Cerebrospinal Fluid Hypocretin-1 (Orexin-A) Level Fluctuates with Season and Correlates with Day Length

<div><p>The hypocretin/orexin neuropeptides (hcrt) are key players in the control of sleep and wakefulness evidenced by the fact that lack of hcrt leads to the sleep disorder Narcolepsy Type 1. Sleep disturbances are common in mood disorders, and hcrt has been suggested to be poorly regulated in depressed subjects. To study seasonal variation in hcrt levels, we obtained data on hcrt-1 levels in the cerebrospinal fluid (CSF) from 227 human individuals evaluated for central hypersomnias at a Danish sleep center. The samples were taken over a 4 year timespan, and obtained in the morning hours, thus avoiding impact of the diurnal hcrt variation. Hcrt-1 concentration was determined in a standardized radioimmunoassay. Using biometric data and sleep parameters, a multivariate regression analysis was performed. We found that the average monthly CSF hcrt-1 levels varied significantly across the seasons following a sine wave with its peak in the summer (June—July). The amplitude was 19.9 pg hcrt/mL [12.8–26.9] corresponding to a 10.6% increase in midsummer compared to winter. Factors found to significantly predict the hcrt-1 values were day length, presence of snow, and proximity to the Christmas holiday season. The hcrt-1 values from January were much higher than predicted from the model, suggestive of additional factors influencing the CSF hcrt-1 levels such as social interaction. This study provides evidence that human CSF hcrt-1 levels vary with season, correlating with day length. This finding could have implications for the understanding of winter tiredness, fatigue, and seasonal affective disorder. This is the first time a seasonal variation of hcrt-1 levels has been shown, demonstrating that the hcrt system is, like other neurotransmitter systems, subjected to long term modulation.</p></div

Summary of variables in the dataset.

<p>Summary of variables in the dataset.</p

CSF hcrt-1 correlates with day light levels and presence of snow.

<p>A) The relationship between CSF hcrt-1 levels and average day length the preceding three weeks, divided in groups dependant on the presence of snow. In the samples taken on days without snow, there was a significant correlation (linear regression p = 0.0001) between day light and the hcrt-1 level, which was not found in the samples taken on days with snow. B) The effect of snow on hcrt-1 levels in winter month (Dec-Feb). The average hcrt-1 level is significant higher (student t-test, p = 0.009) when CSF sampling was performed on days with snow than on days without snow.</p

CSF hcrt-1 levels around Christmas vacation in Denmark.

<p>Shown are linear regression fits of data excluding days of snow and divided into three parts: Nov-Dec (non-significant), Jan (p = 0.03), and Feb-March (non-significant). The Christmas vacation is marked with red and data sampled on days with snow are marked with a blue circle.</p

Seasonal variation in CSF hcrt-1 levels in human individuals.

<p>Sinusoidal fitted curve to monthly average of patient CSF hypocretin-1 levels, excluding January. Dashed lines represent 95% confidence interval. The inserted numbers above x-axis equals the number of data points for each month.</p

Predictors of CSF hcrt-1 level.

<p>Summary of Multiple Regression Analysis.</p

Association signal at the mapping intervals flanking rs34593439 and rs7553711.

<p>Association scores at 15q25.1 (panel A) and 1q25.1 (panel B). Genotyped (diamonds) and imputed (circles) SNPs are indicated and the top genotyped SNP in the interval is outlined in orange. A SNP in 15q25.1 previously associated with Diabetes is outlined in blue. The degree of red color in each diamond or circle indicates the strength of LD with the top SNP (on a scale shown in the legend at the upper left hand corner of the plot). The X-axis shows the chromosome and physical distance (kb) from the human genome reference sequence (hg19), the left Y-axis shows the negative base ten logarithm of the p-value and the right Y-axis shows recombination rate (cM/Mb) as a navy line. The genome-wide significance threshold (P<5×10⁻⁸) is given by the dashed grey line. Genes in the regions are annotated at the bottom as green arrows. Also indicated in 1q25.1 is a ∼130 kb region with no SNPs on the ImmunoChip.</p

Non-HLA narcolepsy risk variant loci reaching genome-wide significance.

<p>Chr.: Chromosome; BP: position according to NCBI build 36 (Hg18) coordinates; MAF_N: minor allele frequency in narcolepsy (_N) and controls (_C); P: P value according to variance component model (EMMAX). EMMAX does not provide OR (Odds Ratio) or adjusted allele frequencies, therefore MAF, OR, and 95% confidence intervals (CI) were calculated with Plink on subset of 8,474 samples with the greatest PCA homogeneity (see Figure S2; EV 11.21<0.004, EV 4.12<0.01).</p

Sample collections.

*<p>Numbers of samples by country of origin are listed in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003270#s3" target="_blank">Methods</a> section.</p><p>Case cohort names represent location of genotyping, and do not reflect country of origin of samples.</p

Manhattan Plot of association statistics.

<p>The significance threshold used (blue line) was P = 5×10⁻⁸; The insets depict plots of 1) association results in a broad region encompassing the HLA locus (chr 6:24,067–35,474 kb) that were excluded from the present analysis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003270#s3" target="_blank">Methods</a>) and 2) QQ plot of results for 109,777 markers after excluding a 1 Mb window surrounding the associated loci (λ = 1.004). The inflation statistic for all 111,240 tested markers is 1.04.</p