| Mental disorders including drug addiction are complex diseases with strong genetic components. A number of candidate genes have been intensely studied, but the effects of genetic variants for most of them remain elusive. Clinical studies have demonstrated associations of genetic variants in candidate genes with susceptibility to several common mental diseases such as schizophrenia, depression, bipolar disorders, and drug addiction, but little was known about the underlying mechanisms. Single nucleotide polymorphisms (SNPs), naturally occurring nucleotide substitutions in DNA sequence, are the most common genetic variants, sometimes reaching high frequency in target populations. Genome-wide survey studies have shown SNPs in noncoding regions are more prevalent than those SNPs that alter protein sequence (nonsynomous SNPs). This suggests a pervasive effect of those SNPs on inter-individual variability. A large number of studies has shown that SNPs in coding and noncoding regions of a gene can modulate gene expression at the level of mRNA and protein by affecting transcription, mRNA processing, pre-mature RNA splicing, mRNA stability, or translation. Moreover, functional SNPs can affect alternative splicing, which is controlled by cis and trans regulation, another important mechanism causing inter-individual variability. My objectives were to test the hypothesis that functional SNPs regulating mRNA expression and processing of the mu opioid receptor and D2 dopamine receptor exist at frequencies that have the potential to contribute to differential susceptibility to mental disorders in the human population.;To address this goal, I first determined whether there is mRNA expression imbalance (AEI) between two alleles of OPRM1 and DRD2 in relevant target tissues (postmortem brain samples). Primer extension assays (SNaPShot) was used to detect allelic mRNA expression in human postmortem brain tissues using indicator SNP A118G for OPRM1 and three SNPs (rs6275, rs6277, and rs6279) for DRD2, respectively. Allelic expression imbalance (AEI) was observed for both OPRM1 and DRD2. For OPRM1 (Chapter 2), in 8 heterozygous samples measured, the A118 mRNA allele was 1.5- to 2.5-fold more abundant than the G118 allele. Transfection into CHO cells of a cDNA representing only the coding region of OPRM1, carrying A, G, C, or T in position 118, resulted in 1.5-fold lower mRNA levels only for OPRM1-G118, and more than tenfold lower OPRM1 protein levels, measured by Western blotting and receptor binding assay. After transfection and inhibition of transcription with actinomycin D, analysis of mRNA turnover failed to reveal differences in mRNA stability between A118 and G118 alleles, indicating a defect in transcription or mRNA maturation. These results indicate that OPRM1-G118 is a functional polymorphism with deleterious effects on both mRNA and protein yield. Clarifying the functional relevance of polymorphisms associated with susceptibility to a complex disorder such as drug addiction provides a foundation for clinical association studies.;In chapter 3, among 68 heterozygous samples tested for AEI in DRD2, 7 of them displayed higher expression of the major allele (C allele) than T allele, while another 8 samples had the opposite trend. To search for regulatory polymorphisms, I performed SNP scanning of the gene locus, which identified a novel regulatory SNP (rs12364283) located in a conserved suppressor region, with the minor allele (∼7% allele frequency) causing enhanced expression. Moreover, differences in allelic mRNA expression of the long and short DRD2 splice isoforms---showing distinct pre- and post-synaptic functions---revealed two linked intronic SNPs (rs2283265 and rs1076560, 17% minor allele frequency) strongly associated with reduced formation of DRD2-short. Exploratory clinical studies supported an association of the promoter variant with schizophrenia, while the two intronic SNPs significantly associated with enhanced cognitive processing (done by collaboration with Dr. Bertolino from the University of Bari). These results indicate that DRD2 variants appear to have strong effects on dopaminergic transmission in vivo.;In chapter 4, I investigated and characterized the direct binding of G protein coupled receptor derived peptides with calmodulin using a quantitative assay (S-Tag). Calmodulin binding to receptor regions involved in G protein coupling appears to play a role in the regulation of signaling, and at least one naturally occurring polymorphisms in the mu opioid receptor had previously been shown to affect CaM binding. CaM binding of peptides derived from the third intracellular loop (i3) of the mu opioid receptor (MOR) was confirmed and the CaM-binding motif refined. A MORi3 peptide with a Lys>Ala substitution---shown to reduce CaM-binding of intact MOR---bound 5-fold less avidly than the wild-type peptide. Screening peptides derived from i3 loops of other GPCR families confirmed 5HT1A, and identified muscarinic receptor 3, and melanocortin receptor 1, as proteins carrying CaM-binding domains. The use of S-Tag labeling can serve for rapid screening of putative CaM-binding domains in GPCRs.;This study examines the extent and nature of regulatory polymorphisms in candidate genes, and provides a foundation for clinical association studies by clarifying the functional relevance of polymorphisms associated with susceptibility to mental disorders. |
