Molecular Autopsy with Pharmacogenomics - A Multi-Center Study for Certifying Methadone Deaths: 2006 Update on Data Acquisition and Multiplex Genotyping CYP 450 2D6, 2C9, 2C19, 3A4 and 3A5 by Pyrosequencing(TM)

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S. H. Wong1, B. C. Schur2, J. M. Jentzen3, S. Gock3, P. Jannetto2, R. Z. Shi1, R. J. Schneider1, R. Winecker4, B. Logan5, B. Hepler6, L. Langman7, M. Lehrer8, C. V. Wetli8, A. Jenkins9, M. Wagner10, et al. 1 Medical College of Wisconsin and Milwaukee County Medical Examiner's Office, Milwaukee, WI, 2 Medical College of Wisconsin, Milwaukee, WI, 3 Milwaukee County Medical Examiner's Office and Medical College of Wisconsin, Milwaukee, WI, 4 Office of the Chief Medical Examiner of North Carolina, Chapel Hill, NC, 5 Forensic Lab Services Bureau - Washington State Patrol, Seattle, WA, 6 Wayne County Medical Examiner's Office, Detroit, MI, 7 Mayo Clinic, Rochester, MN, 8 Suffolk County Medical Examiner's Office, Hauppauge, NY, 9 Cuyahoga County Coroner Office, Cleveland, OH, 10 Department of Safety of New Hamsphire, Concord, NH,

Methadone has been used in the treatment of heroin addiction and for pain management. In selected regions of the United States, methadone intoxication due to abuse and diversion has recently increased. This is partially influenced by the concerted effort by governmental agencies to lower oxycodone diversion and abuse, resulting in the alternate use abuse of methadone. Further, methadone is also used for the treatment of opioids addiction such as oxycodone. The multi-pathways metabolism of methadone is partially mediated by CYP 2B6, 2C9, 2C19, 2D6, and 3A4/5 and are encoded by their respective polymorphic genes. This multi-center study was organized to enroll an adequate number of methadone cases to evaluate the potential contribution of genetic variation to methadone related deaths Pharmacogenomics as an aspect of Molecular Autopsy. Personnel from eleven medical examiner coroner offices and several academic departments formed the Forensic Pathology Toxicology Pharmacogenomics Methadone Study Group (FPTPMSG) and received IRB approval. Two other academic centers would provide additional data analysis and case review. In 2005, one site withdrew while another added, maintaining the total number of participating offices sites to be 11. Inclusion criteria were: cases from 2002 to 2003, certified as methadone toxicity or as methadone related, and methadone and for its metabolites present and quantified. The MS-ACCESS database was redesigned in 2005. Critical design changes included restructuring and securing forms and data, and adding a data merging utility. This final version of the database was released July 2005. Of the 11 copies distributed, 3 have been completed and returned to the core laboratory where case history and toxicology results will be combined with genotyping data to form a master database for detailed statistical analysis. Of the 1100 samples expected, all but 50 have been received without incident and are stored in the core laboratory. Approximately 200 of these samples have been processed and genotyped for CYP 2D6, 2C9, 2C19, and 3A4/5 by Pyroscquencing(TM) With the current analysis of some of the samples, the following prevalence were established: 2D6 (n=187): *3, 0.8%., *4, 15.2%., *5, 5.1%., *6, 1.1%., *7 and *8, 0%., 2C9 (n=135); *2, 9.3% and *3, 5.6%., 2C19 (n=152); *2, 14.8%, *3 and *4, 0%., 3A4* 1B (n=184): 6.0%., and 3A5*3 (n=184): 90.2%. For the 2006 update, the study demonstrated: the feasibility of coordinated planning of the eleven sites and two academic centers., sample collection without incident., the need for modification of the database tor the initial data entry and transfer to the core pharmacogcnomics laboratory via Internet by several centers., and the feasibility of routine multiplex genotyping reliably performed on previously collected postmortem whole blood samples.