INTRODUCTION:

Asthma is one of the diseases of chronic respiratory tract infection characterized by obstruction of the airways and lack of function of the lungs leading to and shortness of breath (1). Is different in its causes etiology, severity, pathogenicity and response to treatment (2). Asthma is a disease with a complex genetic background that occurs as a result of the interplay between genetic and environmental factors as well as the race that plays an important role in the readiness of individuals to cause the disease so that genetic predisposition genes may vary from one place to another (3).

Its diverse and frequent symptoms involve many cells cellular elements include mast cells, T-cell cells, eosinophil's cells, neutrophils, macrophages, and epithelial cells (2).

Genetic changes in more than 100 genes play essential role in pathogenesis of asthma, despite the variability in the roles of these genes (4). The identification of genes for the genetic predisposition of asthma is very important" because it can help in diagnose of disease and identify the vital pathways of the used drugs which can lead to reduced disease severity (5). With the completion of the human genome Project, studies on single nucleotide polymorphism SNP in genes related to asthma that play an important role in pathogenesis have increased, as well as their importance in determining the quality and quantity of therapy (6).

Many genes are involved in asthma pathogenesis, most notably is ADAM33 as a genetic predisposition to asthma. The ADAM33 gene is highly expressed in sub epithelial fibroblasts and smooth muscle of lung, which supports its participation in airway hyper-responsiveness (7). Thus, any change in the effectiveness of the protein can affect the function of these cells, importantly bronchial hyper-reaction and airway obstruction induced by the presence of airway remodeling is closely correlated with asthma (8). The ADAM33 gene is located on chromosome 20p13, length of about 14 kilo base pair, containing 22 exons and 21 introns and its encoded to a protein consisting of 813 amino acid (8). The ADAM33 gene contain more than (SNPs 37), many of which are closely associated with the genetic predisposition of asthma in many populations (9,10). Several studies have been performed on the relationship between ADAM33 (SNPs) and asthma especially (V4, rs2787094 C/G), but some have given conflicting results and this can be because of ethnic diversity (11). ADAM33 is associated with bronchial hyper responsiveness and airway remodeling, but its association with asthma (12). ADAM33 is a subgroup of metalloproteinases, a group of enzymes that are involved in cell fusion, proteolysis, cell adhesion and cell signaling growth factors and receptors that are associated with inflammation. ADAM33 lead to hypertrophy of airway smooth muscles that produce of activation of the Th2-mediated allergic reaction and bronchial hyper-responsiveness (13), ADAM33 plays a significant role in regulating the immune response by T helper 2 cells (9).

Immunoglobulin E (IgE) is a glycoprotein that characterized by high of concentration in asthmatic patients, although its normal level about (0.001%) of the total serum immunoglobulin (14). IgE plays an important role in the immunopathogenesis of asthma. Elevated IgE concentration is an indicator of allergic inflammation atopy and a marker of disease progression (15). Asthma patients with a higher level of IgE are more likely than those with low concentrations (16). Increased of IgE production in asthmatics patients promotes acute hypersensitivity responses and chronic acid-allergic allergic rhinitis with the production of T2 helper cytokines (17).

Antioxidants are one of the chemicals that can inhibit the oxidation of a molecule and can negate the pathogenic effects of oxidation caused by free radicals in living organisms (18). Increase of oxidative stress in asthma refer to that active oxygen and nitrogen may affect lung and airways function, including the contraction of smooth muscles of the airways, the response of airway tracts, mucosal hyperplasia, dysplasia and vascular perfusion (19). The presence of airway inflammation is an important biochemical feature of asthma. Oxidative stress with the reactive oxygen species is an essential component of inflammation, although the antioxidant of the host should remove reactive oxygen species. Genetic factors play important role in ability of the person to deal with oxidation leading to differences in the continuity of inflammation, activation of the mechanisms of the trachea and accelerate the onset of symptoms of asthma (20). Non-enzymatic antioxidants such as glutathione are one of the main protective antioxidants in the lungs that play a key role in regulating inflammatory responses. Glutathione plays a key role in defense against a variety of diseases. It removes toxins from carcinogens, free radicals and peroxides, regulates the immune system, immunohistochemical function and structure of protein structure (21). Reduced glutathione is the main molecule in an antioxidant system, it is able to deletion free radicals either directly, non-enzymatically or enzymatically by glutathione peroxidase thereby maintaining the cellular molecules in reducing stress. Some sources indicated that glutathione levels in the liquid of the endothelial respiratory tract were 100 times higher than plasma levels (22). Serum albumin is a multi-functional, negatively charged protein with transport properties and antioxidant functions. It has many anti-oxidant activities resulting from its ability to ligand-binding. Also it can connect with metals which might undergo oxidation reduction reactions (23).

As a result of the lack of studies in Iraq, which include the relationship between ADAM33 polymorphism and the immunological and antioxidant markers, the aim of this study was to evaluated the relationship between ADAM33 (V4) polymorphism and the level of IgE and some antioxidants of asthma patients in Iraqi population.

MATERIALS AND METHODS:

Subjects and Sample Collection:

The study was conducted on 70 patients with allergic asthma from the Zahra Center for Allergy and Asthma in Baghdad, who were diagnosed by specialist doctors and forty healthy selected as control group. The age of participants ranged between 18-75 years, some information has been taken from patients and healthy people such as age, sex, region and family history. Under complete aseptic conditions, five ml of blood were collected from all participants during the period from September to October 2017, each sample was divided into two parts: first (2ml) was collected in EDTA tube for DNA extraction and kept immediately at -20 °C, while the second part (3 ml) was collected in a normal test tubes until the serum is separated for measurement of biochemical tests.

Measurements of immunological and antioxidant parameters:

Serum IgE levels using Enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Abcam-UK). Glutathione, total protein and albumin were measured by using spectrophotometer for all asthmatic patients and controls.

DNA extraction and genotyping of ADAM33 (V4):

Genomic DNA was extracted from whole blood using a method described by (24), quality of the DNA was determined using 1 % agarose gel electrophoresis and its concentration was determined by using the Nanodrop (Thermo scientific, Germany), then ADAM33(V4) polymorphism was determined by Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR - RFLP) technique , using the following primers (USA): 5'-ACA CAC AGA ATG GGG GAG AG-3' (Forward) and 5'-CCA GAA GCA AAG GTC ACA CA-3' (Reverse) according to (25). PCR amplification was performed in a total volume of 20 μL including 10 μL of 2X Go Taq green master mix supplied by Promega company (USA), (100 ng) of genomic DNA and (10) picomole of each primer showed the best results, then complete the volume of reaction with distilled water to 20 μl. Components of the reaction were mixed well and cycling parameters were as follows: one cycle for 5 minutes at 94 ⁰C followed by 35 cycles, each cycle includes 94 ⁰C, 54 ⁰C and 72 ⁰C for 30 sec, with a one cycle of 5 minutes at 72 ⁰C for a final extension. The PCR product (374 bp size) was visualized on a 2% agarose gel electrophoresis stained with red save and imaging using Gel Documentation System. 10 units of the PstI restriction enzyme was added to 5 μl of the PCR product and incubated at 37 ⁰C for one hour and the digestion product were visualized on 3% agarose gel electrophoresis stained with Redsave in the presence of 50 bp DNA ladder (NEW ENGLAND Biolabs) as a molecular marker. Three types of genotypes were shown, which is single fragment (374 bp) as a CC homozygous, 2 fragments (206 and 168 bp) as a GG homozygous and 3 fragments (374, 206, 168 bp) as a GC heterozygous according to(25).

Statistical analysis:

Statistical analyses were done using SPSS version 20 computer software. The mean, standard deviation (SD) and of p-value of IgE and antioxidant parameters were calculated using student’s t-test (which considered significant when p<0.05 and highly significant when p<0.01) for the patients and healthy group. Hardy–Weinberg equilibrium and frequency of alleles and genotypes in addition of odds ratios (OR) and their 95% confidence intervals (CI) of the patients and healthy group were calculated by chi square test. Levels of IgE among asthma patients according to the CC, GC and GG genotypes of ADAM33 (V4) polymorphism were estimated using the student’s t-test.

RESULTS:

The ages of both groups ranged from 18-75 years with average of 42.3, there was no statically difference in the distribution of sex among patients (31 males, 39 females) and healthy (12 males, 8 females) in the patients and healthy group.

The comparison of IgE and antioxidant levels of the asthmatic patient and control groups participate in this study were presented in table 1.

Table (1): Comparison between IgE and antioxidant levels of asthma patients and control group

Parameter	Groups		P value
Parameter	Patients No. (Mean ± SD)	Control No. (Mean ± SD)	P value
IgE	70 (212.0±22.3)	20 (58.2±4.77)	0.001**
Glutathione	70 (10.8± 2.01)	20 (16.73± 4.87)	0.01**
Total Protein	70 (6.11± 0.39)	20 (8.82± 1.74)	0.01**
Albumin	70 (4.16± 1.32)	20 (5.21± 0.63)	0.01**

** significant P ≤ 0.01.

The results shown in table (1) indicate a significant increase (P <0.01) in the IgE levels in asthma patients compared to control group  this results agreement with (26), while there were a significant decrease (P <0.01) in the levels of antioxidants (glutathione, total Protein and albumin) of the asthma patients compared to the control group.

Table (2): Comparison between IgE and antioxidant levels of asthma patients and control group according to age and gender

Parameter	Groups		P value
Parameter	Patients No. (Mean ± SD)	Control No. (Mean ± SD)	P value
IgE (Gender)	Male 31 (209.0±21.3)	Female 39 (215.0± 23.0)	0.25
IgE (Age)	≥ 40 Year (213.0±26.2)	≤ 40 Year (212.0±17.5)	0.299
Glutathione (Gender)	Male 31 (11.43±1.77 )	Female 39 (10.3±2.07 )	0.01**
Glutathione (Age)	≥ 40 Year (10.78±1.5 )	≤ 40 Year (10.81±2.41 )	0.947
Total Protein (Gender)	Male 31 (5.92±0.84 )	Female 39 (6.26±0.97 )	0.136
Total Protein (Age)	≥ 40 Year (6.22±1.02 )	≤ 40 Year (6±0.82 )	0.320
Albumin (Gender)	Male 31 (4.1±0.81 )	Female 39 (4.2±0.46 )	0.586
Albumin (Age)	≥ 40 Year (6.22±1.02 )	≤ 40 Year (4±0.49 )	0.235

** significant P ≤ 0.01,

Also, the results shown in table (2) indicate there were insignificant differences in IgE and antioxidants levels expect the glutathione levels that decreased at a significant value (P <0.01) according to sex and age between patients and healthy group.

After PCR-RFLP analysis of the ADAM33 (V4) polymorphism, there are three types of genotypes (CC, CG, GG) with band sizes of 374 bp (Homozygous wild) 168/206 bp (Heterozgous), and 168/206/374 bp 374 bp (Homozygous mutant) (Fig 1).

The results in table (3) showed that number of GC heterozygous and GG mutant homozygous were higher (P value 0.0003, < 0.0001 respectively), while there was on any different in the number of wild homozygous genotype CC (P value 0.834) in asthma patients compared with control group. Also the number (90 with 64.3%) and frequency of mutant G-allele was higher (0.643) in asthma patient compared with control group (0.175) at P value < 0.0001, on the contrary the number of C allele (31 with 77.5%) and frequency (0.357) of patients vs (0.825) for control group at p value < 0.0001. The genetic differences between patients and control demonstrated that the presence of GG genotype (OR 0.23 and 95% CI 0.07 – 0.73) seems to confer a risk for asthma. Also high value of OR of G allele (6.200) with (95% CI 3.49 - 20.57) appeared as risk factor while C allele seems to be protective for the asthma in Iraqi society. The results of this study were consistent with the results which obtained by (27) that found that number of patients with GG was 31 (45%) which is higher than in the healthy 1 (5%). The number of patients with CG 29 (42%) was higher than in the healthy 4 (20%) and the number of patients with CC 9 patients (13%) was lower than compared with healthy 15 (75%). Also the results was agreed with (28), they found number of patients with GG was 248 (62%), higher than those with CC (19) with 4.8% without significant differences (P = 0.65).

To evaluate effect of both C and G alleles in ADAM33 (V4) polymorphism on the IgE concentration, the mean IgE was compared according to the genotype of patients group as in figure 2.

Figure (2): Shows comparison between IgE levels of patients group according to genotype of ADAM33 (V4)

When comparison of IgE levels between asthma patients according to the genotypes of ADAM33 (V4) polymorphism as shown in and figure 2.

There were statically differences between mean of IgE levels of CC genotype (199.0 ± 12.5) in comparison to the GG genotype (216.0 ± 22.5 ) with p value 0.023,

although the difference in mean of IgE levels of GG genotypes (216.0 ± 22.5) in comparison to the CG genotype (213.0 ± 22.6) but P value (0.60) was nonsignificant,

also when comparison of CC genotype had low mean of IgE levels (199.0 ± 12.5) with CG genotype (213.0 ± 22.6) no significant differences were observed (0.064), these may be due to the small size of the patients sample. The results confirm influence of ADAM33 (V4) polymorphism on IgE levels and the IgE levels were highest in GG and GC in comparison with CC genotypes, thus may be G allele is a genetic marker for increased of IgE levels of asthma patients.

DISCUSSION:

IgE play an important role in the pathogenesis of allergic disorders. Increased levels of total serum IgE have been implicated in the clinical expression asthma (15). This agreement with our results that shown in tables 1, we found significant differences (P< value 0.01) in levels of IgE between asthma patients and control groups. IgE is responsible for the release of many inflammatory mediated in asthma from mast cells such as histamine, prostaglandins. These inflammatory mediated further narrow the airways by causing excessive excretion of mucus, smooth muscle spasm in the airways (29).

Glutathione is an antioxidant that protects cells and tissues from oxidation. Thus, the airways of the lungs have an antioxidant system that protects them from exposure to harmful oxidants (30). In antioxidant reactions, glutathione is converted to its oxidized form, GSSG, that severe asthma causes low levels of glutathione and that its lowness leads to overreaction of airways,. glutathione reduces muscle contraction and therefore low levels lead to increased oxidative stress (31). There is evidence that allergies such as bronchial asthma, allergic rhinitis, and allergic skin dermatitis cause oxidative stress (32).

Albumin is the most abundant protein in blood plasma acting as an important antioxidant contributing up to 28% of antioxidant effectiveness (33). That its antioxidant properties are effective because of its structure, so because its anti-oxidant properties albumin will consume and therefore plasma levels decrease in asthmatic patients (34). Albumin works through multiple binding sites and has properties in capturing free radicals from asthma (35). Low levels were reduced in a study of japanese adults and adults with asthma (36,37)

ADAM33 has a close association with asthma, where it has been observed to have high intracranial expression and fibroblasts of the airway. The polymorphism of ADAM33 causes dysfunction of the smooth cells of the airways of the lungs and a decline in the lining of the lungs (38). The genetic variations of ADAM33 play important role in asthma progression and pathogenesis because of these variations lead to abnormal changes in fibroblasts and bronchial smooth muscle cell functions that is correlated with the advance of airway inflammation (39). The ADAM33 gene association with asthma has been studied in many societies and some results suggest it is associated with lung dysfunction. ADAM33 protein can also be considered as a biomarker of disease severity because it plays an "important" role in airway remodeling (40). ADAM33 is a polymorphic gene with large number of SNPs, several studies showed a significant associations of rs2787094/V4 polymorphism with asthma in the many populations (41).

Although many studies have been conducted in many communities around the role of ADAM33 as one of the genetic predisposition genes for asthma, a few studies have found a close correlation between the genetic differences of the gene and asthma, this is still in its early stages (42). The present study focused on SNP (rs2787094/V4) of ADAM33 gene, detection of ADAM33 (V4) SNP was successfully amplified and the results showed this SNP was associated with asthma in Iraqi population, these results are consistent with previous results such as (42,43) in Iraqi population and other countries such as China population (8). For our knowledge, can be said that this study is the first that evaluates the relationship between the polymorphisms of ADAM33(V4) gene and the concentration of IgE and some antioxidants levels with asthma Iraqi patients. Our findings have supported the importance of ADAM33 (V4) gene polymorphism in Iraqi society and number of other studies should be conducted to establish the relationship between the genetic differences of this gene and asthma, also to clarify the functional role of ADAM33 which will help to understand the causes of the disease and its pathological aspects.These studies should include a large number of specimens preferably for different locations in Iraq, including the effect of the allele on the protein of this gene.

In conclusion, our results demonstrate that: there was a significant correlation between elevated IgE and decreased antioxidant levels in addition to ADAM33(V4) gene polymorphism were associated with asthma patients compared to controls in Iraqi population. The homozygous mutant genotype GG and the mutant allele G of ADAM33(V4) can be considered as an marker of genetic predisposition to asthma and the presence of G allele can lead to increased IgE, And thus lead to increased severity and progression of the disease.

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Received on 20.10.2018 Modified on 30.11.2018

Research J. Pharm. and Tech 2019; 12(2):516-522.

DOI: 10.5958/0974-360X.2019.00091.X

Genotypes	Patients No. (70)		Control No. (20)		c2	*P valu*e	OR	(95% CI)
Genotypes	No.	%	No.	%	c2	*P valu*e	OR	(95% CI)
CC	11	15.8	12	60	0.043	0.834	1 Ref	-
CG	28	40	7	35	12.6	0.0003**	0.13	(0.01 – 1.12)
GG	31	44.2	1	5	28.125	0.0001**	0.23	(0.07 – 0.73)
Alleles
C	50	35.7	31	77.5	4.368	0.0001**	1 Ref.	-
G	90	64.3	9	22.5	4.368	0.0001**	6.200	3.49 - 20.57