Pharmacogenetic Study Design Considerations Pharmacogenetic Measures

A pharmacogenetic trait is any measurable or discernible trait associated with a drug, including enzyme activity, drug or metabolite levels in plasma or urine, effects on blood pressure or lipid levels, and drug-induced gene expression patterns. Directly measuring a trait (e.g., enzyme activity) has the advantage that the net effect of the contributions of all genes that influence the trait is reflected in the phenotypic measure but the disadvantage is that it also reflects nongenetic influences (e.g., diet, drug interactions, diurnal, or hormonal fluctuation) and thus, may be "unstable." For example, if a patient is given an oral dose of dextromethorphan, and the urinary ratio of parent drug to metabolite is assessed, the phenotype reflects the CYP2D6 genotype. If dextromethorphan instead is given with quinidine, a potent CYP2D6 inhibitor, the ratio may indicate a poor metabolizer genotype even though the subject carries wild-type CYP2D6 alleles. In other words, quinidine coadministration may result in a drug-induced enzymatic deficiency, and the false assignment to a CYP2D6 poor metabolizer phenotype. Lack of consistency for a given subject in a phenotypic measure, such as the erythromycin breath test for CYP3A, indicates that the phenotype is influenced by nongenetic factors and may indicate a multigenic or weakly penetrant effect of a monogenic trait. Because most pharmacogenetic traits are multigenic rather than monogenic (Figure 4-4), considerable effort is being made to identify the important genes and their polymorphisms that influence variability in drug response.

Most genotyping methods use genomic DNA that is extracted from somatic, diploid cells, usually white blood cells or buccal cells due to their ready accessibility. DNA is extremely stable if appropriately extracted and stored; unlike many laboratory tests, genotyping need be performed only once because DNA sequence is generally constant throughout an individual's lifetime. Although tremendous progress has been made in molecular biological techniques to determine genotypes, relatively few pharmacogenetic tests are used routinely in patient care. Genotyping tests are directed at each specific known polymorphic site using a variety of strategies that generally depend at some level on the specific and avid annealing of at least one oligonucleotide to a region of DNA flanking or overlapping the polymorphic site. Because genomic variability is so common (with polymorphic sites every few hundred nucleotides), "cryptic" or unrecognized polymorphisms may interfere with oligonucleotide annealing, thereby resulting in false-positive or false-negative genotype assignments. Full integration of genotyping into therapeutics will require high standards for genotyping technology, perhaps with more than one method required for each polymorphic site.

Monogenic trait

Multigenic trait

1 a —O— low activity 1 b —■—high activity

Possible Alleles

Enzyme activity

Genotypes

Trait histogram

Thrombosis risk

Enzyme activity

Thrombosis risk

FIGURE 4-4 Monogenic versus multigenic pharmacogenetic tratis. Possible alleles for a monogenic trait (upper left), in which a single gene has a low-activity (1a) and a high-activity (1b) allele. The population frequency distribution of a monogenic trait (bottom left), here depicted as enzyme activity, may exhibit a trimodal frequency distribution with relatively distinct separation among low activity (homozygous for 1a), intermediate activity (heterozygous for 1a and 1b), and high activity (homozygous for 1b). This is contrasted with multigenic traits (e.g., an activity influenced by up to four different genes, genes 2 through 5), each of which has 2, 3, or 4 possible alleles (a through d). The population histogram for activity is unimodal-skewed, with no distinct differences among the genotypic groups. Multiple combinations of alleles coding for low activity and high activity at several of the genes can translate into low-, medium-, and high-activity phenotypes.

Because polymorphisms are so common, the allelic structure that indicates whether polymorphisms within a gene are on the same or different alleles (their haplotype) may also be important. Experimental methods to unambiguously confirm whether polymorphisms are allelic are technically challenging so statistical probability is often used to assign putative or inferred haplotypes.

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