Bronchial asthma (BA) is a common, clinically diverse, multifactorial disease resulting from complex interactions among multiple genetic and environmental factors that collectively contribute to its pathogenesis [1, 2, 3, 4]. The pathogenesis of BA involves a complex interplay of multiple immune cells—including mast cells, eosinophils, T lymphocytes, macrophages, neutrophils, and dendritic cells—and their inflammatory mediators, such as cytokines, chemokines, cysteinyl leukotrienes, histamine, nitric oxide, and prostaglandin D2 [5, 6]. Dysregulated T helper type 2 (Th2) inflammation, predominantly driven by cytokines IL-4, IL-5, and IL-13, underlies the core pathophysiological features of asthma: airway inflammation, hyperresponsiveness, and remodeling [6].

A causal role in asthma development within the general population has been unequivocally established for several environmental factors, including vaccination practices, reduced immune stimulation by bacterial products, occupational exposures, and contact with particulate matter [7, 8]. Bronchial asthma is a genetically heterogeneous disorder with a substantial heritable component, as unequivocally demonstrated by familial aggregation, higher concordance rates in monozygotic compared to dizygotic twins, and findings from large-scale population-based genetic studies [1, 3, 4]. Despite significant advances in identifying asthma susceptibility genes through genome-wide association studies (GWAS) and candidate gene approaches, the immunogenetic mechanisms underlying disease risk and progression remain incompletely understood [2, 3, 9, 10, 11]. Multiple signaling pathways have been identified as contributing factors in the pathogenesis and progression of asthma. The Smad signaling pathway, in which the SMAD3 protein functions as an intracellular signal transducer and transcriptional modulator, is activated by transforming growth factor beta (TGF-$\beta$) and regulates cell proliferation, differentiation, and apoptosis [12, 13]. Aberrant or excessive activation of SMAD3 signaling pathways can induce structural remodeling of the airways, thereby contributing to airway obstruction and hyperresponsiveness—hallmark features of asthma [13, 14].

Numerous multi-ancestral GWAS have identified specific single nucleotide polymorphisms (SNPs) within the SMAD3 gene that are associated with the development and progression of bronchial asthma by modulating airway structure, immune responses, and inflammatory pathways [15, 16, 17]. Although GWAS have produced significant findings across diverse global populations, many SNPs identified within the SMAD3 gene require further validation in independent cohorts to confirm their clinical relevance and practical applicability. The present study aimed to investigate the association between two common SNPs, rs17293632 and rs2033784, located within the SMAD3 gene—previously identified through GWAS [11, 17]—and susceptibility to atopic asthma and sensitivity to allergens in pediatric populations from the Russian Federation.

The present study found, for the first time, that the rs17293632 and rs2033784 polymorphisms of the SMAD3 gene are significant markers of increased genetic susceptibility to bronchial asthma in Russian children in a sex-specific manner. However, both polymorphisms were found to be associated with asthma risk exclusively in girls. Furthermore, the observed associations were exclusively present among children residing in urban environments, while in children from rural areas, these genetic variants showed no significant influence on disease risk. Furthermore, we identified significant correlations between polymorphisms in the SMAD3 gene and multiple allergy phenotypes, demonstrating increased susceptibility to certain allergens while concurrently exhibiting reduced sensitivity to others.

Multiple polymorphisms within the SMAD3 gene have been identified as being associated with an increased risk of asthma and other allergic disorders, including atopic dermatitis, allergic rhinitis, and hay fever [15, 16, 17, 24, 25]. SMAD3 gene polymorphisms, including those examined in this study, are also linked to the development of various immune-mediated diseases such as ankylosing spondylitis, Crohn’s disease, psoriasis, ulcerative colitis, autoimmune thyroid disease, systemic lupus erythematosus, and type 1 diabetes mellitus [26, 27].

The availability of bioinformatics tools and genomic-transcriptomic databases, such as the GTEx Portal and the eQTLGen Consortium, which are derived from large population samples, enables the assessment of the functionality of genetic polymorphisms in relation to their impact on gene expression. This, in turn, facilitates the interpretation of genotypic correlations. The studied polymorphisms are located in intronic regions of the SMAD3 gene. The functional effects of the polymorphisms were evaluated using genome-transcriptome data available at the GTEx Portal (https://gtexportal.org). The asthma-associated alleles rs17293632-T and rs2033784-G are associated to decreased SMAD3 gene expression in thyroid tissue and esophageal mucosa, and, to a lesser extent, in the lung (only for the rs17293632-T allele). Additionally, a decreased SMAD3 gene expression in the carriers of the rs17293632-T and rs2033784-G alleles was observed in blood, according to data from the eQTLGen Consortium portal (https://www.eqtlgen.org/cis-eqtls.html). According to Ensembl data (https://www.ensembl.org/) on genes and regulation obtained from the ENCODE database, SNP rs17293632 participates in epigenetic regulatory functions within an enhancer region located in the lung and airway tissues. This activity is particularly prominent in bronchial epithelial cells, which are characterized by the presence of H3K27me3, H3K4me3, CTCF, and DNase I markers. It is also notable in lung fibroblasts, which exhibit markers such as H3K27ac, H3K27me3, H3K4me1, H3K4me3, CTCF, and DNase I.

We hypothesize that the association between SMAD3 gene polymorphisms and susceptibility to asthma and sensitization to food allergens may result from a disruption of TGF-$\beta$1 signaling mediated by SMAD3. Transforming growth factor-beta plays a critical role in regulating immune responses triggered by allergens, thereby contributing to allergic and asthmatic inflammation [28, 29]. It controls key cellular processes involved in allergic reactions, including cell differentiation, proliferation, and migration. Smad proteins, particularly Smad3, mediate TGF-$\beta$ signaling. Impaired Smad3 signaling is known to reduce Th2 cytokine expression, alter allergen-induced IgG1 antibody responses, and affect mucus production [28, 30, 31]. The TGF-$\beta$/Smad3 signaling axis also exacerbates inflammatory hypersensitivity reactions and disease progression by regulating chemokines and cytokines, facilitating inflammatory cell recruitment, promoting cell proliferation, and modulating antigen-specific antibody responses [31]. In particular, in response to a respiratory chemical sensitizer, Smad3 was found to regulate cytokine and chemokine expression as well as specific antibody production in mice [31]. This experiment may partially explain why the associations of SMAD3 gene polymorphisms identified in this study were observed exclusively among children living in urban environments, where levels of chemical pollution are significantly higher than in rural areas. It is known that the increased exposure to pollutants in urban settings may contribute to the higher incidence of allergic diseases [32, 33]. The deficiency of Smad3 resulted in a pronounced shift toward Th2 differentiation and a significant impairment in IgG production [31]. In particular, Smad3-deficient mice did not form TGF-$\beta$1-induced Smad-containing DNA-binding complexes and failed to mediate the effects of TGF-$\beta$, as demonstrated in experiments with culture-activated hepatic stellate cells [34]. In experiments involving normal mouse airways, Smad3-deficient (Smad3-/-) mice exhibited significantly elevated levels of proinflammatory cytokines and interleukin-4 (IL-4), as well as increased expression of the Th2-associated transcription factor GATA-3 in lung tissue and bronchoalveolar lavage fluid, compared to wild-type mice [30]. Functional studies have also been conducted in patients with atopic dermatitis, a condition pathogenetically related to atypical bronchial asthma [1]. Increased expression of SMAD3 mRNA in the blood has been associated with heightened sensitization to house dust mites in patients with atopic dermatitis [35]. A functional genetic study of patients with atopic dermatitis conducted by Shafi and colleagues observed a significant decrease in the expression of the SMAD3 gene in affected skin, accompanied by an upregulation of TGF-$\beta$1 mRNA expression [36].

Our study has several limitations. The observed sex-specific associations between SMAD3 polymorphisms and asthma in our study are likely attributable to the relatively limited sample size, which is considerably smaller compared to the genome-wide association studies performed by large international consortia. Although the phenomenon of sexual dimorphism in the relationship between SNPs and predisposition to bronchial asthma has been identified in various studies [10, 37, 38], future genetic association studies investigating the SMAD3 gene should be conducted with substantially larger sample sizes. This will provide more robust and reliable estimates of the gene’s association with asthma development, particularly considering the observed sexual dimorphism in disease susceptibility. Since the associations between SMAD3 polymorphisms and various types of allergies were weak, further studies with larger sample sizes are necessary to confirm these findings. Furthermore, the lack of direct functional experimental data in our study limits the mechanistic explanations of the observed associations between SMAD3 polymorphisms and asthma or allergy. An interesting finding was that polymorphisms in the SMAD3 gene are associated with an increased risk of asthma in urban children, suggesting that specific environmental factors—particularly air pollution—may underlie this relationship. Toxicogenomic studies in animal models can help elucidate the underlying mechanisms and assess whether exposure to air pollutants modulates SMAD3 gene expression, thereby contributing to the development of asthma and allergies.

The present study of Russian children confirmed that the rs17293632 and rs2033784 polymorphisms of the SMAD3 gene are significant genetic markers associated with susceptibility to bronchial asthma. However, the association of SMAD3 variants with asthma was observed only among girls, marking the first time this finding has been reported. The associations between polymorphisms and asthma, as well as various types of allergies that we identified, confirm the important role of SMAD3 in the development of immunopathological disorders underlying asthma and sensitization to multiple allergens. Based on available genomic and transcriptomic data, there is strong evidence that the asthma-associated loss-of-function alleles rs17293632 and rs2033784 reduce SMAD3 gene expression, thereby disrupting Smad3-mediated TGF-$\beta$ signaling. This disruption, particularly in the lungs and airways, may contribute to various immunopathological disorders, including decreased Th2 cytokine levels, altered allergen-induced specific IgG1 responses, and the overproduction of proinflammatory cytokines, as demonstrated by several experimental studies. Further studies are necessary to determine whether the SMAD3 gene constitutes a viable therapeutic target for treating asthma and allergic diseases.

BA, bronchial asthma; GWAS, genome-wide association study; Th2, T helper type 2; OR, odds ratio; CI, confidence intervals.

The data presented in this study are available upon reasonable request from the corresponding author.

AS and AP designed the research study. AP conceptualized and supervised the research. AS and AB examined the patients. AS, AP, and MF analyzed the data. AS, OB, and VP performed the laboratory genetic studies. AS drafted the manuscript. AP reviewed and edited the final manuscript. All authors contributed to the critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was conducted in accordance with the guidelines of the Declaration of Helsinki. Informed consent for children’s participation in the study was obtained from their parents or legal guardians. The study protocol was approved by the regional ethics committee of Kursk State Medical University (protocol No. 4, dated 09.04.2018).

We thank all the children and their parents who participated in this study.

This research was conducted with the financial support of Kursk State Medical University.

The authors declare no conflict of interest. Given his role as the Editorial Board member, Alexey V Polonikov had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Gustavo Caetano-Anollés.