Polymorphism in ADAM33 gene associated with asthmatics in West Bengal, India - An investigation by in-silico analysis.

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.

In silico analysis of single nucleotide polymorphism (rs34377097) of TBXA2R gene and pollen induced bronchial asthma susceptibility in West Bengal population, India.

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.

Assessment of Co-Sensitization between Pollen and Food Allergen Sources among Bengali Population, West Bengal, India.

INTRODUCTION: Secondary food allergies due to cross-reactivity between pollen and food allergens are a significant health burden in India. Prevalence of cross-reactivity varies by region, and particularly in West Bengal, it remains unexplored. The present study investigated sensitivities of pollen-related food allergy (PFA) patients of West Bengal by empirically determining co-sensitization patterns. METHODS: The present study retrospectively analysed 1,310 pollen-allergic (PA) patients from the Allergy and Asthma Research Centre, West Bengal. A questionnaire survey was performed to record patients' demographic and clinical features and categorized into seasonal and perennial ones. All patients were subjected to the skin prick test and specific IgE for evaluation of sensitivity against 16 pollen and 16 food allergens. Co-relation and principal component analysis (PCA) was performed to determine co-sensitivity. RESULTS: Seasonal allergic patients were comparatively higher than perennial ones. PFA patients were mostly sensitive to eggplant (n = 387), while Azadirachta indica (n = 341) was predominant sensitizer among pollen allergen sources. Cynodon dactylon showed maximum significant positive correlation with banana, orange, peanut, pineapple, cucumber, eggplant, potato, tomato, and wheat. In the seasonal group, Cynodon dactylon-specific IgE in PFA patients (median = 4.60 kU/L) was significantly higher from that of PA ones (median = 3.44 kU/L). Among 32 allergens tested, 27 were placed in first two dimensions of the PCA biplot. CONCLUSION: The present study established that co-sensitivity between pollen and food allergen sources were predominant in allergic patients of West Bengal. The co-sensitization patterns among unrelated allergen sources may be due to possible expression of biologic cross-reactivity to similar allergens.

Assessment of egg and milk allergies among Indians by revalidating a food allergy predictive model.

Background: The recent upsurge in food allergy indicates the need for accurate medical diagnostics. The application of predictive diagnostic models can envisage the outcome of oral food challenge (OFC), reducing cost and time. A logistic regression model was developed by DunnGalvin for children predicting OFC outcome using six predictors viz: sex, age, history, specific IgE, total IgE minus specific IgE, and skin prick test. This model was later updated by Klemans, reducing the number of predictors enhancing the calibration and discrimination of outcome. Objective: Our aim was to revalidate both the models for assessment of egg and milk allergies among Indians in the age group 0-19 years and to determine regression coefficients for our study population. Methods: Revalidation was done at the allergy clinic using OFC outcomes of egg and milk allergic patients. Precise values of the predictors were set up for which calibration (predicted against observed outcome) and discrimination (area under curve [AUC] of receiver operator characteristic curve [ROC]) would be better. Results: The Klemans model with reduced number of predictors showed better accuracy, calibration and discrimination than the DunnGalvin. Best calibration for egg allergy was achieved in the Klemans model with correlation coefficient (r2) of 0.90 and accuracy of 97%. The AUC of ROC was 0.90. For milk allergy, the coefficient was 0.94 with accuracy of 98%. The AUC was 0.91. Conclusion: The present study showed that mathematical models are non-invasive and can be successfully used as appropriate alternative to OFC in Indian population after proper validation.

Association of HLA-DQ and IL13 gene variants with challenge-proven shrimp allergy in West Bengal, India.

Little is known about genetic factors and mechanisms underlying shrimp allergy. Genome-wide association studies identified HLA class-II and IL13 genes as highly plausible candidates for shrimp allergy. The present study was designed to investigate potential associations of HLA-DQ rs9275596, IL13 rs20541, and IL13 rs1800925 polymorphisms with challenge-proven shrimp allergy using the data from 532 people of West Bengal, India; selected on basis of positive skin prick test, elevated specific IgE and medical history. Risk genotypes, i.e., HLA-DQ rs9275596 CC, IL13 rs20541 AA, and IL13 rs1800925 TT, were found to be significantly associated with challenge positive shrimp allergy (P = 0.04, 0.01, and 0.03, respectively). Distribution of genotypes for HLA-DQ and IL13 polymorphisms in allergic and control subjects showed significant difference between younger (20-40 years) and older (> 40 years) age group (P = 0.006). Risk genotypes significantly associated with elevated shrimp-specific IgE. IL13 TA haplotype significantly associated with shrimp allergy and elevated specific IgE (P = 0.02). Synergistic effect of IL13 TA haplotype-HLA-DQ rs9275596 CC genotype interaction significantly elevated specific IgE (P = 0.03). The present study suggests that HLA-DQ and IL13 polymorphisms pose major risk for shrimp allergic patients in West Bengal, India and thus could be helpful for early target-specific therapeutic intervention in near future.

Phytochemical analysis of mite-infested tea leaves of Darjeeling Hills, India.

INTRODUCTION: Darjeeling tea of India is one of the most famous beverages globally. However, a large amount of tea crop is damaged every year by the attack of mites. OBJECTIVES: The study aimed to determine the changes in different pigments and biochemical components of tea leaves due to mite infestation. MATERIALS AND METHODS: We used UV-visible and Fourier-transform infrared (FTIR) spectroscopy simultaneously to understand the damages in pigment contents of the leaves of tea (Camellia sinensis (L.) Kuntze) due to attack of phytophagus mite, Oligonychus coffeae Nietner. Furthermore, chemical analysis of infested tea leaves was also performed to compare the nutrients of the plants, namely total phenol, protein and soluble sugar. RESULTS: The UV-visible study reveals severe reduction of the pigments such as chlorophyll (Chl), carotenoids and xanthophylls in the tea leaf due to mite infestation. The findings of the FTIR study, also shows variation in different physiochemical components in the leaf Chl. The sugar and protein content of the infested leaves have been reduced compared to uninfested ones. Results in the case of tea leaves plucked during first (March) and third (November) flushes show similar trends. CONCLUSION: The increase in phenolic and alcoholic components and decrease in Chl contents may affect the quality of Darjeeling tea. The extent of damages done by the pests measured here could aid the pest management in tea gardens.