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A-1607
2004 |
| False-Positive Non-Synonymous Single Nucleotide Polymorphism (SNP) of Human Opioid-Like Receptor (OP4) Gene |
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Piotr K. Janicki, M.D., Ph.D., Gregg Schuler, B.A., Victor Ruiz-Velasco, Ph.D. Anesthesiology, Penn State Hershey Medical Center, Hershey, Pennsylvania. |
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Introduction The G protein coupled receptor (GPCR) subfamily represents one of the largest families of genes in the human genome and the genetic variations and polymorphisms of GPCR can be associated with disease states. Some of these genetic variations have been ascribed to non-synonymous single nucleotide polymorphisms (SNP), which result in a change of amino acid sequence. The four major types of opioid receptors, OP1-OP4 which have been cloned, belong to the GPCR family. The most recently discovered opioid receptor, opioid like receptor 1 or OP4, has been implicated in a wide range of physiological as well as pathophysiological conditions relevant to anesthesia and pain management. Analysis of the recent entries into the NCBI SNPs database revealed that 45 different SNP candidates were associated with the human OP4 gene (OPRL1). However, only two of them are associated with the OPRL1 coding region. One of these SNPs (NCBI SNP cluster ID: rs6062627) should result in a non-synonymous change of the OP4 receptor coding sequence. The SNP (G873T) should result in an amino acid change from Gln to His in the OP4. The aim of this study was to determine the occurrence and frequency of the coding, non-synonymous OP4 receptor gene polymorphism, G873T, in humans. Methods With the IRB approval genomic DNA derived from randomly selected 100 adult patients undergoing anesthesia and surgery in the major academic hospital was used for the study. In addition, the additional DNA sample presumably containing the SNP G873T mutation was obtained from the Coriell Institute for Medical Research (Coriell DNA NA07340). The DNA samples were analyzed using the specific, custom-designed TaqMan real-time polymerase chain reaction and 5’ nuclease assay (Assay-on-Design, Applied Biosystems). PCR primers for both real-time PCR and verification sequencing assays were designed on the reference sequence using the Primer3 software. The primers used in the TaqMan assay consisted of forward 5’GCCTGTCCAGGTCTTCGT3’, reverse 5’AATGGCCACGGCAGTCT3’ oligonucleotides and fluorescent reporters VIC-CTCGGCTGAACCC (wild-type allele1) and FAM-CTCGGATGAACCC (mutated allele2). Results The putative non-synonymous SNP G873T was not observed in any of the analyzed clinical samples using the TaqMan assay. In addition the Coriell NA07340 DNAsample (previously described as containing the analyzed SNP mutation in the GenBank database) was also negative in the TaqMan assay. In order to ascertain that the lack of the detection was not due to the faulty assay, the direct double-strand sequencing of the Coriell sample DNA was performed using 5’GAGAGAAGGACCGGAACC3’ (forward) and 5’GCAGATGCACAGCAGAACT3’ (reverse) primers using a ABI Prism BigDye Terminator protocol run on an ABI3700 automatic sequencer. The resulted sequence of the 221 bases PCR product was compared with the reference complete coding sequence of the human OPRL1 (GenBank accession:AY268428) using the MACVector7 software (Accelrys). No presence of the putative G873T SNP was, once again, confirmed using this direct sequencing reaction. Conclusions Based on these results, we suggest that the variability of the OP4 gene is not well defined, and we caution against exclusive reliance on databases for selection of candidate OPRL1 polymorphisms for disease association and pharmacogenetic studies. (Study supported by NIH R01HL74311) Anesthesiology 2004; 101: A1607 |