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\begin{document}

\title{The Relationship Between Arginase Genes Polymorphisms and Preschool
Wheezing Phenotypes}



\author[1]{pinar Gokmirza Ozdemir}%
\author[1]{Damla Eker}%
\author[2]{velat çelik}%
\author[3]{Burcin Beken}%
\author[2]{Hakan Gürkan}%
\author[2]{Necdet Süt}%
\author[2]{Mehtap Yazicioglu}%
\affil[1]{Trakya University}%
\affil[2]{Trakya University Faculty of Medicine}%
\affil[3]{Trakya Universitesi}%


\vspace{-1em}



  
  \date{May 19, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Background: The association between arginase I (ARG1) and arginase II
(ARG2) genes and asthma has been reported in previous studies, but
associations between polymorphisms in ARG genes and preschool wheezing
(PSW) phenotypes are unknown. Objective: To examine the association
between genetic variation in ARG1 and ARG2 genes and PSW phenotypes and
to compare results with asthmatic patients. Methods: We enrolled 102
patients and 86 healthy controls. The patient group included three
subgroups: episodic wheezing (EW) (n = 42, median age 41 months),
multiple-trigger wheezing (MW) (n = 41, median age 39 months), and
asthma (n = 19, median age 72 months). We genotyped six single
nucleotide polymorphisms (SNPs) in ARG1 and six SNPs in ARG2. Eighteen
haplotypes for ARG1 and 31 haplotypes for ARG2 were constituted, and the
distributions of SNPs and haplotypes in patients and controls were
analyzed. Results: The frequency of homozygote cytosine-cytosine
genotype of the ARG1 rs2781667T\textgreater{}C SNP in the EW group was
significantly lower than controls (p = 0.006) {[}OR (95\% CI): 0.26
(0.10-0.66){]}, the MW group (p = 0.002) {[}OR (95\% CI): 0.19
(0.06-0.52){]}, and asthmatics (p = 0.025) {[}OR (95\% CI): (0.22
(0.06-0.75){]}. ARG1 haplotype 4 was more frequent in the MW group,
asthmatics, and controls than in the EW group (p \textless{} 0.0001)
{[}OR (95\% CI): 7.77 (2.54-23.7){]}, (p = 0.036) {[}OR (95 \%CI): 4.31
(1.15-16.15){]}, and (p = 0.030) {[}OR (95\% CI): 3.44 (1.20-10.0){]}.
Conclusion: Variations in ARG1 gene may be useful in discriminating PSW
phenotypes.%
\end{abstract}%



\sloppy


\textbf{MAIN TEXT}

\section*{INTRODUCTION}

{\label{introduction}}

Preschool wheeze (PSW) is a highly prevalent clinical
problem\textsuperscript{1} that presents within a wide spectrum of
severity and is associated with considerable healthcare
costs\textsuperscript{2}. Asthma has its origins in early childhood;
however, different patterns of childhood wheezing vary in their
association with subsequent asthma, atopy, and bronchial
hyper-responsiveness. Wheezing phenotypes used in epidemiological
studies are frequently defined retrospectively and are therefore not
usable in clinical practice\textsuperscript{3}. In 2008, the European
Respiratory Society's preschool study group divided wheezing phenotypes
into episodic (viral) wheeze (EW) and multiple-trigger wheeze
(MW)\textsuperscript{4}. According to this classification, wheezing
attacks triggered only by viral respiratory tract infections with no
complaints between attacks are classified as EW. Wheezing attacks
triggered by allergens, exercise, cold weather conditions, or smoke, in
addition to viral respiratory tract infections, with complaints between
attacks are classified as MW\textsuperscript{4}. However, wheeze
patterns in young children vary over time and with treatment, rendering
the distinction between EW and MW unclear in many
patients\textsuperscript{5}. Therefore, we need predictive markers that
are useful for differentiating between wheezing phenotypes and
predicting asthma development in children with PSW as a guide for
clinicians in early treatment decision-making.

There has been increasing interest among researchers to find a
relationship between different genes and wheezing phenotypes and
persistence and the development of childhood
asthma\textsuperscript{6,7}. Arginase (\emph{ARG} ) genes have been
investigated in this context. Arginase, an essential enzyme in the
hepatic urea cycle, is involved in L-arginine homeostasis by catalyzing
the reaction in which L-arginine is converted to L-ornithine and urea.
Arginase is also expressed in nonhepatic tissues, including in the
airways. In humans, two arginase isoenzymes have been identified,
arginase 1 and arginase 2, and both are constitutively expressed in the
airways. Arginase and nitric oxide synthases (NOS) compete for their
common substrate, L-arginine. Increased arginase expression and low
L-arginine levels have been found to be associated with asthma and
airway remodeling\textsuperscript{8-12}. After defining the important
contributions of arginase in asthma, genetic studies have attempted to
identify an association between genetic variation in \emph{ARG} genes
and susceptibility for asthma, atopic sensitization, and bronchodilator
response (BDR) in different populations\textsuperscript{13-21}. These
studies have reported many associations between different single
nucleotide polymorphisms (SNPs) in \emph{ARG1} or \emph{ARG2} genes and
atopic sensitization\textsuperscript{13, 15}, risk of
asthma\textsuperscript{13, 16,21}, asthma severity\textsuperscript{16},
bronchodilator/steroid response in asthma\textsuperscript{14, 16,
19,20}, and the arginase-NOS pathway \textsuperscript{17,18}, although
some of these results have not been replicated.

To date, associations between polymorphisms in \emph{ARG} genes and PSW
phenotypes are unknown. Therefore, this study aimed to investigate
whether there is any relationship between polymorphisms in
\emph{ARG}genes and PSW phenotypes among Turkish children and compare
these results with those of asthmatic children over five years old.



\section*{METHODS}

{\label{methods}}

\subsection*{Study population}

{\label{study-population}}

This cross-sectional study was conducted at the Pediatric Allergy
Department of Trakya University between May 2017 and May 2019 and
included 102 patients and 86 healthy controls.

The patient group included three subgroups: EW (n = 42; median age 41
months), MW (n = 41; median age 39 months), and asthma (n = 19; median
age 72 months). Asthma diagnoses were based on the Global Initiative for
Asthma (GINA) guidelines\textsuperscript{22}. Wheezing phenotypes in
young children were discriminated, as proposed by the European Task
Force\textsuperscript{4}. The control group included 86 (median age 20
months) non-allergic, unrelated, healthy outpatients with no history of
recurrent wheezing and no individual or family history of allergic
illness (e.g., asthma, atopic dermatitis, allergic rhinitis, allergic
rhinoconjunctivitis, and food allergy). The inclusion criteria were the
presence of [?] 4 EW or MW episodes in a year for children up to five
years of age or having asthma for children over five years of age.
Patients with any other chronic disease or wheezing-associated diseases,
such as cystic fibrosis, ciliary dyskinesia, anatomic abnormalities, and
immunodeficiency disorder, and preterm-born children were excluded from
the study.

Study forms were completed by pediatric allergists during patient visits
at our pediatric allergy outpatient clinic. These forms included
demographic data (i.e., age and gender) tobacco exposure, wheezing or
asthma exacerbations per year, history of atopic dermatitis, presence of
allergic rhinitis symptoms (i.e., nasal congestion, nasal itching,
rhinorrhea, and sneezing), parental asthma, or other allergic diseases.

\subsection*{Laboratory tests}

{\label{laboratory-tests}}

Serum total IgE levels in the patient group were measured by
chemiluminescent immunometric assay (AU5800; Beckman Coulter~Inc., CA,
USA). Complete blood count analysis was performed by an automatic
analyzer (UniCell DxH 800 Coulter Cellular Analysis System; Beckman
Coulter Inc.) within one hour of blood sampling. The original
manufacturer's kits were utilized for the laboratory tests.

\subsection*{Skin tests}

{\label{skin-tests}}

Aeroallergen and food allergen sensitivity were determined using a skin
prick test. The following aeroallergens, common in Turkey, and food
allergens were used: Dermatophagoides pteronyssinus and farinae, Felis
domesticus, Canis familiaris, Blattella germanica, Dactylis glomerata,
Festuca pratensis, Poa pratensis, Lolium perenne, Phleum pratense),
Corylus avellana, Betula verrucosa, Alnus glutinosa, Artemisia vulgaris,
Chenopodium album, Plantago lanceolata, Parietaria judaica, Alternaria
alternata, Aspergillus fumigatus, Cladosporium herbarum, Penicillium,
milk, egg white, egg yolk, wheat, hazelnuts, peanuts, walnuts, almond,
and cacao (ALK-Abell\selectlanguage{ngerman}ó, Madrid, Spain). Histamine was used as a positive
control and saline as a negative control. Skin reactions were evaluated
15 minutes after the skin test. A positive reaction was defined as wheal
diameter \selectlanguage{english}[?] 3 mm.

\subsection*{Genetic analyses}

{\label{genetic-analyses}}

DNA isolations and real-time PCR studies were carried out in the Genetic
Diseases Diagnosis Center's Molecular Genetics Laboratory of Trakya
University Medical Faculty.

\subsection*{SNPs selection}

{\label{snps-selection}}

We searched PubMed and open-access databases\textsuperscript{10-12} for
the keywords ``arginase, asthma genetics, atopy, asthma severity, SNP,
bronchodilator response.'' SNPs in \emph{ARG1} and \emph{ARG2} were
found associated with an increased risk of atopic sensitization, risk of
asthma, asthma severity, and bronchodilator/steroid response in asthma
in previous studies\textsuperscript{13,16,20}\textsubscript{.} These
were selected for investigation in our study.

\subsection*{Genomic DNA isolation}

{\label{genomic-dna-isolation}}

Peripheral blood samples from both the patient and control groups were
collected using tubes containing EDTA. Genomic DNA isolation from
peripheral blood samples was performed using a PureLink\selectlanguage{english} Genomic DNA
Mini Kit (Invitrogen\selectlanguage{english}, Catalog number:~K182001; ThermoFisher Scientific)
according to the optimized protocol specified in the manufacturer's
manual. Quality control and the purity of the isolated genomic DNA
samples were determined using a DNA spectrophotometer (NanoDrop 2000C;
ThermoFisher Scientific Inc., MA, USA). High-quality samples that had
A260/280 values of 1.8--2.0 were included in the study, while
low-quality samples were re-isolated from the blood samples.

\subsection*{Genotyping with real-time
PCR}

{\label{genotyping-with-real-time-pcr}}

The real-time polymerase chain reaction (RT-PCR) method was used for
allelic discrimination for the genotyping. The discrimination of the
genotypes in the SNPs was performed using a PCR machine (Applied
Biosystems StepOnePlus\textsuperscript{TM} Real-Time PCR system;
ThermoFisher Scientific) and
TaqManAssays\textsuperscript{(r)}(ThermoFisher Scientific). The RT-PCR
reaction was performed according to the manufacturer's recommended
protocols for six SNPs in the\emph{ARG1} gene and six \emph{SNPs} in the
\emph{ARG2} gene, respectively (rs2781668, rs3756780, rs2781659,
rs2781665, rs2781667, and rs2246012 and rs3759757, rs742869, rs3742879,
rs17249437, rs3742880, and rs6573788) (see Figure 1).

Each PCR reaction was prepared in a 20 \selectlanguage{greek}µ\selectlanguage{english}l total volume of 10 \selectlanguage{greek}µ\selectlanguage{english}l
2xPrecision MasterMix, 1 \selectlanguage{greek}µ\selectlanguage{english}L TaqManAssay, 4 \selectlanguage{greek}µ\selectlanguage{english}L RNAse/DNAse free water,
and 5 \selectlanguage{greek}µ\selectlanguage{english}L genomic DNA. The PCR conditions were 95 \selectlanguage{ngerman}°C for eight minutes
for enzyme activation, 95 °C for 10 s for denaturation, and 60 °C for 60
s for the first extension in 10 cycles followed by 95 °C for 10 s for a
second denaturation and 68 °C for 60 s for the second extension in 35
cycles. The ROX\textsuperscript{TM} channel and
VIC\textsuperscript{TM}channel of the RT-PCR machine were coded for the
wild-type probe or mutant-type probe with sequences determined by the
TaqManAssay\textsuperscript{®} manufacturer. According to the signals
from the canals, the SNPs were considered either homozygous mutant,
homozygous wild, or heterozygous. Fluorogenic data for each of the 12
polymorphisms were collected through the ROX and VIC channels at the end
of each cycle of the second extension and determined individually.

\subsection*{Haplotype analysis}

{\label{haplotype-analysis}}

After determining the homozygote and heterozygote genotypes for the
selected SNPs in \emph{ARG1} and \emph{ARG2} , 18 haplotypes
for\emph{ARG1} and 31 haplotypes for \emph{ARG2} were constituted. The
GVSc names and H19 localizations of the selected SNPs and haplotypes in
the\emph{ARG1} and \emph{ARG2} genes are shown in Figure 2.

\subsection*{Ethical considerations}

{\label{ethical-considerations}}

This study was approved by Trakya University's ethical committee
(approval number 2016/264). Written informed consent was obtained from
either parents or children more than seven years old.

\subsection*{Statistical analyses}

{\label{statistical-analyses}}

Mean, standard deviation, median, minimum, maximum, and number
(percentage) values were used for the descriptive statistics. Variable
distribution was checked using the one-sample Kolmogorov--Smirnov test.
Kruskal--Wallis and Mann--Whitney U tests with Bonferroni correction
were used for a comparison of the quantitative data. Comparisons of
genotype and haplotype frequencies and other categorical data were
performed using Pearson's chi-squared test or Fisher's exact test when
the sample size was small. The Hardy--Weinberg equilibrium test was used
to calculate genotype frequencies. \emph{P} -values \textless{} 0.05
were considered statistically significant.~IBM SPSS Statistics for
Windows, Version 20.0 (Armonk, NY: IBM Corp) was used for the
statistical analyses.



\section*{RESULTS}

{\label{results}}

\subsection*{Demographic characteristics and laboratory
results}

{\label{demographic-characteristics-and-laboratory-results}}

The mean age of the total patient group was 46.3 ± 25.9 months (median
46), and the mean age of the control group was 23.9 ± 13.4 months
(median 20) (\emph{p} \textless{} 0.001). In the patient group, 37
(36.3\%) of the cases were female, and 35 (40.7\%) of the patients in
the control group were female; (\emph{p} = 0.534). A comparison of the
demographic characteristics and laboratory results of the patient
subgroups is shown in Table 1.

\subsection*{Genetic workup with real-time
PCR}

{\label{genetic-workup-with-real-time-pcr}}

\subsubsection*{1. Genotype analysis of the studied
SNPs}

{\label{genotype-analysis-of-the-studied-snps}}

1a. Comparison of total patient group and controls

{\label{a.-comparison-of-total-patient-group-and-controls}}

The genotypes were grouped as homozygote, heterozygote, or wild. After
determining the genotypic distribution, we performed a genetic
comparison according to the autosomal recessive inheritance pattern of
the \emph{ARG1} and \emph{ARG} genes between the patients, controls, and
patient subgroups. There was no difference between the total patient
group and the healthy controls in terms of the homozygote genotype of
the SNPs in the \emph{ARG1} and \emph{ARG2} genes (Table 2).

1b. Comparison of patient subgroups

{\label{b.-comparison-of-patient-subgroups}}

The homozygote cytosine-cytosine (CC) genotype of the
\emph{ARG1}rs2781667T\textgreater{}C SNP in the EW group was
significantly lower than for the controls (\emph{p} = 0.006) {[}OR (95\%
CI): 0.26(0.10--0.66){]}, the MW group (\emph{p} = 0.002) {[}OR (95\%
CI): 0.19(0.06--0.52){]}, and the asthmatics (\emph{p} = 0.025) {[}OR
(95\% CI): 0.22(0.06--0.75){]}. There was no difference in the
distribution of the other studied SNPs in the \emph{ARG1} and
\emph{ARG2} genes between the patient subgroups and the controls (Table
2).

1c. Association between the SNP analysis and allergic rhinitis,
aeroallergen sensitivity, number of aeroallergens detected as positive
in the skin prick test, and tobacco exposure

{\label{c.-association-between-the-snp-analysis-and-allergic-rhinitis-aeroallergen-sensitivity-number-of-aeroallergens-detected-as-positive-in-the-skin-prick-test-and-tobacco-exposure}}

The homozygote CC genotype of the \emph{ARG1} rs2781667T\textgreater{}C
SNP was more frequent in patients with AR than in those without AR
(49.1\% and 21.3\%, respectively, \emph{p} = 0.007) {[}OR (95\% CI):
3.56(1.48--8.56){]}.

There was no difference in the presence of aeroallergen sensitivity, the
number of aeroallergens detected as positive in the skin prick test,
tobacco exposure, and homozygote genotype distribution of any SNPs among
all the patient groups.

\subsubsection*{2. Haplotype analysis}

{\label{haplotype-analysis-1}}

2a. Comparison of total patient group and controls

{\label{a.-comparison-of-total-patient-group-and-controls-1}}

There was a statistically significant difference in terms of \emph{ARG}
1 haplotype 5 (CT/T/G/T/T/CT) and \emph{ARG} 1 haplotype 9 (C/T/G/T/T/T)
between the healthy controls and the patient group. The frequency of
the\emph{ARG1} haplotype 5 in the patient group was 9.8\% and 1.2\% in
the control group (\emph{p} = 0.028) {[}OR (95\% CI):
9.23(1.15--73.70){]}. The frequency of \emph{ARG1} haplotype 9 in the
patient group was 2\% and 11.6\% in the control group (\emph{p} = 0.016)
{[}OR (95\% CI): 0.15(0.03--0.71){]}. There was no difference between
the patient groups and the controls in terms of other haplotypes in the
\emph{ARG1} and\emph{ARG2} genes (see Tables 3A and 3B).

2b. Comparison of patient subgroups

{\label{b.-comparison-of-patient-subgroups-1}}

The frequency of the \emph{ARG1} haplotype 2 (CT/T/AG/AT/CT/CT) was
significantly higher in the EW group than in the healthy control and MW
groups (\emph{p} = 0.041) {[}OR (95\% CI): 2.76(1.13--6.76){]} and
(\emph{p} = 0.014) {[}OR (95\% CI): 5.88(1.49--25.0){]}, respectively.
The frequency of haplotype 5 was significantly higher in the EW group
than in the controls (\emph{p} = 0.005) {[}OR (95\% CI):
14.16(1.64--121.93){]}. The \emph{ARG1} haplotype 4 (C/T/A/A/C/T) was
more frequent in the MW, asthmatics, and control groups than in the EW
group (\emph{p} \textless{} 0.0001) {[}OR (95\% CI):
7.77(2.54--23.7){]}, (\emph{p} = 0.036) {[}OR (95\% CI):
4.31(1.15--16.15){]}, and (\emph{p} = 0.030) {[}OR (95\% CI):
3.44(1.20--10.0){]}, respectively (see Tables 3A, 3B).

2c. Association between SNP analysis and allergic rhinitis, aeroallergen
sensitivity, number of aeroallergens detected as positive in the skin
prick test, and tobacco exposure

{\label{c.-association-between-snp-analysis-and-allergic-rhinitis-aeroallergen-sensitivity-number-of-aeroallergens-detected-as-positive-in-the-skin-prick-test-and-tobacco-exposure}}

\emph{ARG1} haplotype 4 was more frequent in patients with allergic
rhinitis than those without allergic rhinitis, 41.8\% and 21.3\%,
respectively (\emph{p} = 0.046) {[}OR (95\% CI): 2.65(1.10--6.41){]}.

There was no difference in terms of haplotype distribution and presence
of aeroallergen sensitivity, the number of aeroallergens detected as
positive in the skin prick test, and tobacco exposure among all the
patient groups.



\section*{DISCUSSION}

{\label{discussion}}

To our knowledge, this is the first study to look at PSW phenotypes
and\emph{ARG} gene polymorphisms. We hypothesized that there would be
relationships between a PSW phenotype and polymorphisms of
the\emph{ARG1} and \emph{ARG2} genes. Our study revealed that the
frequency of the homozygote C-allele rs2781667 in \emph{ARG1} was
significantly higher in the MW and asthma groups than in the EW group.
Li et al.\textsuperscript{13} reported that four SNPs in
\emph{ARG1}(rs2781659, rs2781665, rs2781667, rs2246012) and rs 3742880
in\emph{ARG2} were associated with the number of positive skin tests,
and carrying two copies of the minor allele for rs3759757 and rs6573788
SNPs in \emph{ARG2} were found to be associated with the presence and
severity of asthma. Litonjua et al.\textsuperscript{14} reported that
rs2781659 in \emph{ARG1} was significantly associated with BDR in
asthmatic children. In another study\textsuperscript{15}, each variant
allele of rs3742879 in \emph{ARG2} was found to be associated with
increased asthma risk. Vonk et al.\textsuperscript{16} reported that two
polymorphisms in \emph{ARG2} (rs17249437 and rs3742879) were associated
with both asthma and more severe airway obstruction, and increased
airway hyperreactivity (AHR) and lower beta-2-agonist reversibility were
reported as associated with both \emph{ARG1} and \emph{ARG2.} Hing Yee
et al.\textsuperscript{20} reported that rs7216389 in \emph{ORMDL3} and
rs3756780 in \emph{ARG1} interactions might be associated with a risk of
asthma, and rs2749935 in \emph{ARG1} and rs2190242 in
\emph{CRHR2}interactions might be associated with BDR. A recent study
from India showed that high arginase activity and IL-13 concentration in
serum and the \emph{ARG1} rs2781666 G/T genotype might increase the risk
of asthma\textsuperscript{21}. Vonk et al.\textsuperscript{16} reported
that the protective effect of inhaled corticosteroids (ICS) against
annual FEV1 decline was significantly lower in homozygote carriers of
the C-allele of rs2781667 in \emph{ARG1} in a longitudinal cohort of 200
adult asthma patients. Based on this, our finding of a higher frequency
of homozygote C-allele rs2781667 in \emph{ARG1} in the MW and asthma
groups may be important for the management of patients with PSW. We
think it might be a useful marker for the differentiation of the PSW
phenotype. In addition, the association between this SNP and ICS or BDR
could be addressed in future studies.

We also found that the homozygote CC genotype of the
\emph{ARG1}rs2781667T\textgreater{}C SNP was more frequent in patients
with allergic rhinitis than in those without allergic rhinitis (\emph{p}
= 0.004). While it is known that the arginase-NOS pathway is associated
with allergic rhinitis \textsuperscript{23,24}, we were unable to find
any study related to the association of allergic rhinitis and
\emph{ARG}genes until now. Furthermore, we have no data for whether
\emph{ARG1}rs2781667 is a functional polymorphism and associated with
changes in arginase expression in the nasal mucosa.

Our haplotype analyses revealed that the frequencies of the
\emph{ARG1}haplotype 2 and haplotype 5 were significantly higher in the
EW group than in the control group. Two recent studies suggest an
interaction between arginase expression and viral airway
infections\textsuperscript{25,26}. Santiago-Olivares et
al.\textsuperscript{25} investigated the impact of Respiratory Syncytial
Virus (RSV) infection on nitric oxide production in an experimental
model and found that \emph{ARG1} overexpression contributes to the
maintenance of the RSV genome in the host in persistent infection.
Wieczfinska et al.\textsuperscript{26} found that the expression of
TGF-\selectlanguage{greek}β\selectlanguage{english}1 and arginase was increased due to rhinovirus infection. To date,
we have no data for viral-induced wheezing and genetic variations
in\emph{ARG} genes, but new studies could advance this. The
\emph{ARG1}haplotype 4 was more frequent in the MW group than in the EW
group. Interestingly, \emph{ARG1} haplotype 4 carried the variant
C-allele for rs2781667 in \emph{ARG1} , so it may be useful for
differentiating between EW and MW. Salam et al.\textsuperscript{15}
reported that the association between arginase variants and childhood
asthma may be related with haplotypes rather than SNPs. Duan et
al.\textsuperscript{19} studied haplotypes were identified according to
the SNPs (rs2781659, rs2781663, rs2781665, and rs60389358) in
the\emph{ARG1} and they have reported that two haplotypes may alter BDR
and differentially regulate gene expression. The haplotypes that we
estimated according to the studied SNPs were different from those used
in these studies, so it is impossible to make a comparison between
haplotype-based results.

The diagnoses of asthma cases and the classification of patients
according to a PSW phenotype were performed by pediatric allergists at a
tertiary pediatric allergy specialty clinic, these were strengths of the
our study. However, there were some limitations. First, the sample size
was smaller than previous studies have referred to; additionally, it
does not represent all Turkish children. Second, this was a case-control
study; thus, we have no longitudinal data about the progress and
bronchodilator or ICS responses of patients with a homozygote CC
genotype of the \emph{ARG1} rs2781667T\textgreater{}C and/or
\emph{ARG1}haplotype 4. Third, we included no information about the
functionality of the homozygote CC genotype of the
\emph{ARG1}rs2781667T\textgreater{}C and/or \emph{ARG} 1 haplotype 4 in
lung and nasal mucosa.

In conclusion, our study revealed that
\emph{ARG1}rs2781667T\textgreater{}C and/or \emph{ARG} 1 haplotype 4
were more frequent in patients with MW and asthma than in the EW group.
The frequencies of the \emph{ARG1} haplotype 2 and haplotype 5 were
significantly higher in the EW group, while haplotype 9 was more
frequent in the healthy controls. Taken together, we conclude that
variations in \emph{ARG1} gene may be potentially important for
differentiating PSW phenotypes. Our findings need to be confirmed in
larger, different PSW populations, preferably with different ethnic
backgrounds.

\textbf{Acknowledgement}

The study was funded by the Research Council of Trakya University (STUDY
NUMBER 2017/86).

\textbf{REFERENCES}

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