Association of IL-13 Gene Polymorphisms in Atopic Dermatitis Patients

Background: The genetic predisposition of atopic dermatitis is one important factor that leads to cell expression and releases of interleukin (IL)-13. IL-13 plays an important role in the induction of immunoglobulin E (IgE) and in the pathogenesis of atopic dermatitis (AD).

Objective: Since IL-13 is a major component in the expression of atopic diseases, analysis of distribution of G4257A and G4738A polymorphisms of IL-13 in atopic dermatitis, their relationship with atopic markers and the functional of impact of single nucleotide polymorphisms (SNP/SNP) interaction within the same gene are the main issue of the present study.

Methods: PCR-based restriction fragment length polymorphism assay was used to investigate IL-13 gene polymorphisms at + 4257 & + 4738 in atopic dermatitis patients. Furthermore, the serum IL-13 and total IgE levels were measured by ELISA.

Results: The studied parameters showed a significant association of genotyping and allele frequencies of G4257A mutation in atopic dermatitis group as compared with control group (Allele frequency was 28% in atopic dermatitis, 8% in control) with OR 4.85 for atopic dermatitis, P <0.05. No significant difference in allele frequency of IL-13 at G4738A in atopic dermatitis patients when compared with control (Allele frequency was 18% in atopic dermatitis, 10% in control), P >0.05. The mean value of total serum IgE and IL-13 were significantly higher in atopic dermatitis group as compared with control group, P <0.05. High significant increase in the total serum IgE and IL-13 levels were towards homozygous A/A than homozygous G/G in atopic dermatitis group. Linkage disequilibrium was observed between G4257A and G4738A variants, P <0.05 in atopic dermatitis. No significant association between allele and genotype frequency of G4257A and atopic dermatitis severity index. Significant increase of IL13 concentration and total serum IgE were also detected in the atopic dermatitis patients when compared with control. Significant association of IgE with atopic dermatitis severity index was found, P<0.001. A positive correlation was detected between serum IL13 level and total serum IgE level in atopic dermatitis group.

Conclusion: It could be concluded that a significant elevation of IL13 and total IgE level in sera of patients and the positive correlation between them support the effect of IL13 on induction of total IgE. The G4257A substitution of IL13 only leads to atopic susceptibility

Association of IL-13 Gene Polymorphisms in Atopic Dermatitis Patients

Ola El-Asar*, Manal El-Sayed*, Inas Al Makhzangy*, Samia Ibraheem*, Eman Nofal* & Hossam Salah**

Departments of Dermatology & Venereology* and Biochemistry**, Faculty of Medicine, Zagazig University

Abstract

Introduction

The cause of atopic dermatitis (AD) is multifactorial. Immunological abnormalities as well as physiological and biochemical defects of the skin barrier structure are involved in the pathogenesis. Furthermore, genetic and environmental factors can modify these abnormalities⁽¹⁾.

Several genome-wide searches had provided evidence for the linkage of atopy to loci on multiple autosomal chromosomes of particular interest is the 5q31-33 region, which contains the Th2 cytokine gene cluster (Interleukin (IL)-4, IL-13, IL-5 and IL-9) and had been linked to atopy in many studies⁽²⁾.

IL-13 is a 12-kd cytokine cloned from activated T cells. IL-13 is produced at relatively high levels by both Th1 and Th2 CD4 T cells after activation. In addition both Th0 and CD8⁺ T cells produce considerable quantities of it. Unlike IL-4 it is also produced by native CD 45RA T cells. Thus, IL-13 does not behave as a classical Th2 cytokine. IL-13 is also produced by a number of non T-cell populations such as keratinocytes, mast cells and basophils⁽³⁾.

The functional IL-13 receptor complex is heterodimer composed of IL 4-Ra chain and a 65- to 70 Kd protein termed IL-13 Ra1 and IL-13 Ra2. This receptor binds IL-13 not IL 4 but does not import in signaling, currently called decoy receptor since it is found in soluble form. The receptor complex formed by IL-4Ra and IL-13 Ra1, also acts as a receptor for IL-4 γc in cell lacking IL4Ra chain⁽⁴⁾. IL-13 is a central mediator of allergic inflammation and is sufficient to induce most, if not all, of the key features of atopy⁽⁵⁾. IL-13 produced by Th2, promotes class switching in the B cell causing it to synthesize IgE antibodies⁽⁶⁾.

In support of the notion that IL-13 is a major component in the expression of asthma and atopy, several studies examining the roles that the polymorphisms in the IL-13 gene have effect on the activity or expression levels of IL-13 and, perhaps more importantly, down-stream responses to these changes will provide valuable insight on the overall mechanisms that cause susceptibility to atopy.

IL-13+4257G®A is of particular interest because that single nucleotide polymorphism (SNP) is found in approximately 25% of the general population⁽⁷⁾ and is expected to result in the non-conservative replacement of a positively charged arginine (R) with a neutral glutamine (Q) at position 130 signaling peptide:

The R130 Q substitution occurs in a-helix D, the region of IL-13 that is thought to interact with IL-4Ra/IL-13 Ra1 heterodimers. Therefore, IL-13+ 4257 G®A has a potential to affect IL-13 dependent signaling events⁽⁸⁾.

In detailed analysis of the gene encoding IL-13, Howard et al.⁽⁹⁾ have identified the polymorphism at 3^׳ untranslated region (3'UTR) (at position 4738) and have showed a significant association between that polymorphism and atopy.

Aim of the Present Study

This work had been designed for identification of the allelic distribution of G4257A and G4738A polymorphisms of IL-13 in atopic dermatitis, their relationship with atopic markers and the functional of impact of SNP/SNP interaction within the same gene.

Subjects and Methods

This study was carried out at the Medical Biochemistry Department and Dermatology Clinics, Faculty of Medicine, Zagazig University. The study included 25 patients with atopic dermatitis of both sexes with an age ranging from 8-22 years and 13 normal control subjects (age and sex matched). All patients met the diagnostic criteria for atopic dermatitis, as defined by Hanifin and Rajka⁽¹⁰⁾. None of these patients had other atopic conditions (asthma, rhinitis or conjunctivitis) nor received antihistamines, or systemic or topical corticosteroids during the period of 3 weeks before clinical evaluation. The severity of atopic dermatitis was measured by using the SCORAD index⁽¹¹⁾. AD was considered mild, moderate, and severe forms in which the SCORAD index was less than 25, between 25 and 50 and 50 respectively.

Both patients and healthy control were subjected for the following assessment:

· Full history taking and clinical examination.

· Stool & urine analysis to exclude parasitic infestations.

· Determination of serum total IgE level by ELISA according to Dorrington and Bennich⁽¹²⁾.

· Determination of serum IL-13 by ELISA according to Huang et al.⁽¹³⁾.

· Determination of Arg 130GIn polymorphism by PCR-based restriction fragment length polymorphism(RFLP) according to Graves et al.⁽¹⁴⁾

· Detection of IL-13 gene polymorphism at +4738 by PCR-based RFLP according to Howard et al.⁽⁹⁾

Genetic analysis

Determination of Arg 130 GIn polymorphism by PCR-based restriction fragment length polymorphism:

DNA extraction was performed according to Bubbon⁽¹⁵⁾ and using the PUREGENE DNA isolation Kit purchased from Gentra and DNA purity and quantity was determined as described by Surzychi⁽¹⁶⁾.

Amplification of Arg 130 GIn polymorphism primers: (from Biosynthesis)

· 5'-CTT CCG TGA GGA CTG AAT GAG ACG GTC-3'(sense)

· 5'-GCA AAT AAT GAT GCT TTC GAA GTT TCA GTG GA-3' (antisense)

PCR Reaction: PCR was performed in a final volume of 15 ml that contained: DdH₂O 8.9 ml 10X PCR Buffer 1.5 ml 10 mM dNTPs 0.30 ml for each, 100 pmol/ul 5' Primer 0.15 ml 100 pmol/ul 3' Primer 0.15 ml 50 mM Mg Cl₂ 0.5 ml 5 U/ml TAQ Polymerase and 0.6 ml Genomic DNA 2 ml.

PCR Conditions: The amplification was carried out by using thermal cycler PTC-100 machine (MJ Research, Inc., Watertown, Mass. USA) according to the following protocol; 1 cycle of 94°C for 2 minutes, then 33 cycles of 94°C for 40 sec, 55°C for 40 sec, 72°C for 50 sec, and then 1 cycle 72°C for 10 minutes. Then it was stored at 4°C till use.

Restriction Digestion Reaction: The digestion was performed in a total volume of 35ml that contained: 10X NEBuffer 4(3.5 ml), 100X BSA (0.35 ml), 1 U/ml 5 ml of Nla IV from (New England Biolabs, Boston, Mass). DdH₂O (11.15 ml), PCR Product (15 ml) and incubate at 37°C for no less than 8 hours.

Gel electrophoresis for PCR-digested products: Each digested product sample was prepared and electrophoresed on 1.5% agarose then the gel was visualized under UV trans-illuminator with 100 base pair DNA ladder and photographed.

Genotyping of interleukin 13 gene at +4738 by PCR-based restriction fragment length polymorphism⁽⁹⁾

Amplification of interleukin 13 Polymorphism at position 4738:

Primers: From Biosynthesis

· 5'-CTT TGC TAA CAT ATT TAA TAT TTA AAT ACG-3' (sense)

· 5'-GTC ACC GTT GGG GAT TGG GGA AG-3' (antisense)

PCR Reaction: PCR was performed in a final volume of 10 ml that contained: DdH₂O 4.3 ml, 10X PCR Buffer, 1 ml 10 mM dNTPs 0.30 ml for each, 100 pmol/ul 5' Primer 0.15 ml, 100 pmol/ul 3' Primer, 0.15 ml 50 mM MgCl₂, 0.5 ml 5 U/ml TAQ Polymerase and 0.2 ml Genomic DNA 2.5 ml.

PCR Conditions: The amplification was carried out using pervious thermal cycler PTC-100 machine according to the following protocol; 1 cycle of 94°C for 4 minutes, then 30 cycles of 94°C for 30 sec, 68°C for 30 sec, 72°C for 30 sec, and then a final extension step at 72°C for 6 minutes. Then it was stored at 4°C till use.

Restriction Digestion Reactions: The digestion was performed in a 30 ml total volume that contained" 10X NE buffer, 2, 3.0 ml 100X BSA (0.3 ml), 1 ml of 10 U/ml Nhe 1 from 0 New England Bio Labs, Boston, Mass 1 ml Dd H₂O 15.7 ml and PCR Product 10 ml Incubated at 37°C for less than 8 hours.

The alleles were resolved by electrophoresis on a 1.5% agarose gel. Then the gel was visualized under UV transilluminator with 100 base pair DNA ladder and photographed.

Statistical analysis: Was done with Microsoft WinPEP statistical program. Parametric data we compared using mean±SD and ANOVA test for expression of them. Nonparametric data was analyzed by Chi-Square test.

Results

The study included 25 patients with atopic dermatitis of both sexes. The demographic data for the studied groups was shown in Table (1).

Arg 130 GIn IL-13 polymorphism:

Genotyping frequency in studied groups: In control group, none had homozygous AA, while atopic dermatitis group showed 8%. There was significant difference of AA genotype distribution in atopic dermatitis patients when compared with control group P <0.05 (Table 2).

Allele frequency in studied groups: In atopic group, there was a significant association of A allele than in control group (P <0.05) (Table 3).

IL-13 gene polymorphism at +4738 genotyping frequency in studied groups: In control group, none had homozygous AA, while AD group showed 8% of AA (mutant form). There was no significant difference of all forms of genotypes in atopic dermatitis when compared with the control group (P = 0.34) (Table 4).

Allele frequency in studied groups: There was no significant difference in A allele and G allele between atopic dermatitis group and those in control group (P >0.05) (Table 5).

Association of allele and genotype frequencies with atopic dermatitis severity in G4257A polymorphism: There was no significant difference in genotype frequency between atopic dermatitis patients with mild disease and those with severe disease (P = 0.05). There was also no significant difference in allele frequency between atopic dermatitis patients with mild disease and those with severe disease (P = 0.24) (Table 6).

The relation between genotyping of both polymorphism as indicator for the linkage disequilibrium: Association of G-A substitution in the two sites of the IL-13 gene was significant in atopic dermatitis group (P <0.05) where as there was no significant association between A 4257G A4738G in the control group (Tabes 7 & 8).

The relation on between serum IgE, serum IL-13 and different alleles of G 4257A in atopic dermatitis group: In studied group patients, simple analysis of variance revealed a significant difference of the mean value of serum total IgE level, serum IL-13 level among different allelic variants (F = 9.2, 6.7 respectively) and P <0.05. There was a highly significant increase in the serum level of total IgE towards homozygous AA in atopic dermatitis (P <0.001) as shown by LSD test. There was also, a significant increase in the serum level of IL-13 towards homozygous AA in atopic dermatitis (P<0.05) as shown by LSD test (Table 9).

The relation between serum IgE, serum IL-13 and the allelic variants of IL-13 gene polymorphism at +4738: Simple analysis of variance revealed a significant difference of the mean values of serum total IgE level, serum IL-13 level among different allelic variants in atopic dermatitis group (F = 3.5, 7.2 respectively) and P <0.05. There was a highly significant increase in the serum level of total IgE towards homozygous AA in atopic dermatitis (P <0.001) as shown by LSD test, there was also, a significant increase in the serum level of IL-13 towards homozygous AA in atopic dermatitis (P <0.05) as shown by LSD test (Table 10).

Serum IL-13 and total IgE levels in the studied groups: Comparing with the control group it showed a highly significant increase of the mean values of serum IL-13 and atopic dermatitis group (P <0.001). There was no statistically significant correlation between serum IL-13 levels and severity of atopic dermatitis (P <0.05). Comparing serum total IgE level between control group and atopic dermatitis group, showed a highly significant increase of serum total IgE in AD (P <0.001) as compared with the control. A significant relation of IgE and disease severity (P <0.001) was also detected (Tables 11 & 12).

Eventually there was a significant positive correlation between serum IL-13 and serum total IgE levels in the studied groups (Table 13).

Table 1. Demographic data of atopic dermatitis (AD) group versus control group.

	Control (n = 13)	AD (n = 25)	P
Age (yrs):
Range	8-26	8-22
Mean ± SD	16±4.3	15.8±3.1	0.31
Sex:
Male	7	14
Female	6	11	0.78

P value versus control group P >0.05.

Table 2. Genotyping frequency for G 4257A polymorphism of IL-13 gene

	Control group		Atopic dermatitis group
	No	Percent	No	Percent
AA	0	0 %	2	8%
AG	2	16%	10	40%
GG	11	84%	13	52%
X²			6.45
P			0.039*

* P <0.05 when compared with control.

Table 3. Allele frequency and odds ratio for G4257A polymorphism of IL-13 gene for A4257 allele in studied groups

	Control group (n = 26)		Atopic dermatitis group (n = 50)
	No	Percent	No	Percent
A allele	2	8 %	14	28%*
G allele	24	92%	36	72%
Odds ratio (95% CI)			4.85 (1.1-22.87)^*

* p <0.05 when compared with control.

Table 4. Genotyping frequency for IL-13 polymorphism at +4738.

	Control group		Atopic dermatitis group
	No	Percent	No	Percent
AA	0	0 %	2	8%
AG	3	20%	5	20%
GG	10	80%	18	72%
X²			2.11
P			0.34

* p <0.05 when compared with control.

Table 5. Allele frequency for IL-13 gene polymorphism of +4738

	Control group (n = 26)		Atopic dermatitis group (n = 50)
	No	Percent	No	Percent
A allele	3	10 %	9	18%*
G allele	23	90%	41	82%

* p <0.05 when compared with control.

Table 6. Association of allele and genotype frequencies with atopic dermatitis severity in G4257A polymorphism.

	Mild	Moderate	Severe	X²	P-value mild vs severe
AA	-------	-------	2 (40%)	8.7	0.012
AG	4 (40%)	6 (60%)	-------
GG	6 (60%)	4 (40%)	3 (60%)
A	4 (20%)		4 (40%)	1.36	0.24
G	16 (80%)		6 (60%)

* (X²) -test.

Table 7. Genotyping of both polymorphisms as indicators for the linkage disequilibrium in control group

	A4257A	A4257G	G4257G	Total
A4738A	---------	---------	---------	---------
A4738G	---------	1	1	2
G4738G	---------	2	9	11
Total	---------	3	10	13

Agreement = 0.8, Kappa coefficient = 0.32±0.19, Z = 1.64, P >0.05

Table 8. Genotyping of both polymorphisms as indicators for the linkage disequilibrium in atopic dermatitis group

	A4257A	A4257G	G4257G	Total
A4738A	1	1	---------	2
A4738G	1	4	5	10
G4738G	---------	---------	13	13
Total	2	5	18	25

Agreement = 0.72, Kappa coefficient = 0.48±0.14, Z = 3.24, p >0.001, (X²) = 14.53, P <0.05

Table 9. Serum IL-13 (ng/mL) and total IgE levels (1U/ml) and different alleles of 4257 in atopic dermatitis group (n = 25).

	AA	AG	GG	F	P
IL-13	(n = 2)	(n = 10)	(n = 13)	6.7	<0.05
	16.63±1.08	13.98±1.9	12.6±1.3	6.7	<0.05
IgE	(n = 2)	(n = 10)	(n = 13)	9.2	<0.05
	452.3±30.3	259.7±48.5	271.3±67.5	9.2	<0.05

Table 10. Serum IL-13 (ng/mL) and total IgE levels (1U/ml) and different alleles of A4738 in atopic dermatitis group (n = 25).

	AA	AG	GG	F	P
IL-13	(n = 2)	(n = 5)	(n = 18)	7.2	<0.05
	15.37±0.7	15.42±1.9	12.73±1.4	7.2	<0.05
IgE	(n = 2)	(n = 5)	(n = 18)	3.5	<0.05
	452.3±30.2	274.7±46.3	264±63	3.5	<0.05

Table 11. Serum IL-13 levels (ng/ml) and total IgE levels (IU/ml) in the studied groups.

				Control (n = 13)		AD (n = 25)		F		P
		IL-13:
	Range		3.73-7.55		10.94±17.4		214.3		<0.001
Mean ± SD			5.22±1.04		13.44±1.9
		IgE:
	Range		67.85-146.0		188.1-473.7		56.157		<0.001
Mean ± SD			104.84±25.84		281.25±76.65

Table 12. Relation of serum IL-13 levels (ng/ml) and total IgE (IU/ml) in atopic dermatitis patients according to the severity of the disease.

	Mild (n = 10)	Moderate (n = 10)	Severe (n = 5)	F	P
IL-13:	12.79±1.3	13.55±1.9	14.54±2.5	1.6	0.05
IgE:	219.6±18.05	280.98±37.7	404.98±51.9	47.43	<0.001

Table 13. Correlation between serum total IgE and serum IL-13 in studied groups.

	r	p
Control group	0.6	< 0.05
Atopic dermatitis group	0.5	< 0.05

Association of IL-13 Gene Polymorphisms in Atopic Dermatitis Patients

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