Polymorphism in the PER3 promoter associates with diurnal preference and delayed sleep phase disorder

Abstract

Study objectives: 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.

Design: 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.

Setting: N/A.

Patients or participants: 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).

Interventions: N/A.

Measurements and results: 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).

Conclusions: Polymorphisms in the PER3 promoter could affect its expression, leading to potential differences in the observed functions of PER3.

Figure 1

Location of the three promoter SNPs genotyped in this study relative to the novel 1 or 2 repeat VNTR. The promoter allele combinations PH1 – PH4 are shown, together with the aligned sequence from the chimpanzee (release 2.1).

Figure 2

Predicted frequencies for the TA2G haplotype in the extreme morning, intermediate, and extreme evening preference groups, and DSPD patients. P = 0.033 (*), P = 0.002 (**).

Figure 3

Relative induction of luciferase gene expression, compared to vector control with no insert, for the 4 different allelic combination constructs. N = 9 for each construct. P < 0.05 (*), P < 0.01 (**).

Figure 4

The table part of the figure shows predicted transcription factor binding sites (TFBS), promoter position relative to the transcriptional start site, matrix sequence similarity scores (maximum = 1.00) in the different deletion constructs. To the right is shown relative induction of luciferase gene expression, compared to the vector control with no insert, for the 5 promoter serial deletion constructs. N = 6 for each construct. P < 0.0001 (***).