P96. New laboratory method to mirror individual circadian rhythms and sleep preference in idiopathic hypersomnia

Abstract

Introduction The circadian regulation is based on a molecular genetic network of transcriptional feedback loops of clock genes. The nucleus suprachiasmaticus is the central pacemaker and integrates environmental information and synchronizes the phase of oscillators in peripheral cells. Numerous studies had proven that the molecular clock mechanism is cell-autonomous and that circadian period and phase is associated with circadian and sleep preference. Material and methods We measured clock gene expression in fibroblasts from healthy controls and patients suffering from idiopathic hypersomnia who were polysomnographically screened in our sleep laboratory and diagnosed according to the ICSD2-criteria. Two complementary molecular genetic methods to quantify the dynamic of clock gene expression were applied. Human fibroblasts derived from punch biopsies were transfected with a circadian reporter gene, the Bmal1 promoter-driven luciferase gene, using a lentiviral system. The luciferase activity showed a circadian rhythm of lumincescence expression. The individual period length over a time course of 5days was detectable by measuring the bioluminescence every ten minutes in vitro. Previous studies had proven that the physiological period length of human circadian clock in vivo is directly proportional to period in human fibroblast. To test whether differences in the magnitude of oscillatory amplitude of the clock genes Bmal1, Per1 and Per2 might account for period and phase differences, we quantified the transcriptional gene expression of clock genes in dermal fibroblasts by a different method because the measurement of the bioluminescent oscillation of clock genes which is sensitive to the degree of viral infection is not suitable to quantify the transcriptional amplitude. Thus we measured RNA levels of clock genes by means of quantitative real-time PCR (qPCR) and complemented these data with the bioluminescence assay. Total RNA was extracted from fibroblast cell lines every 6h over 72h and totalRNA was reverse transcribed into cDNA followed by qPCR using the primer for the clock genes. Results In fibroblasts from patients suffering from idiopathic hypersomnia clock gene expression exhibit a diminished amplitude of BMAL1 which was significantly reduced by 63% ( P =0.004) compared to healthy controls. The amplitude of PER1 was reduced by 45% ( P =0.048) compared to the control group so was the amplitude of PER2 reduced by 32% ( P =0.032). In the luciferase assay using the same fibroblast cell lines,an averaged period length of 24.6h could be detected in dermal fibroblasts obtained from healthy volunteers. Conclusion Due to the significantly diminished amplitude of clock gene expression in the group of idiopathic hypersomniacs, our data suggest a disturbed chronobiology as a heterogeneous aspect in a primary sleep disorder. The implementation of this combined strategy to measure the dynamics of clock gene expression permits a reliable assessment – in objective statistical terms – of both the period length and the absolute magnitude of oscillatory amplitude. The coupling of the circadian gene regulation with individual EEG-parameter following the principles of the two-process-model of sleep regulation can be a useful tool to quantify therapeutic agents and regimens modifying circadian disruption (e.g. period length, phase and amplitude of circadian rhythms) and to develop possible personalized therapeutic options.