IL-13 and STAT 6 Gene Variants and Atopic Disorders-A Case Control study from the Northern State of India

Syed Mudassar^1*, Taha Ashraf Qurashi¹, Gulzar Ahmad Bhat¹, Mosin Saleem Khan¹ and Roohi Rasool^2
*Correspondence: Syed Mudassar, Department of Clinical Biochemistry, SKIMS University, Srinagar, India, Tel: 9419016201, Email:

Keywords

Atopy • IL-13 • STAT 6 • Gene variants • Kashmir

Introduction

Allergic diseases are of great health concern with very wide geographical difference in its incidence. Though, the disease is being considered as an epidemic in developed countries and has doubled in past 2 to 3 decades but, currently the incidence is increasing alarmingly both in developing and developed world [1]. India is experiencing an increasing trend, which otherwise was considered as low endemic zones for such disorders. The disease is multi etiological without a single factor being established as a causal agent. Therefore, etiology remains unclear rather than conclusive. Epidemiological research over the last years has helped to identify possible environmental factors associated with allergic diseases [2]. Among the various risk factors like smoke in different forms, pollution, microbial infections, occupational exposures, diet, pesticide use etc have been associated with allergies. However, the worldwide variation in its incidence suggests a complex interplay of multiple environmental, socioeconomic, and cultural factors. Further research regarding the role of various risk factors in modulating differences in allergic disease prevalence between migrant and native populations will enhance our understanding of the complex gene-environment interactions involved in the pathogenesis of allergic disorders [3,4].

The condition that result due to exposure of body to environmental substances is characterized by raised IgE levels a feature of allergic diseases. It results due to immune reaction to a substance called an allergen, which is normally harmless. Allergen-specific IgE-antibodies cause most allergic reactions. A number of atopy susceptibility interleukin genes have been identified which regulate the production of IgE. Genome Wide Association Studies (GWAS) have identified strong linkage of different genes with atopy in different populations. IL-13 and STAT 6 genes have been found to be involved in the regulation of IgE synthesis and manifestation of allergic disorders [5,6].

A number of studies regarding the different polymorphic variants of inflammatory markers and different allergic phenotypes are available. However, such studies are lacking in this part of the world. With a suitable environment for boarding a huge variety of allergens, Kashmir valley has a significant number of atopic patients. On the basis of our outpatient department experience, there has been an increasing flow of atopic patients seeking medical attention for complaints ranging from seemingly mild symptoms such as watery and itchy eyes, cough and cold to serious asthmatic attacks. There is a general perception that visible particles in air such as fluffy seeds from poplars and blooms cause a rise in allergies in Kashmir but there is no concrete scientific data to support such observations [7].

Since, in allergic disorders an increased level of IgE and mutations in different inflammatory genes like IL4, IL-13 etc. are a common biochemical manifestation as reported elsewhere with none of the studies available in study population, it has created the opportunity to develop novel clinical strategies for the management and treatment of these patients in study population. Therefore, we intend to identify the patients having atopy and elucidate the role of IL-13 and STAT 6 gene variants alone as well as their combinational effect on atopy in presence of different life style and environmental factors.

Materials and Methods

Patients and controls

A total of 528 confirmed cases diagnosed with “atopy” attending allergy clinics, Sher-i- Kashmir Institute of Medical Sciences (SKIMS), department of dermatology, SKIMS Medical College, Srinagar and department of dermatology, SMHS Hospital, Srinagar were included in this study. Blood samples were collected from all 528 cases. Besides blood, samples were obtained from 610 no-atopic subjects from the out patients departments of above departments. A written pre-informed consent was obtained from all cases and controls. The study was approved by the Institutional Ethical Clearance Committee of SKIMS, Srinagar vide order No. SIMS 1 131/IEC-SKIMS/2016-133 [8,9].

Data collection

Detailed information on age, sex, place of residence, ethnicity, religion, education, dietary data including and other potential confounding factors of interest was collected using a questionnaire specifically designed for the study population. Detailed information on life-long history of use of several tobacco products was obtained. Information on family history of any allergic disorder was obtained from all the participants. To assess the Socio-Economic Status (SES) of the subjects, information on potential parameters of SES was obtained including education level (highest level attained), monthly income (INR), house type and ownership of several household appliances. Similarly, the information regarding, second hand smoking was also acquired from all the subjects. For certain parameters, due to logistic constraints (or not required in some parameters) we could not analyze them in control group. The parameters which we were missed in control group include PFT, SPT, time of peak allergies, history of allergies in families, comorbidities, collateral diseases and diagnosis. For PFT parameters, we followed the guidelines as per reports published elsewhere. Briefly, In we calculated the basic parameters like Vital Capacity (VC) the maximal volume of air that can be exhaled after a maximal inhalation or the maximal volume of air that can be inhaled after a maximal exhalation; Forced Expiratory Volume (FEV), Forced Vital Capacity (FVC) and FEV/VC% (calculated as FEV/VC × 100). In most cases these variables suffice to provide all the information needed to interpret a spirogram. The normal limits of FEV and FVC is considered to be 80 to 120%, while as the normal reference range for FEV/VC% should fall within 5% of the predicted ratio [10].

Genetic analysis of IL-13 and STAT 6

Five milliliters of venous blood was collected from each subject in sterilized plastic vials containing EDTA (0.5 M; pH=8.0) and stored at -80ºC before DNA extraction. Genomic DNA was extracted from collected blood samples using the phenol chloroform method and kit based method (DNASure Blood Mini Kit from Nucleo-pore) [11].

The details of PCR conditions, primers, restriction enzyme, and length of expected fragments on digestion, minor alleles and change in nucleotide position of the above genes are given in Table 1.

It is pertinent to mention that two different SNPs were simultaneously studied in case of IL-13 gene based on their reported association with different inflammatory diseases which is common manifestation among patients with allergic diseases [12].

Gene polymorphism analysis

RFLP for IL-13–C1055 T missense mutation (rs20541): PCR was setup to amplify the 372 bp product followed by restriction enzyme digestion by Hpy 199 I restriction enzyme (New England Biolabs, USA). The details of the PCR product and digestion details are given in Figure 1.

RFLP for IL-13–C1512A SNP (rs1881457): PCR was setup to amplify the 214 bp product, which was subjected to restriction digestion with Bsh 1236I restriction enzyme (Thermo Fisher Scientific, USA). Details provided in Figure 2 and Table 1 polymorphism pattern of IL13-C1512A using Bsh1236I restriction enzyme. Lane M: 50 bp ladder, Lane 6 and 7 represent the Homozygous wild (C/C) genotype (size 192 and 22 bp), Lane 3 and 4 represent homozygous variant (A/A) genotype (size 214 bp uncut product), Lane 1 and 2 represent C/A heterozygous genotype (size 214, 192 and 22 bp), Lane 5 represents negative control.

RFLP for STAT 6 G2964A (3’UTR) SNP (rs 324015): PCR was setup to amplify the 93 bp product. Enzyme digestion was carried out using BsaHI or Hin1I restriction enzymes (New England Biolabs, USA). The details are given in Figure 3.

The European Molecular Genetics Quality Network (EMQN) good practice guidelines have been followed. A few PCR vials with all the PCR contents except the DNA, were also included per PCR batch as ‘‘negative controls’’. No contamination was observed and there were no ‘‘false positives’’. To minimize the risk of contamination, sterilized and autoclaved solutions and equipment were used during DNA isolation. The ingredients for PCR were well stored at -20°C and were thawed just before use. Retyping of samples was done at random to check for the homology of results.

Statistical analysis

Categorical variables were set for presenting and calculating numbers and percentages for different genotypes of IL-13 and STAT 6. Test for Hardy-Weinberg Equilibrium (HWE) were conducted by comparing observed and expected genotype distributions by the χ² goodness of fit. Statistical significance for departure of a genotype frequency from its expected frequency under the HWE model was set at P ≤ 0.05. Conditional logistic regression models were used to calculate Odds Ratios (ORs) and corresponding 95% Confidence Intervals (95% CIs) to assess the association of the genotypes with allergies and to assess the analysis of effect modification of genegene and Gene-Environment Interaction (GEI). All statistical analysis was done using STATA software, version 14 (STATA Corp., College Station, TX, USA). Two sided P values <0.05 were considered as statistically significant.

Results

Characteristics of study subjects

A total of five hundred twenty-eight (n=528) consecutive patients diagnosed with atopy and six hundred ten (n=610) no atopic subjects (at least free from any sort of Allergy) were included for the polymorphic analysis of IL-13 and STAT 6 genes. Almost all the cases had attended the hospital with a clinical presentation of atopy.

Among cases 60.2% (n=318) were females and 39.8% (n=210) were males with a male: female ratio of 0.7:1. In controls 58.2% (n=355) were females and 41.8% (n=255) were females. On the basis of age, the patients were grouped into two categories, less than 40 years (<40) and greater or equal to 40 years of age (≥ 40). The number of cases in the age group of <40 years (n=414; 78.4%) exceeded than ≥ 40 years (n=114; 21.6%) with a mean age of 29.7 ± 13.9 years in cases while as in control subjects in the age group of <40 (n=459; 75.0%) exceeded than ≥ 40 years (n=151; 25.0%). The mean age of the control subjects was 29.45 ± 14.2 years. Based on the smoking status, 63 (12%) patients were smokers as compared to 38 (6.2%) smokers among control group. Among cases we found 525 (99.4%) patient with elevated IgE levels while as only 123 (20.2%) controls presented with higher IgE levels. Other study variables of the study subjects did not showed any statistically significant association with atopy (P>0.05) Table 2.

Polymorphic analysis of IL-13

Analysis of I0L-13-C1055T polymorphism (rs20541): The representative gel pictures showing amplified products of IL-13 5’ UTR and PCR-RFLP of the IL-13–C1055T polymorphism are shown in Figure 1. The frequency of variant allele (CT+TT) was found to be higher in cases (n=321; 60.8%) than controls (n=284; 46.4%), a difference which is statistically significant (OR= 1.8 (1.4-2.2), P<0.0001) suggesting that the presence of variant allele predisposes a person to allergic disorders (Table 3).

On further analyzing IL-13–C1055T genotypes and clinicopathological variables for possible association with, it was found that patients with <40 years of age and were having higher frequency of variant allele (CT+TT) were significantly associated with the disease (OR=2.0, P<0.0001). Female showed increased risk of atopy when they harbor variant allele (CT+TT) (OR=2.1, P<0.0001). Rather unusually we observed increased risk of atopy among passive and non-smokers when they also contain variant form of IL-13–C1055T polymorphism (P<0.0001). Majority of the patients with elevated IgE levels were also harboring variant genotype (CT+TT), hence elevated IgE levels were strongly associated with the IL-13–C1055T polymorphism (P<0.0001). We observed a higher frequency of variant alleles of IL-13 gene in patients with normal eosinophil count (P<0.0001). Fascinatingly, the subjects with no family history and harbor CT+TT alleles had higher risk of developing atopy than patients with family history of the disease (P=0.02).

Association between IL-13–C1055T genotypes and demographic/ clinico-pathological characteristics is shown in Table 4. The majority of clinic-pathological characteristic showed a statistically significant difference when compared cases and controls (P<0.05). However, majority of these factors did not showed any significance with IL-13– C1055T polymorphism when compared among cases only (P>0.05) except peak time and diagnosis of atopy (P<0.05).

Analysis of IL-13–C1512A polymorphism (rs1881457): The representative gel pictures showing amplified products of IL-13 5’ UTR and PCR-RFLP of the IL-13–C1512A polymorphism are shown in Figure 2. The genotype distributions of IL-13–C1512A polymorphism in the cases and controls are shown in Table 3. The frequency of variant allele (CC+AC) was found to be higher for cases (n=429; 81.2%) than controls (n=265; 43.5%), a difference which is statistically significant (P<0.0001) suggesting that the presence of variant allele predisposes a person to atopy.

On stratifying IL-13–C1512 A genotypes with clinico-pathological variables of study subjects, it was found that the frequency of wild (AA) and variant genotypes (CA+AC) between cases and controls with respect to all the subgroups of age, gender, dwelling, smoking status, eosinophil count and vitamin D levels did not showed any significant difference statistically. However, the presence of variant genotype (CA+AC) was significantly associated with elevated IgE levels (OR=3.5, P<0.0001), similarly, the frequency of CA+AC genotypes was greater in patients having morning allergic attacks as compared to patients with no usual peak time for atopy (OR=4.6, P=0.0006). Similarly, IL-13–C1512 A genotypes were significantly associated with all the differential diagnosis of atopy cases (P<0.0001) Table 5.

Analysis of STAT 6 G2964A 3’UTR polymorphism (rest 324015): The representative gel pictures showing amplified products of IL-13 5’ UTR and PCR-RFLP of the STAT6 G2964 A polymorphism are shown in Figure 3. The genotype distributions of STAT6 G2964 A polymorphism in the cases and controls are shown in Table 3. There was a significant difference in the genotypic variants among cases and controls (P<0.0001). The frequency of variant allele (GA+AA) was found to be significantly higher for cases (n=372; 70.5%) than controls (n=245; 40.2%) (P<0.0001) suggesting that the presence of variant allele predisposes a person to allergic disorders (Table 3).

On further classification of STAT6 G2964 A genotypes and clinicopathological variables was analyzed, it was found that there was a statistically significant (P<0.0001) difference in the frequency of wild (GG) and variant alleles (GA+AA) between cases and controls with respect to all the subgroups of age, gender, dwelling, smoking status, eosinophil count and vitamin D levels. Majority of the Atopy patients with elevated serum IgE levels were harboring variant genotype (GA +AA), thus suggesting that an elevated IgE levels in combination with STAT6 G2964A polymorphism could act as a common feature among atopy subjects (OR=3.5, P<0.0001). STAT6 G2964A genotypes were significantly associated with all the triggers (P<0.0001) and comorbidities (P=0.01) associated with atopy. Similarly, STAT6G2964A genotypes were significantly associated with all the differential diagnosis of atopy patients (P=0.002) (Table 6).

Discussion

Different cytokines as modulating factors of the immune response and inflammatory reactions are involved in the pathogenesis of Atopy. It is an inflammatory disorder of upper airways and skin resulting from environmental and genetic factors individually as well as complex interactions between the two. Among various clinico-pathological characteristics, the IgE levels were substantially increased among cases. Similarly, a good number of cases were having a history of atopy among blood relatives. Among studied genotypes, variant genotypes were significantly associated with increased risk of atopy. The above risk predisposing genotypes proved to be important modifiers of environmental exposure and life style factors in determining the risk of atopic disorders. Additionally, there was an increased serum IL-13 and STAT6 levels among atopic patients as compared to controls [13-15].

IgE plays an important role in the development of allergic disorders. The generation of IgE requires the activation of the adaptive immune system. B cells in the secondary lymphoid organs are stimulated to produce mature antibodies. Current evidence shows that high-affinity IgE in blood stream of allergic individuals derives mainly from the mucous membrane. Also, mucosal synthesis of IgE can occur in the absence of systemic atopy. Higher serum IgE levels have been reported to be correlated with the clinical expression of allergy and asthma. During allergic responses, respiratory mucosa is a site of IgE production. Evidences of presence of sIgE in AR are numerous. Studies have identified presence of 90% specific IgE in nasal secretions of allergic patients. A study from early 1990 s demonstrated that house dust mite-specific IgE increased faster in the nasal secretions than in serum after allergen exposure. Similarly AR patients on exposure to pollen showed significant changes in IgE repertoires in nasal mucosa [16,17].

Our results are completely in agreement with earlier epidemiological studies which reported higher IgE levels are associated with bronchial responsiveness, a major component of the asthma phenotype, rhinitis and AD. The stimulus upon exposure of allergens in the mucosa might produce a sequential switching to IgE from IgG or IgA. In recent years, a high serum levels of IgE is used to predict the development of asthma, independent of other allergic factors. Therefore, an understanding of the mechanism regulating total serum IgE levels will valid efforts to scrutinize the components of different atopic disorders.

Although reports are available regarding heritability of serum IgE levels, however, the precise mode of genetic control has remained indefinable. There have been multiple complex segregation analyses investigating the mode of inheritance of total serum IgE within families. Several studies have found evidence for a recessive gene regulating IgE levels, with different estimates of gene frequencies and mean IgE levels [18].

described to change the binding strength of IL-13 to its receptor, causing higher activity of IL-13 variants. Computer modeling of rs20541 variant has shown that this substitution affects the signal strength between interleukin 13 and its receptor. In atopic patients, Th2 phenotype predominates, leading to increased production of IL-13 and class-switching in B lymphocytes to synthesize IgE antibodies. Arima et al. have found, using animal model, that IL-13 with Gln110 variant has higher activity and is present in blood for a longer time. This was confirmed by Chen et al., who performed the experiments on mouse cell line with stable expression of human IL-13R, that had the ability to bind human IL13. This polymorphism was associated with asthma and AD in number of studies including few GWAS. A meta-analysis study which was further confirmed in cohort studies reported an association of variant genotype of IL-13 with atopic disorders. An association between AD and higher IgE serum levels was also indicated by earlier reports.

Signal Transducer and Activator of Transcription (STAT6) is a central molecule in the signal transduction pathway used by IL-4 in IgE isotype switching and Th2 cytokines production 53. STAT6 2964 G/A polymorphism is located in the 3’UTR region of the STAT6 gene at chromosome 12 q13.3-q14 and has been reported to be associated with allergic diseases. Another common polymorphism is located in intron 2 of STAT6 gene; 2892 C/T conferred risk for asthma and elevated serum IgE levels in various ethnic groups [19,20].

The current study results were reported by different studies, were variant genotypes of STAT6 gene were significantly associated with the pathogenesis of different atopic disorders. Another study reported an association between G2964A polymorphism and mild atopic asthma in Japanese population and childhood nut allergy in the United Kingdom. However, few studies could not reproduce our results and showed no association between the variant of the STAT6 gene and AR and asthma in Korean and Chinese population respectively. Similarly, no association was observed in Japanese British, and Caucasian populations. These findings suggest that there are marked differences not only between different ethnic groups but also within one ethnic group, indicating genetic heterogeneity of allergic diseases.

Furthermore, our study demonstrated significant increase in the levels of serum IgE in variant genotype (GA+AA) as compared to homozygous wild GG genotype. This result indicates that genetic variants of STAT6 gene significantly contribute to the regulation of serum IgE levels. In agreement with our result, other studies indicated that STAT6 3’-UTR polymorphisms significantly contributed to elevated serum IgE levels. However, Pykalainen, et al. reported results which were in disagreement with our results. In this study variant genotypes of STAT6 gene among Korean AR patients and Finnish asthmatic families did not showed any significant association with serum IgE levels.

The strength of the study is that previous studies have mostly reported the effect of various environmental and genetic factors on atopy individually. However, the current study reported the effect of such factors individually as well as in combination Other major strength is comparatively larger sample size and confounding of the results with the probable allergy risk factors. However, selection or recall bias could be the weak point of this study, although the same hospital setting and only a single interviewer lessen this bias.

Conclusion

In conclusion, the variant forms of IL-13 and STAT6 genes showed a strong association towards atopy which was further increased among subjects harboring these variant genotypes in presence of different clinico-epidemiological and clinico-pathological characteristics.

References

1. Asher MI, Montefort S, Bjorksten B and Christopher KW Lai.Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys. Lancet 368 (2006): 733-743.

Google Scholar, Crossref, Indexed at

2. Deckers IA, McLean S, Linssen S and Mommers M, et al. Investigating international time trends in the incidence and prevalence of atopic eczema 1990-2010: a systematic review of epidemiological studies. PloS one 7 (2012): 39803.  

Google Scholar, Crossref, Indexed at

3. Paramesh H. Epidemiology of asthma in India. Indian J Pediatr 69 (2002): 309-312.

Google Scholar, Crossref

4. Barr RG, Aviles-Santa L and Davis SM, et al. Pulmonary Disease and Age at Immigration among Hispanics. Results from the Hispanic Community Health Study/Study of Latinos. Am J Respir Crit Care Med 193 (2016): 386-395.

Google Scholar, Crossref, Indexed at

5. Cabieses B, Uphoff E, Pinart M and Anto JM, et al. A systematic review on the development of asthma and allergic diseases in relation to international immigration: the leading role of the environment confirmed. PloS one 9 (2014): 105347.

Google Scholar, Crossref, Indexed at

6. Eldeirawi K, McConnell R and Furner S. Associations of doctordiagnosed asthma with immigration status, age at immigration, and length of residence in the United States in a sample of Mexican American School Children in Chicago. J Asthma 46 (2009):796-802.

Google Scholar, Crossref, Indexed at

7. Yao J and Sbihi H. Prevalence of non-food allergies among nonimmigrants, long-time immigrants and recent immigrants in Canada. Can J Public Health 107 (2016): 461-466.

Google Scholar, Crossref, Indexed at

8. Akdis CA. Allergy and hypersensitivity: mechanisms of allergic disease. Curr Opin Immunol 18 (2006): 718-726.

Google Scholar, Crossref, Indexed at

9. Chatzi L, Alegakis A, Tzanakis N and Siafakas N, et al. Association of allergic rhinitis with pesticide use among grape farmers in Crete, Greece. Occup Environ Med 64 (2007): 417-421.

Google Scholar, Crossref, Indexed at

10. Marsh DG, Hsu SH and Roebber M. HLA-Dw2: a genetic marker for human immune response to short ragweed pollen allergen Ra5. I. Response resulting primarily from natural antigenic exposure. J Exp Med 155 (1982): 1439-1451.

Google Scholar, Crossref, Indexed at

11. Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol 8 (2008): 183-192. 

Google Scholar, Crossref, Indexed at

12. Hirota T, Takahashi A and Kubo M. Genome-wide association study identifies eight new susceptibility loci for atopic dermatitis in the Japanese population. Nat Genet 44 (2012): 1222-1226.

Google Scholar, Crossref, Indexed at

13. Munir S, Ber Rahman S and Rehman S. Association analysis of GWAS and candidate gene loci in a Pakistani population with psoriasis. Mol Immunol 64 (2015): 190-194.

Google Scholar, Crossref, Indexed at

14. Weidinger S, Willis-Owen SA and Kamatani Y. A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis. Hum Mol Genet 22 (2013): 4841-4856.

Google Scholar, Crossref, Indexed at

15. Weidinger S, Klopp N and Wagenpfeil S. Association of a STAT 6 haplotype with elevated serum IgE levels in a population based cohort of white adults. J Med Genet 41 (2004): 658-663.

Google Scholar, Crossref, Indexed at

16. Liu X, Beaty TH and Deindl P. Associations between total serum IgE levels and the 6 potentially functional variants within the genes IL4, IL13, and IL4RA in German children: the German Multicenter Atopy Study. J Allergy Clin Immunol 112 (2003): 382-388.

Google Scholar, Crossref, Indexed at

17. Liu X, Nickel R and Beyer K. An IL13 coding region variant is associated with a high total serum IgE level and atopic dermatitis in the German multicenter atopy study (MAS-90). The J Allergy Clin Immunol 106 (2000):167-170.

Google Scholar, Crossref, Indexed at

18. Basehore MJ, Howard TD and Lange LA. A comprehensive evaluation of IL4 variants in ethnically diverse populations: association of total serum IgE levels and asthma in white subjects. J Allergy Clin Immunol 114 (2004): 80-87.

Google Scholar, Crossref, Indexed at

19. Liu S, Li T and Liu J. Interleukin-4 rs2243250 polymorphism is associated with asthma among Caucasians and related to atopic asthma. Cytokine 59 (2012): 364-369.

Google Scholar, Crossref, Indexed at

20. Mitsuyasu H, Izuhara K and Mao X-Q. Ile50Val variant of IL4Rα upregulates IgE synthesis and associates with atopic asthma. Nat Genet 19 (1998): 119.

Google Scholar, Crossref, Indexed at