Transcriptomic analysis identifies phosphatases as novel targets for adenotonsillar hypertrophy of pediatric obstructive sleep apnea

Abstract

Rationale: Obstructive sleep apnea (OSA) is a highly prevalent disorder in children, in which enlarged adenotonsillar tissues (AT) play a major pathophysiologic role. Mechanisms leading to the proliferation and hypertrophy of AT in children who subsequently develop OSA remain unknown, and surgical extirpation of AT is associated with potential morbidity and mortality.

Objectives: We hypothesized that a computationally based analysis of gene expression in tonsils from children with OSA and children with recurrent tonsillitis without OSA can identify putative mechanistic pathways associated with tonsillar proliferation and hypertrophy in OSA.

Methods: Palatine tonsils from children with either polysomnographically documented OSA or recurrent infectious tonsillitis were subjected to whole-genome microarray and functional enrichment analyses followed by significance score ranking based on gene interaction networks. The latter enabled identification and confirmation of a candidate list of tonsil-proliferative genes in OSA.

Measurements and main results: In vitro studies using a mixed tonsil cell culture system targeting one of these candidates, phosphoserine phosphatase, revealed that it was more abundantly expressed in tonsils of children with OSA, and that pharmacological inhibition of phosphoserine phosphatase led to marked reductions in T- and B-lymphocyte cell proliferation and increased apoptosis.

Conclusions: A systems biology approach revealed a restricted set of candidate genes potentially underlying the heightened proliferative properties of AT in children with OSA. Furthermore, functional studies confirm a novel role for protein phosphatases in AT hypertrophy, and may provide a promising strategy for discovery of novel, nonsurgical therapeutic targets in pediatric OSA.

Figure 1.

Schematic overview of the experimental approach for identifying and confirming candidate targets in adenotonsillar hypertrophy of pediatric obstructive sleep apnea (OSA). RI = recurrent tonsillar infection.

Figure 2.

Gene product interaction network in tonsillar proliferation of obstructive sleep apnea. Candidate genes reaching significance based on our scoring algorithm (cutoff > 4.86) are highlighted (red: up-regulated relative to RI; green: down-regulated relative to recurrent tonsillar infection). Phosphoserine phosphatase and dual-specificity phosphatase 1 have been labeled. See text for details.

Figure 3.

Immunohistochemical staining for phosphoserine phosphatase (PSPH) of palatine tonsils from (A, A1) a child with obstructive sleep apnea (OSA) and (B, B1) a matched child with recurrent tonsillar infection (RI). In children with OSA, PSPH preferentially localizes to germinal centers, where it is less abundant in children with RI.

Figure 4.

Proliferative rates in tonsil cell cultures from children with obstructive sleep apnea after administration of okadaic acid, calyculin A, and protein phosphatase inhibitor 2 (PPI2) at increasing concentrations. In recurrent tonsillar infection–derived tonsil cell cultures, no significant differences emerged for any of the compounds.

Figure 5.

(A) Representative example of bromodeoxyuridine (BrDU)-based proliferation assay in tonsil cell cultures from a 6-year-old child with obstructive sleep apnea (OSA) treated with vehicle (left) or calyculin (right). Marked reductions in global cell proliferation and in T-cell and B-cell proliferation are apparent. (B) Representative example of annexin V–based apoptosis assay in a 4-year-old child with OSA treated with vehicle (left) or calyculin (right). Marked increases in global cellular apoptosis and in T-cell and B-cell apoptosis are apparent.