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Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting

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Abstract

Paraoxonase-1 (PON1) and butyrylcholinesterase (BCHE) are natural bioscavengers of organophosphate acetylcholinesterase inhibitors in the human body, which can determine individual sensitivity to organophosphate toxicity. Interindividual differences in activity of PON1 (catalytic bioscavenger) and substrate specificity are strongly associated with the substitution of two amino acids: Leu/Met (L/M) at position 55 (rs854560) and Gln/Arg (Q/R) at position 192 (rs662). In the case of BCHE (stoichiometric bioscavenger) substitution, Ala/Thr (A/T) at position 539 produces the so-called “K-variant” of the enzyme (rs1803274). Threonine allele is often co-inherited with an atypical BCHE allele (rs1799807). The atypical variant of BCHE displays a lower affinity for cholinesterase inhibitors. Genotyping rs662 and rs1803274 single-nucleotide polymorphisms (SNP) by high-resolution melting (HRM) is facilitated by the nucleotide substitution A>G (G>A), which resulted in a changed number of hydrogen bonds in the PCR product and, consequently, shifted T m. In the case of rs854560, genotyping is complicated by the nucleotide substitution T>A, which has no significant effect on the T m of the PCR product. An addition of a small quantity of LL homozygote DNA into the reaction mixture before PCR discriminates the three genotypes by the melt curves due to different amounts of heteroduplexes formed in the LM and MM samples. HRM analysis can be applied for genotyping human rs854560, rs662, and rs1803274 SNPs.

Difference curve pattern of amplicons containing SNP rs1803274

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Acknowledgments

We thank Dr. N.S. Khlebnikova for critically reading the manuscript. This work was supported by the Russian Federal Medical Biological Agency (contract nos. 25.442.12.0 and 25.441.13.0).

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Authors and Affiliations

  1. Research Institute of Hygiene, Occupational Pathology and Human Ecology (RIHOPHE), Russian Federal Medical Biological Agency, g/p Kuzmolovsky, St. Petersburg, Russia, 188663

    Ivan Kurdyukov, Andrey Radilov & Vladimir Babakov

  2. Nikiforov Russian Center of Emergency and Radiation Medicine (NRCERM, EMERCOM of Russia), Acad. Lebedev St., 4/2, St. Petersburg, Russia, 194044

    Gennady Rodionov

Authors
  1. Ivan Kurdyukov
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  2. Gennady Rodionov
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  3. Andrey Radilov
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  4. Vladimir Babakov
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Correspondence to Ivan Kurdyukov.

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Published in the topical collection Analysis of Chemicals Relevant to the Chemical Weapons Convention with guest editors Marc-Michael Blum and R. V. S. Murty Mamidanna.

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Kurdyukov, I., Rodionov, G., Radilov, A. et al. Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting. Anal Bioanal Chem 406, 5087–5092 (2014). https://doi.org/10.1007/s00216-014-7734-7

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  • DOI: https://doi.org/10.1007/s00216-014-7734-7

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