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Analysis of T-cell Immunoglobulin and Mucin 1 Gene -416G

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Association analysis of T-cell immunoglobulin and mucin 1 gene -416G > C polymorphism with asthma in center of Iran.

Background

TIM1, One of the gene family members of T cell immunoglobulin (Ig) domain and mucin domain (TIM) expressing on TH2 cells, promotes producing of Th2 signiture cytokines and increases a series of responses in this cells which could be one of the causes of asthma or asthma-related phenotypes.We are determined to investigate whether a TIM-1 promoter single nucleotide polymorphism (SNP), -416G > C, associated with asthma in Chaharmahal va Bakhtiari and Isfahan, Iran.

Method

In a case-control study existence of the -416G > C polymorphism was examined using polymerase chain reaction (PCR) and restriction fragment length polymorphism in 130 healthy control and 130 asthmatic patient. Additionally, the relationship between this polymorphism genotypes and total serum IgE levels in this iranian population was evaluated.

Results

We discovered no association between the -416G > C polymorphism and asthma susceptibility in this population (p>0.05). But our results showed this significant relation between this polymorphism and serum IgE levels (p<0.05).

Conclusions

These results suggest that -416G > C polymorphism in TIM-1 gene, could not be a predisposing factor for asthma susceptibility in Chaharmahal va Bakhtiari and Isfahan but CC genotype of this SNP can be effective in increasing blood sugar levels in patients with asthma in a subset of Iranian population.

Key words

asthma, T-cell immunoglobulin and mucin domain molecule-1, polymorphism, single nucleotide polymorphism

introduction

Asthma is a chronic inflammatory disorder of the airway that result from abnormal (irregular) immune responses and induced by environmental factors in genetically susceptible individuals (p1 Palmer & Cookson, 2000). Asthma is a serious problem worldwide, with an estimated 300 million affected individuals (human being). (.2004 Masoli.Bateman 2008) People of all ages in countries throughout the (in every part of (…† world are affected by this disorder that, when uncontrolled, can (may be) place severe (some) limits on (daily life and occasionally is fatal (Beasley R The Global Burden of Asthma Report; ( Global Initiative for Asthma (GINA)www.ginasthma.org/ReportItem.asp?l1 = 2&l2 = 2&intId = 94 Date last updated.2012, Bateman 2008 ). It is proved that CD4+T cells plays a determinant role in the development of allergen-induced airway hyper-reactivity (AHR) and the pathogenesis ( pathological processes) of asthma Naive CD4+T helper (TH) cells after activation (When activated) can differentiate into TH1and TH2 cells. (p1Mosmann et al., 2005)Th1 cells are a subset of CD4 T cells which (and ) secrete (generate) the potent macrophage activating cytokine, IFN-γ. CD4 Th2 cells produce a different panel of cytokines, including IL-4, IL-5, and IL-10 ({Mosmann, 1986, Cohn, 1997). Th2 cells are potent (have a crucial role essential) in activating) B cells to (and then B cells) make antibody, particularly (especially) IgE (Kuhn, 1991 , Finkelman, 1986, Stevens1988) . The balance between TH1 and TH2 cells is vital in the normal immune response to pathogens, tumour antigens, and allergens If their balance was interrupted, both kinds of cells could result in pathological effect and diseases) ( in (for) the (a) normal immune response to pathogens, tumour antigens, and allergens, It is important that Balance between TH1 and TH2 cells to be maintained. Otherwise both kinds of cells could leads to pathological (detrimental) effect and diseases.) ŒTH1 cells are responsible for delayed hypersensitivity and organ specific autoimmune diseases, such as type 1 diabetes,while TH2 cells mediate asthma and other allergic diseases (TH1 cells can cause mediate hypersensitivity and organ specific autoimmunity whilst TH2 cells has the ability to cause asthma and other allergic diseases (Kuchroo 1995; Hofstra., 1998).

Results of several human linkage analyses have been (performed, and the results) implicate (Imply) at least 15 genetic loci in human atopy and asthma (p1.1, Steinke, 2003, De Souza, 2005) One human genetic locus that has received a great deal of attention (gained a lot of attention) is 5q23–35. The conserved syntenic region of mouse chromosome 11 has also been involved in the development of atopic asthma.. Further analysis identified a family of similar (alike) Tim genes (similar genes named Tim) consisting of (comprising of) eight genes (Tims1–8) on mouse chromosome 11B1.1 and three membersof this gene family (TIMs 1, 3 and 4) on human chromosome 5q33.2 (p1.1McIntire 2001// Kuchroo, 2003). TIM genes encode surface glycoproteins with common (usual) structural features including (containing) an extracellular immunoglobulin (Ig)-like domain, a mucin domain close to the membrane, a single transmembrane domain, and cytoplasmic regions of various lengths (McIntire 2004, Kuchroo 2003). on naïve CD4+ T cells TIM-1 is not expressed (emerge…. Umetsu2005, McIntire2001), but becomes upregulated within hours of TCR stimulation (During the time that TCR is stimulating) (emergMcIntire2001) and this molecule preferentially expressed on Th2 cells (emergUmetsu2005, Meyers2005) hence, TIM-1 has become a good (competent) candidate susceptibility gene for TH2-driven diseases (th2 related diseases), such as asthma ( p1). Recent population studies have demonstrated (indicated) that genetic variations in TIM-1 were associated with susceptibility to allergic diseases. (Chae2003, Wu Q 2009, Gao, 2005) polymorphisms in both murine and human TIM-1 linked with relative asthma susceptibility are found in the mucin domain. Found that both murine and human have polymorphisms associated with asthma susceptibility in the mucin domain of TIM-1 molecule.) (McIntire., 2004). Furthermore some Polymorphisms of the human TIM-1 gene found to)seem) be linked with the allergic response.

For instance It was reported that the frequency of the homozygous deletion variant (157delMTTTVP) in the 4 exon of the TIM-1 gene contribute to (effective in) asthma susceptibility in a African American population (Gao et al., 2005)while in a Japanese pediatric patient family study it was showed that none of genotype insertion/deletion polymorphisms in TIM-1 was preferentially transmitted to asthmatic children (p3Noguchi 2003). a other polymorphism study performed on the TIM-1 promoter region in a Korean population have identified, −416G>C and −1454G>A variation sites in the human TIM-1 promoter region, to be associated with development of allergic rinitis in a Korean population (Chae 2005). Furthermore it was found a promoter polymorphism, –416G>C is associated with asthma susceptibility in a Chinese Han population. In the present study, in order to determine whether the –416G>C SNP in TIM-1 promoter in Iranian population is significantly linked with asthma susceptibilityusing using population-based analyses. and, we evaluated the allele frequency of –416G>C. In this work we also investigated (surveyed) the relationship between the genotypes of this polymorphism and total serum IgE levels in asthma patients.

Material and method

The study was conducted on 300 patients including , and 309 controls from Hajar and Amin Hospital in Chaharmahal va Bakhtiari and Isfahan Provinces respectively, Provinces in center of Iran. As controls, we enrolled age-matched healthy individuals.

These healthy subjects did not have any of the symptoms or personal or family history of allergic and respiratory disease in their previous history or past physical check-up. Furthermore Both of these groups, including patients and healthy enrolled in the study were living in the same area of (the)Chaharmahal va Bakhtiari and Isfahan provinces in central Iran. All subjects with asthma were diagnosed according to the criteria of the National Institutes of Health (National Heart, Lung, and Blood Institute. Guidelines for the diagnosis and management of asthma. National Asthma Education Program, Expert Panel Report.

J Allergy Clin Immunol 1991;88:425-534.

The average ages of the patients with asthma and helthy controls were 42.84 years and 40.36 years, respectively. Our study was approved by the ethical committee of the university, and informed consent was earned from all control and patient subjects.

DNA extraction

Four milliliter whole blood anti-coagulated with EDTA obtained from all individuals in groups of patients and healthy. and stored at 4CËš.then Genomic DNA was extracted from leukocytes of peripheral blood using a Genomic DNA isolation kit (feldan,Canada) according to the manufacturer’s directions provided by the kit.

PCR Amplification and Sequencing

The genotyping of the -416G > C polymorphism was performed by restriction fragment length polymorphism (RFLP). A 879 bp fragment in the TIM1promoter region of was amplified by PCR. The primers used to amplify the fragment containing the SNP were: forward 5′- AGTTGGTTGATTCATATGAGCC-3′ and reverse 5′-GGAGGTGTAGTCTGAAGCATG-3′ .. PCR was carried out in a total volume of 25 µL containing The PCR mixture contained 150 ng of genomic DNA and 2.5 µL of 10X PCR buffer, 1 U of Taq DNA-polymerase, 200 µmol/L of dNTPs and 400 nmol/L of each primer. Amplification was carried out with an initial denaturation step at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at optimum temperature for 45 s, and extension at 72 °C for 45 s. And lastly a final extension was performed at 72 °C for 5 min. PCR amplification was carried out in a programmable PCR in an Eppendorf thermal cycler (Eppendorf Germany).

For genotyping the SNP, was used TaqI restriction enzyme (Fermentas) And for this purpose PCR products containing the SNP were incubated (overnight , 65ºC) with TaqI restriction enzyme (Fermentas) according to the manufacturer’s instructionsand and electrophoresed on 1.5% agarose gel and stained with DNA green viewer and visualized under UV light. TaqI cut the 879 bp PCR product into fragments 488 and 391 bp in size. Fragments of 488 and 391 bp indicated the presenceof homozygous -416GG genotype, a single 879 bp fragment (band) represented the presence (appearance) of homozygous -416CC genotype and three fragments of 879, 488 and 391 bp displayed the presence of heterozygous-416GC (Fig.1) Genotyping results (Accuracy of results) were approved by several randomly selected samples In addition to the forward primers used in the PCR for direct sequencing (Fig.2). The results of sequencing were 100% (completely) accordant with PCR-RFLP genotyping .

Staistical analyze

The SPSS 20 software package (SPSS company, Chicago, IL, USA) was used to carry out statistical analyses.The chi square test was first applied to compare the frequency distribution of gender, and smoking status between cases and controls. In addition, this test was used to compare the genotype distributions, allele frequencies between two groupe of patients and controls. Association between this polymorphism and asthma was expressed (declared) as odds ratios (OR) estimates with 95% confidence intervals (95% CI). Logistic regression analysis was used to predict (prophesy) the relation of the - 416G>C SNP with susceptibility to asthma. Furthermore age, eosinophils percentage and total serum IgE levels between two groups were compared by means of Independent T-Test and for compartion mean serum IgE levels and eosinophils percentage. between genotype groups standard analysis of variance (ANOVA) and analysis of covariance (ANCOVA) were used. And Pvalue <0.05 was considered significant in all of these tests. In addition to having a normal distribution for the analysis IgE levels were change to log10 values.

Result

Table1. frequency distributions of selected variables and characteristics of the study population in case and control

case (N = 300) control (N =309) P-value

Age (mean±SD) 42.76±14.66 42.94±14.68 0. 189

Gender

male 121 (40.3%) 120 (38.8%) 0.705

female 179 (59.7%) 189 (61.2%)

Eosinophils (mean±SE)103per µl 0.255±0.017 0.857±0.003 0.000*

total serum IgE log10 (mean±SD) 1.727±0652 0.773±0.390 0.000 *

Smoking

no 270 (90%) 276 (89.3%) 0.783

yes 30 (10%) 33 (10.7%)

In the present study, we analysed -416G > C SNP of TIM-1 gene in 300 patients with asthma and 309 controls from a Iranian population. Selected characteristics of two groupes and the relationship with asthma are shown in Table1. According to this table the matching found on two variables, age and gender, was sufficient and there were no major variances in mean the two variables distribution between patients and controls. Compared with the controls, the cases had higher eosinophils percentage and total serum IgE levels (Pvalue = 0.000). In contrast smoking status was not statistically different between two groups (Pvalue =0.783). A successful genotyping for the SNP in the all individual performed (Figure1) and verified by sequence analysis of PCR products. The genotype distribution in all of the subjects was agreement with that expected by Hardy-Weinberg equilibrium.

Table2. Genotype and allele frequencies of TIM-1 -416 G >C polymorphism

 

Control (N/%)

Patients (N/%)

Crude Odds Ratio

(95% CI)

P-value

Allel

       

C

157 (25.4%)

172 (28.6%)

1.007 (0.728-1.329)

0.965

G

461 (74.6%)

428 (71.3%)

0.601 (0.381-0.947)

0.027*

Genotype

       

CC

36 (11.7%)

54 (18%)

1.665 (1.056-2.625)

0.027*

GG

188 (60.8)

182 (60.7%)

0.993 (0.717-1.374)

0.965

GC

85 (27.5%)

64 (21.3%)

0.492-1.037))0.715

0.076

The frequency of -416C/C, C/G and G/G genotypes in patients was 18% % and ,60.7 %, 21.3% respectively and the frequency of C and G alleles was 28.6%and 71.3%, respectively. The multivariate logistic regression analysis was applied to investigate the association between the -416G > C polymorphism genotypes and asthma. As shown in Table 3 none of the genotype forms had an risk or protective impact on asthma. However the CC genotype was associated with the risk of asthma before OR adjustment. (adjusted OR = 0.477, 95%, CI = 0.214-1.063, P value =0.070 , crude OR = 1.665, 95%, CI = 1.056-2.625, P value =0.027 ) but the GG, GC genotypes had no effect in relation to asthma in any way (in no way). According to table 2 G allel showed protective effect on asthma, but after or adjustment this allel had no correlation with asthma.

Furthermore total serum IgE log10 difference between genotype groups investigate by ANOVA test and a significant difference in serum IgE log10 was observed between these groups (table 4). Bonferroni correction showed that this difference was related to CC genotype. In other words ins/ins genotype has a significant association with total serum IgE level. After adjusting for variables age, gender and smoke status variables, using ANCOVA test, this association survived (table 5).

Table 3. Adjusted Odds Ratios with 95% Confi dence Interval (CI) in TIM-1 -416 G >C genotypes with adjustment for age, gender,smoke statuse, eosinophil count and total ige.

 

adjusted Odds Ratios

P-value

Groups

CC

0.477 (0.214-1.063)

0.070

GG

1.593 (0.929-2.734)

0.091

GC

0.857 (0.470-1.565)

0.616

Alells

C

0.628 (0.366-1.077)

0.091

G

2/099 (0.941-4.680)

0.075

Table4. Association between total serum Immunoglobulin (Ig) E levels and Tim-1 -416G > C polymorphism genotype.

 

Total Serum IgE, log10 values

Mean ± SD

F

P-value

Genotype group

     

CC

2.089 ± 0.685

11.59

0.000*

GG

1.619 ± 0.606

   

GC

1.730 ± 0.647

   

Table5.analysis of covariance of the effect of 416G > C on Total Serum Immunoglobulin (Ig) E Levels in

Patients With Atopic Asthma after Adjustment for Age and gender and smoke status.

 

Total Serum IgE, log10 values

Mean ± SD

F

P-value

Genotype group

     

CC

2.089 ± 0.685

11.49

0.000*

GG

1.619 ± 0.606

   

GC

1.730 ± 0.647

   

Discussion

In developed countries, atopic diseases such as asthma are the main causes of morbidity and moreover, their frequency has been increasing (Noguchi1,2). Asthma,a complex (multifactorial) disorder, is affected by environmental and genetic factors and other agents. (2pdf 28). through genomewide screens several loci have been discovered for Predisposing to asthma (noguchi3-9), such as 5q31-33 region. The T-cell immunoglobulin and mucin domain (TIM) gene family, located at 5q33, involve in regulating T-cell proliferation and TH differentiation.

Numerous studies have investigated the TIM-1 polymorphism association with risk of various allergic diseases such asthma. A few studies search for possible connection -416G > C with asthma in different populations, but with contradictory findings. Until now, no researches have been Undertaken on Iranian population to evaluate (appraise) the correlation of this SNP with athma risk. In current study, we investigated this TIM-1 gene promoter polymorphism population with asthma in a subset of Iranian population. No significant association was found between aleel genotype frequencies of -416G > C with asthma in the target population. None of CC, GG, GC genotypes were not affect the risk of asthma. Our finding were inconsistent with a research in Chinese Han (liu2007 14 ¯± pdf3) that showed a association between -416G > C and asthma. And also with a report in Chinese Han indicated that this SNP linked to susceptibility to allergic rhinitis. But seemingly present result consistent with a lake association between -416G > C with asthma in Korean population. The divergent finding may perhaps be due to difference in sample size and or ethnic diversity with particular genetic background (base) and interaction (Interactivity) of distinct environmental factors may be affect the efficacy of this polymorphism. Moreover we investigate The effect of this polymorphism on serum total IgE level in patients that our result showed -416G > C CC genotype can affect total serum IgE level increasing. To our knowledge, None of pervious studies have been yet conducted this survey in patients with asthma. Overall no association was observed between -416G > C and the risk of asthma in Iranian population but this polymorphism can affect the level of total serum IgE level.


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