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Supplement: 3rd Amino Acid Workshop

Pharmacogenetics and Individual Variation in the Range of Amino Acid Adequacy: The Biological Aspects¹

The University of Liverpool, Liverpool L69 3GA, UK

² To whom correspondence should be addressed. E-mail: jcc{at}liv.ac.uk.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
There have been major developments in our understanding of the ways in which genetic variation among individuals in a population can affect responses to drugs, and use of such information can improve the safety and efficacy of medicines. The brief review summarizes the emergence of modern pharmacogenetics, illustrating the importance of the field using the CYP 2D6 polymorphism as a paradigm. These pharmacogenetic polymorphisms are compared and contrasted with the long-established inborn errors of amino acid biochemistry, exemplified by phenylketonuria, suggesting ways in which the approaches of pharmacogenetics might inform the safe and effective use of amino acids as food additives and supplements.

KEY WORDS: • pharmacogenetics • CYP 2D6 • debrisoquine • adverse drug reactions • amino acidurias • phenylketonuria • inborn errors of amino acid biochemistry

Preamble

The greatest of 19^th century physicians, Sir William Osler, professor of medicine at Johns Hopkins Medical School in Baltimore, MD and later Regius professor of medicine at Oxford University, Oxford, UK, said in 1892, "If it were not for the great variability among individuals, medicine might as well be a science and not an art" (1).

Almost certainly the first demonstration that Osler's conundrum might be resolved came from Archibald Garrod (2), with his work on alkaptonuria and the demonstration of the individuality of biochemistry and the subsequent development of knowledge on a series of hereditable inborn errors of metabolism of endogenous compounds, most notably the amino acids (3). At the time, these were viewed as "metabolic sports" but it is now appreciated that, as well as being important in their own right, they provide a series of paradigms for a much wider range of disease etiology.

The first demonstration of marked individual differences in response to a drug was the association of malignant hyperthermia with general anesthesia in the early 1950s by Kalow (4). The term "pharmacogenetics" was originally defined in 1959 as "clinically important hereditary variation in response to drugs" by Vogel (5) and the discipline was established by Kalow's monograph "Pharmacogenetics" in 1962 (6). A small number of further examples accrued from the 1950s onward, generally involving a small number of related individuals showing aberrant responses to a number of specific agents (7). The field underwent explosive growth from the mid-1970s with the discovery of what is now recognized as the genetic polymorphism of the microsomal monooxygenase CYP 2D6 (8) with work on the adrenergic neurone blocker debrisoquine by Smith in London and on the oxytocic alkaloid sparteine by Eichelbaum in Bonn. This was the first genetic polymorphism whose frequency was such as to make it of relevance in the general population and also the first to affect a number of therapeutically significant drugs.

The huge growth in recent years of knowledge of the genome and the development of technologies that have made tractable the sequencing of genes and the expression of their products has revolutionized both these fields, the inborn errors of amino acid biochemistry and pharmacogenetics. It is the purpose of this brief review to summarize how pharmacogenetics has impacted upon therapeutic drug use and to examine to what extent these developments teach how to deal with variability in response to amino acids with particular reference to their use as food additives and supplements. It is common now to see two terms being used apparently interchangeably in discussions of this type, namely pharmacogenetics and pharmacogenomics. A consideration of working definitions (9,10), i.e., pharmacogenetics, the study of variability in drug response attributable to hereditary factors, and pharmacogenomics, the analysis of genomes and genomic expression as they relate to drug responses, provides a separation for present purposes. Although these two fields overlap, especially in terms of the technologies that they both employ, it is clearly pharmacogenetics that is the more relevant to this discussion.

This survey will first provide an indication of the importance of pharmacogenetics in general, illustrate this with the paradigm of CYP 2D6, and then consider what parallels might exist between the issues that have been informed by knowledge of this polymorphism and its implications and the issues of the use of certain amino acids as food additives and supplements in human populations.

The impact of pharmacogenetics on drug therapy

Differences in our genes can affect the way in which we respond to medicines in two ways, by determining variation in the way in which: i) the same symptoms can have the different origins (differentiating diseases), and ii) the body processes a medicine (an understanding of the ways in which people are differentiated).

Many diseases that appear as single clinical conditions have a number of underlying causes and an enhanced understanding of the genetic basis of such diseases will provide information about drugs that might be effective in the different classes of the disease. In a number of cases, notably cancers, diseased cells have different genotypes from those in normal tissue. This is seen in the case of breast cancer, where a particularly aggressive form of the disease in characterized by overexpression of the protein Her2 (11). High levels of Her2 expression are associated with a good response to the drug trastuzumab (Herceptin) but this agent is essentially ineffective against other breast tumors (12).

Genetic variation in the activities of enzymes and transporters responsible for the absorption, distribution, metabolism, and excretion of drugs, the traditional remit of pharmacogenetics, underlies many examples of interindividual differences in drug response. Rapid metabolism and clearance makes a drug ineffective whereas slow metabolism leads to excess levels and potential accumulation, often associated with adverse events and frank toxicity.

We may summarize the potential benefits of increased understanding of pharmacogenetic variation as follows (13,14):

1. Improving drug safety. If a specific genotypic variant is found to be associated with an adverse reaction, doctors could avoid prescribing the medicine to patients with this genotype,
   
2. Adjusting dosage. Knowledge of genotypes with specific pharmacokinetic characteristics could be used to adjust the dosage of affected drugs, reducing the trial-and-error approach of prescribers to determine the most effective dose,
   
3. Enhancing efficacy. Many widely used treatments for major diseases, such as diabetes, depression, and asthma, are effective in only half the population. Rather than using trial and error with all its attendant problems, pharmacogenetics may identify responsive and nonresponsive patients or offer new medicines designed on the basis of the genetics of the disease.
   

There are now a series of well-established polymorphisms of the drug-metabolizing enzymes (15,16): oxidation (CYP 2D6, CYP 2C19, and CYP 2A6), conjugation [N-acetyltransferase (NAT)³, UDPglucuronyltransferase (UGT), and thiopurine methyltransferase], and hydrolysis (pseudocholinesterase).

Of these, it is the CYP 2D6 polymorphism that is the most clearly understood in terms of both its genetic origins, biochemical mechanisms, and therapeutic consequences and it therefore provides a paradigm for a wide range of pharmacogenetic polymorphisms.

The CYP 2D6 polymorphism

Debrisoquine is an isoquinoline derivative with adrenergic neurone blocking activity, formerly used in the treatment of essential hypertension. It showed marked intersubject variability in clinical response, with a small number of patients showing excessive hypotension. Studies in volunteers revealed a very marked intersubject variability in the metabolism of the drug, which involves hydroxylation to form 4-hydroxydebrisoquine (Fig. 1).

View larger version (8K): [in this window] [in a new window]   FIGURE 1  The metabolism of debrisoquine by CYP 2D6 to 4-hydroxydebrisoquine.

View larger version (20K): [in this window] [in a new window]   FIGURE 2  Histogram of the "debrisoquine metabolic ratio" in a typical Caucasian population.

As the "debrisoquine polymorphism" was revealed by these studies in the mid-1970s, it became clear that it showed remarkable similarities to another polymorphism revealed by the oxytocic alkaloid sparteine. This is also metabolized by oxidation and both the incidence of poor metabolizers and the consequences of the polymorphism were uncannily similar to findings with debrisoquine. Crossover studies showed that they were indeed the same phenomenon, and the polymorphism is now known to affect the metabolism and therapeutic effect of >50 major drugs (14,16). These include: ß-blockers, e.g., propranolol and metoprolol; tricyclic antidepressants, e.g., amitriptyline and nortriptyline; codeine; dextromethorphan; guanoxan (an adrenergic neurone blocker similar to debrisoquine); encainide; flecainide; perhexiline; phenformin; phenacetin; and propafenone.

At the time of the discovery of the polymorphism, knowledge of the multiplicity of the cytochrome P450 family, the mixed function oxidases responsible for the oxidation of the great majority of drugs and other xenobiotics entering the animal body, was scant. These discoveries with debrisoquine and sparteine were one of the major driving forces behind the rapid growth in knowledge of the molecular biology and subsequently molecular genetics of this important enzyme family, now known to have >500 members in 70 families. Debrisoquine, sparteine, and the other affected drugs listed above are all substrates for CYP 2D6.

The consequences of the CYP 2D6 polymorphism for the PM phenotype vary with the drug affected and its individual characteristics. Three main groups may be identified (17).

1. Where impaired metabolism results in excessive exposure to unchanged drug, leading to adverse reactions: a) after single doses, e.g., the excessive hypotensive response to debrisoquine described above; b) upon accumulation after many doses, e.g., perhexiline, where the PM phenotype was associated with fatal hepatic damage; and c) in defined "at risk" groups, e.g., flecainide in those with impaired renal function.
   
2. Where impairment of a normally major pathway results in "metabolic switching" to normally minor metabolites mediating toxicity, e.g., phenacetin, where 2-hydroxyphenetidine, formed when CYP 2D6–mediated O-deethylation is impaired, is responsible for nephrotoxicity.
   
3. Where the affected pathway is responsible for the formation of pharmacologically active metabolites, such that the affected phenotype is relatively or absolutely unresponsive to the drug.
   

Although the population genetics of the CYP 2D6 polymorphism are relatively straightforward, its molecular genetics are more complex (16). More than 70 different CYP 2D6 alleles have been reported, which encode at least 37 different proteins. The great majority of these show either markedly reduced or no activity relative to the wild-type enzyme, but three alleles give proteins with enhanced activity. It is thus evident that numerous distinct genotypes give rise to the small number of phenotypes to be discerned in the population: there are at least seven different genotypes each representing >1% of a Caucasian population in the EM phenotype whereas the 7% of PMs are predominantly of two different genotypes. It has thus taken some time before genotyping has been able to predict phenotype with sufficient reliability for the purposes of guiding drug development or treatment.

Consequences of genetic polymorphisms for drug therapy

The CYP 2D6 polymorphism provides a paradigm to assess the overall significance of pharmacogenetic polymorphisms for drug development and use. It is now evident that this and the related polymorphisms of other enzymes, affecting other important drugs in comparable ways, are of major significance for affected individuals. We may thus identify a series of legacies of pharmacogenetic variability for these individuals and for affected drugs (13,14,17), which include: i) failure late in clinical development, e.g., the ß-blocker bufurolol; ii) iatrogenic disease (drug-associated morbidity and mortality), e.g., tricyclic antidepressants; iii) restrictions on drug use; and iv) drugs withdrawn from the market, e.g., perhexiline, phenformin, and phenacetin.

It is very important to view pharmacogenetics in a positive light, in that the understanding it brings to drug development and the wider availability of both genotyping and phenotyping offer the promise of safer, more efficacious, and more easily prescribed drugs to large populations. These benefits have been summed up as offering "the right drug at the right dose to the right patient." Although this may not be possible at present, it is now seen as more than the hyperbole it may have seemed some years ago and we stand at the beginning of the exploitation of this knowledge for widespread patient benefit.

The genetics of amino acid metabolism and transport

Having summarized the impact of pharmacogenetics on improving drug development and use, let us now turn to consider what this knowledge might teach us in considering the safe and effective use of amino acids as food supplements in large and diverse populations. A series of major inborn errors of biochemistry affecting amino acid metabolism and/or transport exist, knowledge of which built up over the last century starting with the seminal work of Garrod. These include: alkaptonuria, phenylketonuria, tyrosinaemia, Canavan disease, Hartnup disease, cystinuria, maple syrup urine disease, Isovaleric acidaemia, and congenital pyridoxine deficiency.

These disorders attracted a great deal of attention because they include a number of serious disorders, which are frequently fatal. It is clearly outside the scope of the present summary to cover these in detail, but further consideration will be given to one important disorder of metabolism: phenylketonuria (PKU).

Phenylketonuria

Phenylketonuria is a disorder characterized by the presence of abnormal levels of phenylalanine in the blood and urine. PKU is an inherited error of metabolism caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH), so that phenylalanine is not converted to tyrosine (Fig. 3).

View larger version (7K): [in this window] [in a new window]   FIGURE 3  The metabolism of phenylalanine to tyrosine by phenylalanine hydroxylase.

PKU was first reported in 1933 and was the fifth inborn error of metabolism to be documented. It has emerged subsequently as one of the most important inborn errors and provides a useful paradigm for a series of related disorders. The majority of cases are due to a deficiency in phenylalanine hydroxylase activity, but some are cofactor (tetrahydrobiopterin, BH₄) dependent (18). In recent years, >440 mutations in the PAH gene have been reported, half of which involve missense changes (19). A more detailed study of the most common alleles showed that these combine to give three distinct groups of genotypes (20). These variously lead to: i) inactive enzyme, with little or no protein expression, ii) moderate (10–70%) wild-type activity and protein expression, or iii) substantial (80–100%) enzyme activity and protein expression.

A detailed study of the genotype-to-phenotype associations of PKU (20) showed that of 7 genotypes of the first type, 5 were associated with classical PKU and 2 with moderate PKU. Of 7 genotypes of the second type, 1 had classical PKU, 1 had moderate PKU, and 5 had mild PKU. Of 5 genotypes of the third type, 2 had mild PKU and 3 had HPA.

Sorting out the genotype-to-phenotype puzzle

It is evident that the discovery of genetic variability in the enzymes or transporters determining the disposition of either an amino acid, a drug, or an exogenous chemical is no guarantee of the existence of a separate, somehow disfavored phenotype in the population. The discussions above have shown clearly that a series of steps are required to establish the phenotypic consequences of genotypic variation between individuals in a population, listed below: 1) high-quality phenotyping of subjects, with a full biochemical and clinical description of the case; 2) appropriate genetic analysis, concentrating on candidate genes likely to underlie the observed disorder; 3) isolation and cloning of the mutated gene; 4) transfection of the gene variants and expression of the proteins encoded by the gene; 5) functional analysis of the expressed proteins; and 6) correlation of functional results with clinical characteristics of phenotyped subjects.

Each of these steps is critical to the proper establishment of a link between genotype and phenotype.

Comparison of pharmacogenetic polymorphisms and amino acid defects

The population incidences of the two types of genetic polymorphisms are variable. The inborn errors of amino acid biochemistry are rare disorders, with incidences of one in several thousand subjects (21). Thus, cystinuria occurs at 1 in 7000 subjects, PKU in 1 in 10,000, Hartnup disease in 1 in 24,000, and maple syrup urine disease in from 1 in 30,000 to 1 in 90,000. In comparison, the major polymorphisms of drug metabolism are more common (15). CYP 2D6 poor metabolizers comprise 7% of Caucasian populations and 2% are CYP 2C19 poor metabolizers. Fifty percent of Caucasians are NAT2 slow acetylators, whereas for the bilirubin UGT variants, 2% have Gilbert's syndrome but only 1 in 50,000 have the very severe and generally fatal Crigler-Najjar syndrome.

The discovery of a number of genetic polymorphisms of drug metabolism in recent years is due to the development of new technologies but also to the large numbers of new chemicals introduced into the body. The range of chemical diversity to which the population is exposed has grown substantially. In comparison, the number of amino acids in the diet is a constant, although they may be presented in new ways, in novel combinations, and at different levels. The exposure to amino acids from their incorporation into food supplements is generally small relative to their consumption in a diet containing adequate amounts of protein. It thus seems unlikely that new polymorphisms of amino acid biochemistry of population significance remain to be discovered, even in response to new challenges.

The defects in amino acid biochemistry are highly specific to particular amino acids, notably the essential amino acids, which must be taken in from the diet. In comparison, the polymorphisms of the cytochrome P450 and other drug metabolizing enzymes affect many drugs. In nearly every case, these defects may be readily detected by a study of the enzymes themselves. However, the errors of amino acid metabolism frequently have an impact on complex biochemical pathways sometimes some distance removed from the amino acid itself.

The range of variability encountered in the population for the two types of polymorphisms is similar, with 50–500-fold variability in the amounts of active enzyme protein between the different phenotypes. The incidence of the amino acid defects is much lower in the general population and their sequelae of amino acid defects are often more severe and harder to control.

A comment is also appropriate on the existence in populations of individuals heterozygous for affected alleles. For the polymorphisms of drug metabolism, these heterozygotes may handle normal doses of a drug well but become at risk at higher doses. They may well also be more liable to drug-drug interactions as a consequence of their limited metabolic capacity. In comparison, there is little knowledge of the consequences of such heterozygosity with respect to amino acid metabolism or transport. The existence of heterozygous unaffected genetic carriers is well known and it can be surmised that they may be at risk from the higher than normal consumption of the relevant amino acid, e.g., in a food supplement.

Concluding comments

The best examples of the genetic basis of human disease are monogenic disorders, where a single mutation in a single gene is causatively related to the phenotype. These are typified by a number of inborn errors of amino acid biochemistry, such as phenylketonuria discussed here. This is also the case in pharmacogenetics for those polymorphisms of drug metabolism that are a major determinant of the pharmacokinetic profile of affected drugs. The CYP 2D6 polymorphism (which is inherited as an autosomal recessive trait) provides a paradigm here, because it influences the metabolism of a wide variety of drugs, including important cardiovascular and psychotropic agents.

However, the majority of genetic predispositions to disease and pharmacogenetic traits are polygenic and multifactorial. Polygenic traits are influenced by a number of different contributing genes, which may be additive or interactive in their effects. Genetic influences are often overlaid by environmental factors so that the overall phenotype is truly multifactorial and highly variable both between individuals and within individuals across time. The growth of genetic knowledge has the potential to provide important knowledge relevant to the safety-in-use of many drugs, food additives, and other chemicals to which humans are deliberately, coincidently, or accidentally exposed.

Pharmacogenetics offers the prospect of the individualization of drug treatment and promises to provide a rational basis for prescribing drug treatment based on a specific patient's genetic profile. In recent years, these approaches broadened to include novel genomic approaches to issues in chemical safety, with the emergence of toxicogenetics and toxicogenomics. The interaction of genetic variation with dietary factors leads now to the new field of nutritional genetics (nutrigenomics), which relates the role of genetics to nutritional requirements and nutrition-mediated susceptibility to chronic disease, with, it is suggested, comparable benefits.

	FOOTNOTES

 
¹ Presented at the conference "The Third Workshop on the Assessment of Adequate Intake of Dietary Amino Acids" held October 23–24, 2003 in Nice, France. The conference was sponsored by the International Council on Amino Acid Science. The Workshop Organizing Committee included Vernon R. Young, Yuzo Hayashi, Luc Cynober, and Motoni Kadowaki. Conference proceedings were published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Vernon R. Young, Dennis M. Bier, Luc Cynober, Yuzo Hayashi, and Motoni Kadowaki.

³ Abbreviations used: EM, extensive metabolizers; HPA, hyperphenylalaninaemia; NAT, N-acetyltransferase; PAH, phenylalanine hydroxylase; PKU, phenylketonuria; PM, poor metaboloizers; UGT, UDPglucuronyltransferase.

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