Identification and validation of endogenous control miRNAs in plasma samples for normalization of qPCR data for Alzheimer's disease.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNAs that are highly relevant as disease biomarkers. Several studies that explored the role of miRNAs in Alzheimer's disease (AD) demonstrated their usefulness in clinical identification. Nevertheless, miRNAs that may act as endogenous controls (ECs) have not yet been established. The identification of ECs would contribute to the standardization of these biomarkers in AD. The objective of the study was to identify miRNAs that can be used as ECs in AD. METHODS: We evaluated 145 patients divided into two different cohorts. One was a discovery cohort of 19 women diagnosed with mild to moderate AD (Mini-Mental State Examination (MMSE) score ≥ 20) and with confirmed pathologic levels of Aß42 in CSF. The stability assessment cohort consisted of 126 individuals: 24 subjects without AD or any kind of dementia and negative for all core CSF biomarkers of AD, 25 subjects with MCI and negative for CSF biomarkers (MCI -), 22 subjects with MCI and positive for CSF biomarkers (MCI +), and 55 subjects with AD and positive for CSF biomarkers. In the discovery cohort, a profile of 384 miRNAs was determined in the plasma by TaqMan low-density array. The best EC candidates were identified by mean-centering and concordance correlation restricted normalization methods. The stability of the EC candidates was assessed using the GeNorm, BestKeeper, and NormFinder algorithms. RESULTS: Nine miRNAs (hsa-miR-324-5p, hsa-miR-22-5p, hsa-miR-103a-2-5p, hsa-miR-362-5p, hsa-miR-425-3p, hsa-miR-423-5p, hsa-let-7i-3p, hsa-miR-532-5p, and hsa-miR-1301-3p) were identified as EC candidates in the discovery cohort. The validation results indicated that hsa-miR-103a-2-5p was the best EC, followed by hsa-miR-22-5p, hsa-miR-1301-3p, and hsa-miR-425-3p, which had similar stability values in all three algorithms. CONCLUSIONS: We identified a profile of four miRNAs as potential plasma ECs to be used for normalization of miRNA expression data in studies of subjects with cognitive impairment.

Circulating MicroRNA Profile Associated with Obstructive Sleep Apnea in Alzheimer's Disease.

The diagnosis of obstructive sleep apnea (OSA) in Alzheimer's disease (AD) by polysomnography (PSG) is challenging due to the required collaboration of the patients. In addition, screening questionnaires have demonstrated limited usefulness with this subpopulation. Considering this, we investigated the circulating microRNA (miRNA) profile associated with OSA in AD patients. This study included a carefully selected cohort of females with mild-moderate AD confirmed by biological evaluation (n = 29). The individuals were submitted to one-night PSG to diagnose OSA (apnea-hypopnea index ≥ 15/h) and the blood was collected in the following morning. The plasma miRNA profile was evaluated using RT-qPCR. The patients had a mean (SD) age of 75.8 (5.99) years old with a body mass index of 28.6 (3.83) kg m-2. We observed a subset of 15 miRNAs differentially expressed between OSA and non-OSA patients, of which 10 were significantly correlated with the severity of OSA. Based on this, we built a prediction model that generated an AUC (95% CI) of 0.95 (0.88-1.00) including 5 of the differentially expressed miRNAs that correlated with OSA severity: miR-26a-5p, miR-30a-3p, miR-374a-5p, miR-377-3p, and miR-545-3p. Our preliminary results suggest a plasma miRNA signature associated with the presence of OSA in AD patients. Further studies will be necessary to validate these findings.

Role of primary care in the follow-up of patients with obstructive sleep apnoea undergoing CPAP treatment: a randomised controlled trial.

OBJECTIVE: To evaluate whether follow-up of patients with obstructive sleep apnoea (OSA) undergoing CPAP treatment could be performed in primary care (PC) settings. DESIGN: Non-inferiority, randomised, prospective controlled study. SETTINGS: Sleep unit (SU) at the University Hospital and in 8 PC units in Lleida, Spain. PARTICIPANTS: Patients with OSA were randomised to be followed up at the SU or PC units over a 6-month period. MAIN OUTCOMES MEASURED: The primary outcome was CPAP compliance at 6 months. The secondary outcomes were Epworth Sleep Scale (ESS) score, EuroQoL, patient satisfaction, body mass index (BMI), blood pressure and cost-effectiveness. RESULTS: We included 101 patients in PC ((mean±SD) apnoea-hypopnoea index (AHI) 50.8±22.9/h, age 56.2±11 years, 74% male) and 109 in the SU (AHI 51.4±24.4/h, age 55.8±11 years, 77% male)). The CPAP compliance was (mean (95% CI) 4.94 (4.47 to 5.5) vs 5.23 (4.79 to 5.66) h, p=0.18) in PC and SU groups, respectively. In the SU group, there were greater improvements in ESS scores (mean change 1.79, 95% CI +0.05 to +3.53, p=0.04) and patient satisfaction (-1.49, 95% CI -2.22 to -0.76); there was a significant mean difference in BMI between the groups (0.57, 95% CI +0.01 to +1.13, p=0.04). In the PC setting, there was a cost saving of 60%, with similar effectiveness, as well as a decrease in systolic blood pressure (-5.32; 95% CI -10.91 to +0.28, p=0.06). CONCLUSIONS: For patients with OSA, treatment provided in a PC setting did not result in worse CPAP compliance compared with a specialist model and was shown to be a cost-effective alternative. TRIAL REGISTRATION NUMBER: Clinical Trials NCT01918449.

Impact of obstructive sleep apnea on the 24-h metabolic hormone profile.

OBJECTIVE: Obstructive sleep apnea (OSA) has been associated with metabolic disorders. Sleep-disordered breathing could generate an altered rhythm in the expression of metabolic hormones, which could predispose to metabolic disorders. The aim of this study was to evaluate the effect of sleep apnea on diurnal variations in metabolic hormones. METHODS: Thirty-seven male, newly diagnosed, patients with OSA with an apnea-hypopnea index (AHI) > or = 20/h and 11 male controls (AHI <10/h) matched for body mass index (±3 kg/m2) were included. Six different samples were obtained from each subject during a period of 24h. Levels of the metabolic hormones ghrelin, leptin, resistin, and adiponectin were measured in plasma by immunoassay. RESULTS: Patients with OSA (AHI (mean±SD) 46±26/h) were older than the controls (42±9 vs. 33±9 years, P=0.01). Differences in metabolic hormones between groups did not reach statistical significance at any point in the evaluation. No significant differences were observed in the area under the curve for any of the hormones analysed. Likewise, we did not detect diurnal variations in metabolic hormones. CONCLUSIONS: The results of this study indicate that the day-night variations in the levels of several metabolic hormones are not influenced by the presence of sleep apnea.

Day-night variations in endothelial dysfunction markers and haemostatic factors in sleep apnoea.

Patients with sleep apnoea have a significant alteration in the day-night pattern of myocardial infarction and sudden cardiac death observed in the general population. The aim of this study was to investigate the influence of sleep apnoea on the diurnal variations in various haemostatic parameters (factor VII, von Willebrand factor and plasminogen activator inhibitor (PAI)-1) and markers of endothelial dysfunction (asymmetric dimethylarginine (ADMA) and soluble CD40 ligand (sCD40L)). We studied 26 male patients with obstructive sleep apnoea syndrome (OSAS; 13 patients with severe OSAS (apnoea/hypopnoea index (AHI) >30 events · h(-1)) and 13 patients with mild-to-moderate OSAS (AHI <30 events · h(-1))) and 12 controls of similar body mass index (BMI) and waist circumference. In each subject, six different samples were obtained over 24 h. Although all the markers values tended to be higher in patients with severe OSAS, differences did not reach statistical significance at any time. PAI-1 levels were significantly related to BMI (p<0.001), mean (p<0.001) and minimal (p = 0.047) nocturnal oxygenation saturation. ADMA levels were significantly related to arousal index (p = 0.046). The results of this study suggest that day-night variations in factor VII:antigen (Ag), von Willebrand factor:Ag, PAI-1, sCD40L and ADMA levels may be dependent on either the obesity index or metabolic dysfunction rather than on sleep apnoea alone.

Plasma levels of neuropeptides and metabolic hormones, and sleepiness in obstructive sleep apnea.

BACKGROUND: Obstructive sleep apnea (OSA) is related to obesity and metabolic disorders. The main clinical symptoms are excessive daytime sleepiness (EDS) and snoring. However, not all patients with OSA manifest EDS. Hypocretin-1, neuropeptide Y, leptin, ghrelin and adiponectin are implicated in both metabolic and sleep regulation, two conditions affected by OSA. We hypothesized that levels of these peptides may be related to EDS in OSA patients. METHODS: We included 132 patients with EDS, as defined by an Epworth Sleepiness Scale (ESS) score ≥ 13 (mean ± SD, 15.7 ± 2.3) and 132 patients without EDS as defined by an ESS score ≤ 9 (6.5 ± 1.9). All patients had an apnea-hypopnea index (AHI) ≥ 20 h(-1). Both groups were matched for gender (males; 83.3% vs. 85.6%), age (50.15 ± 11.2 yrs vs. 50.7 ± 9.9 yrs), body mass index (BMI) (31.8 ± 5.6 kg m(-2) vs. 32.1 ± 4.8 kg m(-2)), and apnea-hypopnea index (AHI) (45.5 ± 19.1 h(-1) vs. 43 ± 19.2 h(-1)). RESULTS: OSA patients with EDS showed significantly higher plasma hypocretin-1 levels (p < 0.001) and lower plasma ghrelin levels (p < 0.001) than OSA patients without EDS. There were no statistically significant differences in neuropeptide Y (p = 0.08), leptin (p = 0.07) and adiponectin (p = 0.72) between the two groups. In the multiple linear regression model ESS score was associated with plasma levels of hypocretin-1, ghrelin and total sleep time. CONCLUSION: Our study shows that EDS in patients with OSA is associated with increased circulating hypocretin-1 and decreased circulating ghrelin levels, two peptides involved in the regulation of body weight, energy balance, sympathetic tone and sleep-wake cycle. This relationship is independent of AHI and obesity (two key phenotypic features of OSA).

Free fatty acids and the metabolic syndrome in patients with obstructive sleep apnoea.

Obesity and metabolic syndrome (MS) occur frequently in patients with obstructive sleep apnoea syndrome (OSAS). We hypothesised that circulating free fatty acids (FFAs) are elevated in OSAS patients independently of obesity. This elevation may contribute to the development of MS in these patients. We studied 119 OSAS patients and 119 controls. Participants were recruited and studied at sleep unit of our institution (Hospital Universitari Son Dureta, Palma de Mallorca, Spain) and were matched for sex, age and body mass index (BMI). The occurrence of MS was analysed by clinical criteria. Serum levels of FFAs, glucose, triglycerides, cholesterol, high-density lipoprotein-cholesterol, aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, C-reactive protein and 8-isoprostanes were determined. Prevalence of MS was higher in OSAS than in the control group (38 versus 21%; p=0.006). OSAS patients had higher FFAs levels than controls (mean±sd 12.2±4.9 versus 10.5±5.0 mg·dL(-1); p=0.015). Among subjects without MS, OSAS patients (OSAS+ MS-) showed higher levels of FFAs than controls (OSAS- MS-) (11.6±4.7 versus 10.0±4.4 mg·dL(-1); p=0.04). In a multiple regression model, after adjustment for age, sex, BMI and the presence of MS, FFAs were significantly associated with apnoea/hypopnoea index (p=0.04). This study shows that FFAs are elevated in OSAS and could be one of the mechanisms involved in the metabolic complications of OSAS.