Pharmacogenetics: from history to today

Pharmacogenetics, the study of hereditary variations in drug response, is a term that first appeared in the anesthesia literature in the mid-1950s. Historically most relationships involving pharmacogenetics were associated with pre-existing disease states. The first genetic variants associated with variable, sometimes fatal response to anesthesia included cholinesterase variants. Conditions such as Malignant Hyperthermia were subsequently observed when a small set of otherwise healthy people were exposed to certain inhaled anesthetics. More recently, with the rapid pace of discoveries in genetics and the availability of the complete human genome, variant alleles have become the focus of pharmacogenetics research. These variants often occur in enzymes that are not essential to development or normal metabolism and not required for vital functions, therefore, deficiencies are unnoticed until a medication proves ineffective or even harmful at standard dosing. The best-known examples of these types of enzymes are members of the cytochrome P450 monooxygenase (CYP) gene family. By predicting metabolizing phenotype in advance, when drugs which are substrates for the variant CYP enzymes are medically indicated, the most effective and safest dose can be started from the beginning. This could save time, money and perhaps most importantly reduce adverse drug reactions.

Pdseudocholinesterase deficiency

In 1 in 1500 persons, plasma pseudocholinesterase (butyrylcholinesterase, BChE) deficiency decreases succinylcholine inactiviation. In these people, the plasma cholinesterase has a 1/100th affinity for succinylcholine, and a standard does of succinylcholine causes prolonged paralysis, hours vs. minutes, which may require mechanical ventilation (1). There are over 20 variants known in the BChe encoding gene (BCHE, 2q26.1-q26.3). The two most common variants are the A-variant in exon 2 (A209G, Asp70Gly) and the K-variant in exon 4 (G1615A, Ala539Thr), with allele frequencies of 0.02 and 0.128-0.21 respectively. AA homozygous individuals have the greatly decreased activity described above. Wild-type patients take 30 minutes to recover from mivacurium, homozygous A-variant patient show a 6-8 hour recovery time, and heterozygous patient recovery time is increased by 50%. In a study by Gätke (2) the A-variant was found in 45% of patients referred to the cholinesterase research unit for evaluation of prolonged postoperative respiratory insufficiency. The heterozygous genotype is found in 4% of Northern Europeans (3) and the homozygous variant in 1/2000-2/3000 (4,5). In most cases the A-variant has been found together with the K-variant, suggesting that these mutations along with two others (at amino acid -116 and 914) are in linkage disequilibrium (6). Phenotypic consequences of the K-variant are not well defined due to the previous difficulty of typing someone with phenotypic testing alone (7). Pharmacogenetic testing for both the A- and K-variant is available. More studies are needed to correlated BCHE-genotyping with prediction of prolonged blockade. However, based on prevalence data BCHE-genotyping could prevent prolonged recovery in >4% of patients.

Malignant hyperthermia susceptibility (MHS)

Malignant hyperthermia susceptibility (MHS) is a life-threatening elevation in body temperature resulting from a hypermetabolic response to a combination of drugs. Usually the drugs are a depolarizing muscle relaxant such as succinylcholine and a potent, volatile inhaled general anesthetic such as halothane, isoflurane or sevoflurane. An MHS crisis can cause generalised skeletal muscle rigidity, very high body temperature, acidosis, hypoxia and rhabdomyolysis (8). This reaction, seen in 1 in 20,000 seemingly normal individuals, can also be seen in patients with muscular dystrophy and myotonia. The consequences of malignant hyperthermia can be grave, with a 7% mortality rate in developed countries. Malignant hyperthermia is thought to be caused by a variant in the sarcoplasmic reticulum (SR) calcium release channel, also known as the ryanodine receptor (RYR, 19q13.1), this variant is present in (50% of MHS families are genetically linked to variants in the RYR-1 gene. In the European population, twenty-three mutations have been identified within RYR1 which are found together with MHS. Fourteen mutations occur in isolated MHS families and are rare, the incidence of the remaining nine were each found in 2 - 10% of the total number of MHS families tested (9,10,11). Recently through genetic linkage studies other DNA regions have also been implicated as modifiers of the phenotype including the alpha 2/delta subunit of voltage-dependent calcium channel (CACNA2D1), and 2 genetic loci of undetermined function proteins MHS4 (located at 3q13.1) and MHS6 (located at 5p )(12).

Testing for MHS

There is a phenotypic test for MHS, in-vitro caffeine-halothane contracture test from muscle biopsy specimens (IVCT). This test is invasive, and not widely available. There are only twelve test centers in North America (13). There are six MHS genetic variants that are prevalent in the UK MHS population, with a variant from these six detected in 25% of families tested. Genetic testing for MHS is accomplished in one of 3 ways (14). The first is genetic screening a member of a family with a known mutation. The second is screening for the six most common RYR1 alleles. The third is the most time consuming, and is only done after the first two, is looking for additional rare or discovery of novel variants in RYR1 or the other candidate genes. Genetic testing in the general population for a susceptibility to malignant hyperthermia is not economically feasible, but testing in individuals from families with affected individuals has the potential to greatly reduce morbidity and mortality (15). Genetic testing as a screen for MHS cannot currently stand alone in the absence of contracture testing. However genetic testing of affected individuals and their families must continue along with the IVCT. Due to the finite nature of the set of genetic variants that most likely contribute to this phenotype, this approach will eventually allow for the development of the MHS diagnostic pharmacogenetics profile that can replace the IVCT altogether.

CYP2D6

CYP2D6 is a member of the supergene family of heme-thiolate proteins and is an excellent example of the influence of genetic polymorphism on drug metabolism. This enzyme is responsible for the metabolism of a large number of therapeutic compounds, and there are proposed PG dose modifications for the following; amitriptyline, clomipramine, desipramine, fluoxetine, fluvoxamine, imipramine, maprotiline, miaserin, nortriptyline, paroxetine, and venlafaxine (see Table 1). The drug-metabolism phenotype of this enzyme is defined by the debrisoquine metabolic ratio, which is the ratio of unchanged administered drug (debrisoquine) to the metabolite (4-hydroxydebrisoquine) recovered in the urine following a standard dose. The dramatic range of CYP2D6 activity within the Caucasian population is illustrated in Figure 3. When the CYP2D6 activity is very low, virtually no unchanged drug is recovered in the urine sample and the majority of drug is in the metabolized form, indicating extremely rapid or ultra-rapid metabolism (UM). In contrast, when the majority of drug recovered is in the unchanged state, the metabolic ratio is very high indicating poor metabolism (PM). Metabolic rates intermediate to the UM and PM phenotypes make-up the extensive metaboliser (EM) phenotype which is characteristic of the majority of the population. Heterozygous individuals are commonly grouped with the homozygous EMs. However, it is apparent that these heterozygous EMs have partially impaired metabolic activity and have been designated in certain circumstances as intermediate metabolisers (IM). Deficiency in CYP2D6, PM or IM phenotypes, is most prevalent among people of Caucasian descent. An example of the striking differences in serum levels resulting from a single metropolol dose in CYP2D6 PM and EM individuals is shown in Figure 4. Metropolol succinate is used as a treatment for hypertension and angina pectoris. Therapeutic effect is proportional to serum levels, and these levels differ after the same dose due to CYP2D6 phenotype.

The relationship between the CYP2D6 enzyme metabolic rate and the CYP2D6 genotype has been extensively characterized (16,17) and was elegantly illustrated by the work of Agundez et al. (18) illustrated in Figure 5. Individuals homozygous for two inactive alleles show the least metabolic capacity (PM) as indicated by an excessively high ratio of unchanged drug to metabolite recovered in the urine. Heterozygous individuals with only one active CYP2D6 allele demonstrate metabolic activity intermediate between PM's and subjects homozygous for two active CYP2D6 alleles (EM's). In contrast, approximately 5 to 10% of subjects have a duplication of the CYP2D6 on one chromosome. These individuals illustrate the highest metabolic capacity and constitute the ultra-rapid metaboliser phenotype (UM). Thus, there is a relationship between the CYP2D6 genotype and the resulting drug metabolism phenotype.

Based on the effect and prevalence of the various alleles multiple genotypes (allele combinations) can yield a common phenotype. The best CYP2D6 pharmacogenetic screening protocol includes combination testing to provide > 97.5% reliability in predicting the PM or UM phenotypes (19). PG testing for CYP2D6 genotype is already available in a clinical setting.

Interpretation of CYP2D6 genotyping.

An important aspect of pharmacogenetic information is how to apply this information to the therapeutic management of an individual for a given drug. Testing laboratories, along with providing the results of genetic analysis should provide a detailed interpretation of those results for practitioners to make well informed decisions on dosing. Therefore, we will only provide general guidelines for dosing here. As a consequence of decreased metabolism associated with homozygosity for CYP2D6 PM alleles, PM subjects have been successfully treated by reducing the dose of the therapeutic compound. For example, reduction in the dose of the antidepressant imipramine in poor metabolisers approximately 6-fold was required in order to attain typical therapeutic concentrations of imipramine and the active metabolite desipramine and the appropriate response (20). In UMs' increased metabolism of active drug results in subtherapeutic drug concentrations at standard dosages and may lead to toxicity resulting from over-production and accumulation of toxic metabolites (21). As a consequence of increased metabolism of CYP2D6 substrates resulting from gene duplication, therapeutic failure may result because plasma concentrations of active drug at standard doses are far to low. To correct for increased metabolism, UM subjects have been successfully treated with megadoses (2 to 12-fold greater that the standard dose) of certain drugs in order to obtain therapeutic efficacy (22). Several examples of therapeutic management in light of pharmacogenetic information are illustrated in Table 1.

To illustrate the effects of decreased CYP2D6 activity on a therapeutic compound we will discuss the effect of CYP2D6 on codeine metabolism. Codeine is an alkaloid obtained from opium or prepared from morphine by methylation. Codeine phosphate is a commonly prescribed oral analgesic medication. To obtain analgesia from codeine however requires metabolism of codeine to morphine by CYP2D6. Lack of CYP2D6 enzymatic capacity leads to no analgesia, but increased side effects from the parent drug, including fatigue. A study by DG Williams (23) on codeine metabolism in children found that 47% of the children had reduced CYP2D6 enzymatic capacity, and in 36% of the children no morphine or morphine metabolites were detected one hour after taking codeine. In these children another analgesic may have been a better choice.

References

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