Prolonged Mild Sleep Restriction Changes Epigenome-Wide DNA Methylation in Peripheral Blood Cells: A Randomized Crossover Trial.

DNA methylation (DNAm) is implicated in age-related disease susceptibility. Some studies have reported alterations in DNAm patterns with sleep deprivation, yet this has not been demonstrated in long-term studies. We aimed to analyze whether prolonged mild sleep restriction (SR) results in differentially methylated loci (DML) in selected candidate circadian genes and explore changes in DML epigenome-wide. We conducted a pooled analysis of two randomized crossover trials of SR. Healthy adults (n=60; 65% women, age ≥20y) habitually sleeping 7-9h/night completed two 6-wk (week) intervention periods (condition): maintenance of habitual adequate sleep (AS, ≥7h/night) and SR (-1.5h/night), separated by a washout interval. We determined DNAm levels in morning fasting blood samples collected at baseline and endpoint using EPICv.2 array and multivariable adjusted models for repeated measures and analyzed the sleep condition x week interaction. Pathways and biological processes from the most significant DML were explored. In the candidate core circadian gene approach, sleep condition x week interactions were at cg02394126 (ARNTL; p˂0.001), cg23506964 (CLOCK; p=0.001), cg03701037 and cg06606972 (NPAS2; both p=0.009). In the exploratory EWAS, suggestive top DML were cg23738833 (SNHG3-RCC1; p=1.34E-06), cg13280380 (FAF1; p=2.25E-05), and cg03179866 (MMP12; p=2.78E-05). All but one (cg23738833) showed hypermethylation after SR vs AS. The most significant pathways associated with SR were aging-related genes involved in TGF-beta signaling, glucagon signaling, and fatty acid degradation. These findings reveal that prolonged mild SR is associated with DNAm in core clock candidate genes and suggests DML in other genes across the epigenome suggesting a potentially plastic epigenetic mechanism. Studies are needed to replicate these preliminary findings.