Fig. 2

The PER2 variant identified in this patient. a Structure of the PER2 protein and the position of the p.Arg468Gln. NES, nuclear export signal; PAS, PER-ARNT-SIM; CKIε, casein kinase Iε; NLS, nuclear localization signal; and CRY, cryptochrome. b Extreme rarity of the c.1403G>A variant in representative public databases and in-house database, and high pathogenicity of this variant predicted by different methods (for URLs, see Methods). c Electrochromatograms showing the maternally inherited c.1403G>A substitution (marked with red asterisks). Direct sequencing denotes that the area under curve is much larger for the variant (VT) ″A″ allele than for the wildtype (WT) ″G″ allele in the patient, while it is similar between the ″A″ and ″G″ alleles in the mother. d The position of PER2 on chromosome 2 and the frequency of the variant allele. PER2 is present on the four BAF band region, and the observed frequencies of the VT and WT alleles in PER2 are similar to the predicted frequencies of the VT and WT alleles in a gene on the four BAF band regions (see Additional file 1: Fig. S2). e Protein modelling analysis. Dashed green lines indicate hydrogen bonds. f Subcellular distribution analysis. Shown are representative subcellular distribution patterns of the GFP-labelled p.Arg468Gln-PER2 protein, and the number of cells assessed as "N", "N + C", and "C" distribution patterns. DAPI, 4′,6-diamidino-2-phenylindole; N, nucleus-dominant distribution; N + C, both nuclear and cytoplasmic distribution; and C, cytoplasm-dominant distribution. g Simplified schematic representation of the circadian cycle [19, 20, 24, 25]. PER2 protein contains at least two functionally different phosphorylation sites: one hitherto unidentified site that primarily mediates proteasomal degradation (target site-1, dark orange) and the other site for the nuclear retention that is phosphorylated by CKIε (target site-2, dark green). Left part indicates the circadian cycle with WT-PER2 (highlighted with yellow). At the beginning of the circadian cycle (morning – midday), the CLOCK-BMAL1 heterodimer activates PER2 and CRY expressions by binding to their regulatory elements (E-box), and PER2 with phosphorylation at the target site-1 and CRY are exported to the cytoplasm. At the later phase of the circadian cycle (afternoon – early night), PER2 with phosphorylation at the both target sites-1 and -2 forms a heterodimer with CRY, and the PER2-CRY heterodimer is imported to the nucleus where it represses the transactivation function of the CLOCK-BMAL1 heterodimer for PER2 and CRY. At the end of the circadian cycle (late night), the repression function of the PER2-CRY heterodimer is decreased because of the degradation of PER2 with phosphorylation at the target site-1 in the cytoplasm. This permits the operation of the circadian rhythm. Right part indicates the circadian cycle with the p.Arg468Gln-PER2 variant (highlighted with dark gray). In this case, it is predicted that nuclear export of PER2 and PER2-CRY heterodimer is compromised (indicated with thin red dotted arrows) because of the position of the variant at NES3, leading to a relative accumulation of the PER2-CRY heterodimer in the nucleus (shown with a red vertical arrow) and, consequently, a prolonged repression by the PER2-CRY heterodimer for the CLOCK-BMAL1 transactivation function (indicated with thick red bars). This would decelerate the production of PER2 and CRY, leading to IH