Abstract
Background/aim: A variety of behavioral consequences and physiological processes are under the influence of the core clock genes. Mammalian species have distinct transcription-translation feedback loops which oscillate for approximately 24 hours that entrains peripheral tissues to the dark-light cycle. Consequently, species have dissimilar activity patterns regarding their daily actions. For instance, diurnal species are known to sleep in dark periods, meanwhile being active during light periods. Throughout evolution, phenotypic traits are susceptible to diverge along with subtle changes in protein sequences. Taken together, the study aims to assess the residues of core clock proteins whether there are significant amino acid sites that may be the cause of being diurnal or non-diurnal from an evolutionary perspective. Moreover, the involvement of these residues with sleep disorders will be inspected with a reverse phenotyping approach.

Methodology: Multiple sequence alignment of vertebrate core clock proteins has been conducted through the Constraint-based Multiple Alignment Tool (COBALT) of the National Center for Biotechnology Information (NCBI). Species are divided into two groups which are “diurnals” and “non-diurnals” in the alignment to observe the amino acid residues that are separately distributed in a non-random manner. Significance levels of each residue in the multiple sequence alignmenthave been assigned with Fisher’s exact score. Therefore, the selective distribution of an amino acid only in one group yielded a lower p-value. The threshold level of significance for each protein calculated using Bonferroni Correction. Seven well-known core clock proteins are evaluated for their significant amino acid sites. Corresponding nucleotide loci of the evolutionary important amino acid residues will be examined through cohort sequencing data to reach individuals who are the carriers of a variant on such sites.

Results: ARNTL (Aryl hydrocarbon receptor nuclear translocator-like protein), CLOCK (Circadian locomoter output cycles protein kaput), PER1/2/3 (Period circadian protein homolog), CRY1/2 (Cryptochrome) proteins are evaluated. Preliminary analysis revealed those amino acid residues whose distribution may non-randomly be associated with sleep patterns in terms of being diurnal or not. Bonferroni correction yields 6 residues of CRY1, 7 residues of CRY2, 9 residues of PER1, 15 residues of PER2, 68 residues of PER3, 21 residues of CLOCK, and 7 residues of ARNTL as statistically significant. The usual accumulation of these significant sites relies on the tail regions of the proteins. Mutations in such regions could disrupt folding events or post- translational mechanisms.

Conclusion: The preliminary research shows that there is a variety of significant amino acids among core clock proteins to examine regarding functionality. As expected, complete conservation or non-conservation would not be observed among both groups. Overall, this study will be pioneer research on the phenotypic impact of core clock proteins about their direct relation to behavioral traits with the evolutionary patterns. Analysis of the cohort data enables us to gain an insight into genomic medicine as well.