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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 16  |  Issue : 4  |  Page : 292-295

The relationship between −330 interleukin-2 gene polymorphism and its plasma levels in patients with alopecia areata


1 Department of Biochemistry, College of Medicine, University of Babylon, Hillah, Iraq
2 Department of Internal Medicine, College of Medicine, University of Babylon, Hillah, Iraq

Date of Submission21-Jun-2019
Date of Acceptance06-Sep-2019
Date of Web Publication23-Dec-2019

Correspondence Address:
Prof. Abdulsamie Hassan Alta'ee
Department of Biochemistry, College of Medicine, University of Babylon, Hillah
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/MJBL.MJBL_46_19

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  Abstract 


Background: Alopecia areata (AA) is an autoimmune, dermatological, chronic, inflammatory disease that attacks hair follicles and causes hair loss. Hair loss usually occurs on the scalp, but it can also affect the beard, eyebrows, and other areas of the body. Interleukin-2 (IL-2) is a cytokine that contributes to the regulation of the immune system and is classified as a proinflammatory factor. IL-2 is an autocrine secretary element produced from activated T-cells, exhibiting growth factor characteristics. Objective: The objective of this study was to investigate the effect of the −330 IL-2 gene polymorphism (rs2069762) on plasma IL-2 levels in Iraqi patients with AA. Materials and Methods: In this study, 100 patients with AA and 100 ethnicity-, age-, and sex-matched healthy controls were selected. Blood samples of all individuals were collected in EDTA tubes. The restriction fragment length polymorphism–polymerase chain reaction method was applied to determine various alleles and genotypes in these individuals. Plasma concentration of IL-2 was measured in all the samples using human IL-2 kit. Results: The frequency of −330 G/T IL-2 genotype was higher in patients with AA compared to normal individuals. Accordingly, the plasma levels of IL-2 were significantly higher (P < 0.0090) in patients when compared to the control group. Conclusion: In case of patients with AA, the −330 G/T IL-2 genotype is associated with higher plasma levels of IL-2.

Keywords: −330 interleukin-2 polymorphism, alopecia areata, hair loss, interleukin-2


How to cite this article:
Al-Rubaye SI, Alta'ee AH, Al-Fadhily ZS. The relationship between −330 interleukin-2 gene polymorphism and its plasma levels in patients with alopecia areata. Med J Babylon 2019;16:292-5

How to cite this URL:
Al-Rubaye SI, Alta'ee AH, Al-Fadhily ZS. The relationship between −330 interleukin-2 gene polymorphism and its plasma levels in patients with alopecia areata. Med J Babylon [serial online] 2019 [cited 2020 Jan 22];16:292-5. Available from: http://www.medjbabylon.org/text.asp?2019/16/4/292/273772




  Introduction Top


Alopecia areata (AA) is a dermatological, chronic, inflammatory, autoimmune disease, which targets the hair follicles, causing hair loss. The hair loss usually occurs on the scalp, but can also affect the beard, eyebrows, and other areas of the body.[1] Alopecia primarily affects hair follicles, but it can also affect fingernails, causing small indentations and roughness. Most individuals experience onset of alopecia by the age of 40 years, with nearly half experiencing onset before the age of 20 years. In the United States, approximately 500,000 individuals have AA. AA tends to occur in three different patterns: focal, totalis, and universalis [2] in addition to androgenetic alopecia.[3] For patients with alopecia totalis and universalis, onset is typically before the age of 30 years. In children, the mean age of onset is between 5 and 10 years of age.[2]

Focal pattern alopecia consisting of one or multiple hairless patches appears on the scalp. Alopecia universalis consists of complete hair loss on all parts of the body, whereas alopecia totalis consists of total hair loss on the scalp. Patients with alopecia may experience periods of hair regrowth and hair loss throughout the course of the disease.

Interleukin-2 (IL-2) is classified as a pro-, anti-inflammatory factor; it is a cytokine contributed with immunological response regulation.[1] It acts as a signal molecule, which participates in the natural immune response by binding to receptors of IL-2 that are expressed by the lymphocytes which are the cells responsible for immunity. IL-2 gene is a major functional factor, which is involved in immune regulation and function.[4]

IL-2 is an autocrine secretary element produced by activated T-cells and has growth factor properties. IL-2 has an exciting effect on the T-cell proliferation and differentiation. IL-2 has a critical role in reservation of peripheral T-cell tolerance; it has a critical role in regulatory T-cell (Treg) equilibrium, and deterioration of Treg cells could be involved in the autoimmunity as a result of IL-2 disturbance.[1] IL-2 plays a major role during T-cell development in the thymus for the maturation of a unique category of T-cells called Tregs;[5] Treg function prevents other T-cells from identifying and reacting with the self-antigens, which may lead to autoimmunity by preventing the responding cells from producing IL-2. Considering these informations, IL-2 distinguishes between self and non-self, which is one of the immune system characteristics.[6]

Many studies have demonstrated that many of IL-2 gene polymorphisms and other genes that participate in immune system are involved in autoimmune diseases.[7] Because the impact of −330 IL-2 polymorphism on the plasma concentration or expression of IL-2 in patients with AA has not been reported, the present study aims to report the impact of the −330 IL-2 gene polymorphism (rs2069762) on the IL-2 plasma levels of Iraqi patients with AA.


  Materials and Methods Top


Patients and control groups

One hundred distinct patients with AA were participated in the present study. These patients were selected from Baghdad Teaching Medical City and were diagnosed by a neurologist based on the McDonald criteria.[8] The mean and standard deviation age was 26.00 ± 9.47 for males and 23.68 ± 11.65 for females in the patient group, and the age range was 15–48 years.

Furthermore, one hundred ethnicity-, gender-, and age-matched healthy individuals without any personal/family history of any autoimmune diseases were selected. The mean age was 29.42 ± 12.93 for males and 31.53 ± 7.078 for females in the control group, and the age range was 15–48 years.

DNA extraction and genotyping

Five milliliters of blood sample was taken from each participant in an EDTA tube. The DNA was extracted from the blood samples using a DNA extraction kit. Then, the DNA samples were subjected to a polymerase chain reaction (PCR)–restriction fragment length polymorphism. One hundred nanograms of extracted DNA were amplified using specific primers: direct 5'-ATTCACATGTTCAGTGTAGTTCT-3' and reverse 5'-GTGATAGCTCAATTCATGC-3'. PCR conditions were 95°C for 3 min, followed by 35 cycles of 45 s at 95°C, 45 s at 56°C, 45 s at 72°C, and a final extension step of 7 min at 72°C.[9]

PCR amplification gave a band of 131 bp. After digestion with the restriction enzyme Bfa-1 (Takara), the PCR products were digested into 110 and 21 bp fragments. Two percent agarose gel electrophoresis was used. The plasma concentration of IL-2 was detected using a human IL-2 kit purchased from eBioscience (http://www.eBioscience.com). The kit has been used in accordance with the manufacturer's instructions. Finally, a comparison was made between the genotype −330 IL-2 and the plasma level of IL-2.

Statistical analysis

To investigate the effect of IL-2 −330 polymorphism and plasma IL-2 concentration, the independent Student's t-test was performed. The differences between the parameters determined in patients with AA and in the control group are compared, and P < 0.05 was considered statistically significant. Version 6 of GraphPad Prism (GraphPad Software Inc., La Jolla, California, USA) was used for Windows software.[10]

Ethical consideration

The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki. It was carried out with the patient's verbal and analytical approval before the sample was taken. According to this approval, all the samples were collected and the tests were carried out.


  Results Top


The frequency of the T allele at the −330 IL-2 polymorphism was insignificantly higher in patients than controls (odds ratio (OR): 0.6321, confidence interval (CI): 0.2687–1.487, and P = 0.3889). Moreover, the G/T genotype was more frequent in patients than that in controls (36% vs. 10%, OR = 5.063, CI = 0.9491–27.00, and P = 0.0476) as shown in [Table 1]. Allelic association of (−330 G/T) IL-2 Gene Polymorphism in Promoter AA patients and healthy controls is shown in [Table 2]. The relationship between IL-2 and genotyping (−330G/T) IL-2 gene of polymorphism in promoter is shown in [Table 3].
Table 1: The genotype and percentage of interleukin-2

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Table 2: Allelic association of (-330 G/T) interleukin-2 gene polymorphism in promoter alopecia areata patients and healthy control

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Table 3: Relationship between interleukin-2 plasma level and genotyping (-330G/T) interleukin-2 gene of polymorphism in promoter

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The patients who carried T/G genotype had higher plasma concentration of IL-2 compared to that measured in controls (P = 0.0138). The concentration of serum IL-2 of the patient group was found to be significantly increased in patients with AA when compared with control groups (P = 0.0090), as shown in [Table 4] and [Figure 1].
Table 4: Serum interleukin-2 and cytotoxic T-lymphocyte antigen-4 concentration in patients with alopecia areata and healthy controls

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Figure 1: Serum interleukin-2 in patients with alopecia areata and healthy controls

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  Discussion Top


Because IL-2 helps maintain the immune response and plays a fundamental role in self-tolerance activity, its alteration could be implicated in the etiology of autoimmune diseases such as AA. Therefore, different genotypes of −330 IL-2 may affect plasma levels of IL-2 in patients with AA.

Several lines of study have concluded that IL-2 is involved in the pathogenesis of AA. IL-2 levels have been reported to be elevated in the serum of patients with autoimmune diseases, including patients with AA.[11] John et al. (1998) detected the single-nucleotide polymorphism of the IL-2 −330 promoter and reported that “both −330 IL-2 G/T and T/T genotypes of the IL-2 gene had an association with susceptibility to progressive secondary progression of multiple sclerosis.” They observed that the IL-2 promoter in the cell line exhibited significantly higher levels of gene expression at −330. In contrast, they found that IL-2 expression in lymphocytes increased in carriers of G/T and T/T genotypes.[7]

The present study examined the relationship between the −330 G/T genotype of the IL-2 gene and the plasma concentrations of IL-2 in Iraqi patients with AA and revealed that the genotype −330 T/G genotype was significantly more frequent in patients with AA and was related to a higher plasma concentration of IL-2 comparing to controls, suggesting that −330 G/T genotype of IL-2 gene could have an effect on increasing IL-2 plasma concentration. The study showed that plasma levels are significantly higher in patients with AA than that in healthy controls, and these levels are increased in patients with AA who carried G/T genotype, suggesting that there would be a relation between G/T genotype and plasma levels of IL-2 in patients with AA.

These results are agreed with a study that concluded that the serum levels of IL-2 were significantly elevated in patients with AA, indicate that IL-2 may play a role in the pathogenesis of AA, assume that the elevated serum levels of IL-2 may reflect the inflammatory symptoms in AA, and that control of IL-2 production may be important to the management of this disease.[11]

The present study agreed with another previous similar study by Sayad et al. that reported a significantly increased levels of serum IL-2 in cases of another autoimmune disease (multiple sclerosis) compared with healthy individuals.[7] The results of the present study suggest that IL-2 family cytokines might be involved in the pathogenesis of AA and that levels of IL-2 family cytokines might reflect the activity of the disease; these results support the hypothesis that IL-2 family cytokines are involved in the pathogenesis of AA.


  Conclusion Top


A higher plasma concentration of IL-2 was observed in patients with AA with G/T genotype.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Prie BE, Voiculescu VM, Ionescu-Bozdog OB, Petrutescu B, Iosif L, Gaman LE, et al. Oxidative stress and alopecia areata. J Med Life 2015;8:43-6.  Back to cited text no. 1
    
2.
Areata A. The Voice of the Patient: Alopecia Areata. Center for Drug Evaluation and Research (CDER) U.S. Food and Drug Administration (FDA); 2017. Available from: http://www.fda.gov/ForIndustry/User Fees/PrescriptionDrugUserFee/ucm326192.htm. [Last accessed on 2018 Jul 20].  Back to cited text no. 2
    
3.
Martel JL, Badri T. Hair Follicle. National Center for Biotechnology Information, U.S. National Library of Medicine. Bethesda MD: Rockville Pike; 2018.  Back to cited text no. 3
    
4.
Dakshinamurthi AK, Chidambaram MV, Manivel VA, Detchanamurthy S. Site directed mutagenesis of human Interleukin-2 gene to increase the stability of the gene product. Int J Bioinform Res 2009;1:4-13.  Back to cited text no. 4
    
5.
Thornton AM, Donovan EE, Piccirillo CA, Shevach EM. Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J Immunol 2004;172:6519-23.  Back to cited text no. 5
    
6.
Alanzy AK, Alta'ee AH, Alrubiae SJ. Serum tumor necrosis factor alpha and Gene polymorphisms in rheumatoid arthritis patients in Babylon province, Iraq. J Global Pharm Technol 2018;10:387-95.  Back to cited text no. 6
    
7.
Sayad A, Movafagh A. The Association of − 330 Interleukin-2 Gene Polymorphism with Its Plasma Concentration in Iranian Multiple Sclerosis Patients. Scientifica 2014;Article ID 724653:4.  Back to cited text no. 7
    
8.
Alta'ee AH, Shemran KA, Chabuk SK. Role of testosterone on waist to hip ratio in women with hirsutism. J Global Pharm Technol 2017;9:163-8.  Back to cited text no. 8
    
9.
Abdullah RG, Alta'ee AH, Joodi MR. Association of -34 T > C CYP17 gene polymorphism with benign prostatic hyperplasia in Babylon province, Iraq. J Global Pharm Technol 2018;10:154-8.  Back to cited text no. 9
    
10.
Al-Mokhtar AM, Alta'ee AH, Al-Mokhtar NJ, Al-Hattab MK. Clinical association of thyroid hormones and psoriatic severity in patients with chronic plaque psoriasis. Biochem Cellular Arch 2018;18:415-8.  Back to cited text no. 10
    
11.
Kasumagić-Halilovic E, Cavaljuga S, Ovcina-Kurtovic N, Zecevic L. Serum levels of interleukin-2 in patients with alopecia areata: Relationship with clinical type and duration of the disease. Skin Appendage Disord 2018;4:286-90.  Back to cited text no. 11
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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