ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil

https://zancojournals.su.edu.krd/index.php/JPAS

ISSN (print ):2218-0230, ISSN (online): 2412-3986, DOI: http://dx.doi.org/10.21271/zjpas

RESEARCH PAPER

Molecular Study of IL-7R Gene Polymorphism and their Associations with

Male Multiple Sclerosis Patients in Erbil Province.

Shukur W. Smail1, Mukhlis H. Aali1, Fikry A. Qadir1, Ahmed O. Pirdawid2, Abdulla A.

Shareef1, Abdulkarim Y. Karim1, Roza T. Yaseen3, Mohammed Q. Mustafa3, Kovan F.
Jalal3 and Abbas Salihi1,3
1
Department of Biology, College of Science, Salahaddin University-Erbil, Kurdistan Region, Iraq
2
Department of Biology, College of Science - University of Duhok, Kurdistan Region, Iraq
3
Department of Medical Analysis, Tishk International University, Erbil, Kurdistan Region, Iraq

A B S T R A C T:
Multiple sclerosis (MS) is an autoimmune disease in which immune cells attacks the body cells mistakenly; it is
characterized by chronic inflammation that leads to demyelination and conduction of nerve impulse is affected negatively. The
cause of the disease is unknown, but it may be partially under the control of genetics, including interleukin 7 receptor alpha
(IL7Rα). In this case-control study, 40 relapsing-remitting MS (RRMS) male patients, which fulfills McDonald criteria and 40
healthy controls, with matched sex, were compared depending on the rs6897932 polymorphism within the exon 6 of IL7Rα gene
by Tetra-amplification refractory mutation system polymerase chain reaction (Tetra-ARMS-PCR) method. The frequency of T
allele of IL7Rα rs6897932 was considerably higher in male MS patients than healthy control males (31.25 vs 17.5%). Genotype
distributions of the single nucleotide polymorphism (SNP) rs6897932 deviated from Hardy-Weinberg equilibrium with a p-value
of 0.80. Both homozygous (TT) and Heterozygous (CT) were non-significantly positively associated with MS male patients (OR
= 6.75, 95%CI = 0.73-62.4, p = 0.059, OR = 1.68, 95%CI = 0.64-4.38, p = 0.28) respectively. The distribution of the rs6897932
polymorphism is not significantly different in our case/control study in the Erbil province.

KEY WORDS: Multiple Sclerosis, Interleukin 7 Receptor, Polymorphism, Relapsing-Remitting Multiple Sclerosis
DOI: http://dx.doi.org/10.21271/ZJPAS.33.1.3
ZJPAS (2021) , 33(1);21-26 .

1. INTRODUCTION

Multiple sclerosis (MS) is a long-standing

autoimmune disease that occurs by the The disease is expected to influence
inflammation in the central nervous system (CNS) approximately two and a half million people in the
and results in demyelination and impairment of world (Rosati, 2001). Furthermore, MS is more
axons (Valcarcel et al., 2018, Gold et al., 2012). It common in females than in males. Currently, the
can be classified as an autoimmune disease that rate of females with this disease to males is 2–1
impacts CNS and distinguished by numerous (Ascherio and Munger, 2016).
brain and spinal cord lesions (Rosati, 2001, MS etiology is unclear, but several studies have
Hafler, 2004, Wilkins, 2018). already shown that causative agents of MS is
reliant on a strong genetic factor (Akkad et al.,
2009). Several different methods have been used
* Corresponding Author: to determine the genetic bases of MS, such as
Shukur Wasman Smail
genetic linkage, gene expression and candidate
E-mail: [email protected]
Article History:
Received: 16/03/2020
Accepted: 08/09/2020
Published: 20/02 /2021
Smail. Sh. et al. /ZJPAS: 2021, 33 (1): 21-26
22

gene association (Gregory et al., 2007, Rizvi et interaction of this SNP with MS (Čierny et al.,
al., 2020). 2015).
Amongst the association study is a viable To arrive at a definite conclusion, we decided to
approach toward recognizing the risk genetic loci have this study in Erbil province-Iraq to evaluate
of genes as a marker such as SNP. Among these the importance of the IL7Rα gene polymorphisms,
was reported the relationship of many IL-7 rs6897932 in such community and investigate
receptor SNPs with MS in various ethnic groups their influence on IL7Rα gene expression
(Sahami-Fard et al., 2020, Zhang et al., 2019). concerning MS pathogenesis.
The position of IL7R is chromosome 5 short arm
2. MATERIALS AND METHODS
13 (5p13). This gene consists of γ chain (IL7Rγ)
and α chain (IL7Rα) usually recognized as CD132 2.1. Sample Collection
and CD127 respectively. The SNPs throughout the Forty males with MS patients enrolled in this
alpha chain of IL7R gene have a big role in the study who visited department of neurology,
impairment of immune system homeostasis and Rzgari hospital in Erbil city-Iraq from April to
this prone to MS easily (Zhang et al., 2005, June of 2015, and they were diagnosed according
Čierny et al., 2015). to 2005 revised McDonald criteria. Forty control
Appropriately, association studies for the variants males who were age-matched with MS patients.
of IL7Rα gene revealed that rs6897932 in the exon 2.2. Molecular Technique Analysis
6 might be a causative factor for MS evolving in 2.2.1. Genomic DNA Extraction from Human
many Japanese and European communities Blood Samples
(Gregory et al., 2007, Weber et al., 2008, Fang et Human Blood samples were taken from peripheral
al., 2011). veins using the five-milliliter syringe. The blood
In the exon number 6 of IL7Rα gene there is SNP put into K2EDTA (5.4mg) tube for direct DNA
called rs6897932 induces non-conservative amino extraction. The Genomic DNA extracted from
acid transition at location 244 in which isoleucine whole blood samples by using spin column
shifted to threonine 244 (Ile → Thr) (ATC / ACC) method (AccPrep Genomic DNA extraction Kit-
(Teutsch et al., 2003). This amino acid shift Bioneer, South Korea), depending on the
affects the expression product of IL7Rα which manufacturer’s instructions. Then the
results in the variation amount of membrane- concentration and purity of genomic DNA
bound isoform and soluble form. Such extracted from each human blood samples were
modifications are accompanied by the regulation determined using Nano-Drop ™ (Thermo
of the IL7 signaling pathway and a direct Scientific, USA) spectrophotometer by recording
association MS with this SNP (Gregory et al., the concentration ranged (11.05-61.60 ng/µl) and
2007). purity (1.69-2.27) for each sample.
Before many types of researches have been done, 2.2.2. Genotyping IL7Rα Gene Polymorphism
most of them have assessed the relationship of (PCR Amplification)
T244I variant, rs6897932 with MS pathology, Genotyping of the SNP rs6897932 C>T that
while other researches couldn’t find any located in exon 6 of IL7Rα gene performed by a
connection between them. Studies that recently rapid and cost-effective technique called the
done have shown that the C allele is slightly more Tetra-ARMS-PCR method in Tishk International
prevalent than the T allele. Also, it has been University. In this polymorphism one sequence-
suggested in region rs6897932 the C allele will specific forward primers: 5′-
lead to a higher threat of MS in some population AAGAAGGGAAGAGAGCATTGG-3′, and three
(Gregory et al., 2007, Weber et al., 2008, Fang et sequence-specific reverse primers that two of
al., 2011). Therefore, the opportunity to have MS them: 5′-GAAAAAACTCAAAATGCTGATGG-
will increase by 1.5 and 1.3 folds for the 3′ (for C allele) and 5′-AGAAAAAACT
genotypes CC and CT respectively, if we CAAAATGCTGATGA-3′ (for T allele) and one
deliberated that genotype TT as a normal risk. reverse were a primer for internal control 5′
Dependent on many types of researches with TTACTTTGGGGACAGCGTTT-3′ used
varied results about the polymorphism rs6897932, (Majdinasab et al., 2014).
it is difficult to know for sure concerning the The total of 25 µl volume of PCR master mix
reaction achieved containing 3µl of genomic

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23

DNA, 12.5 µl of Taq DNA Polymerase 2x Master 1T) has TT genotypes, sample 2 (lane 2C and 2T)
Mix RED (Ampliqon, Danish) and 1µl was added has CT genotype and sample 3 (lane 3C and 3T)
for each of the forward and reverse primers for has CC genotype, M= 250 bp DNA ladder.
IL7Rα gene. Then the mixture was completed by
adding 3.5 µl of nuclease-free water (Čierny et al., 2.3. Statistical Analysis
2015). GraphPad Prism 6 statistical software used for
The program of PCR amplification consisted of an statistical analysis. A student t-test used to
initial denaturation at 94 °C for 5 minutes compare the ages between MS patients and
followed by the next 35 cycles 94 °C for 30 healthy controls. Genotype and allele frequencies
seconds (denaturation), 62 °C for 30 seconds of MS patients and healthy controls were analyzed
(annealing),72 °C for 30 seconds (elongation) and using the Chi-square (χ2) test. Both genotype and
the final step at 72 °C for 5 minutes to extend all allelic odds ratio (ORs) and 95% confidence
PCR fragments (Majdinasab et al., 2014). After interval (CI) calculated to determine the
PCR amplification, the PCR DNA amplicons association of the SNP in exon 6 of the IL7Rα
separated on 1.5% agarose gel (after applying 5 receptor gene polymorphisms with MS. A p-value
V/cm) (Magdeldin, 2012), then the separated of less than 5% ( p <0.05) set to be statistically
bands were stained with ethidium bromide to significant.
visualize under UV light (Bio-Rad UV-
transilluminator) (Brown, 2016). The 301 bp mean 3. RESULTS
the appearance of IL-7R gene polymorphism in
Demographic profiles of both male MS
exon 6 (Figure 1).
patients and healthy control males had shown in
table 1. The mean age of MS patients was 34.85 ±
1.442 years, and of the control group was 32.25 ±
1.375 years; yielding no statistically significant
difference in their ages. In this case-control study,
40 MS patients and 40 healthy controls compared
depending on the rs6897932 polymorphism within
the exon 6 of IL7Rα gene by Tetra-ARMS-PCR
method. The genotype and allele frequencies of
IL7Rα rs6897932 are given in (Table 2). The
frequency of the T allele of IL7Rα rs6897932 was
not significantly changed in male MS patients
than healthy control males (31.25 vs 17.5%).
Genotype distributions of the SNP rs6897932
deviated from Hardy-Weinberg equilibrium with a
p-value of 0.80. The significance association was
not found in both homozygous (TT) and
Heterozygous (CT) in MS male patients (OR =
6.75, 95%CI = 0.73-62.4, p = 0.059, OR = 1.68,
Figure 1. Gel electrophoresis for the IL7Rα 95%CI = 0.64-4.38, p = 0.28) respectively (Table
rs6897932 gene showing the T and C alleles in 2).
some MS patients. Band 301bp indicates the
presenece of T or C alleles. Sample 1 (lane 1C and

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24

Table 1. Age of MS patients and controls

Variable Age Range (years) Mean Age ± SE

MS Patients 20-50 34.85 ± 1.442

Controls 20-50 32.25 ± 1.375

Table 2. Association of MS with carriage of alleles/genotypes of IL7Rα rs6897932 SNP

Polymorphism MS (n=40) Control (n=40) OR 95% CI p-value HWE

No. % No %

CC 20 50.0 27 67.5 1.0 - - 0.80

CT 15 37.5 12 30.0 1.688 0.64- 0.28

4.38

TT 5 12.5 1 2.5 6.75 0.73- 0.059

62.4

C-Allele 55 68.75 66 82.5 2.143 1.016- 0.0428

T-Allele 25 31.25 14 17.5 4.518

development and severity of the MS. Non-HLA

4. DISCUSSION genes have a role in regulation and modulation of
Multiple sclerosis (MS) is an autoimmune disease, cytokine, cytokine receptor, transcription factor
which is characterized by demyelination of the and T lymphocyte receptor (Rounachi et al.,
axon of neurons in the CNS and predominant of 2009). Non-HLA genes that are usually involved
inflammation. The etiology of the disease is not in MS are CD 25, EVI5, CD58, CD154 and
well known, but it may be under genetics and IL7Rα.
environmental factors (Dyment et al., 1997). The Interleukin 7 receptor alpha (IL7Rα, also known
genes, which are involved and increased as CD127), is one of the most critical genes which
susceptibility to the disease, are divided into two has a role in developing MS. It located on
groups: HLA genes (Sawcer et al., 2011) and non- chromosome 5p13.2. It expressed on B and T
HLA genes (Rounachi et al., 2009). The most lymphocytes, it has essential in maturation,
HLA gene which is related to MS is HLA- DRB1* developing and survival of them (Manolio et al.,
1501 class II allele (Alcina et al., 2010), it is 2009). There are many SNP of IL7Rα but, the
located on chromosome 6p21. Besides HLA genes most critical SNP in MS is (rs6897932, p.T244I)
there are many genes which are proposed by which is vital for developing IL7Rα in the form of
GWAS that candidates be involved in the membrane-bound or the way of the soluble

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 Smail. Sh. et al. /ZJPAS: 2021, 33 (1): 21-26
25

receptor, in this manner it is involved in Variation in the IL7RA and IL2RA genes in
developing MS (Booth et al., 2005, Zuvich et al., German multiple sclerosis patients. Journal of
autoimmunity, 32, 110-115.
2009). Since, rs6897932 polymorphism of IL7Rα
controlling post-transcription modification and ALCINA, A., FEDETZ, M., NDAGIRE, D., FERNÁNDEZ,
alternative splicing the exon 6 of pre-mature O., LEYVA, L., GUERRERO, M., ARNAL, C.,
mRNA of IL7Rα (Booth et al., 2005). The allele DELGADO, C. & MATESANZ, F. 2008. The
of “C” has a role in skipping alternative splicing T244I variant of the interleukin-7 receptor-alpha
gene and multiple sclerosis. Tissue Antigens, 72,
the exon 6, and developing a soluble form of 158-61.
IL7Rα while, The allele of “T” has a role
alternative splicing the exon 6 and developing a ALCINA, A., FERNÁNDEZ, Ó., GONZALEZ, J. R.,
membrane-bound type of IL7Rα (Gregory et al., CATALÁ-RABASA, A., FEDETZ, M.,
2007). The carriers of the "T" allele believed to be NDAGIRE, D., LEYVA, L., GUERRERO, M.,
ARNAL, C. & DELGADO, C. 2010. Tag-SNP
a predisposing factor for developing MS because analysis of the GFI1-EVI5-RPL5-FAM69 risk
it leads to the formation of the more membrane- locus for multiple sclerosis. European journal of
bound form of IL7Rα and the creation of more human genetics, 18, 827-831.
auto-reactive T and B lymphocyte (Gregory et al.,
2007). ASCHERIO, A. & MUNGER, K. L. 2016. Epidemiology of
Multiple Sclerosis: From Risk Factors to
Another explanation for the role of IL7Rα in Prevention-An Update. Seminars in Neurology, 36,
the pathogenesis of RRMS is that IL7Rα down- 103-14.
regulates the forkhead box P3 (FoxP3) which is,
in turn, is the transcription factor for developing BOOTH, D., ARTHUR, A., TEUTSCH, S., BYE, C.,
CD4+CD25+ regulatory T (T reg) cells, T reg RUBIO, J., ARMATI, P., POLLARD, J., HEARD,
R., STEWART, G. & CONSORTIUM, S. M. G.
cells is crucial in promoting tolerance and 2005. Gene expression and genotyping studies
suppress autoreactive T lymphocyte. IL7Rα by implicate the interleukin 7 receptor in the
decreasing FoxP3 can breakdown tolerance and pathogenesis of primary progressive multiple
developing an autoimmune disease, e.g. MS . The sclerosis. Journal of molecular medicine, 83, 822-
results of the current analysis are inconsistent with 830.
O'Doherty et al. (2008), Alcina et al. (2008) and BROWN, T. A. 2016. Gene cloning and DNA analysis: an
Majdinasab et al. (2014) who found that introduction. New York, United States: John Wiley
rs6897932 polymorphism of IL7Rα was related & Sons.
with MS in UK, Spain and Iran, respectively.
ČIERNY, D., HÁNYŠOVÁ, S., MICHALIK, J.,
KANTOROVÁ, E., KURČA, E., ŠKEREŇOVÁ,
M. & LEHOTSKÝ, J. 2015. Genetic variants in
CONCLUSIONS interleukin 7 receptor α chain (IL-7Ra) are
associated with multiple sclerosis risk and
In summary, there is no association between disability progression in Central European Slovak
rs6897932 polymorphism of IL7Rα and RRMS population. Journal of neuroimmunology, 282, 80-
patients in the Erbil province, by comparing this 84.
variant in control and MS groups. This study DYMENT, D., SADOVNICK, A. & EBERS, G. 1997.
confirmed that rs6897932 polymorphism of IL7Rα Erratum: Genetics of multiple sclerosis (Human
is not a predisposing factor for developing the Molecular Genetics (1997) 6 (1693-1698)). Human
disease in the Erbil province. Molecular Genetics, 6.

Acknowledgment FANG, L., ISOBE, N., YOSHIMURA, S., YONEKAWA,

We thank MS center in Rzgari hospital in Erbil T., MATSUSHITA, T., MASAKI, K., DOI, H.,
OCHI, K., MIYAMOTO, K. & KAWANO, Y.
city-Iraq for their support for taking permission 2011. Interleukin-7 receptor alpha gene
from MS patients to give blood for conducting the polymorphism influences multiple sclerosis risk in
research. Asians. Neurology, 76, 2125-2127.

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