Abstract
To screen the PER3 promoter for polymorphisms and investigate the phenotypic associations of these polymorphisms with diurnal preference, delayed sleep phase disorder/syndrome (DSPD/DSPS), and their effects on reporter gene expression. Interspecific comparison was used to define the approximate extent of the PER3 promoter as the region between the transcriptional start site and nucleotide position -874. This region was screened in DNA pools using PCR and direct sequencing, which was also used to screen DNA from individual participants. The different promoter alleles were cloned into a luciferase expression vector and a deletion library created. Promoter activation was measured by chemiluminescence. N/A. DNA samples were obtained from volunteers with defined diurnal preference (3 x 80, selected from a pool of 1,590), and DSPD patients (n=23). N/A. We verified three single nucleotide polymorphisms (G -320T, C -319A, G -294A), and found a novel variable number tandem repeat (VNTR) polymorphism (-318 1/2 VNTR). The -320T and -319A alleles occurred more frequently in DSPD compared to morning (P = 0.042 for each) or evening types (P = 0.006 and 0.033). The allele combination TA2G was more prevalent in DSPD compared to morning (P 0.033) or evening types (P = 0.002). Luciferase expression driven by the TA2G combination was greater than for the more common GC2A (P < 0.05) and the rarer TA1G (P < 0.001) combinations. Deletion reporter constructs identified two enhancer regions (-703 to -605, and -283 to -80). Polymorphisms in the PER3 promoter could affect its expression, leading to potential differences in the observed functions of PER3.
Highlights
TWO DISTINCTIVE BIOLOGICAL TIMING PROCESSES INTERACT IN THE CREATION OF A REST-ACTIVITY CYCLE - THE CIRCADIAN OSCILLATOR AND THE SLEEP homeostat.[1]
Associations with extreme diurnal preference, which is a much more obtained correlate of intrinsic period and sleep timing,[10,11] have been reported for polymorphisms in CLOCK,[12] PER1,13 PER2,14 and PER3.15-17 In contrast to ASPD, delayed sleep phase disorder/syndrome (DSPD/DSPS) is not generally believed to necessarily reflect a longer intrinsic period
We have previously reported an association between the PER35 allele and extreme morning preference, and between the PER34 allele and extreme evening preference as well as DSPD.[15]
Summary
Promoter Expression Constructs Fragments representing the four different observed promoter allelic combinations (PH1–PH4 in Figure 1) (–874 to +130 in the canonical sequence) were inserted into the pGL3-Enhancer vector (Promega). The existence of these four combinations on single chromosomes was confirmed by sequencing of TAcloned PCR products amplified from individual participants. The difference between DSPD approximate PER3 promoter region sequence was ana- patients and evening types with respect to the −320T allele lyzed for the existence of potential transcription factor binding remained statistically significant after Bonferroni correction sites (TFBS) (MatInspector, Genomatix, Munich, Germany).
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