Mutation of the Human Circadian Clock Gene CRY1 in Familial Delayed Sleep Phase Disorder

Abstract

Patterns of daily human activity are controlled by an intrinsic circadian clock that promotes ∼24 hr rhythms in many behavioral and physiological processes. This system is altered in delayed sleep phase disorder (DSPD), a common form of insomnia in which sleep episodes are shifted to later times misaligned with the societal norm. Here, we report a hereditary form of DSPD associated with a dominant coding variation in the core circadian clock gene CRY1, which creates a transcriptional inhibitor with enhanced affinity for circadian activator proteins Clock and Bmal1. This gain-of-function CRY1 variant causes reduced expression of key transcriptional targets and lengthens the period of circadian molecular rhythms, providing a mechanistic link to DSPD symptoms. The allele has a frequency of up to 0.6%, and reverse phenotyping of unrelated families corroborates late and/or fragmented sleep patterns in carriers, suggesting that it affects sleep behavior in a sizeable portion of the human population.

Keywords: DSPD; circadian clock; circadian rhythm; sleep.

Figure 1. Circadian behavior of a control subject ‘TAU18’ (left) and the DSPD proband ‘TAU11’ (right)

(A) Double-plotted home actigraphy records. Red and blue triangles indicate in-bed/out-of-bed times, respectively, according to sleep logs. Asterisks indicate Sundays. (B) In-laboratory protocol: entrainment conditions on the first four days with sleep log-based, habitual sleep times on entrainment days (EN) 1 and 2 and enforced times in bed from 23:00 to 07:00 on EN3 and 4. Saliva samples for DLMO estimation were collected beginning at 18:00 on EN1 every 30 minutes until bedtime. From the fifth day on until the end of the study (free-run days FR1 to 14), subjects were kept under time isolation conditions with instructions to sleep whenever so inclined. Polysomnographic (PSG) sleep and core body temperature were recorded continuously throughout the study. (C) Double-plotted sleep/wake behavior during the in-laboratory study. Colors denote sleep stage derived from PSG records (N1 turquoise, N2 green, N3 blue, REM red). Grey areas indicate periods of missing PSG data during log-based time in bed. On the first and last study day, grey shading marks the beginning and end of data acquisition. Arrow denotes DLMO. Asterisk denotes the beginning of the free-run. (D) Analysis of sleep rhythmicity. Circadian rhythm parameters during the free-run were analyzed by Χ² periodogram and fast Fourier transform (FFT) analysis, which yielded period and amplitude, respectively.

Figure 2. Core body temperature of a control subject ‘TAU18’ (left) and the DSPD proband ‘TAU11’ (right)

(A) Double-plotted core body temperature during the in-laboratory study. The scale of the y-axis for each individual study day is 2.3 °C. Data shown as grey fill are interpolated from raw data shown as black dot overlay (see STAR Methods for details). Red asterisk in the DSPD proband denotes the beginning of the free-run. In the control subject, the indicated free-run start time corresponds to the time used for analysis of rhythmicity and differs from the actual free-run start time due to a preceding ~12-hour gap in the temperature record. (B) Double-plotted sub-mean core body temperature. The mean temperature of the entire data series was calculated from outlier-corrected, interpolated data for each subject and data points below the mean are plotted as black fill. (C) Analysis of core body temperature rhythmicity. Circadian rhythmicity during the free-run was analyzed by Χ² periodogram and FFT analysis to measure period and amplitude, respectively. See also Figure S1.

Figure 3. Mutation of CRY1

(A) The core molecular circadian clock in mammals. Transcriptional activity of Clock and Bmal1 leads to expression of Per and Cry family genes, whose products undergo posttranslational modification, translocate to the nucleus and inhibit Clock/Bmal1-mediated transcription with Cry1 acting as the main repressor. (B) Exon organization of the human CRY1 gene with the encoded protein regions shown above. Box represents the region enlarged in (C). Arrows indicate primer binding sites used in (D). (C) Primary sequencing trace of the region immediately following exon 11 in the proband’s genomic DNA (left) and schematic diagram depicting the expected consequences of the A to C transversion on CRY1 mRNA splicing (right). (D) RT-PCR analysis of the CRY1 mRNA between exons 10 and 13. Samples 03 to 21 are amplified from primary fibroblast cell lines from 19 different subjects with number 11 belonging to the proband. Controls on the right are amplified from cloned CRY1 full-length and Δ11 cDNA. Expected product sizes are indicated. (E) CRY1 protein expression in the 19 subject-derived fibroblast cell lines. TUBULIN levels are shown as a loading control.

Figure 4. Effect of the CRY1 mutation on human sleep timing and clock oscillation

(A) Segregation of the CRY1 c.1657+3A>C allele with delayed sleep in the proband’s family. Genotype is shown inside symbols. Color code and symbols are explained in the legend. Numbers represent midsleep point on free days (MSF) (Roenneberg et al., 2003). See also Table S1 for details. (B) Deletion of CRY1 exon 11 affects circadian period length. CRY1 fl or Δ11 cDNAs were expressed in Bmal1-luc DKO MEFs using a lentiviral expression system that preserved the regulatory elements necessary to recapitulate endogenous CRY1 expression. Cells were synchronized with 20 M forskolin and bioluminescence output was recorded for approximately seven days. Traces show average detrended bioluminescence counts normalized to the first peak for each genotype (CRY1 fl blue, CRY1 Δ11 red). Period was calculated from bioluminescence recordings of quadruplicate samples from quadruplicate CRY1 infections (circles: fl 1–4, diamonds: Δ 1–4). Data from three independent experiments are shown (grey shading). Mean periods from each infection (indicated by horizontal lines) were used to assess statistical significance between genotypes. The overall mean period was 31.6 hrs for full-length CRY1 and 32.1 hours for Δ11 CRY1. Steady-state CRY1 levels for infections 1–4 from each experiment were measured by Western Blot with Tubulin shown as loading control. See also Figures S2 and S3 and Table S1.

Figure 5. Sleep behavior in CRY1 c.1657+3A>C carrier families of Turkish descent

(A–F) Sleep behavior in families DSPD-4, -6, -14, -1, -9 and -7 assessed through sleep and chronotype questionnaires and personal interview. Genotype is shown inside symbols. Numbers represent midsleep point on free days (MSF) (Roenneberg et al., 2003). See also Table S1 for details. (G) Legend for colors and symbols used in (A-F). (H) MSF from subjects in (A–F) as well as Figure 4A are plotted on a discontinuous clock face from 22:00 to 9:00 for carriers (left, red) and non-carriers (right, black). No subject data fell within the gap time (9:00 to 22:00) not represented in the plot.

Figure 6. Exon 11 deletion enhances CRY1 function in the molecular circadian clock

(A) CRY1 expression was assessed in fractionated extracts prepared from the proband’s fibroblasts at the indicated times following synchronization. TUBULIN and TBP were used as loading controls for cytoplasmic and nuclear extracts, respectively, and to assess fractionation purity. (B) Same as (A) except proband fibroblasts were replaced with CRY1 fl/Δ MEFs and the sampling interval was adjusted to account for the longer circadian period in this cell type. (C) Co-immunoprecipitation of CRY1 with ARNTL and CLOCK in unsynchronized whole cell (left) and nuclear (right) extracts from the proband’s fibroblasts. (D) Co-immunoprecipitation of CRY1 with Clock, Bmal1 and K538 Acetyl-Bmal1 in nuclear extracts from CRY1 fl/Δ MEFs at 30 or 45 hours post-synchronization as well as from unsynchonized CRY1 fl/Δ and empty vector control DKO MEFs. (E) Co-immunoprecipitation of Clock, Bmal1, K538 Acetyl-Bmal1 and Per2 with CRY1 from nuclear extracts of CRY1 fl or Δ11 MEFs at 30 or 45 hours post-synchronization as well as from unsynchonized CRY1 fl or Δ11 MEFs and the empty vector control. (F) Levels of CRY1, pre-Bmal1, pre-Per2, pre-Per1, pre-Dbp and pre-Lrwd1 were assessed by real-time quantitative RT-PCR in synchronized CRY1 fl (blue) or Δ11 (red) MEFs. Graphs show mean expression levels from five independent experiments with the shaded area indicating the standard error. Statistically significant differences in gene expression between genotypes are indicated. n.s. = not significant See also Figure S4.

Figure 7. CRY1 Δ11 affects the occupancy of CRY1, Bmal1 and Clock at target gene promoters

(A–C) CRY1, Bmal1, Clock and H3K4me3 were immunoprecipitated from CRY1 fl (blue) or Δ11 (red) MEFs at 30 or 45 hours post-synchronization following chromatin crosslinking. The amount of Per2- (A), Dbp- (B) and Lrwd1-promoter DNA (C) in the immunoprecipitates was assessed by real-time quantitative PCR. The background signal (dashed line) corresponds to the respective real-time quantitative PCR values of a control reaction using a CRY1 chromatin-immunoprecipitate from Cry-deficient cells as template. Data in each experiment were normalized to the amount in the CRY1 fl sample, which was set to 1. Error bars represent the standard error from three independent experiments.