Received for publication January 31, 2006.
Revised April 27, 2006.
Accepted for publication April 27, 2006.

New cytochrome P450-2D6*56 allele identified by genotype/phenotype analysis of cryopreserved human hepatocytes

Abstract

Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2;*3,*4,*5,*6,*7,*8,*9,*10,*11,*14,*15,*17,*18, *19,*20,*25,*26,*29,*30, *35,*40,*41,*43 and various multiple copy CYP2D6 alleles (*1xn, *2xn and *4xn) by the AmpliChip CYP450 prototype microarray. Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C>T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested, not just those with access to private liver banks.

Key words: CYP2D, cytochrome P450, cytochrome P450 function, genotype, hepatocytes, human CYP enzymes, human genetics, pharmacogenetics