RESUMO
Introduction: Asthma is one of the common chronic polygenic inflammatory diseases. Genome wide association studies have identified ADAM33 as an asthma candidate gene. The present study investigated possible association of rs2280090 (T1), rs2280091 (T2) and rs3918396 (S1) single nucleotide polymorphisms (SNPs) of ADAM33 with aeroallergen induced asthma in West Bengal population, India. In addition, in-silico analysis was performed to find out changes in protein function. Methods: Forced expiratory volume in 1 second (FEV1)/Forced vital capacity (FVC), peak expiratory flow rate (PEFR) were assessed using spirometry in 1039 participants. Allergic sensitivity of 619 spirometry positive asthma patients was assessed by skin prick test (SPT) against 22 aeroallergens. For genotyping of T1, T2, and S1 SNPs in 540 allergic asthma patient and 420 control subjects, polymerase chain reaction-based restriction fragment length polymorphism was performed. Total Immunoglobulin-E (IgE) level was measured in both patients and controls. ADAM333 haplotype blocks were constructed using Haploview software v.4.2. Structural model of transmembrane and cytoplasmic domains of ADAM33 was generated using RaptorX. Protein-protein interaction was analysed using the STRING server. Results: Highest number of patient sensitivity was observed towards Cocos nusifera (n = 215) and Dermatophagoides farinae (n = 229). Significant difference in sensitivity was observed between child and late adult (P = 0.03), child and early adult (P = 0.02), adolescent and late adult (P = 0.02) and adolescent and early adult (P = 0.01). Genotypic frequencies differed significantly between patients and controls (P < 0.05). rs2280090 GG, rs2280091GG and AG genotype, and rs3918396 AA carried significant risk for asthma (P = 0.02, P = 0.008, P = 0.04, P = 0.01 respectively). ADAM33 T1, T2, and S1 polymorphisms were in high Linkage Disequilibrium (D = 0.98). Haplotype consisting of rs2280090G, rs2280091G and rs3918396A alleles were found significantly higher in patient population in comparison with controls (OR = 2.03). IgE level differed significantly among different genotypes for T1, T2, and S1 SNPs analysed in pair (P < 0.0001). FEV1/FVC ratio differed significantly among different genotypes for T1, T2 and S1 SNPs analysed in pair (P < 0.0001). Significant difference of FEV1/FVC was also found between GGA and AAG haplotype (P < 0.0001). In-silico analysis revealed T1 and T2 polymorphisms are located in cytoplasmic domain of ADAM33 may cause bronchial smooth muscle cell mobility and cellular hyperplasia as well as cytoskeletal remodelling by altered interaction with different cytoplasmic proteins found by string analysis. Conclusion: Present study showed significant association of T1, T2, and S1 polymorphisms of ADAM33 with aeroallergen-induced asthma in West Bengal, India. These polymorphisms may be used as prognostic markers and possible targets for therapeutics in future.
RESUMO
Introduction: Prevalence of asthma is increasing steadily among general population in developing countries over past two decades. One of the causative agents of broncho-constriction in asthma is thromboxane A2 receptor (TBXA2R). However few studies of TBXA2R polymorphism were performed so far. The present study aimed to assess potential association of TBXA2R rs34377097 polymorphism causing missense substitution of Arginine to Leucine (R60L) among 482 patients diagnosed with pollen-induced asthma and 122 control participants from West Bengal, India. Also we performed in-silico analysis of mutated TBXA2R protein (R60L) using homology modeling. Methods: Clinical parameters like Forced expiratory volume in 1 second (FEV1), FEV1/Forced vital capacity (FVC) and Peak expiratory flow rate (PEFR) were assessed using spirometry. Patients' sensitivity was measured by skin prick test (SPT) against 16 pollen allergens. Polymerase chain reaction-based Restriction fragment length polymorphism was done for genotyping. Structural model of wild type and homology model of polymorphic TBXA2R was generated using AlphaFold2 and MODELLER respectively. Electrostatic surface potential was calculated using APBS plugin in PyMol. Results: Genotype frequencies differed significantly between the study groups (P=0.03). There was no significant deviation from Hardy-Weinberg equilibrium in control population (χ2=1.56). Asthmatic patients have significantly higher frequency of rs34377097TT genotype than control subjects (P=0.03). SPT of patients showed maximum sensitivity in A. indica (87.68%) followed by C. nusifera (83.29%) and C. pulcherima (74.94%). Significant difference existed for pollen sensitivity in adolescent and young adult (P=0.01) and between young and old adult (P=0.0003). Significant negative correlation was found between FEV1/FVC ratio and intensity of SPT reactions (P<0.0001). Significant association of FEV1, FEV1/FVC and PEFR was observed with pollen-induced asthma. Furthermore, risk allele T was found to be clinically correlated with lower FEV1/FVC ratio (P=0.015) in patients. Our data showed R60L polymorphism, which was conserved across mammals, significantly reduced positive electrostatic charge of polymorphic protein in cytoplasmic domain thus altered downstream pathway and induced asthma response. Discussion: The present in-silico study is the first one to report association of TBXA2R rs34377097 polymorphism in an Indian population. It may be used as prognostic marker of clinical response to asthma in West Bengal and possible target of therapeutics in future.