Elsevier

Sleep Medicine

Volume 10, Issue 1, January 2009, Pages 75-86
Sleep Medicine

Original Article
Genome-wide gene expression profiling in children with non-obese obstructive sleep apnea

https://doi.org/10.1016/j.sleep.2007.11.006Get rights and content

Abstract

Background

Obstructive sleep apnea (OSA) is a multi-factorial and highly prevalent disorder in which both genetic and environmental factors may be involved. If left untreated, OSA may lead to significant cardiovascular and neurocognitive and behavioral morbidities. We hypothesized that pediatric OSA would lead to altered gene expression in circulating leukocytes.

Methods and results

Oligonucleotide-based microarray technology was used to identify mRNAs that may be differentially regulated in non-obese children with polysomnographically-established OSA compared to matched control children. Total morning blood RNA from 40 children (20 OSA and 20 controls) was extracted, labeled, and hybridized onto independent oligonucleotide-based microarrays. Of the 44,000 transcripts, 1217 transcripts were differentially expressed in OSA (p-value <0.05), with 68 transcripts (38 RefSeq accession numbers, 30 ESTs) fulfilling high stringency criteria. False Discovery rate (FDR) was used to determine the significance-difference of OSA vs. normal samples. Microarray data were further validated using quantitative RT-PCR techniques. Biological pathways pertinent to the differentially expressed genes were explored and revealed prominent involvement of inflammatory pathways.

Conclusions

RNA derived from peripheral leukocytes confirms the presence of altered expression of functionally relevant gene clusters in pediatric OSA. Large-scale genomic approaches may provide further insights into adaptive and end-organ injury related mechanisms in the context of OSA in children.

Introduction

Obstructive sleep apnea (OSA) is a relatively prevalent disorder across the lifespan, in which both genetic and environmental factors may be involved [1]. OSA is characterized by episodic partial or complete upper airway obstruction during sleep in association with loud snoring, altered gas exchange, and sleep fragmentation. This condition may affect up to 3% of otherwise healthy school-aged children [2], and has been associated with substantial cardiovascular, metabolic, and neurocognitive morbidities [3], [4], [5], [6]. Indeed, schooling and behavioral problems such as restlessness, inattention and impulsiveness, aggressive behavior, excessive daytime sleepiness, and poor test performances have been repeatedly reported in children with OSA [6], [7], [8], [9], [10]. In addition, systemic and pulmonary hypertension, reduced somatic growth, mood disturbances, and decreased quality of life may develop in pediatric OSA patients [11], [12]. In children, hypertrophy of the tonsils and adenoids in the upper airway is the most frequent and prominent abnormality associated with OSA, such that surgical extirpation of the enlarged upper airway lymphoid tissue is usually the initial management approach [13], [14]. The definitive diagnosis of OSA currently requires an overnight polysomnographic evaluation in a sleep laboratory, and is therefore an onerous and labor intensive procedure, such that delays in the timely diagnosis and treatment are frequent occurrences. Although much has been learned on the pathophysiology and consequences of pediatric OSA in the last 30 years, the mechanisms and specific genes associated with such processes remain poorly defined.

In the last decade, development of high-throughput technologies, such as gene expression profiling using microarrays, has become a fundamental approach for identifying potential diagnostic and therapeutic targets for many diseases. The information gained by such a non a priori approach offers an unprecedented opportunity to fully characterize biological processes, since DNA microarrays yield a simultaneous measurement of gene expression levels for thousands of genes or for the whole genome, thereby allowing for analysis of differential gene expression patterns [15], [16]. Several microarray platforms are currently available [15], [16], [17], [18] and use different approaches to the construction, layout, optimization, hybridization, image acquisition and data extraction methods. Several reviews using different microarray platforms have been reported and summarize the major and most salient points related to their inherent advantages and limitations [19], [20]. Although microarrays have been used to identify molecular signatures for many diseases, such as asthma [21], and heart disease [22], there have been only a few reports on the use of this technology in sleep-related issues [23], [24]. Furthermore, we are aware of only one published study in four adult patients with OSA in whom microarrays were employed. In this report, differential expression of genes mediating oxidative stress was found and postulated as playing an important role in end-organ injury associated with OSA [25].

Clearly, the characterization of specific genes and particular biological pathways in children with OSA using high-throughput gene expression may further enhance our knowledge of this condition in the pediatric age range and allow for reliable and convenient clinical approaches for diagnosis and treatment of these children in the future. Royce et al. [26] reported that the applications of microarrays span from the bench to the bedside, thereby providing tools that require less effort, expense, and sample amount than any other technology. Based on aforementioned considerations, we hypothesized that specific changes in gene expression would occur in children with OSA. Therefore, the aims of the present study were to examine global changes in gene expression profiles in children with OSA, identify differentially expressed genes, and validate some of the latter using other molecular techniques such as QRT-PCR.

Section snippets

Subjects

Approval for the study was obtained from the institutional review board of the University of Louisville School of Medicine. Parental informed consent and child assent, in the presence of a parent, were obtained. Consecutive pre-pubertal non-obese children between the ages of four and nine years of age with a polysomnographic diagnosis of OSA (see below) were identified and recruited to the study. Control subjects were recruited from an ongoing large-scale population study and were initially

Demographic and polysomnographic characteristics

The demographic characteristics of children with non-obese OSA and their matched control groups are shown in Table 1 and their polysomnographic findings are presented in Table 2. In this cohort of non-obese children with OSA, evidence for enlarged adenotonsillar tissues in the upper airway was present in all children and considered as the major pathophysiological mechanism contributing to the development of OSA. However, we can not exclude that subtle changes in craniofacial anthropometrics or

Discussion

To the best of our knowledge, this study represents the first attempt to characterize genome-wide gene expression profiling analysis of circulating leukocytes among non-obese children with OSA. The major findings support the activation and regulation of several functionally-related pathways in children with OSA, a significance that, while completely unexplored, is clearly of great potential interest for future discovery. Among such pathways, up-regulation and modulation of inflammation was

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    Funding sources: A.K. is supported by University of Louisville Institutional Research Grant E0606. D.G. is supported by NIH Grants HL-65270 and HL-83075, The Children’s Foundation Endowment for Sleep Research, and by the Commonwealth of Kentucky Challenge for Excellence Trust Fund. L.K.G. is supported by a grant from the National Space Agency (NNJ05HF 06G).

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