The obstructive sleep apnea syndrome (OSAS) is a frequent sleep disorder that constitutes an independent risk factor for the development of metabolic syndrome and cardiovascular disease (1). Most of the gene components of critical metabolic pathways and their regulators exhibit circadian variation at the mRNA and protein levels (2). They are regulated by core clock genes, which in turn are modulated by sleep/vigilance cycles (3). Importantly, single genetic disruption of the core clock genes CLOCK (4) and BMAL1 (5, 6) cause full-blown metabolic syndrome phenotypes in mice. Milder metabolic syndrome phenotypes have also been observed in double or triple, but not single, inactivation of additional core clock genes, including Per1, 2, 3, and Cry1 and 2 (7). In this work, we investigated whether the expression level of mRNA coding for clock genes is altered in non-treated OSAS patients and if it can be corrected by continuous positive airway pressure (CPAP) treatment.

Using Wilcoxon signed-rank test, we found statistically significant differences between patients and control volunteers in the ESS (W = 51.5, p-value = 0.007) and in polysomnography parameters (Table 1) including in AHI (W = 91, p-value < 0.0001), oxygen desaturation index (W = 91, p-value < 0.001), SpO₂ time < 90% in minutes (W = 90, p-value < 0.001), minimal SpO₂ (W = 1, p-value < 0.0001), and average SpO₂ (W = 1.5, p-value < 0.001). Statistically significant differences were also found for SWS in minutes (W = 0, p-value < 0.001), REM sleep in minutes (W = 0, p-value = 0.0001), and arousal index (W = 60, p-value < 0.002), but not for sleep efficiency (W = 29.5, p-value = 0.310) or total sleep time (W = 34, p-value = 0.892).

On the comparison between control volunteers and patients before treatment initiation, we found statistically significant differences for CLOCK (W = 15, p-value = 0.022) expression in PBMCs of patients, but not for Cry 1 and 2 or the three Period genes tested (Table 2). The differences of BMAL1 expression between control volunteers and patients nearly reached statistical significance (W = 20, p-value = 0.068), an outcome that is likely, should the population size be larger.

Patient treatment with CPAP did not change abnormal expression of clock genes (Table 3). It also did not improve the reduced levels of SWS observed in non-treated OSAS patients (p-value = 0.875, Table 4), contrary to REM sleep. The persistence of the abnormal expression pattern of the CLOCK gene cannot be attributed to the lack or sub-optimal adherence levels to CPAP treatment as demonstrated by the metrics of CPAP adherence of our patients (Table 5; Table S1 in Supplementary Material). Our data show that adherence to CPAP treatment is well above the minimum required level recommended by the most recent proposal of the American Thoracic Society (12) that points to the need of CPAP use in more than 70% of nights and for more than 4 h per night.

In our study, as expected, control volunteers and OSAS patients could be easily distinguished by the statistical analysis of the ESS results and polysomnography parameters. They could also be distinguished on the basis of expression levels of CLOCK, but not any of the other core clock genes, with the possible exception of BMAL1. Interestingly, these results are strikingly reminiscent of metabolic syndrome phenotypes found in the knockout of the corresponding genes in mice as described earlier. The levels of expression of the CLOCK gene were altered in OSAS patients before and after CPAP treatment, suggesting that either the standard therapeutic management of these patients is insufficient to correct the disruption of the circadian patterns of genes with expression conditioned by cycles of sleep/vigilance or changes in severe sleep apnea patients are structural, for example, as a consequence of irreversible cerebral vascular lesions.

Before patients were treated, we observed consistently and substantially reduced levels of REM and SWS content as compared to healthy controls. Perhaps significantly, while all the patients who initiated CPAP recovered to normal levels of REM sleep, SWS content remained invariably abnormal, at much lower levels than controls (Table 4). It was previously shown that even in young healthy adults, selective suppression of SWS causes pronounced decrease of insulin sensitivity, reduced glucose tolerance, and higher diabetes risk (13).

While it is clear that CPAP substantially improves cognitive and behavioral aspects of the disease (14), metabolic parameters and other OSAS-related risk factors are only partially modified (15), which might explain the incomplete reduction of cardiovascular risk in OSAS. These facts point to the need to complement CPAP therapy with other therapy interventions that are aimed at the sleep structure and circadian rhythm normalization and are likely to have a substantial impact on the natural history of the disease. Drugs that effectively improve the SWS content of patients might constitute prime candidates for this goal.

Informed consent was obtained from all individual participants included in the study.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional (Centro Hospitalar Lisboa Norte, EPE) and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

SM and LM conceived and designed the study; SM, RR, NP, ANC, DP, CP, DF, JR, and CB collected the data. RR, AB, and LM analyzed the data. LM wrote the manuscript.

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. The reviewer J H-R and handling editor declared their shared affiliation.