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PHARMACOGENETICS - Mary V. Relling and Kathleen M. Giacomini
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PHARMACOGENETICS: INTRODUCTION

Introduction

Pharmacogenetics is the study of the genetic basis for variation in drug response. In this broadest sense, pharmacogenetics encompasses pharmacogenomics, which employs tools for surveying the entire genome to assess multigenic determinants of drug response. Until the technical advances in genomics of the last few years, pharmacogenetics proceeded using a forward genetic, phenotype-to-genotype approach. Drug response outliers were compared to individuals with "normal" drug response to identify the pharmacologic basis of altered response. An inherited component to response was demonstrated using family studies or imputed through intra- vs. intersubject reproducibility studies. With the explosion of technology in genomics, a reverse genetic, genotype-to-phenotype approach is feasible whereby genomic polymorphisms can serve as the starting point to assess whether genomic variability translates into phenotypic variability.

Historical Context. In the pre-genomics era, the frequency of genetic variation was hypothesized to be relatively uncommon, and the demonstration of inherited drug-response traits applied to a relatively small number of drugs and pathways (Eichelbaum and Gross, 1990; Evans and Relling, 2004; Johnson and Lima, 2003). Historically, uncommon severe drug-induced phenotypes served as the triggers to investigate and document pharmacogenetic phenotypes. Prolonged neuromuscular blockade following normal doses of succinylcholine, neurotoxicity following isoniazid therapy (Hughes et al., 1954), and methemoglobinemia in glucose-6-phosphate dehydrogenase (G6PD) deficiency (Alving et al., 1956) (see Chapter 39) were discovered to have a genetic basis in the first half of the 20th century. In the 1970s and 1980s, debrisoquine hydroxylation and exaggerated hypotensive effects from that drug were related to an autosomal recessive inherited deficiency in the cytochrome P450 isoenzyme 2D6 (CYP2D6) (Evans and Relling, 2004). Since the elucidation of the molecular basis of the phenotypic polymorphism in CYP2D6 (Gonzalez et al., 1988), the molecular bases of many other monogenic pharmacogenetic traits have been identified (Meyer and Zanger, 1997).

Individuals differ from each other approximately every 300 to 1000 nucleotides, with an estimated total of 3.2 million single nucleotide polymorphisms (SNPs; single base pair substitutions found at frequencies ³1% in a population) in the genome (Sachidanandam et al., 2001; The International SNP Map Working Group, 2001). Identifying which of these variants or combinations of variants have functional consequence for drug effects is the task of modern pharmacogenetics.

Importance of Pharmacogenetics to Variability in Drug Response

Drug response is considered to be a gene-by-environment phenotype. That is, an individual's response to a drug depends on the complex interplay between environmental factors and genetic factors (Figure 4-1). Variation in drug response therefore may be explained by variation in environmental and genetic factors, alone or in combination. What proportion of drug-response variability is likely to be genetically determined? Classical family studies provide some information (Weinshilboum and Wang, 2004). Because estimating the fraction of phenotypic variability that is attributable to genetic factors in pharmacogenetics usually requires administration of a drug to twins or trios of family members, data are somewhat limited. Twin studies have shown that drug metabolism is highly heritable, with genetic factors accounting for most of the variation in metabolic rates for many drugs (Vesell, 2000). Results from a twin study in which the half-life of antipyrine was measured are typical (Figure 4-2). Antipyrine, a pyrazolone analgesic, is eliminated exclusively by metabolism and is a substrate for multiple CYPs. There is considerably greater concordance in the half-life of antipyrine between the monozygotic (identical) twin pairs in comparison to the dizygotic (fraternal) twin pairs. Comparison of intra-twin vs. inter-pair variability suggests that approximately 75% to 85% of the variability in pharmacokinetic half-lives for drugs that are eliminated by metabolism is heritable (Penno et al., 1981). It has also been proposed that heritability can be estimated by comparing intra-subject vs. inter-subject variability in drug response or disposition in unrelated individuals (Kalow et al., 1998), with the assumption that high intra-subject reproducibility translates into high heritability; the validity of this method across pharmacologic phenotypes remains to be established. In any case, such studies provide only an estimate of the overall contribution of inheritance to the phenotype; because multiple gene products contribute to antipyrine disposition, most of which have unelucidated mechanisms of genetic variability, the predictability of antipyrine disposition based on known genetic variability is poor.

Another approach to estimating the degree of heritability of a pharmacogenetic phenotype uses ex vivo experiments with cell lines derived from related individuals. Inter- vs. intrafamily variability and relationships among members of a kindred are used to estimate heritability. Using this approach with lymphoblastoid cells, cytotoxicity from chemotherapeutic agents was shown to be heritable, with about 20% to 70% of the variability in sensitivity to 5-fluorouracil and docetaxel estimated as inherited, depending upon dose (Watters et al., 2004).

For the "monogenic" phenotypic traits of G6PD deficiency, CYP2D6 or thiopurine methyltransferase (TPMT) metabolism, it is possible to predict phenotype based on genotype. Several genetic polymorphisms of drug metabolizing enzymes result in monogenic traits. Based on a retrospective study, 49% of adverse drug reactions were associated with drugs that are substrates for polymorphic drug metabolizing enzymes, a proportion larger than estimated for all drugs (22%) or for top-selling drugs (7%) (Phillips et al., 2001). Prospective genotype determinations may result in the ability to prevent adverse drug reactions (Meyer, 2000).

Defining multigenic contributors to drug response will be much more challenging. For some multigenic phenotypes, such as response to antihypertensives, the large numbers of candidate genes will necessitate a large patient sample size to produce the statistical power required to solve the "multigene" problem.

GENOMIC BASIS OF PHARMACOGENETICS

Phenotype-Driven Terminology

Because initial discoveries in pharmacogenetics were driven by variable phenotypes and defined by family and twin studies, the classic genetic terms for monogenic traits apply to some pharmacogenetic polymorphisms. A trait (e.g., CYP2D6 "poor metabolism") is deemed autosomal recessive if the responsible gene is located on an autosome (i.e., it is not sex-linked) and a distinct phenotype is evident only with nonfunctional alleles on both the maternal and paternal chromosomes. For many of the earliest identified pharmacogenetic polymorphisms, phenotype did not differ enough between heterozygotes and homozygous "wild-type" individuals to distinguish that heterozygotes exhibited an intermediate (or codominant) phenotype (e.g., for CYP2D6-mediated debrisoquine metabolism). Other traits, such as TPMT, exhibit three relatively distinct phenotypes, and thus were deemed codominant even in the premolecular era. With the advances in molecular characterization of polymorphisms and a genotype-to-phenotype approach, additional polymorphic traits (e.g., CYP2C19 metabolism of drugs such as mephenytoin and omeprazole) are now recognized to exhibit some degree of codominance. Some pharmacogenetic traits, such as the long QT syndrome, segregate as dominant traits; the long QT syndrome is associated with heterozygous loss-of-function mutations of ion channels. A prolonged QT interval is seen on the electrocardiogram, either basally or in the presence of certain drugs, and the individual is predisposed to cardiac arrhythmias (see Chapter 34).

In an era of detailed molecular characterization, two major factors complicate the historical designation of recessive, codominant, and dominant traits. First, even within a single gene, a vast array of polymorphisms (promoter, coding, noncoding, completely inactivating, or modestly modifying) are possible, making the assignment of "variant"vs. "wild-type" to an allele a designation that depends upon a complete survey of the gene's polymorphisms and is not necessarily easily assigned. Secondly, most traits (pharmacogenetic and otherwise) are multigenic, not monogenic. Thus, even if the designations of recessive, codominant, and dominant are informative for a given gene, their utility in describing the genetic variability that underlies variability in drug response phenotype is diminished, because most phenotypic variability is likely to be multigenic.

Types of Genetic Variants

A polymorphism is a variation in the DNA sequence that is present at an allele frequency of 1% or greater in a population. Two major types of sequence variation have been associated with variation in human phenotype: single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) (Figure 4-3). In comparison to base pair substitutions, indels are much less frequent in the genome and are of particularly low frequency in coding regions of genes (Cargill et al., 1999; Stephens et al., 2001). Single base pair substitutions that are present at frequencies of 1% or greater in a population are termed single nucleotide polymorphisms (SNPs) and are present in the human genome at approximately 1 SNP every few hundred to a thousand base pairs, depending on the gene region (Stephens et al., 2001).

SNPs in the coding region are termed cSNPs. cSNPs are further classified as nonsynonymous (or missense) if the base pair change results in an amino acid substitution, or synonymous (or sense) if the base pair substitution within a codon does not alter the encoded amino acid. Typically, substitutions of the third base pair, termed the wobble position, in a three base pair codon, such as the G to A substitution in proline shown in Figure 4-3, do not alter the encoded amino acid. Base pair substitutions that lead to a stop codon are termed nonsense mutations. In addition, about 10% of SNPs can have more than two possible alleles (e.g., a C can be replaced by either an A or G), so that the same polymorphic site can be associated with amino acid substitutions in some alleles but not others.

Polymorphisms in noncoding regions of genes may occur in the 3' and 5' untranslated regions, in promoter or enhancer regions, in intronic regions, or in large regions between genes, intergenic regions (Figure 4-4). Polymorphisms in introns found near exon-intron boundaries are often treated as a separate category from other intronic polymorphisms since these may affect splicing, and thereby affect function. Noncoding SNPs in promoters or enhancers may alter cis- or trans-acting elements that regulate gene transcription or transcript stability. Noncoding SNPs in introns or exons may create alternative exon splicing sites, and the altered transcript may have fewer or more exons, or shorter or larger exons, than the wild-type transcript. Introduction or deletion of exonic sequence can cause a frame shift in the translated protein and thereby change protein structure or function, or result in an early stop codon, which makes an unstable or nonfunctional protein. Because 95% of the genome is intergenic, most polymorphisms are unlikely to directly affect the encoded transcript or protein. However, intergenic polymorphisms may have biological consequences by affecting DNA tertiary structure, interaction with chromatin and topoisomerases, or DNA replication. Thus, intergenic polymorphisms cannot be assumed to be without pharmacogenetic importance.

A remarkable degree of diversity in the types of insertions/deletions that are tolerated as germline polymorphisms is evident. A common glutathione-S-transferase M1 (GSTM1) polymorphism is caused by a 50-kilobase (kb) germline deletion, and the null allele has a population frequency of 0.3 to 0.5, depending on race/ethnicity. Biochemical studies indicate that livers from homozygous null individuals have only ~50% of the glutathione conjugating capacity of those with at least one copy of the GSTM1 gene (Townsend and Tew, 2003a). The number of TA repeats in the UGT1A1 promoter affects the quantitative expression of this crucial glucuronosyl transferase in liver; although 4 to 9 TA repeats exist in germline-inherited alleles, 6 or 7 repeats constitute the most common alleles (Monaghan et al., 1996). Cystathionine b-synthase has a common 68 base pair insertion/deletion polymorphism that has been linked to folate levels (Kraus et al., 1998). Although in many of these cases the local sequence context of these insertions/deletions strongly suggests mechanisms underlying the genomic alterations (e.g., homologous recombination sites bracket the GSTM1 deletion), high allele frequencies are maintained due to Mendelian inheritance.

A haplotype, which is defined as a series of alleles found at a linked locus on a chromosome, specifies the DNA sequence variation in a gene or a gene region on one chromosome. For example, consider two SNPs in ABCB1, which encodes for the multidrug resistance protein, P-glycoprotein. One SNP is a T to A base pair substitution at position 3421 and the other is a C to T change at position 3435. Possible haplotypes would be T3421C3435, T3421T3435, A3421C3435, and A3421T3435. For any gene, individuals will have two haplotypes, one maternal and one paternal in origin, which may or may not be identical. Haplotypes are important because they are the functional unit of the gene. That is, a haplotype represents the constellation of variants that occur together for the gene on each chromosome. In some cases, this constellation of variants, rather than the individual variant or allele, may be functionally important. In others, however, a single mutation may be functionally important regardless of other linked variants within the haplotype(s).

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