Advertisement

Detecting Polymorphisms in G Protein-Coupled Receptor Genes

  • Dmitri ProudnikovEmail author
  • Vadim Yuferov
  • Mary Jeanne Kreek
Protocol
Part of the Neuromethods book series (NM, volume 60)

Abstract

The genes for G protein-coupled receptors (GPCRs) including those encoding the classical mu, delta, and kappa opioid receptors (MOR, DOR, and KOR); cannabinoid receptors (CB1); ACTH receptor (melanocortin receptor type 2, MC2R); and serotonin receptors (5HT1B) have been a focus of the studies of our group for a number of years since these receptors are involved in specific addictions. Genetic variants of GPCR genes have been associated with vulnerability to stress, anxiety, depression, and predisposition to develop drug addiction. To study these variants including single nucleotide polymorphisms (SNPs) and their allocation on alleles (haplotypes), our group developed special techniques (genotyping assays using polyacrylamide gel pad technology, molecular haplotyping assays based on the use of fluorescent PCR) and also used commercially available techniques and methodologies. Although these novel technologies allow rapid and reliable high-throughput analysis, in order to use them, the precise position of the polymorphic site should be known in advance. The contemporary genetic databases contain copious information on genetic variants. However, we found that some important functional variants are still unreported. Therefore, resequencing of the genes studied in specific populations is necessary. Each technology that we use has specific advantages that we will discuss below.

Key words

Genotyping SNP Molecular haplotyping Microarrays Single nucleotide extension Polymorphism 

Notes

Acknowledgments

We thank Drs. Ann Ho and Orna Levran for a critical reading of this chapter and Susan Russo for editorial assistance. This work was supported by the NIH Grant P60-DA05130 (MJK) and Clinical and Translational Science Award Grant UL1 RR024143 (NCRR, Rockefeller University).

References

  1. 1.
    Sadée W, Dai Z (2005) Pharmacogenetics/genomics and personalized medicine. Hum Mol Genet 14:R207–214PubMedCrossRefGoogle Scholar
  2. 2.
    Knight JC (2005) Regulatory polymorphisms underlying complex disease traits. J Mol Med 83:97–109PubMedCrossRefGoogle Scholar
  3. 3.
    Bond C, LaForge KS, Tian M et al (1998) Single nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: Possible implications for opiate addiction. Proc Natl Acad Sci USA 95:9608–9613PubMedCrossRefGoogle Scholar
  4. 4.
    Bart G, LaForge KS, Borg L et al (2006) Altered levels of basal cortisol in healthy subjects with a 118G allele in exon 1 of the mu opioid receptor gene. Neuropsychopharmacology 31:2313–2317PubMedGoogle Scholar
  5. 5.
    Bart G, Heilig M, LaForge KS et al (2004) Substantial attributable risk related to a functional mu-opioid receptor gene polymorphism in association with heroin addiction in central Sweden. Mol Psychiatry 9:547–549PubMedCrossRefGoogle Scholar
  6. 6.
    Yuferov V, Fussell D, LaForge KS et al (2004) Redefinition of the human kappa opioid receptor gene (OPRK1) structure and association of haplotypes with opiate addiction. Pharmacogenetics 14:793–804PubMedCrossRefGoogle Scholar
  7. 7.
    Proudnikov D, LaForge KS, Hofflich H et al (2006) Association analysis of polymorphisms in serotonin 1B receptor (HTR1B) gene with heroin addiction: a comparison of molecular and statistically estimated haplotypes. Pharmacogenet Genomics 16:25–36PubMedCrossRefGoogle Scholar
  8. 8.
    Proudnikov D, Hamon S, Ott J et al (2008) Association of polymorphisms in the melanocortin receptor type 2 (MC2R, ACTH receptor) gene with heroin addiction. Neurosci Lett 435:234–239PubMedCrossRefGoogle Scholar
  9. 9.
    Proudnikov D, Kroslak T, Sipe JC et al (2010). Association of polymorphisms of the cannabinoid receptor (CNR1) and fatty acid amide hydrolase (FAAH) genes with heroin addiction: impact of long repeats of CNR1. Pharmacogenomics J 10:232–242PubMedCrossRefGoogle Scholar
  10. 10.
    Briant JA, Nielsen DA, Proudnikov D et al (2010) Evidence for association of two variants of the nociceptin/orphanin FQ receptor gene OPRL1 with vulnerability to develop opiate addiction in Caucasians. Psychiatr Genet 20:65–72PubMedCrossRefGoogle Scholar
  11. 11.
    Proudnikov D, LaForge KS, Kreek MJ (2004) High-throughput molecular haplotype analysis (allelic assignment) of single-nucleotide polymorphisms by fluorescent polymerase chain reaction. Anal Biochem 335:165–167PubMedCrossRefGoogle Scholar
  12. 12.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  13. 13.
    Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560PubMedCrossRefGoogle Scholar
  14. 14.
    Proudnikov D, Mirzabekov A (1996) Chemical methods of DNA and RNA fluorescent labeling. Nucleic Acids Res 24:4535–4542PubMedCrossRefGoogle Scholar
  15. 15.
    Proudnikov D, Timofeev E, Mirzabekov A (1998) Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips. Anal Biochem 259:34–41PubMedCrossRefGoogle Scholar
  16. 16.
    Bart G, Kreek MJ, Ott J et al (2005) Increased attributable risk related to a functional mu opioid receptor gene polymorphism in association with alcohol dependence in central Sweden. Neuropsychopharmacology 30:417–422PubMedCrossRefGoogle Scholar
  17. 17.
    Slawik M, Reisch N, Zwermann O et al (2004) Characterization of an adrenocorticotropin (ACTH) receptor promoter polymorphism leading to decreased adrenal responsiveness to ACTH. J Clin Endocrinol Metab 89:3131–3137PubMedCrossRefGoogle Scholar
  18. 18.
    Nielsen DA, Ji F, Yuferov V et al (2008) Genotype patterns that contribute to increased risk for or protection from developing heroin addiction. Mol Psychiatry 13:417–428PubMedCrossRefGoogle Scholar
  19. 19.
    Nielsen DA, Ji F, Yuferov V et al (2010) Genome-wide association study identifies genes that may contribute to risk for developing heroin addiction. Psychiatr Genet 20:207–214PubMedCrossRefGoogle Scholar
  20. 20.
    Khrapko KR, Lysov YP, Khorlyn AA et al (1989) An oligonucleotide hybridization approach to DNA sequencing. FEBS Lett 256:118–122PubMedCrossRefGoogle Scholar
  21. 21.
    Fotin AV, Drobyshev AL, Proudnikov DY et al (1998) Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips. Nucleic Acids Res 26:1515–1521PubMedCrossRefGoogle Scholar
  22. 22.
    Chechetkin VR, Turygin AY, Proudnikov DY et al (2000) Sequencing by hybridization with the generic 6-mer oligonucleotide microarray: an advanced scheme for data processing. J Biomol Struct Dyn 18:83–101PubMedGoogle Scholar
  23. 23.
    Proudnikov D, Kirillov E, Chumakov K et al (2000) Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips. Biologicals 28:57–66PubMedCrossRefGoogle Scholar
  24. 24.
    Guschin DY, Mobarry BK, Proudnikov D et al (1997) Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology. Appl Environ Microbiol 63:2397–2402PubMedGoogle Scholar
  25. 25.
    LaForge KS, Shick V, Spangler R et al (2000) Detection of single nucleotide polymorphisms of the human mu opioid receptor gene by hybridization or single nucleotide extension on custom oligonucleotide gelpad microchips: potential in studies of addiction. Am J Med Genet 96:604–615PubMedCrossRefGoogle Scholar
  26. 26.
    Witcombe D, Brownie J, Gillard HL et al (1998) A homogeneous fluorescence assay for PCR amplification to real-time single-tube genotyping. Clin Chem 44:918–923Google Scholar
  27. 27.
    Nielsen DA, Barral S, Proudnikov D et al (2008) TPH2 and TPH1: association of variants and interactions with heroin addiction. Behav Genet 38:133–150PubMedCrossRefGoogle Scholar
  28. 28.
    Drgon T, Zhang PW, Johnson C et al (2010) Genome wide association for addiction: replicated results and comparisons of two analytic approaches. PLoS One 5:e8832PubMedCrossRefGoogle Scholar
  29. 29.
    Hodgkinson CA, Yuan Q, Xu K et al (2008) Addictions biology: haplotype-based analysis for 130 candidate genes on a single array. Alcohol Alcohol 43:505–515PubMedGoogle Scholar
  30. 30.
    Levran O, Londono D, O’Hara K et al (2008) Genetic susceptibility to heroin addiction: a candidate gene association study. Genes Brain Behav 7:720–729PubMedCrossRefGoogle Scholar
  31. 31.
    Levran O, Londono D, O’Hara K et al (2009) Heroin addiction in African Americans: a hypothesis-driven association study. Genes Brain Behav 8:531–540PubMedCrossRefGoogle Scholar
  32. 32.
    Li J, Burmeister M (2005) Genetical genomics: combining genetics with gene expression analysis. Hum Mol Genet 14:R163-169PubMedCrossRefGoogle Scholar
  33. 33.
    Jansen RC, Nap JP (2001) Genetical genomics: the added value from segregation. Trends Genet 17:388–391PubMedCrossRefGoogle Scholar
  34. 34.
    Serre D, Gurd S, Ge B et al (2008) Differential allelic expression in the human genome: a robust approach to identify genetic and epigenetic cis-acting mechanisms regulating gene expression. PLoS Genet 4:e1000006PubMedCrossRefGoogle Scholar
  35. 35.
    Yuferov V, Ji F, Nielsen DA et al (2009) A functional haplotype implicated in vulnerability to develop cocaine dependence is ­associated with reduced PDYN expression in human brain. Neuropsychopharmacology 34:1185–1197PubMedCrossRefGoogle Scholar
  36. 36.
    Branch AD, Unterwald EM, Lee SE et al (1992) Quantitation of preproenkephalin mRNA levels in brain regions from male Fischer rats following chronic cocaine treatment using a recently developed solution hybridization assay. Brain Res Mol Brain Res 14:231–238PubMedCrossRefGoogle Scholar
  37. 37.
    Guschin D, Yershov G, Zaslavsky A et al (1997) Manual manufacturing of oligonucleotide, DNA, and protein microchips. Anal Biochem 250:203–211PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Dmitri Proudnikov
    • 1
    Email author
  • Vadim Yuferov
  • Mary Jeanne Kreek
  1. 1.Laboratory of the Biology of Addictive DiseasesThe Rockefeller UniversityNew York CityUSA

Personalised recommendations