We hypothesized that genetic variation among receptors excitatory neurotransmitter receptors, growth factor, and growth factor receptor genes play an important role in modifying neuronal plasticity. It is also plausible that allelic variation in shared signal transduction pathways among different neurotransmitters (such as glutamate and serotonin neurotransmitters) and their receptors are critical to creating and maintaining neuronal connectivity may also play a role in mediating early responses (sensitization) or conditioned responses (addiction) to alcohol or other drugs. The NMDA class of glutamate receptors, named for the specific compound (or agonist) that activates the receptor, is directly inhibited by ethanol (an antagonist) and it is thought that changes in NMDA receptor activity is involved in ethanol-induced adaptations, such as tolerance and dependence. Recent findings have bolstered the idea that chronic ethanol changes NMDA receptor subunit composition, particularly of NR2 subunits in the nucleus accumbens and amygdala (brain regions known to have critical roles in anxiety behaviors, emotion, response to stress, and drug dependence). In addition, a characteristic or so-called ?intermediate phenotype,? associated with increased risk of alcohol consumption and dependence in family history positive individuals is the reduced sensitivity of individuals to the dysphoric effects of the NMDA receptor antagonist ketamine, which may signal a reduced response to ethanol. To capture sequence variant data at glutamate receptor genes, we initially surveyed the entire family of ionotrophic glutamate receptors (IGR), composed of the NMDA, AMPA, and KA subtypes. We used screening technologies, including denaturing high-performance liquid chromatography, DNA melting analysis, and direct sequencing as well as information mining of public (e.g. dbSNP, ENSEMBL) and private (i.e. Celera Discovery System) sequence databases to discover novel coding region and promoter regions single nucleotide polymorphisms (SNPs). A collection of 38 missense variants were identified by the informatics and resequencing approaches in several of these receptor genes. Of particular interest were eight SNPs in NR1 and NR2B subunit genes. Four of the variants resulted in an amino acid change (missense variants). Three are candidate promoter variants. All of the variants were previously unidentified. In particular the NR1 gene variant 8396G>A resulted in a cysteine to tyrosine substitution at position 744. When expressed in human embryonic kidney cells with a native NR2A subunit, this receptor variant demonstrated prolonged desensitization to agonist (glutamate) and showed increased resistance to NMDA-mediated excitotoxcity. The three GRIN2B missense variants, Ser116Thr, Leu120Ile, and Thr275Ala are localized to the extracellular ligand binding or modulatory domain of the NR2B subunit. Of the variants we identified by resequencing approaches, only NR2B Thr275Ala had a minor allele frequency above 1% (0.02 in U.S. Caucasians). This variant, however, may be of particular interest since the amino acid residue involved maps near the ifenprodil binding site on the NR2B subunit. Also of note was the low rate of SNP confirmation using computational SNPs from public databases. Using direct sequence analysis, on 50 individuals, only 18% of the computational SNPs could be confirmed. Each of the variants discovered and validated must now be functionally characterized in order to determine its potential impact on IGR function and to determine it contribution to alcoholism-related phenotypes.
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