Functional screening of drug target genes: m1 muscarinic acetylcholine receptor phenotypes in degenerative dementias.
OBJECTIVE
A number of recent studies surveying single nucleotide polymorphisms within the exonic regions of human genes have revealed a significant number of such variants, including many non-synonymous variants. This highlights the need to directly identify, within individual clinically well-defined patients, those variants that alter protein function as well as structure. We report on the development of a novel phenotypic screening process that combines high-throughput molecular cloning techniques with functional expression utilizing the cell-based assay R-SAT.


METHODS
We applied the phenotypic screening process to an analysis of the m1 muscarinic acetylcholine receptor (CHRM1) gene in a cohort of 74 individuals, including 48 diagnosed with neurodegenerative disease, primarily Alzheimer disease, who have been stratified according to their clinical response to the acetylcholinesterase inhibitor donepezil. Phenotypic screening of the CHRM1 gene involved PCR-based amplification from genomic DNA and heterologous expression in mammalian cells.


RESULTS
Phenotypic screening yielded functional responses to the agonist carbachol displaying a mean potency (-pEC(50)+/- standard deviation) of 5.8 +/- 0.2, which did not differ from that observed with expression of the wild-type receptor gene (6.0 +/- 0.3). No altered levels of constitutive receptor activity were observed. Dideoxy sequencing did not reveal any non-synonymous variants in the coding exon of this gene within this clinical cohort, while detecting three synonymous variants.


CONCLUSIONS
The results confirm that the m1 receptor gene (CHRM1) is not highly polymorphic in the human population, suggesting that genetic variation within the coding exon of this gene is not a contributing factor to the clinical variability observed during treatment of dementia with cholinergic enhancement therapies.
Weiner
David M
DM
ACADIA Pharmaceuticals, Inc., San Diego, California 92121, USA. dweiner@arcadia-pharm.com
Goodman
Matilda W
MW
Colpitts
Tonya M
TM
Feddock
Michelle A
MA
Duggento
Kate L
KL
Nash
Norman R
NR
Levey
Allan I
AI
Brann
Mark R
MR
D000368:Aged; D002800:Cholinesterase Inhibitors / Q000627:therapeutic use; D015331:Cohort Studies; D004247:DNA / Q000032:analysis / Q000235:genetics; D003704:Dementia / Q000188:drug therapy* / Q000235:genetics*; D000077265:Donepezil; D016503:Drug Delivery Systems; D005260:Female; D005820:Genetic Testing; D006801:Humans; D007189:Indans / Q000627:therapeutic use; D008297:Male; D019636:Neurodegenerative Diseases / Q000188:drug therapy* / Q000235:genetics*; D018697:Nootropic Agents / Q000627:therapeutic use; D010641:Phenotype; D010880:Piperidines / Q000627:therapeutic use; D011110:Polymorphism, Genetic / Q000235:genetics; D043643:Receptor, Muscarinic M1 / Q000187:drug effects* / Q000235:genetics*; D011976:Receptors, Muscarinic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
C086088:CHRM1 protein, human; D002800:Cholinesterase Inhibitors; D007189:Indans; D018697:Nootropic Agents; D010880:Piperidines; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D000077265:Donepezil; D004247:DNA