Medical
Communication
Biosci. Biotech. Res. Comm. 9(3): 415-420 (2016)
Relationship between interleukin 4 gene promoter
polymorphisms and cutaneous Leishmaniasis cases in
North Eastern Iran
S. Ra zadeh,
1
M. Saraei
2
, MR. Abai
3
, M. Mohebali
4
, H. Bakhshi
3
and Y. Rassi*
3
1
Ministry of Health and Medical Education, NIMAD, Tehran, Iran.
2
Department of Parasitology and Mycology, Cellular and Molecular Research Center, Qazvin University of
Medical Sciences, Qazvin, Iran.
3
Department of Medical Entomology & Vector Control, School of Public Health, Tehran University of Medical
Sciences, Tehran, Iran.
4
Department of Parasitology and Mycology, Tehran University of Medical Sciences, Tehran, Iran.
ABSTRACT
Cutaneous leishmaniasis (CL) is a worldwide public health and a social problem in many developing countries. The
main objective of this study was to investigate the relationship between interleukin 4 (IL-4) gene promoter polymor-
phisms and leishmanial infected people in endemic focus of zoonotic cutaneous leishmaniasis (ZCL), in north east of
Iran. In order to determine of polymorphism of interleukin 4 (IL-4) among patients with different clinical symptoms,
fty human blood samples were prepared. All cases were classi ed into ve groups including: cases with sever and ≤
2 lesions, cases with ≥ 2 lesions, cases with treatment duration of less than two months, cases with treatment duration
of more than two months and healthy samples with no signs. All 50 human samples were tested by PCR and followed
using restriction enzyme of Eco47I. Our study revealed that Eco47I RE could cut the PCR product to an 18 bp and
177 bp if the SNP was C. Among the 50 PCR products we found 4 samples (8%) with CT allele and also 1 sample(2%)
with TT allele. The rest of samples (90%) were CC. In conclusion, there was a signi cant difference on frequencies of
three alleles (CC, CT, TT) in studied group (2=56.4; df=2 ; p=0.000).
KEY WORDS: SNP, INTERLEUKIN 4, POLYMORPHISMS, CUTANEOUS LEISHMANIASIS, IRAN
415
ARTICLE INFORMATION:
*Corresponding Author: rassiy@tums.ac.ir
Received 24
th
Aug, 2016
Accepted after revision 24
th
Sep, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
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Online Contents Available at: http//www.bbrc.in/
416 INTERLEUKIN 4 GENE PROMOTER POLYMORPHISMS IN LEISHMANIASIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Ra zadeh et al.
INTRODUCTION
Leishmaniasis is a parasitic disease with a wide spectrum
of clinical manifestations ranging from a self-healing
skin lesion to lethal form of visceral disease. Accord-
ing to WHO estimate the prevalence of leishmaniasis is
12 million with 0.9–1.6 million new cases each year.
Leishmaniasis occurs in 98 countries worldwide, and
350 million people are at risk of contracting the disease.
Zoonotic Cutaneous Leishmaniasis (ZCL) is the most
common form of leishmaniasis and about one-third
of cases are reported from the Americas, the Mediter-
ranean basin, and Western Asia from the Middle East
to Central Asia. Countries such as Afghanistan, Alge-
ria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan,
Costa Rica and Peru, which together account for 70 to
75% of global estimated ZCL cases. It is shown that the
distribution of ZCL in central and south Asian countries
such as Kazakhstan, Kyrgyzstan, Turkmenistan, Uzbeki-
stan, Tajikistan, Iran, Pakistan, Afghanistan, southern
Mongolia, and north-western China overlaps with the
presence of great gerbils, the main reservoir of ZCL in
Iran (Sosnina, 1979; Mallon, 1985; Yaghoobi-Ershadi
and Javadian, 1996, Strelkova et al., 2001; Molur et al.,
2005; Abai et al., 2010; Smith et al., 2010; Tashbaev and
Mustafaev, 2010; Oshaghi et al., 2011; Alvar et al., 2012;
WHO 2012 and Bakhshi et al., 2014).
Leishmaniasis clinical manifestation depends upon Leish-
mania species and host genetic background which governs
generation of type of immune response (Mohamed et al.,
2003; Salhi and Rodrigues, 2008; ; Sakthianandeswaren
et al., 2009 and 2010; Castellucci et al., 2012; Moravej et
al., 2012, Bakhshi et al., 2013 and 2014).There is correla-
tion between the type of immune response induced and
cytokines production and outcome of the diseases. It is well
known that in murine model of L. major infection dichot-
omy of Th1/Th2 immune response determines the outcome
of the disease (Reiner and Locksley, 1995). In leishmaniasis
usually cytokines such as IFN-, IL-4, IL-5, IL-10, TNF-,
TNF-, etc in the level of protein and genes are titrated to
assess the severity and outcome of CL due to L. major (Habibi
et al., 2001; Mahmoodi et al., 2003; Kamali-Sarvestani et
al., 2006; Sakthianandeswaren et al., 2009; Salhi and Rod-
rigues, 2008).
IL-4 plays an important role in various biological
activities including immune response development and
its polymorphisms is reported from different populations
(Kamali-Sarvestani et al., 2006). Correlations between
IL-4 polymorphisms and different disorders such as vis-
ceral leishmaniasis (VL), CL, and leprosy are reported
(Mohamed et al., 2003; Kamali-Sarvestani et al., 2006;
Yang et al., 2011).There is a report that in human IFN-c
+ 874 A>T polymorphisms in uences the progression of
the disease towards chronic CL while IL-4 -590 C>T pol-
ymorphism increases the risk of developing CL (Kamali-
Sarvestani et al., 2006).
In 2014, a PCR-based assay was developed to amplify
IL-4 promoter gene to possibly de ne IL-4 promoter gene
polymorphism in R. opimus populations with a range of
Leishmania infection and symptoms collected from dif-
ferent foci of the central, north and northeast regions
of Iran. The results showed that the designed primers
amplify 689 bp of the promoter gene and Sequence
analysis of the promoter gene revealed ve polymorphic
sites assembly six haplotypes among the gerbil popula-
tions (Bakhshi et al., 2014).
This is important that the ve polymorphisms cause
different outcome phenotypes following infection with
L. major in R. opimus specimens. In the current study
it was proposed to investigate the relationship between
interleukin 4 (IL-4) gene promoter polymorphisms and
leishmanial infected people in endemic focus of cutane-
ous leishmaniasis in north east of Iran.
MATERIAL AND METHODS
This study were carried out in Esfarayen County dur-
ing 2014-2015. This county is located in North Khorasan
Province, north-east of Iran (Fig.1). At the 2006 census
its population was 51,321, in 13,376 families. Esfarayen
is one of the focal points for residence of Aryan tribes
after entering into Iran. Basis on combination of clini-
cal and parasitological criteria, fty cases were selected
from three villages of Charborj(CH), Kalateh Reza(KR)
and Hossein Abad (HA). All samples were classi ed
into ve groups (each group=10 cases) including: cases
with sever and ≤ 2 lesions, cases with ≥ 2 lesions, cases
with treatment duration of less than two months , cases
with treatment duration of more than two months and
healthy samples with no lesions or scars. Blood samples
were prepared from all selected groups.
Two hundred μL of the frozen blood samples were
thawed and treated with proteinase K, and then the
genomic DNA was extracted using the G-spin Tissue
Spin Kit (G-spin, South Korea) according to the manu-
facturer’s instructions. The sequences of the designed
primers were as follow: F-: 5’-TGG GGA AAG ATA GAG
TAA TA-3’ and R-: 5´- TAA ACT TGG GAG AAC ATG
GT-3’ (Kamali-Sarvestani et al., 2006).
The primers amplify a fragment with size of 195 bp.
Ampli cation was performed on an Eppendorf thermal
c ycler. A touch-down PCR thermal program was carried
out with the following pro le: 94, 53 and 72 degrees
centigrade for 50 seconds respectively. PCR products
were then used for RFLP by Eco47I Restriction Enzyme
and then the ampli ed products were monitored by elec-
trophoresis in 2.5% agarose gel and ethidium bromide
staining.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS INTERLEUKIN 4 GENE PROMOTER POLYMORPHISMS IN LEISHMANIASIS 417
Ra zadeh et al.
The expected band was puri ed from the gel by gel
extraction kit (Bioneer, South Korea). Sequencing was
performed using an ABI 3730 sequencer machine and
Seqlab (Göttingen, Germany). The ambiguous sequences
were corrected using Chromas program and the consensus
sequences obtained using DNASTAR Lasergene (SEQMAN
and EDITSEQ). The sequences were aligned using ClustalW
(Chenna et al., 2003) to explore possible polymorphisms.
RESULTS AND DISCUSSION
The aim of this investigation was to determine of pro-
moter region of IL-4 gene and its correlation with people
infected to Leishmania major. All samples were ampli-
ed and nally the PCR products were used to evalua-
tion by the use of Eco47I restriction enzyme. The results
revealed the presence of 3 genotypes including CC, CT
and TT. The most prevalent genotype belonged to CC
type (90%). Eight percent of samples (4 cases) had an
genotype of CT and nally just one of the samples had
a genotype of TT (2%). In the group of people with no
lesions or scars we only observed CC genotype. Also we
observed a TT genotype in the group of people which had
been treated less than two months. The other samples
out of this group had CC genotype. Also we observed
1/10 CT and 9/10 CC genotypes in the group which
had been treated more than two months. We found 1/9
CT genotype and 9/10 CC genotypes in the group with
severe lesions. Finally we found 1/10 CT genotype and
9/10 CC genotypes in the group belonged to samples wih
small lesions, (Table 1).
FIGURE 1: The map of North Khorasan Province, North-East of Iran, Esfarayen
district has been located in south of the province
Table 1: The observed genotype and allele frequencies for interleukin gene promoter, among ve selected
group in Esfarayen county, North east of Iran, 2015
G1 N (%) G2 N (%) G3 N (%) G4 N (%) G5 N (%)
IL-4 genotype
CC 8 (80) 9 (90) 9 (90) 9 (90) 10 (100)
CT 2 (20) 1 (10) 0 (0) 1 (10) 0 (0)
TT 0 (0) 0 (0) 1 (10) 0 (0) 0 (0)
IL-4 allele
C 18 (90) 19 (95) 18 (90) 19 (95) 20 (5)
T 2 (10) 1 (5) 2 (10) 1 (5) 0 (0)
G1(Acute lesions > 2), G2(Mild lesions ≤2 ), G3(Treatment time ≤ 2 month)
G4 (2 month < Treatment time ≤ 6month) and G5 (Healthy cases without lesion or scar)
418 INTERLEUKIN 4 GENE PROMOTER POLYMORPHISMS IN LEISHMANIASIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Ra zadeh et al.
Our study showed that Eco47I RE could cut the PCR
product to an 18 bp and 177 bp if the SNP was C (Fig 2.).
A single nucleotide polymorphisms (SNPs) in the IL-4
promoter gene of the infected people were identi ed
which might be related to the pathogenesis and clini-
cal outcome of leishmaniasis. Of course the impact of
this SNP on the outcome of the disease should be tested
independently, because all of the mutations in cytokine
genes may not have similar in uence on the clinical
outcomes of leishmaniasis. In human, it is reported that
TNF- -308 G-->A and TNF- +252 G-->A gene pol-
ymorphisms showed no effect on the disease but IFN-
+874 A-->T polymorphism in uences the progression
of the disease towards chronic CL while IL-4 -590 C-->T
polymorphism might increase the risk of developing CL
(Kamali-Sarvestani et al., 2006).
The results of this study were analyzed by SPSS-
Version 18 and X
2
(Non parametric) soft wares. Due to
this analyze, the prevalence of CC, CT and TT alleles
among 5 mentioned groups revealed that these 3 allele
prevalence in the selected population has signi cant dif-
ferences (2=56.4; df=2 ; p=0.000); but we could not
nd a signi cant difference in prevalence of CC, CT and
TT alleles in three selected villages of this investigation
2=5.6; df=2 ; p=0.062). Also we could not observed any
signi cant difference in CC and CT alleles among the 5
groups (2=1.3; df=1 ; p=0.248); but the prevalence of
CC, CT and TT alleles in 4 groups (cases with less than
two lesions, Treatment period < 2 months, Treatment
period > 2 months, Healthy without lesions or scars) had
a signi cant difference (2=4.5; df=1 ; p=0.035).
In a study performed in human visceral leishmania-
sis, it was shown that IL-4 polymorphism but not IL-9
in uences VL incidence and clinical phenotypes due to
L. donovani infection (Mohamed et al., 2003). In con-
trast, IFNGR1 polymorphism was linked and associated
to post Kala-azar dermal leishmaniasis (PKDL) but not
VL. The authors concluded that polymorphism in a type
2 cytokine gene in uences underlying susceptibility to
VL, whereas IFNGR1 is speci cally related to suscepti-
bility to PKDL. In a study performed in CL due to L. bra-
ziliensis, it was shown that a SNP in macrophage inhibi-
tory factor (MIF -173C) favored CL infection and disease
progression (De Jesus Fernandes Covas et al., 2013).
In another study it was shown that a SNP in IL-1
(-511 C/T) is possibly a key player determining the sever-
ity of the disease in diffuse (DCL) patients infected with
L. mexicana (Fernández-Figueroa et al., 2012). Also it
was shown that IL-6 -174 G/C promoter polymorphism
in uences susceptibility to mucosal but not localized CL
(Castellucci et al., 2006).
In another investigation conducted on R. opimus
specimens in Iran, six IL-4 gene promoter region haplo-
types among sequenced specimens observed. Sequence
FIGURE 2: RFLP product of IL-4 gene promoter in leishmanial cases in Esfarayen County, North East of Iran,
2015. (M) 100 bp molecular weight marker (Fermentase), (A) a case with TT allele, (B-G & I) cases with CC allele,
(H) a case with CT allele
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS INTERLEUKIN 4 GENE PROMOTER POLYMORPHISMS IN LEISHMANIASIS 419
Ra zadeh et al.
analysis of these samples revealed ve single nucleotide
polymorphisms (SNPs) assembly six haplotypes among
the gerbil populations .Four out of ve SNPs (80%) were
of transition type (A-G or T-C). The inter population
genetic variations ranges from zero to 1% .There was
no signi cant correlation between the haplotypes and
the geographical origins or subspecies of the great ger-
bils but some speci c geographical haplotype were seen
(Bakhshi et al., 2014).
CONCLUSION
In this study, presence of three different genotypes of
CC, TT, CT among leishmaniasis cases was con rmed the
polymorphism in the promoter of IL-4. Also, the high
frequency of CC genotype (90%) was observed compared
with the other two genotypes. More importantly, healthy
individuals with no ulcers were observed with CC geno-
type, that inconsistent with the results of the other inves-
tigators in Iran. Therefore, it seems that the endemicity of
the disease (Hypo, Meso and Hyper endemic) has a signif-
icant impact on results. Finally, further studies are needed
to discover SNPs impressing the disease in the human
populations and also rodents particularly in R. opimus,
which is the main reservoir host of ZCL. These data may
lead to develop novel control strategy against ZCL.
ACKNOWLEDGEMENT
The authors gratefully thank Esfarayen Health Center,
Northern Khorassan province, north east of Iran for its
eld assistance. This study was nancially supported by
the School of Public Health, Tehran University of Medi-
cal Sciences (Project No.24217) and Qazvin University of
Medical Sciences.
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