Abstract
As a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) plays a key role in pain perception and addiction. Genetic variants of OPRM1 have been implicated in predisposition to drug addiction, in particular the single nucleotide polymorphism A118G, leading to an N40D substitution, with an allele frequency of 10-32%, and uncertain functions. We have measured allele-specific mRNA expression of OPRM1 in human autopsy brain tissues, using A118G as a marker. In 8 heterozygous samples measured, the A118 mRNA allele was 1.5-2.5-fold more abundant than the G118 allele. Transfection into Chinese hamster ovary cells of a cDNA representing only the coding region of OPRM1, carrying adenosine, guanosine, cytidine, and thymidine in position 118, resulted in 1.5-fold lower mRNA levels only for OPRM1-G118, and more than 10-fold 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 variant 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.
Highlights
Because of its central role in drug addiction, numerous studies have addressed potential contributions of polymorphisms in the gene encoding OPRM1 to addiction susceptibility [7, 8]
Genomic DNA and total RNA were isolated from brain tissues, and mRNA was reverse-transcribed to Complementary DNA (cDNA) using a gene-specific primer and oligo(dT). cDNA synthesis using only oligo(dT) failed in brain tissue extracts because the coding region is far removed from the poly(A) tail of OPRM1, and because of partial mRNA degradation
This result indicates that the level of mRNA from the wild-type A118 allele yielded ϳ2-fold higher mRNA levels than the G118 allele in heterozygous brain tissues
Summary
Peak area ratios were measured for both genomic DNA and mRNA (cDNA). MRNA Analysis Using Real-time PCR—To quantify mRNA expression of the genotyped samples, we first set up a standard curve using various amount (50 fg to 100 pg) of plasmid DNA (OPRM1), performed with real-time PCR with the same PCR amplification primers used for SNaPshot, with 12.5 l of 2ϫ PCR SYBR master mixture (3 mM MgCl2, 200 M dATP, 200 M dCTP, 200 M dGTP, 200 M dUTP, 0.625 units of AmpliTaq Gold, and 0.25 units of AmpErase UNG). To detect any possible DNA contamination, real-time PCR was performed in RNA control samples in the absence of reverse transcriptase during cDNA synthesis. Statistical analysis was performed using the program GraphPad Prism version 3.00 for Windows, GraphPad Software (San Diego, CA)
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