Determination of Paraoxonase 1 Status and Genotypes at Specific Polymorphic Sites

Rebecca J. Richter1, Rachel L. Jampsa1, Gail P. Jarvik1, Lucio G. Costa1, Clement E. Furlong1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.12
DOI:  10.1002/0471140856.tx0412s19
Online Posting Date:  May, 2004
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Abstract

The procedures for determining paraoxonase (PON1) status and for determining PON1 genotypes for polymorphisms in coding and important regulatory regions are described. PON1 status is determined by a functional two‐substrate analysis of plasma PON1 activities. Differences in catalytic efficiency of the PON1192Q and PON1192R alloforms result in the clear separation of all three phenotypes at position 192 (Q/Q, Q/R, R/R) and at the same time, the two‐substrate analysis indicates activity levels of PON1. Because the enzyme activity levels are as important as the polymorphic genotypes, this two‐substrate analysis of PON1 status provides the most relevant information for investigating the association of PON1 genetics with susceptibilities to disease, organophosphorus insecticide sensitivity, and pharmacokinetic status of drug metabolism. Genotyping of polymorphic sites alone fails to provide this important information but can be useful for gene frequency determination and forensic analysis. Analytical procedures for determining PON1 status and genotypes are described.

Keywords: PON1 status; PON1 polymorphisms; PON1 regulatory region polymorphisms

     
 
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Table of Contents

  • Basic Protocol 1: Phenotyping Individuals for Paraoxonase Status
  • Basic Protocol 2: Paraoxonase 1 Genotyping for Polymorphism 192QR
  • Alternate Protocol 1: Paraoxonase 1 Genotyping for Polymorphism −909CG
  • Alternate Protocol 2: Paraoxonase 1 Genotyping for Polymorphism −162GA
  • Alternate Protocol 3: Paraoxonase 1 Genotyping for Polymorphism −108TC
  • Alternate Protocol 4: Paraoxonase 1 Genotyping for Polymorphism 55LM
  • Alternate Protocol 5: Paraoxonase 1 Genotyping for Polymorphism 194WX
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Phenotyping Individuals for Paraoxonase Status

  Materials
  • Plasma samples: blood samples collected in lithium heparin (green‐top) tubes, centrifuged 5 min at 500 × g, 4°C, to remove cells
  • Dilution buffer (see recipe)
  • Paraoxon (Chem Service)
  • Assay buffer (see recipe), 37° and 23°C
  • Diazoxon (Chem Service)
  • Standard flat‐bottom 96‐well plates (Dynatech)
  • 50‐ml polypropylene tube (Falcon)
  • 37°C water bath
  • Plate reader (e.g., SPECTRAmax PLUS Microplate Spectrophotometer; Molecular Devices), 37°C
  • UV‐transparent 96‐well plates (Costar)
  • 15‐ml screw‐cap tubes (Falcon)
  • Conventional spectrophotometer (e.g., DU‐70; Beckman), optional
CAUTION: Utmost care must be used with these organophosphate substrates, as they are potent cholinesterase inhibitors. All substrate‐containing waste needs to be hydrolyzed in strong NaOH solution. Wear gloves and goggles for all assays and procedures using diazoxon or paraoxon. In a fume hood, add wastes to 5 N NaOH and let stand overnight. Then wash the wastes down the drain with lots of water (Mueller et al., ).

Basic Protocol 2: Paraoxonase 1 Genotyping for Polymorphism 192QR

  Materials
  • ∼100 ng/µl purified human DNA
  • 10× Opti‐prime buffer #3 (Stratagene)
  • 16 µM forward primer 192: 5′‐TAT TGT TGC TGT GGG ACC TGA G‐3′
  • 16 µM reverse primer 192: 5′‐CAC GCT AAA CCC AAA TAC ATC TC‐3′
  • 10 mM dNTPs
  • Dimethyl sulfoxide (DMSO), molecular biology grade
  • 2 U/µl Vent DNA polymerase (NEB)
  • 5 U/µl Taq DNA polymerase (Promega)
  • 100 mM MgSO 4 ( appendix 2A)
  • 5 U/µl AlwI and NEBuffer #4 (NEB)
  • 6× Blue/orange loading dye (Promega)
  • Low‐molecular‐weight DNA ladder (standards for 25‐ to 100‐bp fragments)
  • 0.1 µg/ml ethidium bromide (see recipe) diluted in water or comparable staining solution
  • 0.2‐ml PCR tubes
  • Thermal cycler (e.g., PE 9700; PE Biosystems)
  • 37°C water bath
  • Camera and UV light box or other instrument for recording the stained gel image
  • Additional reagents and equipment for DNA agarose gel electrophoresis (unit 2.2)

Alternate Protocol 1: Paraoxonase 1 Genotyping for Polymorphism −909CG

  • 10× Taq polymerase buffer (Promega)
  • 25 mM MgCl 2 (Promega or appendix 2A)
  • 25 µM forward primer −909: 5′‐TAT TAT AAT ATA TTA TAT CAT TCA CAG TAA CAG CAG ACA GCA GAG AAA AGA‐3′
  • 25 µM reverse primer −909: 5′‐AAC ATG TCA CTG TGG CAT ATA TAA TGC TC‐3′
  • 5 U/µl BsmAI and NEBuffer #3 (NEB)
  • Low‐molecular‐weight DNA ladder (standards for 50‐ to 250‐bp fragments)
  • 55°C water bath

Alternate Protocol 2: Paraoxonase 1 Genotyping for Polymorphism −162GA

  • 10× Opti‐prime buffer #1 (Stratagene)
  • 25 µM forward primer −162: 5′‐GCT ATT CTT CAG CAG AGG GT‐3′
  • 25 µM reverse primer −162: 5′‐TGA ATC TGT AGC CAG GGC AC‐3′
  • 10 U/µl BstUI (NEB)
  • NEBuffer #2 (NEB)
  • 1× TAE (see recipe)
  • 1‐kb DNA ladder (Promega) or equivalent
  • 60°C water bath

Alternate Protocol 3: Paraoxonase 1 Genotyping for Polymorphism −108TC

  • 10× Taq polymerase buffer (Promega)
  • 25 mM MgCl 2 (Promega or appendix 2A)
  • 25 µM forward primer −108: 5′‐GAC CGC AAG CCA CGC CTT CTG TGC ACC‐3′
  • 25 µM reverse primer −108: 5′‐TAT ATT TAA TTG CAG CCG CAG CCC TGC TGG GGC AGC GCC GAT TGG CCC GCC GC‐3′
  • 10 U/µl BstUI and NEBuffer #2 (NEB)
  • Low–base pair DNA ladder (standards for 50‐ to 125‐bp fragments)
  • 60°C water bath

Alternate Protocol 4: Paraoxonase 1 Genotyping for Polymorphism 55LM

  • 10× Taq polymerase buffer (Promega)
  • 25 mM MgCl 2 (Promega or appendix 2A)
  • 25 µM forward primer 55: 5′‐AGA GGA TTC AGT CTT TGA GGA AA‐3′
  • 25 µM reverse primer 55: 5′‐CTG CCA GTC CTA GAA AAC GTT‐3′
  • 10 U/µl NlaIII and NEBuffer #4 (NEB)
  • 10× BSA (NEB)
  • Low–base pair DNA ladder (suitable for 100‐ to 400‐bp fragments)

Alternate Protocol 5: Paraoxonase 1 Genotyping for Polymorphism 194WX

  • 10 U/µl BstNI and NEBuffer #2 (NEB)
  • 10× BSA (NEB)
  • 60°C water bath
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Figures

Videos

Literature Cited

   Adkins, S., Gan, K.N., Mody, M., and La Du, B.N. 1993. Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: Glutamine or arginine at position 191, for the respective A or B allozymes. Am. J. Hum. Genet. 52:598‐608.
   Blatter Garin, M.C., Abbott, C., Messmer, S., Mackness, M., Durrington, P., Pometta, D., and James, R.W. 1994. Quantification of human serum paraoxonase by enzyme‐linked immunoassay: Population differences in protein concentrations. Biochem. J. 304:549‐554.
   Brophy, V.H., Costa, L.G., Richter, R.J., Hagen, T., Shih, D.M., Tward, A., Lusis, A.J., and Furlong, C.E. 2001. Polymorphisms in the human paraoxonase (PON1) promoter. Pharmacogenetics 11:77‐84.
   Brophy, V.H., Jarvik, G.P., and Furlong, C.E. 2002. In Paraoxonase (PON1) In Health and Disease: Basic and Clinical Aspects (L.G. Costa and C.E. Furlong, eds.) pp. 53‐77. Kluwer Academic Press, Boston.
   Costa, L.G. and Furlong, C.E., eds. 2002. In Paraoxonase (PON1) In Health and Disease: Basic and Clinical Aspects. Kluwer Academic Press, Boston.
   Costa, L.G., Li, W.‐F., Richter, R.J., Shih, D.M., Lusis, A.J., and Furlong, C.E. 2002. PON1 and organophosphate toxicity. In Paraoxonase (PON1) In Health and Disease: Basic and Clinical Aspects (L.G. Costa and C.E. Furlong, eds.) pp. 165‐183. Kluwer Academic Press, Boston.
   Costa, L.G., Cole, T.B., Jarvik, G.P., and Furlong, C.E. 2003. Functional genomics of the paraoxonase (PON1) polymorphisms: Effects on pesticide sensitivity, cardiovascular disease, and drug metabolism. Annu. Rev. Med. 54:371‐392.
   Davies, H.G., Richter, R.J., Keifer, M., Broomfield, C.A., Sowalla, J., and Furlong, C.E. 1996. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin. Nat. Genet. 14:334‐336.
   Furlong, C.E., Li, W.‐F., Richter, R.J., Shih, D.M., Lusis, A.J., Alleva, E., and Costa, L.G. 2000. Genetic and temporal determinants of pesticide sensitivity: Role of paraoxonase (PON1). Neurotoxicology 21:91‐100.
   Gan, K.N., Smolen, A., Eckerson, H.W., and LaDu, B.N. 1991. Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab. Dispos. 19:100‐106.
   Hassett, C., Richter, R.J., Humbert, R., Chapline, C., Crabb, J.W., Omiecinski, C.J., and Furlong, C.E. 1991. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence. Biochemistry 30:10141‐10149.
   Humbert, R., Adler, D.A., Disteche, C.M., Hassett, C., Omiecinski, C.J., and Furlong, C.E. 1993. The molecular basis of the human serum paraoxonase activity polymorphism. Nat. Genet. 3:73‐76.
   Jarvik, G.P., Rozek, L.S., Brophy, V.H., Hatsukami, T.S., Richter, R.J., Schellenberg, G.D., and Furlong, C.E. 2000. Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype. Arterioscler. Thromb. Vasc. Biol. 20:2441‐2447.
   Jarvik, G.P., Jampsa, R., Richter, R.J., Carlson, C.S., Rieder, M.J., Nickerson, D.A., and Furlong, C.E. 2003. Novel paraoxonase (PON1) nonsense and missense mutations predicted by functional genomic assay of PON1 status. Pharmacogenetics 13:291‐295.
   La Du, B.N. 2002. Historical considerations. In Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects (L.G. Costa, and C.E. Furlong, eds.) pp. 1‐25. Kluwer Academic Press, Boston.
   Li, W.F., Costa, L.G., Richter, R.J., Hagen, T., Shih, D.M., Tward, A., Lusis, A.J. and Furlong, C.E. 2000. Catalytic efficiency determines the in‐vivo efficacy of PON1 for detoxifying organophosphorus compounds. Pharmacogenetics 10:767‐779.
   Lusis, A.J. 2000. Atherosclerosis. Nature 407:233‐241.
   Mackness, M.I., Durrington, P.N., and Mackness, B. 2002. The role of paraoxonase in lipid metabolism. In Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects (L.G. Costa and C.E. Furlong, eds.) pp. 79‐88. Kluwer Academic Press, Boston.
   Mueller, R.F., Hornung, S., Furlong, C.E., Anderseon, J., Giblett, E.R., and Motulsky, A.G. 1983. Plasma paraoxonase polymorphism: A new enzyme assay, population, family, biochemical, and linkage studies. Am. J. Hum. Genet. 35:393‐408.
   Navab, M., Hama, S.Y., Wagner, A.C., Hough, G., Watson, A.D., Reddy, S.T., Van Lenten, B.J., Laks, H., and Fogelman, A.M. 2002. Protective action of HDL‐associated PON1 against LDL oxidation. In Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects (L.G. Costa and C.E. Furlong, eds.) pp. 125‐136. Kluwer Academic Press, Boston.
   Richter, R.J. and Furlong, C.E. 1999. Determination of paraoxonase (PON1) status requires more than genotyping. Pharmacogenetics 9:745‐753.
   Shih, D.M., Gu, L., Xia, Y.‐R., Navab, M., Li, W.‐F., Hama, S., Castellani, L.W., Furlong, C.E., Costa, L.G., Fogelman, A.M., and Lusis, A.J. 1998. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 394:284‐287.
   Shih, D.M., Reddy, S., and Lusis, A.J. 2002. CHD and atherosclerosis: Human epidemiological studies and transgenic mouse models. In Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects (L.G. Costa and C.E. Furlong, eds.) pp. 93‐123. Kluwer Academic Press, Boston.
   Watson, A.D., Berliner, J.A., Hama, S.Y., La Du, B.N., Faull, K.F., Fogelman, A.M., and Navab, M. 1995. Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein. J. Clin. Invest. 96:2882‐2891.
Internet Resource
   http://pga.gs.washington.edu
  The web site for the University of Washington–Fred Hutchinson Cancer Research Center Variation Discovery Resource (SeattleSNPs) includes information on the W194X coding‐region polymorphism.
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