Profiling Gene Expression Changes in Mouth Breathing Individuals

Abstract

Background: Mouth breathing has been linked to tonsillar and adenoids hypertrophy and associated with various physiological and pathological consequences including dentofacial dysmorphology, speech, and oral health problems. The molecular basis of mouth breathing is not well understood and could be explored through epigenetics, the study of reversible gene expression changes.

Aims: The aims of this study are to analyze the transcriptomic signature of mouth breathers using RNA sequencing and to identify the alterations in gene expression patterns in affected individuals.

Methods: Twenty-five patients undergoing adenoidectomy and/or tonsillectomy for various medical reasons were categorized into four groups. Groups 1 and 2 comprise individuals with chronic mouth breathing associated with tonsillar and adenoid hypertrophy who were scheduled for surgical removal of these tissues restore nasal breathing. Group 3 involves chronic mouth breathers scheduled for tonsillectomy because of recurrent streptococcus infection. Group 4 includes nasal breathers scheduled for tonsillectomy also for recurrent infection. Following tissue excision and preservation, RNA extraction and isolation were performed. Subsequently, RNA sequencing/transcriptomic analysis was conducted using Illumina TruSeq Stranded total RNA. Cephalometric radiographs were examined for all participants to evaluate sagittal and vertical relationships between the jaws and measure airway patency (shortest distance between adenoid and soft palate -SAD-).

Results: Increased airway obstruction was associated with younger age at surgery, reduced SAD, and progressively increased vertical skeletal divergence. Transcriptomic exploratory analysis revealed tissue identity as the dominant source of variance, with clear separation between adenoids and tonsils. Breathing mode and infection status had limited global transcriptomic effects. Differential gene expression analyses disclose the following patterns: Sex-specific transcriptional differences in adenoids, with females showing upregulation of epithelial differentiation and keratinization pathways, while male-biased genes were predominantly Y-linked. Age-related changes in tonsils, whereby children older than 6 years old exhibited downregulation of pathways related to epigenetic regulation, cell cycle, DNA repair, and immune signaling, possibly reflecting physiological tonsillar involution. Minimal transcriptional differences between infected and non-infected tonsils and between the infected tonsils of mouth and nose breathers. Tissue-specific divergence between adenoids and tonsils. Three premises may be drawn from the findings on tissue differences: the comparison between: − adenoids and tonsils hypertrophy revealed distinct, tissue-specific defense mechanisms, reflecting structural anatomical differences. Adenoids demonstrated strong enrichment of ciliary structure, motility, and mucociliary clearance pathways, whereas tonsils were enriched for epithelial differentiation, keratinization, barrier formation, and immune signaling programs. Consistent FOXE1 downregulation in adenoids suggests a potential molecular link between adenoid hypertrophy and high narrow palatal morphology in chronic mouth breathers. − adenoids and adenotonsillar hypertrophy did not show significant differences. Hypertrophied adenoids displayed stable transcriptomic profiles regardless of tonsillar involvement. − tonsils and adenotonsillar hypertrophy revealed weakening immunity and chromatin remodeling in the tonsils. Isolated tonsillar hypertrophy was characterized by marked downregulation of immune, epigenetic, and proliferative pathways compared with the more transcriptionally active state in adenotonsillar hypertrophy.

Conclusion: This study demonstrates that the hypertrophied adenoids and tonsils of mouth breathers are associated with distinct tissue-specific transcriptomic signatures shaped primarily by tissue identity, age, and sex. Tissue identity: Adenoids exhibit cilia- and secretion-related pathways whether separately enlarged or associated with tonsillar hypertrophy. In contrast, tonsils are enriched for epithelial barrier and immune programs, with more immune and proliferative activity when tonsillar hypertrophy was combined with adenoid enlargement. Age: Non-infected tonsils in patients older than 6 years revealed downregulation of proliferative and immune pathways, possibly reflecting physiological tonsillar involution. This finding suggests that the age of such involution, still controversial in present literature, should be further investigated. Sex: Female adenoids demonstrated enhanced epithelial and barrier differentiation, potentially explaining the reported male predominance of adenoid hypertrophy. Related conclusions regarding breathing mode or infection status may not be formulated within the design of the study. Further studies of adenoids and facial bones’ transcriptomic signatures are required to better elucidate the impact of mouth breathing on gene expression changes in affected individuals and explore whether adenoid hypertrophy sustains a transcriptional and epigenetic influence on tonsillar immune function.