Additional file 1: Figure S1. of Validation of a leg movements count and periodic leg movements analysis in a custom polysomnography system

Settings for computerized detection and analysis of leg movements (LM) and periodic leg movements (PLM). (TIF 349 kb

Additional file 2: Figure S2. of Validation of a leg movements count and periodic leg movements analysis in a custom polysomnography system

Example of computerized detection of periodic leg movements (PLM). Legend: Leg movements are marked with green rectangles, and periodic leg movements with underlining pink bars. An overview of the PLM during the whole night is visible in the upper part of the figure, where PLM are shown as red bars. (TIF 675 kb

Additional file 4: Table S2. of Validation of a leg movements count and periodic leg movements analysis in a custom polysomnography system

Sensitivity and false positive percentages of the computerized LM and PLM analysis, as derived from comparing the automated scoring algorithm to manual scoring on an event-by-event basis. Data are given as median (range) and interquartile range. P values are given for the comparison between RLS patients and controls. IQR, interquartile range; LM, leg movements; LMS, leg movements during sleep; LMW, leg movements during wakefulness; NREM, non-REM sleep; PLM, periodic leg movements; PLMS, periodic leg movements during sleep; PLMW, periodic leg movements during wakefulness; REM, rapid eye movement; TST, total sleep time. * Significant p-values after correction for multiple comparisons according to Bonferroni are given in bold letters. (DOCX 15 kb

SPM "glass brain" representations (upper row) and statistical FA maps that were superimposed on a normalized T1-weighted template (lower row) showing clusters of microstructural damage of the FD patient group compared to healthy controls (ANCOVA, modelling age as a co-variate; p<0.001, corrected for multiple comparison; minimum of 50 contiguous voxels).

<p>Coloured bars represent t-values; display threshold is set at t value >3.16. A: FA values of the complete FD patients group (n = 23) were significantly reduced in WM areas covering widespread parts of the brain, indicating structural WM changes extending beyond the WM lesions that showed up on conventional MRI. B and C: Subgroup analyses of both patient groups (without (1B; n = 18) and with (1C; n = 5) CSA-CSR), relative to the healthy controls (n = 44). Clusters of FA changes in patients with CSA-CSR were most pronounced in the brainstem. By contrast, FD patients without CSA-CSR revealed more widespread FA decreases in supratentorial areas, but only subtle brainstem involvement. SPM  =  Statistical Parametric Mapping; FD = Fabry Disease; FA = Fractional Anisotropy; WM = White Matter; MRI = Magnetic Resonance Imaging; CSA-CSR = Central sleep apnea with Cheyne-Stokes respiration.</p

Demographic and clinical characteristics of patients and controls.

<p>Patients with CSA-CSR had a higher ESS score (P = 0.02). All other clinical and demographic data did not differ among both patient groups (P>0.05, two-sample t-tests).</p><p>CSA = Central Sleep Apnea; MSSI = Mainz Severity Score Index; WML = White Matter Lesions; LVEF = Left Ventricular Ejection Fraction; NYHA = New York Heart Association functional class; eGFR = estimated Glomerular Filtration Rate; ESS = Epworth Sleepiness Scale; ERT = Enzyme Replacement Therapy; LVEDD = Left Ventricular End-Diastolic Diameter; IVSD = Interventricular Septal Wall Thickness; EF = Ejection Fraction; BMI = Body Mass Index; *Mean±Standard Deviation.</p

Altered Dynamics in the Circadian Oscillation of Clock Genes in Dermal Fibroblasts of Patients Suffering from Idiopathic Hypersomnia

<div><p>From single cell organisms to the most complex life forms, the 24-hour circadian rhythm is important for numerous aspects of physiology and behavior such as daily periodic fluctuations in body temperature and sleep-wake cycles. Influenced by environmental cues – mainly by light input -, the central pacemaker in the thalamic suprachiasmatic nuclei (SCN) controls and regulates the internal clock mechanisms which are present in peripheral tissues. In order to correlate modifications in the molecular mechanisms of circadian rhythm with the pathophysiology of idiopathic hypersomnia, this study aimed to investigate the dynamics of the expression of circadian clock genes in dermal fibroblasts of idiopathic hypersomniacs (IH) in comparison to those of healthy controls (HC). Ten clinically and polysomnographically proven IH patients were recruited from the department of sleep medicine of the University Hospital of Muenster. Clinical diagnosis was done by two consecutive polysomnographies (PSG) and Multiple Sleep Latency Test (MSLT). Fourteen clinical healthy volunteers served as control group. Dermal fibroblasts were obtained via punch biopsy and grown in cell culture. The expression of circadian clock genes was investigated by semiquantitative Reverse Transcriptase-PCR qRT-PCR analysis, confirming periodical oscillation of expression of the core circadian clock genes <i>BMAL1, PER1/2</i> and <i>CRY1/2.</i> The amplitude of the rhythmically expressed <i>BMAL1, PER1</i> and <i>PER2</i> was significantly dampened in dermal fibroblasts of IH compared to HC over two circadian periods whereas the overall expression of only the key transcriptional factor <i>BMAL1</i> was significantly reduced in IH. Our study suggests for the first time an aberrant dynamics in the circadian clock in IH. These findings may serve to better understand some clinical features of the pathophysiology in sleep – wake rhythms in IH.</p></div

Using total RNA from primary dermal fibroblasts collected at the indicated time points were used to perform real-time PCR to measure the expression of circadian clock genes.

<p>Blue dots indicate actual measurements, the colored lines represent the gene expression profile relative to <i>18S</i> rRNA in the shape of a sine curve to prove circadian gene expression in all examined cell lines. Each colored line indicates an approximated sine wave by least squares method to 6 actual measurements (I) (3h, 6h, 12h, 24h, 27h, 30h), (II) (6h, 12h, 24h, 27h, 30h, 36h), (III) (12h, 24h, 27h, 30h, 36h, 48h). The applied method is based on the multiple components analysis which allows fitting several significant functions to the experimental data. The broken black line indicates the best fit sine curve defined as the average value of the three rhythmic functions fitted to the data. Numbers in parentheses beside the figures indicate the time points used for prediction and numbers at the right indicate root-mean-square errors (MSE). The smaller the MSE, the more accurate is the prediction of rhythmic circulation. Given examples show exemplarily the harmonic expression in two fibroblast cell lines from one healthy control (left) and one idiopathic hypersomniac (right) depicting the flattened circadian amplitude of gene expression profile in the IH group versus healthy control.</p

Clinical characteristics of the study patients and age- gender matched controls with polysomnography findings and laboratory parameters.

<p>Clinical characteristics of the study patients and age- gender matched controls with polysomnography findings and laboratory parameters.</p

A direct comparison of the overall gene expression of the core circadian clock genes at the indicated time points reveal no significant difference in the absolute amount of gene expression with exception in case of <i>BMAL1</i> at time point 12h (P = 0.05) and 36h (P = 0.04).

<p>At these points the overall expressional rate in the group of healthy controls is significantly higher compared to the patient cohort. The x-axis reflects the actual interval of the sampling points. The black dots indicate the averaged overall gene expression values ± SEM of both study groups over the course of 72h.</p

Comparison of the individual overall amplitude difference of circadian gene expression rate during the 1^st 24h-period starting 6h-30h and in case of <i>BMAL1</i> during the 2^nd 24h-period starting 30h-54h between healthy controls and idiopathic hypersomniacs.

<p>On the y-axis dots indicate for each examined individual the overall amplitude defined as the half difference between maximum and minimum of the average value of the multiple sine curves. Black lines indicate the averaged values ± SEM considering P<0.05 as significant. <i>BMAL1</i> reveals the strongest damping in circadian gene oscillation in the group of idiopathic hypersomniacs in comparison to healthy controls over two consecutive 24h-periods. In case of <i>PER2</i> and <i>PER1</i> the overall amplitude in the group of idiopathic hypersomniacs is significantly dampened in the 1^st 24h-period, in the 2^nd period there is no significant damping (data not shown). Though <i>CRY1</i> also shows a clear diminished oscillation in the patient group, the difference remains not significant due to a high SEM whereas <i>CRY2</i> shows no significant amplitude difference between the two study groups.</p