Identification of human Clock gene variants by denaturing high-performance liquid chromatography.
The human Clock gene ( hClock) encodes the CLOCK protein essential for the function of the circadian system. We have screened the entire coding region, including the 5' and 3' untranslated regions (UTRs) and the flanking intronic regions, of the hClock gene for sequence variations in 70 unrelated Chinese Singaporeans with denaturing high-performance liquid chromatography (dHPLC). A total of 15 sequence variations were detected, five of which were novel. All involved single base changes. There were 12 substitutions and three insertions/deletions. All except one were found in the introns or the UTRs. Frequencies of the minor allele for all 15 polymorphisms ranged from 0.7% to almost 50%. For the eight sites whose minor allele frequency was found to be at least 10%, pair-wise comparisons revealed that all except one were in almost complete linkage disequilibrium. Our identification of additional single nucleotide polymorphisms in the hClock gene would provide markers whose frequencies could be established in the selected population and used for further analysis of the phenotypic effects. Our results would also be useful for better planning in the selection of polymorphisms for future genetic association studies involving the hClock gene.