels, for which there are several possible explanations. Although the molecular weight of ETP is not known,
Fig 4. Kaplan-Meier plot showing survival by ETP quartile. 95% CI for quartiles 1 and 4 are represented
by the grey areas. ETP = endotrophin.
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the correlation may reflect a reduction in renal clearance of ETP as kidney function declines. It
may also reflect increased production of fibrotic tissue, and therefore Col-6 and ETP
generation, in patients with CKD: renal fibrosis is known to be a common pathological pathway in
CKD and may therefore be a contributing source of ETP, but the elevated levels are also likely
to reflect a greater systemic fibrotic burden in patients with more advanced CKD. Our data do
not allow us to determine the relative contributions of these potential explanations for the
correlation described. We also recognise the possibility that the assay detects peptides of different
length that all terminate with the 10 amino acid sequence targeted by the ELISA, among which
Higher ETP levels were associated with both an increased risk of progression to ESRD and
an increased risk of death. In multivariable models including conventional risk factors, ETP
was no longer associated with an increased risk of progression to ESRD. However, the
significant association between ETP and mortality remained, which may suggest a novel
contribution of systemic fibrosis to the increased mortality seen in CKD. Further, ETP was selected
into the final multivariable model for mortality rather than eGFR (one of the most important
prognostic factors for mortality in CKD), indicating superior predictive power in this cohort.
There are several possible reasons for the significant independent association between
serum ETP level and increased mortality risk. In addition to reflecting increased systemic
fibrosis and therefore increased mortality risk, both Col6 and ETP have known adverse effects
which may be associated with reduced survival. For example, Col6 has a major role in platelet
adhesion, which is intimately involved in both atherosclerosis and microvascular pathology.
Col6 binds platelets both directly and via von Willebrand factor (vWF), and of the multiple
subendothelial collagens to which vWF binds, Col6 appears to be especially important [
. Col6 also appears to have deleterious effects in the myocardium: Col6 deletion in
knockout mice is associated with improved cardiac function, structure and remodelling after
myocardial infarction, and in humans with hypertrophic cardiomyopathy, increased levels of Col6
positively correlate with cardiac interstitial fibrosis and dysfunction [
]. The hypothesis
that increased Col6 expression may be linked with an increased risk of cardiovascular death
would have been supported had our data revealed a higher serum ETP level in those who died
from CVD. Although this was not the case, the risk of a type II error is significant given the
small numbers in each category of cause of death.
The adverse effects of ETP that might explain its association with increased mortality
include its pivotal role in shaping a metabolically unfavourable microenvironment in adipose
tissue, where it triggers fibrosis and inflammation and ultimately results in systemic elevation
of pro-inflammatory cytokines, insulin resistance, and the metabolic syndrome [
]. We have
recently demonstrated that elevated ETP levels predict response to insulin sensitizers in
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patients with type 2 DM [
]. Further, ETP has been shown to play a role in promoting
tumour growth and metastasis [
3, 4, 27
It is also possible that the relationship between Col6 expression, ETP level, and increased
mortality is not causal. Rather, ETP may be a confounder, increased in response to factors that
in themselves increase mortality risk. For example, increased ECM deposition may be a
consequence of pathogenic triggers such as hypertension and increased vascular sheer stress;
notably, the presence of Col6 has been demonstrated in the thickened intima of atherosclerotic
lesions in humans [
]. Hypertension, DM, and myocardial infarction are also associated with
increased cardiac Col6 expression [
]. However, the role of Col6 in the myocardial fibrosis
seen in patients with CKD is unknown.
The strengths of this study include it being a large prospective cohort, with detailed bio
clinical phenotyping incorporating multiple risk factors for adverse outcomes. The study's
limitations include it being single-centre, with no validation cohort, and the lack of experimental
data to explore the mechanisms underlying the association between ETP and mortality.
However, this work provides the basis for confirmatory and mechanistic studies in this important
In conclusion, this study shows that serum ETP levels are independently associated with mortality risk in CKD after adjustment for confounding factors.
AF would like to acknowledge The Binding Site Ltd (Birmingham, UK) for the funding of his research fellowship.
Conceptualization: AF MDJ CJF MAK PC FG.
Data curation: AF MDJ CJF JS MAK PC FG.
Formal analysis: AF JS FG.
Funding acquisition: PC MAK FG.
Investigation: AF MDJ CJF MAK PC FG.
Methodology: AF MDJ CJF JS MAK PC FG.
Project administration: AF PC FG.
Resources: AF MDJ CJF MAK PC FG.
Supervision: CJF MAK PC FG.
Writing ± original draft: AF PC FG.
Writing ± review & editing: AF MDJ CJF JS MAK PC FG.
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