Asthma is correctly termed the 21st century epidemic and modern age disease and represents a substantial public health burden in many countries including India (1–3). The prevalence of asthma has been dramatically increasing in both developed and under developed countries across the world. According to Zvezdin 2015 (4), about 300 million people of all ages suffered from asthma. The disease severity is unevenly distributed, being mild in most cases, while severe asthma forms are registered in the minority of the patients (around 15%). It is assessed that another 100 million people will be affected by the disease until 2025 (5, 6). This lower airway disorder is not a new discovery, as it had been recognized over two hundred years ago. It is therefore amazing that it took centuries to realize the importance of disease diagnosis and adequate treatment. Estimate suggests that India alone reported 6% of children and 2% of adults suffering from asthma (3, 7). According to Global Burden of Disease (GBD, 1990–2019) study, India has 34.3 million asthmatics which accounts for 13.09% of the global burden.Evidence suggests that in Indian population, asthma accounted for 27.9% of disability-adjusted life years (DALYs) (8). One of the most significant causes of asthma was found to be natural exposure to pollens (9) that play a major role in its pathogenesis (10). An estimate suggested that thepollen induced asthma prevalence in India ranges between 10-40% of the total asthmatic population (11–14). Early detection of genetically predisposed individuals to asthma is important for better management of the disease. Bronchial asthma is characterized by several inflammatory mediators including histamine, bradykinin and arachidonic acid metabolites such as prostaglandins, thromboxane A2 (TBXA2), andcysteinyl leukotrienes (LTC4, LTD4, LTE4); responsible for the clinical and pathological events (15, 16). Over the last few decades, there has been a steady growth in interest in exploring the important roles of TBXA2 in the pathogenesis of asthma. TBXA2 binds to its receptor TBXA2R which is primarily expressed in tissues targeted by TBXA2, including bronchial and vascular smoothmuscle and platelets (17). TBXA2R stimulate broncho-constriction inhuman small airways (18) in response to pollen-induced mast cell activation in atopic asthma patients (19). It is a G protein-coupled receptor (GPCR) encoded by the TBXA2R gene located in Chromosome 19 at position p13.3 (20). Very few studies were recorded regarding the genetic association of TBXA2R with bronchial asthma so far.

So, in the present study, attempts have been made to investigate the association of TBXA2R rs34377097 polymorphism with pollen induced bronchial asthma among population of West Bengal, India. rs34377097 is the only non-synonymous polymorphism within TBXA2R gene reported till date (21). In this study, computational algorithms were performed on TBXA2R missense polymorphism (R60L) to examine the changes in protein structure and function. The change of amino acid from Arginine (R) to Leucine (L) may affect the protein conformation, leading to change in downstream signaling pathway. Therefore, the present study performed in-silico analyses of the mutated TBXA2R protein using homology modeling to explore and confirm the above outcome. Our study also attempted to determine the signaling molecules of TBXA2R pathway which are responsible for elevated asthmatic response in individuals carrying the risk allele.

Asthma is a heterogeneous and chronic airways disease and estimated to affect an average of 4.5% individuals throughout the globe, with substantial variation in different countries (31). Bronchial asthma is influenced by both environmental and genetic components (32). There is a substantial curiosity in upgrading of our understanding regarding biological mechanism of bronchial asthma by expanding genomic approaches. Current investigation is based on the association of TBXA2R rs34377097 polymorphism in an Indian population.

Earlier reports indicated that TBXA2 was a negative regulator of LTC4 synthase, which produces leukotriene (33). Evidence suggested that in absence of TBXA2, LTC4 synthase activity is enhanced through its receptor (TBXA2R), resulting in increased leukotriene biosynthesis and asthma symptoms (34). These results clearly signify that TBXA2R polymorphism might play an important role in bronchial asthma pathogenesis. TBXA2 is synthesized from arachidonic acid by the enzyme, cyclooxygenase (COX) (35). It causes constriction of bronchial smooth muscles in asthma (36–38). Pollen grains are important aeroallergens therefore it is essential to havethe knowledge of locally prevalent pollen allergens for better diagnosis and therapy of pollen induced asthma (11, 39). Allergenic potentials of common pollens are investigated throughout India viz. Cynodon in northern region, Fusarium solani and Curvularia from southern region, Azaridacta in central region, Cocos and Phoenix from eastern region (40). In our study, more than 87% of patients were sensitive to Azadirachta indica, one of the major pollens found in West Bengal. This study also reported Cocos nucifera, Caesalpinia pulcherrima and Brassica nigraas top sensitizer as depicted in Figure 2 which is in accordance to reports from Dey et al. (2019) (14). Podder et al. 2009, 2010 (41, 42) reported that the occurrence of allergic symptoms to various inhalants is similar for both sexes. We also found significant difference in SPT sensitivity between adolescent & young adult and between young & old adult. Dey et al. (2019) (14) also reported similar observations among atopic population of West Bengal, India. This difference in sensitivity may be due to the increased exposure to various pollen allergens in the residential/occupational area encountered by young adults compared to adolescents and old adults. Figure 3 showed significant negative correlation of FEV1/FVC ratio and intensity of SPT reactions (in terms of wheel diameter) in all age groups and in both sexes which clearly indicated a role of atopy in lung function impairment. Various studies were conducted previously showing association of allergen sensitization and asthma severity in terms of spirometric parameters (43, 44). Jaen et al. 2002 (45) reported that asthmatics sensitized to various allergens including pollens have significantly lower FEV1/FVC ratio which is in accordance with our result. Allergens are thought to initiate acute episodes of asthma through inflammatory processes and bronchial hyper-responsiveness that might result into airway obstruction (46). Figure 4 depicted existence of significant difference in FEV1/FVC ratio of both male and female patients residing in different habitats (Urban, semi-urban and rural) while that in control subjects remained non-significant. Some previous studies reported that asthma patients residing in urban and semi urban habitat with high indoor and atmospheric pollution show reduced FEV1/FVC ratio than patients from rural counterparts (47, 48) which supported our observation.

Table 2 showed that 56.43% of our study population inherited asthma from their family (either maternal or paternal or both), which supports the role of genetic predisposition in asthma. Literature suggested that TBXA2R pathway play a significant role in asthma (21, 33). In our study, we found significant association of rs34377097 polymorphic TT genotype in the exon-2 region of the TBXA2R gene with elevated pollen induced asthma response such as acute broncho and tracheal constriction in West Bengal population, India. Table 3A clearly demonstrated that TBXA2R rs34377097 TT genotype was significantly associated with pollen induced asthma (OR=5.81, P=0.03). Table 3B depicted that children of age 5-12 years bearing GT and TT genotype have higher risk of asthma than adolescent, young adult and old adult. Prashanth et al. 2022 (49) reported mean urinary Leukotriene level in asthmatic children as 378.47 Pg/Mg and Green et al. 2004 (50) observed mean urinary Leukotriene level in asthmatic adult as 111.70 Pg/Mg which may be a probable cause of higher asthma risk in children. Besides this, Kim 2004 (51) reported that children spend more time outdoors than adults, mostly in summer during late afternoon. This long-term exposure to various outdoor air pollutants significantly contributes in developing asthma since their immune system and lungs are still immature. Table 3C showed that females bearing TT genotype have significant higher risk of asthma. Pignataro et al. 2017 (52) reported that females have higher risk of developing asthma than males which is in accordance to our result. This difference in asthma risk between female and male may be due to expression of female sex hormone receptors on mast cells (53). In Table 3D, we observed that urban and semi-urban dwelling patients bearing TT genotype have higher risk of asthma. Priftis et al. 2009 (54) reported that children living in urban areas have significantly higher risk of asthma than their rural counterparts which confirmed our result. This may be due to increased exposure to indoor and outdoor air pollutants in urban and semi-urban habitat. Emissions of harmful gases from motor traffic contribute significantly to the development of asthma in urban and semi-urban dwelling patients (55).

As per our knowledge is concerned, our study which is based on both structural and functional aspects, is the first one to report the association of TBXA2R rs34377097 polymorphism in an Indian population. It is also the first extensive in silico analysis that perform both polymorphism study and modeling approach to assess TBXA2R rs34377097 polymorphism clinically. Another study conducted in Japanese population reported lack of association of rs34377097 polymorphism with bronchial asthma (21). The present study also depicted significant associations of several clinical parameters like FEV₁, FEV₁/FVC ratio and PEFR with pollen induced asthma both in male and female patients in all age groups. Furthermore, in patients the risk allele T was found to be clinically correlated with FEV₁/FVC ratio (P=0.015). Figure 6 demonstrated that TBXA2R rs34377097TT genotype bearing individuals showed highest intensity of SPT reactions in terms of wheel diameter than GG and GT bearing individuals which indicate association of atopic status with asthma risk.

STRING analysis showed that TBXA2R are involved in interaction with different known and predicted Gq proteins (guanine nucleotide-binding G protein, subunits alpha, group q) (Figure 8). Out of all Gq proteins, GNA11 acts as an activator of phospholipase C (PLC). PLC hydrolyses phosphoinositides into the two stimulatory second messengers - inositol 1,4,5-triphosphate (IP3) and diacylglycerol (56). IP3enhances cytoplasmic free calcium level and diacylglycerol (DAG) activates protein kinase C (PKC). Activated protein kinase C either directly phosphorylates LTC₄ synthase enzyme and inactivates it or this regulation may involve another regulatory protein which is yet to be discovered. Inactivation of LTC₄ synthase leads to reduction of Leukotriene C₄ (LTC₄) biosynthesis in platelets. However, in the case of risk allele rs34377097 T-bearing individuals, the non-synonymous mutation (R60L) in TBXA2R protein inhibits the interaction between GNA11 and TBXA2R due to the change in the positive charge potentiality at the cytoplasmic domain. This is in line with Hirata et al. 1996 (57), who demonstrated that the R60L mutation significantly reduces PLC activity. That leads to simultaneous inactivation of PKC which ultimately results in LTC₄ synthase enzyme activation. Activation of this enzyme leads to LTA₄ to LTC₄ conversion which results in acute asthmatic response including broncho-constriction, tracheolar constriction and increased mucus secretion as shown in Figure 9.

The present study provided several lines of evidence to support that TBXA2R rs34377097 polymorphism act as a potent risk factor for asthma in West Bengal population, India. More studies are warranted in diverse ethnic groups to validate the above findings. The present in-silico study will be useful for the advancement of gene based therapy for better management and treatment of asthma in near future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: SCV003761534 (ClinVar).

The studies involving human participants were reviewed and approved by Clinical Research Ethics Committee, Allergy and Asthma Research Center, West Bengal, India (CREC-AARC Ref: 62/21). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Collection of samples was done by IG, SS and NS. IG and AL contributed to data compilation and wrote first draft of the manuscript. PB did the structural modeling. SM diagnosed the disease and provided the samples. Statistical analysis was done by IS, HB and AL. SP conceived the study design, supervised the work, critically revised the manuscript and made the final draft. All authors contributed to the article and approved the submitted version.