SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

P60
PHARMACOGENOMICS AS MOLECULAR AUTOPSY: GENOTYPING P450 2D6, 2C9 AND
2Cl9 USING PYROSEQUENCING FOR METHADONE CASES
B. Charles Schur 2, Elvan Laleli-Sahin 1.2, Steven H. Wong 1.2, Susan B. Gock 1.2, Paul J. Jannetto I, Jeffrey
M. Jentzen 1.2
IMilwauke County Medical Examiners Office; 2Department Of Pathology, Medical College Of Wi,
Milwaukee, Wi, U.S.A.
Pharmacogenomics, the study of the impact of heritable traits on pharmacology and toxicology, has the
potential to explain the relationship between drug administration, drug metabolism and adverse drug
reactions. Genetically, single nucleotide polymorph isms (SNP's) are single base-pair changes in the DNA
sequence of a gene. Base deletions or substitutions may result in a change in the amino acid sequence of
the polypeptide. These heritable mutations have the ability to change the structure and specificity of the
enzyme to the extent that it may not metabolize the drug in question. Depending on the variant alleles
present, an individuals' metabolic rate may be phenotypically described as poor (PM), intermediate (1M),
extensive (EM, normal), or ultra-extensive (rapid). PM's have a high risk of adverse effects which can
potentially be fatal, while 1M's have a metabolic rate between PM's and EM's.
In Forensic Pathologyrroxicology, molecular techniques can determine the genotype of a decedent and
may aide in the determination of the cause and manner of drug related deaths. Methadone, for example, is
metabolized in the liver by cytochrome P450 (CYP) lA2, 3A4. 2D6 and, to a certain extent, 2C9 and 2CJ9
enzymes. All of the genes encoding these enzymes are polymorphic. In this study we establish the
technical feasibility of genotyping for known mutations in CYP2D6 (*3, *4, *5, *6, *7 and *8) as well as
CYP2C9 (*2, *3) and CYP2C19 (*2, *3, *4) using Pyrosequencing.
Twelve frozen, archived, blood samples from the Milwaukee County Medical Examiners Office
(MCMEO), were selected as part of a multi-center retrospective analysis of cases from June 2002 to
December 2002, which was approved by Medical College of Wisconsin IRE. These cases listed
methadone as a contributing factor in the cause of death. After DNA extraction, PCR was performed and
amplified product was then interrogated for the presence of specific SNP's using a Pyrosequencing
PSQ96MA. Briefly, a sequencing primer is hybridized to a single stranded PCR product, and incubated
with enzymes, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate (APS)
and luciferin. A deoxynucleotide triphosphate (dNTP) is added to the reaction. If incorporated, a release
of pyrophosphate occurs that is equimolar to the number of incorporated nucleotides. ATP sulfurylase
quantitatively converts PPi to ATP in the presence of adenosine 5' phosphosulfate. This ATP converts
luciferin to oxiuciferin and generates visible light detectable by a CCD camera and evident by a peak in
the Pyrogram M. Each light signal is proportional to the number of nucleotides incorporated. Apyrase
degrades the unincorporated dNTP's and excess A TP prior to the addition of another dNTP. Software
correlates each Pyrogram to a reference sequence and genotype determinations are made. This platform
has been previously validated for clinical samples, with results showing ] 00% concordance with other
platforms including conventional PCR with RFLP analysis, direct sequencing using an ABI 3100, and
rapid-cycle PCR with fluorescent melting curve analysis using a Roche LightCycier.
Genotyping results for CYP2D6 indicated 8 cases (66.6%) of EM's, 3 cases (25%) of 1M's and ] case
(8.3%) of a PM. For CYP2C19 SNP's, 8 samples (66.7%) were found to be EM's and 4 samples (33.3%)
were found to be 1M's. No mutations (0%) were detected for CYP2C9 in any sample. These percentages
are comparable to the expected prevalence ranges found in the general population. In this preliminary
study, we demonstrate the feasibility of genotyping forensic samples for multiple SNP's associated with
methadone metabolism. These protocols will be used in a large (n > 2000) multi-centers methadone study.
Keywords: Pharmacogenomics, Methadone, Pyrosequencing
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