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GLAUCOMA
Role of CYP1B1, p.E229K and p.R368H mutations among 120 families
with sporadic juvenile onset open-angle glaucoma
Viney Gupta
1
&Bindu I. Somarajan
1
&Gagandeep Kaur Walia
2
&Jasbir Kaur
3
&
Sunil Kumar
3
&Shikha Gupta
1
&Abadh K. Chaurasia
1
&Dinesh Gupta
4
&
Abhinav Kaushik
4
&Aditi Mehta
1
&Vipin Gupta
5
&Arundhati Sharma
6
Received: 11 August 2017 /Revised: 6 November 2017 / Accepted: 13 November 2017
#Springer-Verlag GmbH Germany, part of Springer Nature 2017
Abstract
Background To determine the frequency of CYP1B1 p.E229K and p.R368H, gene mutations in a cohort of sporadic juvenile
onset open-angle glaucoma (JOAG) patients and to evaluate their genotype/phenotype correlation.
Methods Unrelated JOAG patients whose first-degree relatives had been examined and found to be unaffected were included in
the study. The patients and their parents were screened for p.E229K and p.R368H mutations. The phenotypic characteristics were
compared between probands carrying the mutations and those who did not carry these mutations.
Results Out of 120 JOAG patients included in the study, the p.E229K mutation was seen in 9 probands (7.5%) and p.R368H in 7
(5.8%). The average age of onset of the disease (p= 0.3) and the highest untreated IOP (p= 0.4) among those carrying mutations
was not significantly different from those who did not have these mutations. The proportion of probands with angle dysgenesis
among those with p.E229K and p.R368H mutations was 70% (11 out of 16) in comparison to 65% (67 out of 104) of those who
did not harbour these mutations (p= 0.56). Similarly, the probands with moderateto high myopia among those with p.E229K and
p.R368H mutations was 20% (3 out of 16) in comparison to 18% (18 out of 104) of those who did not harbour these mutations
(p=0.59).
Conclusion The frequency of p.E229K and p.R368H mutations of the CYP1B1 gene is low even among sporadic JOAG patients.
Moreover, there is no clinical correlation between the presence of these mutations and disease severity.
Keywords Glaucoma .Juvenile glaucoma .CYP1B1 .Angle dysgenesis
Introduction
Juvenile onset primary open-angle glaucoma (JOAG), affects
individuals under the age of 40 years [1]. Patients with JOAG
usually have high intraocular pressure (IOP), advanced
glaucomatous optic neuropathy and severe visual field defects
[1]. There is substantial variability in the presentation of the
disease. The prevalence among different populations varies,
and so does the clinical profile of JOAG patients [2–4].
Studies on a few families of JOAG, that led to the dis-
covery of the Myocilin gene, reported the disease to have an
autosomal dominant pattern of inheritance [5–7]. However,
clinically apparent sporadic forms of JOAG have been seen
to be more common in some populations [8,9]. An autoso-
mal recessive pattern of inheritance was also shown to exist
in 8 out of 23 JOAG pedigrees from Iran possibly due to a
higher rate of consanguineous marriages [10]. Higher rates
of autosomal recessive disorders may be seen in popula-
tions where endogamy is more prevalent and a founder
effect plays a large part [11]. In a recent segregation analy-
sis of 176 JOAG pedigrees, we found the disease to be
genetically heterogeneous [9].
Mutations in the CYP1B1 gene alone have been shown to
be responsible for JOAG [8,10,12–15], though defects in
*Viney Gupta
gupta_v20032000@yahoo.com
1
Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India
Institute of Medical Sciences, New Delhi 110029, India
2
Public Health Foundation of India, Gurgaon, India
3
Department of Ocular Biochemistry, All India Institute of Medical
Sciences, New Delhi, India
4
International Centre for Genetic Engineering and Biotechnology,
New Delhi, India
5
Department of Anthropology, University of Delhi, New Delhi, India
6
Department of Anatomy, All India Institute of Medical Sciences,
New Delhi, India
Graefe's Archive for Clinical and Experimental Ophthalmology
https://doi.org/10.1007/s00417-017-3853-0
CYP1B1 have been reported to be the major cause of primary
congenital glaucoma (PCG) [16]. Pathological mutations of
CYP1B1 have been reported in JOAG patients from differ-
ent geographical regions across the world [12,16–19]. The
frequency of CYP1B1 mutation among JOAG was reported
to be higher in Asia, with 85.7% in Saudi Arabian, 32.5% in
Iranian and 23.3% in Indian populations [8,10,12]. A large
study among French primary open-angle glaucoma
(POAG) patients reported CYP1B1 mutations in 4.6% (11
out of 236) of the early onset POAG patients, who were
tested negative for MYOC mutations [20]. The ones
harbouring the CYPB1 mutations were younger in age.
Vincent et al. demonstrated that CYP1B1 acts as a modifier
locus for POAG and together with MYOC mutation expe-
dites the disease progression from adult onset to a juvenile
form by a digenic mode of inheritance [18]. Souzeau et al.
found that among JOAG patients, those with advanced dis-
easeweremorelikelytoharbourCYP1B1 mutations [21].
Most homozygous mutations in the CYP1B1 gene, associ-
ated with JOAG, have been seen among sporadic cases [21].
Given the higher prevalence of sporadic JOAG in our popu-
lation [9], we aimed to explore the association of the two most
common variants in the CYP1B1 gene, i.e. p.E229K and
p.R368H, with JOAG. In this study, we also aimed to analyse
the frequency of these two mutations and to assess their phe-
notypic correlation among clinically apparent sporadic cases
of JOAG.
Material and methods
Selection and clinical evaluation of study subjects
This study included consecutive JOAG patients being follow-
ed up at our centre and whose first-degree relatives, above
10 years of age, were available for examination. All proce-
dures performed involving human participants in this study
were in accordance with the ethical standards of the
Institutional Research Committee of the All India Institute of
Medical Sciences and with the 1964 Helsinki Declaration and
its later amendments or comparable ethical standards.
After taking a written informed consent, family members
of these probands were screened. Sporadic cases were defined
as the ones in which no family member other than the proband
had glaucoma as determined by examining all first-degree
relatives of three generations of the proband.
JOAG was diagnosed as POAG occurring before the age of
40 years. The following criteria were used for ascertaining the
presence of POAG among the probands: Increased IOP >
22 mmHg on at least two occasions in the presence of
glaucomatous neuropathy in one eye with or without visual
field loss consistent with optic nerve damage with wide open
angles on gonioscopy in both eyes without pigmentation
greater than grade 3. The optic disc was considered to be
glaucomatous in the presence of localised or generalised reti-
nal nerve fibre layer defect with thinning of the neuro-retinal
rim or with the presence of a documented decrease in neuro-
retinal rim confirmed on optical coherence tomography
(OCT). A glaucomatous visual field was diagnosed in the
presence of at least a cluster of three or more non-edged points
with a p< 5% sensitivity with at least one having a sensitivity
that occurs in fewer than 1% of the population on the pattern
deviation probability plot, the points being in the expected
location for glaucomatous defect, a pattern standard deviation
(PSD)thatoccursinlessthan5%ofnormalreliablefieldsor
the glaucoma hemifield test indicating abnormality.
Exclusion criteria were those with a history of steroid use,
presence of any retinal or neurological pathology, evidence of
secondary causes of raised IOP (pigment dispersion,
pseudoexfoliation or trauma), those with any pathology de-
tected on gonioscopy such as angle recession, pigmentation of
the angle greater than grade 3 or peripheral anterior synechiae,
those with a corneal diameter > 12 mm and those who had had
any ocular surgery other than for glaucoma before presenta-
tion. Patients whose first-degree relatives were not available
for examination were excluded.
The phenotypic parameters compared among probands in-
cluded the age of onset, the highest untreated IOP in either
eye, the greatest vertical cup disc ratio (VCDR), the worst
mean deviation (MD) in either eye at presentation and pres-
ence of moderate to high myopia. All probands had been
goniophotographed. The presence or absence of angle dys-
genesis was determined as described before [22]. The pres-
ence of goniodysgenesis was compared between those with
and those without mutations. The refractive error was graded
as no/low myopia (<3 D), moderate myopia (3–6 D) and high
myopia (>6 D).
Screening for E229K and p.R368H mutations
Peripheral venous whole-blood samples were collected for
genomic DNA isolation of 120 unrelated JOAG patients and
their parents. The mutation screening was carried out with a
polymerase chain reaction (PCR)-based restriction fragment
length polymorphism (RFLP) assay. Genomic DNA was ex-
tracted from 500 μl of blood as recommended by the QIAamp
DNA Blood Max Kit (QIAGEN, Hilden, Germany). The ex-
tracted DNA was quantified using a NanoDrop ND-1000
spectrophotometer (NanoDrop Technologies, Wilmington,
DE, USA). For mutation analysis, exons 2 and 3 of CYP1B1
were amplified by PCR using primers described previously
[23,24]. The PCRs were performed on a thermal cycler (Bio
Rad, CA, USA) in a total volume of 25 μl containing 200 ng
of genomic DNA, 0.5 μM of each primer, 200 μM dNTPs,
20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 to 3.0 mM
MgCl2, and 1 U of Taq DNA polymerase (AmpliTaq Gold;
Graefes Arch Clin Exp Ophthalmol
ABI). Dimethyl sulfoxide (DMSO) of 10% was used only
with primer sets for exon 2. The cycling conditions were as
follows: first denaturation step of 3 min at 94 °C, 30 cycles of
denaturation (94 °C for 30 s), annealing (primer-specific an-
nealing temperature for 60 s), elongation (72 °C for 40 s), and
a final single elongation step of 10 min. The PCR products
were electrophoresed on 2% agarose gel and visualised using
a video gel documentation system (Gel-Doc 2000; BioRad
Laboratories, Hercules, CA, USA). The PCR aliquots were
digested with corresponding restriction enzymes (MB1
Fermentas,) and cleaved fragments were separated on 8%
polyacrylamide gel. The ethidium bromide stained gel was
visualised and genotyping was done as described previously
to distinguish the wild and mutant type alleles of E229K and
p.R368H [23,24]. The sequencing reactions were performed
using the ABI Big Dye Terminator Cycle Sequencing kit ver-
sion 3.1 (Applied Biosystems Inc., Foster City, CA, USA).
Reactions were carried out in a total volume of 25 μL which
consisted of purified PCR product (1–2.5 ng/μL), 1.5 pmol
primers and ABI Dye Terminator mix. Further, the sequencing
PCR product was cleaned and analysed on an Applied
Biosystems genetic analyser. The RFLP observations were
found to be consistent with the sequencing results.
Statistical analysis
Data was analysed using SPSS (SPSS ver.20 Chicago, IL,
USA). Significance of difference in mean age of onset, IOP,
mean deviation on perimetry and highest VCDR among dif-
ferent groups was determined by an independent ttest. A chi
squared test was used to compare differences among categor-
ical variables. The variables were considered significant if the
pvalue was <0.05.
Results
The study included clinically sporadic JOAG patients whose
first-degree relatives, above the age of 10 years, had been
examined and found to be unaffected. The demographic and
clinical profile of the probands is given in Table 1.
We screened p.E229K and p.R368H mutations in 120 un-
related JOAG patients and their parents. We found the
p.E229K mutation in nine probands (7.5%) and p.R368H in
seven (5.8%). Table 2shows the clinical profile of the pro-
bands who carried these mutations. Co-segregation of
p.E229K and p.R368H mutation was not observed in any of
the patients. The p.E229K mutation was present in heterozy-
gous condition in all nine patients while homozygous
p.R368H mutation was observed in two patients. In all pro-
bands with p.E229K mutation, the mother (unaffected) had a
heterozygous mutation of p.E229K. Among both those with
p.R368H homozygous mutation, both parents carried a
heterozygous p.R368H mutation. In the other five, with het-
erozygous p.R368H, the mutation was seen among the fathers
of the probands.
A comparison of the phenotypic data is shown in Table 3.
The average age of onset of those carrying the mutations was
25.8 ± 8.1 years in comparison to 27 ± 8.59 years among those
without these mutations (p= 0.3). The highest untreated IOP
among those with mutations was 43± 13.69 mmHg compared
to 38 ± 12.3 mmHg among those without these mutations (p=
0.4). The age of onset among JOAG patients with p.E229K
mutations was lower than those without mutations, though the
difference was not statistically significant (p= 0.18).
Similarly, those with p.E229K mutation presented with a
higher untreated IOP than those without this mutation, though
this also was not statistically significant (p= 0.16). On the
other hand, the age of onset and the highest untreated IOP
were similar among JOAG patients with or without
p.R368H mutation.
The proportion of probands with angle dysgenesis among
those with p.E229K and p.R368H mutations was 70% (11 out
of 16) in comparison to 65% (67 out of 104) of those who did
not harbour these mutations (p= 0.56). Similarly the probands
with moderateto high myopia among those with p.E229K and
p.R368H mutations was 20% (3 out of 16) in comparison to
18% (18 out of 104) of those who did not harbour these mu-
tations (p=0.59).
Discussion
CYP1B1 mutations are associated with broad range of clinical
phenotypes in glaucoma. The prevalence of CYP1B1 genetic
variants observed in JOAG patients of various ethnicities
range from 4–85%. The most prevalent CYP1B1 mutations
reported among Indian PCG and POAG patients were
p.E229K and p.R368H [12,17].
We observed a higher frequency of p.E229K and p.R368H
mutations among sporadic JOAG patients compared to other
studies (Table 4)[12,17,18,25–31]. In comparison to previ-
ous studies, our study includes a much larger number, thus
providing a stronger power to understand the association of
these mutations with JOAG. Although p.E229K appears to be
Table 1 Clinical parameters of all JOAG patients
Parameter
Age at diagnosis (mean ± SD) 26.8 ± 8.67 (years)
Male-to-female ratio 106:14
Highest untreated IOP (mean ± SD) 39.2 ± 12.8(mmHg)
Highest VCDR in either eye (mean ± SD) 0.8 ± 0.11
Worst MD in either eye (mean ± SD) −21.31 ± 12.81
VCDR vertical cup disc ratio, MD mean deviation
Graefes Arch Clin Exp Ophthalmol
the more common mutation in our study, this was noted in
only a single JOAG patient in each of the French, Iranian and
Spanish populations [20,27,31]. The p.E229K mutation has
always been reported in heterozygous condition. This muta-
tion was also reported with higher frequency in late-onset
open-angle glaucoma from Pakistan where it was found in
22 of 190 POAG patients [32]. The frequency of p.R368H
mutations among our JOAG patients was more or less similar
to p.E229K mutation. Most studies have reported p.R368H in
a heterozygous condition among JOAG patients [12,17,31].
However, p.R368H was present in a homozygous as well as
heterozygous condition in JOAG patients in our study, as also
in an Iranian study [10].
Although the exact mechanism of CYP1B1 mutation in
causing glaucoma pathogenesis is not fully understood, the
presence of mutations in both PCG and JOAG indicate that
both forms of glaucoma have a common or overlapping
CYP1B1-mediated pathophysiological mechanism. The
CYP1B1 gene is known to catalyse metabolism of retinoic
acid and 17-βestradiol, and both these endogenous com-
pounds are implicated in glaucoma pathogenesis [33]. It is
already known that dysfunction in the retinoic acid and 17-β
estradiol metabolism can lead to PCG and POAG, respective-
ly, which suggests that differential activity of the enzyme cor-
relates with different clinical phenotype (POAG vs. PCG).
Mutations in CYP1B1 result in loss of its enzymatic activity
or relative abundance of the protein [34,35]. Functional stud-
ies have revealed that the p.E229K variant destabilises the
holoenzyme, involving a decline of 25% in the enzyme activ-
ity as compared to wild type and p.R368H variant has reduced
enzymatic activity only declining by about 10% [36]. The
pathogenicity of CYP1B1 mutation could also be influenced
by the background CYP1B1 haplotype. The reduced enzymat-
ic activity associated with some haplotypes may influence the
Table 2 Clinical parameters of JOAG patients with E229K and R368H mutation
Cases Mutation Baseline parameters Goniodysgenesis
Age of onset
(Yrs)
Highest untreated
IOP (mm Hg)
Greatest
VCDR
Worse eye MD Refractive error
E229K
JG10001AK Heterozygous 31 32 0.95 −35 None Present
JG30001AS Heterozygous 32 50 0.90 −33 None Present
JG30030AK Heterozygous 16 58 0.86 −30 None Absent
JG10010A Heterozygous 28 52 0.8 −8.4 Moderate myopia Present
JG10243BKB Heterozygous 26 41 0.67 −0.70 None Absent
JG10152GS Heterozygous 30 30 0.82 −25.0 None Present
JG10039NK Heterozygous 25 28 0.78 −0.80 None Absent
JG30082P Heterozygous 12 50 1.0 −35.0 None Present
JG30038R Heterozygous 7 60 0.89 −35.0 None Present
R368H
JG10097HK Heterozygous 28 20 0.69 −0.40 High myopia Present
JG10237JKC Heterozygous 16 28 0.90 −16.0 None Absent
JG10261LCG Heterozygous 29 59 1 −35.0 None Present
JG10178PR Heterozygous 40 29 0.91 −27.50 High myopia Present
JG30002SG Homozygous 18 60 0.81 −26.22 None Absent
JG30063SPA Heterozygous 40 34 0.61 −0.20 None Present
JG10260BDY Homozygous 30 45 0.91 −17.60 None Present
VCDR vertical cup disc ratio, MD mean deviation
Table 3 Correlation of baseline parameters among JOAG patients with and without CYP1B1 mutations
Age of onset
(years)
pvalue Highest untreated
IOP (mmHg)
pvalue Greatest
VCDR
pvalue Worse eye MD pvalue
E229K (+) vs.
no mutation
23 ± 9.0 vs. 27 ± 8.59 0.18 44 ± 12.19 vs. 38 ± 12.37 0.16 0.84 ± 0.96 vs.
0.80 ± 0.11
0.40 −25.47 ± 13.69 vs.
−21.24 ± 12.75
0.37
R368H (+) vs.
no mutation
28.71 ± 8.6 vs. 27 ± 8.59 0.62 41 ± 19.28 vs. 38 ± 12.37 0.59 0.78 ± 0.13
0.80 ± 0.11
0.46 −17.5 ± 13.39 vs.
21.24 ± 12.75
0.43
Graefes Arch Clin Exp Ophthalmol
baseline activity of the background haplotype which in turn
may have larger impact on pathogenicity of the hypomorphic
allele like p.E229K [17,37].
In the current study, p.E229K was inherited from the
mothers of the probands. In contrast, the p.R368H mutation
was inherited primarily from the father. For both p.E229K and
p.R368H, the parents carrying the mutations were unaffected.
In an interesting report, the mother of a PCG patient, who was
found to be a compound heterozygote for p.E229K and car-
ried a deletion mutation (c.1064–1076del) in CYP1B1, did not
show any symptom of PCG [37]. This incomplete penetrance
could be attributed to two reasons: (i) the possibility that an
impaired but not completely absent ability to process some
component of intermediary metabolism involved in changes
to eye tissues by these mutant forms or (ii) presence of a
dominant suppressor of the open-angle glaucoma phenotype
that may not be linked to CYP1B1 [5,38]. We can presume
that these mutations could function as risk alleles, especially
in the heterozygous state, for early development of glaucoma.
However, we cannot rule out the possibility of the presence of
modifiers or environmental influence or other unknown mu-
tations acting together in causing the disease phenotype. As
with most previous studies,we did not find any co-segregation
of these two mutations in our patient population [12,17].
There is only one reported case of p.E229K in a compound
heterozygous state with p.R368H in a patient with a mild form
of JOAG [31].
Our JOAG patients were all cases who had been proven to
be clinically sporadic after having examined all the first-
degree relatives of a three-generation pedigree. Mutations in
CYP1B1 are known to cause glaucoma as a recessive trait
[10]. However, CYP1B1 mutations are reported in sporadic
JOAG, PCG as well as POAG patients [8,32,39,40]. In a
Saudi Arabian population, 12 out of 14 sporadic JOAG pa-
tients had CYP1B1 mutations and a majority had homozygous
G61E mutations [8]. The G61E mutation is found at a
relatively low frequency in JOAG patients of our population
[17]. It is known that CYP1B1 mutations are associated with
incomplete penetrance of the glaucoma phenotype [41,42]. In
Iranian and Saudi Arabian PCG and JOAG patients, the aver-
age penetrance of CYP1B1 mutations was found to be 50%
[41,43]. This was found to rise as the family members were
followed up over time [43]. The other important parameter
associated with CYP1B1 mutations is its variable expressivity.
Variability in expression extends from mild to an altogether
different phenotype, or sometimes to an Bunaffected^pheno-
type as evidenced in patients with compound heterozygous
p.R368H/R390H or G61E in Iranian and Saudi Arabian pa-
tients [41,43,44]. This variability could be due to a yet un-
known dominant modifier locus, epigenetic factors or other
events modifying eye development.
We found the p.E229K to be associated with an earlier age
of onset and higher untreated IOP though the results were not
significantly different from the larger group of JOAG patients
who did not have CYP1B1 mutations. Acharya et al. also
found the age of onset in 1 patient with JOAG with
p.E229K mutation to be 17 years as compared to another with
p.R368H mutation at 37 years of age [12]. An earlier study
evaluating p.E229K and p.R368H mutation showed p.E229K
to be associated with a more severe phenotype among con-
genital glaucomas [45]. Hollander et al. reported a moderate
goniodysgenesis associated with p.E229K and p.R368H mu-
tations among six PCG patients [46]. However, a more severe
disease was found with p.E229K mutation among Lebanese
and Israeli PCG patients [47,48]. In our study, the prevalence
of goniodysgenesis was similar among those who harboured
the p.E229K and p.R368H mutations and those who did not.
A recent study that attempted a genotype/phenotype correla-
tion with respect to the prevalent mutation among Indian
(p.R368H) and Brazilian (4340delG) PCG patients found that
neither the clinical details nor the demographic variables indi-
cated susceptibility to the mutations [49]. It is thus difficult to
Table 4 Frequency (percentage)
of E229K and R368H mutations
among JOAG and PCG patients
in different studies
Total number of cases
studied
(n) JOAG, (*n) PCG
JOAG PCG Reference
R368H (%) E229K (%) R368H (%) E229K (%)
Current study (n= 120) 5.8 7.5
Egypt (n=98) ––10.5 El-Ashry et al. [25]
South India (n=30, 3.3 3.3 Chakrabarti et al. [17]
South India (*n= 146) *17.8 *4.1 Reddy et al. [26]
East India (n= 34) 2.9 2.9 Acharya et al. [12]
Iran (n=21; *n= 104) 9.5% 4.76 6.3 2.2 Suri et al. [27]
Saudi Arabia (n=27) –––3.7 Badeeb et al. [28]
Australia (n=37) ––2.7 1.3 Dimasi et al. [29]
Spain (n = 21) 4.7 4.7 20 –Milla et al. [25]
Canada (n= 53) 3.8 –––Vincent et al. [18]
Graefes Arch Clin Exp Ophthalmol
phenotypically correlate from the specific genetic mutations
tested. One could hypothesise that reduced penetrance of the
two mutations p.E229K and p.R368H could be due to varia-
tion in allele dosage or copy number, differential allelic ex-
pression or the modifying influence of other genetic variants.
The strength of our study was that we had a large cohort
with a strict JOAG phenotype and that all first-degree family
members had been screened for glaucoma before we consid-
ered the disease to be sporadic. However, among the siblings
and children of JOAG it is difficult to predict if they would
develop the disease at a later age. Also, due to the study lo-
gistics, we could not screen the siblings and the children of the
probands for the mutations in this study, which limits our
ability to truly assess the penetrance and expression patterns
of these mutations. Other limitations were (i) the study repre-
sents only one ethnic population, (ii) we could not analyse the
background haplotype to determine the pathogenic status of
the studied CYP1B1 mutations in our JOAG patients, and that
(iii) other genetic variations in CYP1B1 and MYOC gene were
not screened in the present study.
However, from the study findings we can conclude that
even among sporadic cases of JOAG in our cohort, given
the low prevalence of these mutations, their low penetrance
and lack of a genotype phenotype correlation, screening for
these mutations would not provide any useful information for
counselling. There is a need to investigate other genes and
disease-causing loci that could have greater association with
JOAG.
Funding The Indian Council of Medical Research provided financial
support in the form of funding. The sponsor had no role in the design
or conduct of this research.
Compliance with ethical standards
Conflict of interest All authors certify that they have no affiliations with
or involvement in any organisation or entity with any financial interest
(such as honoraria; educational grants; participation in speakers’bureaus;
membership, employment, consultancies, stock ownership, or other eq-
uity interest; and expert testimony or patent-licencing arrangements), or
non-financial interest (such as personal or professional relationships, af-
filiations, knowledge or beliefs) in the subject matter or materials
discussed in this manuscript.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institu-
tional and/or national research committee and with the 1964 Helsinki
Declaration and its later amendments. Informed consent was obtained
from all individual participants included in the study.
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