Functional consequences of a CKId mutation causing familial advanced sleep phase syndrome

Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K, Ptácek LJ, Fu YH. Nature. 2005 Mar 31;434(7033):640-4.

To discover the genetic causes of Familial Advanced Sleep Phase Syndrome (FASPS), the authors interviewed a family whose members suffer from FASPS. The authors found an A to G change in the csk1d gene that lead to a threonine to alanine mutation, by genetic screening of family members. The affected residue (amino acid 44) is conserved across species, and its mutation implies a role for csnk1d in the circadian clock.

The authors then assessed the function of the mutated protein by expressing it in bacteria. The mutant protein had a maximum reaction rate 60% that of the wildtype protein, as well as a 12% reduction in its Michaelis constant, as assessed by kinase assays.

The mutation was then introduced to fruit flies, and resulted in a slightly longer circadian period, as measured from periodogram analysis of locomotor activity.

Additionally, transgenic mice were generated via bacterial artificial chromosome cloning, and validated using RT-PCR with human primers. The authors found that the mice with the mutant csnk1d had a circadian period shorter by an estimated 24 minutes, and took longer to re-entrain to an LD 12:12 light cycle, compared to wild type mice.

An hPer2 Phosphorylation Site Mutation in Familial Advanced Sleep Phase Syndrome

Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptácek LJ, Fu YH. Science. 2001 Feb 9;291(5506):1040-3.

The purpose of this study was to determine the genetic and biochemical basis for Familial Advanced Sleep Phase (FASPS) Syndrome. The authors mapped the gene to the qter region of Chromosome 2 via linkage analysis confirmed by PCR. One gene in this region is the human homolog of the Drosophila period gene (as confirmed by bacterial artificial cloning and mapping), mutations of which lead to abberant circadian rhythms.

An S662G mutation was indentified in this region via Single-strand conformation polymorphism. Many of the sampled individuals diagnosed with FASPS while none of the controls had this mutation.

This mutation was then characterized by several experiments. It was found that the mutation occured within the Casein Kinase 1 Epsilon (CSNK1E) binding domain by immunoprecipitation, and the mutated site is a phosphorylation target by electrophoresis. Additionally, the mutated site is C-terminal to many other phosphorylation sites by sequencing, and the authors hypothesize that it is the initiation site of a phosphorylation cascade. This hypothesis was confirmed by comparing a S662D mutant (similar to a phosphoserine) to the wildtype and S662G proteins.