Chapter 9: Pharmacogenetics

Introduction

For hundreds of years it has been known that medications can have different effects on different people. It has also been known that close relatives often have similar reactions. Adverse drug reactions are among the 10 most frequent causes of death. These include dose-dependent, dose-independent, and dose-over-time-dependent (cumulative) effects, as well as withdrawal, paradoxical effects of medications, and resistance to therapy. Many of these unusual effects result from the genetic-biochemical makeup of the individual patient.

Basics of Pharmacological Metabolism

The therapeutic window of a medication is the drug dosage that can treat a disease effectively while staying within the safety range (e.g., giving more desired effects than adverse effects). If the dose is too low or the time span between doses too long, or if bioavailability of the medication is insufficient, the effective dose will not be reached. The same is true if the medication is catabolized too rapidly and/or excreted. If the dose is too high, the time span between doses is too short, or the drug is hardly metabolized and excreted, then the therapeutic window will be exceeded and toxic side effects may occur.


Pharmacokinetics:
The representation of the body’s action on the drug.

Pharmacodynamics:
The effects the drug has on the body, or more exactly, the target tissue.


There may be considerable genetic differences in pharmacokinetics and pharmacodynamics from one patient to another, resulting in extremely different medicinal effectiveness between individuals prescribed the same dose of medication. Processes such as absorption, distribution, biotransformation, metabolism, and excretion are part of pharmacokinetics. The distribution and metabolism of a drug can differ greatly between individuals and sometimes depend considerably on genetic factors.

Drug levels in the blood are used as an indirect measurement of drug concentrations in the target tissue. Pharmacodynamics of a medication describes how the drug molecules bind to the respective receptors and thereby trigger signaling cascades, activate transcription factors, or regulate gene expression. These processes also depend on individual genetic disposition.


Phase I reactions:
Modification of a chemical, usually rendering it (more) polar.

Phase II reactions:
The attachment of a polar, ionizable group to the respective molecule.


The great majority of drugs are metabolized in phase I and phase II reactions. Phase I reactions can lead either to activation or inactivation of the drug. A large and important group of phase I enzymes are the P-450 cytochromes, a class of proteins of which more than 50 have been identified in humans. Cytochromes are monooxygenases that hydroxylate their substrates and create the possibility for conjugation with strong polar agents in a subsequent phase II reaction.

Like most phase I enzymes, cytochromes not only play a role in drug metabolism but also have unwanted functional effects. Some of the strongest carcinogens are metabolically activated in vivo by cytochromes of the P-450 system, which change a precursor substance into a chemically reactive form. Other phase I enzymes include various hydroxylases, peroxidases, monoamine oxidases, dioxygenases, reductases, lipoxygenases, cyclooxygenases, and dehydrogenases.

After the introduction of a functional group, a polar and ionizable group can then be attached to the respective molecule in a phase II reaction. In most cases, this involves conjugation with a strong polar group, typically a glucuronyl group. The introduction of a polar group leads to a molecule devoid of activity with increased water solubility, making it more easily excreted from the body.

Pharmacogenetics in Clinical Practice

Variability of N-Oxygenation

Trimethylaminuria (fish odor syndrome) is a less known, yet clinically relevant, “pharmacogenetic” metabolic disorder. It is caused by a deficiency of the flavin-containing monooxygenase type 3 (FMO3) that is required for the oxygenation of nitrogen in numerous substances and is inherited as an autosomal recessive trait. FMO3 deficiency affects the metabolism of various drugs, such as tyramine, benzydamine, and nicotine.

Clinically, the most relevant symptom is caused by the reduced or absent N-oxygenation of trimethylamine (TMA), a substance that gives old fish its unpleasant odor. TMA N-oxide, the product of the FMO3-mediated reaction, is odorless. In FMO3 deficiency, free TMA accumulates, and large quantities of it are excreted in urine, breath, and sweat, causing a highly unpleasant body odor resembling old fish. Numerous mutations in the FMO3 gene have been described. A complete loss of FMO3 results in permanent body malodor that is very stressful for affected persons, although often it is not recognized as a disorder.

There is also a common hypomorphic allele with reduced, but not absent, FMO3 activity, which occurs at a frequency of 20% in various populations and is homozygous in 3% to 5% of individuals of mid-European descent. Such individuals have “mild” or “intermittent” trimethylaminuria, meaning the unpleasant body odor occurs only in special situations (e.g., after eating fish and certain other food products such as peas, choline, or lecithin-rich substances, or after the administration of carnitine).

Fast and Slow Acetylators

The polymorphism of the N-acetyl transferase gene NAT2 on chromosome 8 is the pharmacogenetic variant par excellence. It was recognized at the beginning of the 1950s, when repeated cases of severe polyneuritis were noted after isoniazid was introduced as a new medication for tuberculosis. The cause was soon discovered: some people catabolize the medication slowly and, as a result, develop a toxic overdose reaction. Isoniazid is detoxified by acetylation caused by the hepatic enzyme N-acetyl transferase. With regard to the metabolism of isoniazid or many other substances, people may be classified into two groups: fast and slow acetylators.

The difference is caused by hypomorphic variants in the NAT2 gene that result in a diminished enzyme activity and, thus, slower acetylation. Like most other inborn errors of metabolism, NAT2 deficiency shows autosomal recessive inheritance; slow acetylators are homozygous (or compound heterozygous) for alleles that mediate diminished N-acetyl transferase activity. The frequency of these alleles varies in their ethnic distribution.

While fewer than 20% of Asians are slow acetylators, the percentage rises to 50% for African Americans and 65% for Europeans (up to 90% in the Mediterranean). It is unknown why this metabolic variant is so frequent in some populations; it is possible that slow acetylation had once represented an evolutionary advantage.

Clinical Relevance

Fast acetylators need (and tolerate) higher doses of certain medications than slow acetylators. Slow acetylators have an increased risk for developing lupus erythematosus after administration of hydralazine (for hypertension). The antihypertensive drug debrisoquine and the antiarrhythmic drug sparteine are overly active in slow acetylators and may be ineffective in fast acetylators. Routine genetic testing is not yet recommended, since the many possible gene variants and interindividual variability do not permit an accurate prognosis; when relevant, the drug concentrations can be measured directly in the blood.

Malignant Hyperthermia

Malignant hyperthermia is, perhaps, the most dramatic complication of anesthesia and frequently results in death of the patient. Malignant hyperthermia is inherited in an autosomal dominant manner. It is diagnosed more frequently in children (incidence, 1 in 12,000 children) than in adults (incidence, 1 in 100,000 adults), and males are affected more frequently.

Clinical Relevance

Malignant hyperthermia is due to an adverse reaction to inhalation anesthetic agents such as halothane, enflurane, isoflurane, sevoflurane, and desflurane, as well as depolarizing muscle relaxants such as succinylcholine. These agents are routinely administered in anesthesia practice and rarely cause side effects in the general population; however, administration of one of these medications to a patient with a predisposition for malignant hyperthermia may cause a life-threatening reaction with hyperthermia above 42° C, muscular rigidity, and massive elevation of creatine phosphokinase and myoglobin in the blood. Massive myoglobinemia can cause renal failure. Treatment in the acute phase includes intravenous administration of dantrolene, which is continued as long as hyperthermia persists.

Genetics

To date, two genes that predispose an individual to malignant hyperthermia susceptibility (MHS) have been identified, and three additional loci have been mapped. MHS1 is associated with mutations in the gene RYR1, which encodes for ryanodine receptor type 1; MHS5 is associated with mutations in the gene CACNA1S, which encodes for the skeletal muscle calcium channel. Up to 70% of cases of malignant hyperthermia are caused by mutations in the RYR1 gene.

The type 1 ryanodine receptor is a calcium channel in the sarcoplasmic reticulum of skeletal muscle. RYR1 mutations can result in the leakage of the channel and an unphysiologically high calcium concentration in myoplasm. Another possible effect of the mutations is that the channel acts independently of the relevant regulatory proteins. Molecular genetic testing for RYR1 and CACNA1S is available on a clinical basis. Mutations in the RYR1 gene can also cause a nonprogressive myopathy, central core disease.

Pseudocholinesterase Variants

Serum cholinesterase, also called pseudocholinesterase, hydrolyzes cholinesters such as acetylcholine and succinylcholine, which is a depolarizing muscle relaxant that functions at the neuromuscular junction and is frequently used during anesthesia. Succinylcholine consists of two molecules of acetylcholine and is metabolized and inactivated by cholinesterase. Negative reaction to succinylcholine is caused by variants of the BCHE (butyrylcholinesterase) gene.

As with other conditions relating to enzymatic function, the variants only cause adverse affects when present in a homozygous or compound heterozygous form (autosomal recessive inheritance). Individuals who are homozygous for “variant A” have pseudocholinesterase deficiency, with respective clinical symptoms. Among persons of European descent, the AA genotype occurs in 1 in 3,300 individuals.

Clinical Relevance

The administration of succinylcholine or succinyldicholine (suxamethonium) to affected individuals often results in apnea that last several hours. The affected patient must be supported with extended mechanical ventilation until the drug is eventually metabolized.

Pharmacogenetic Disorders

Glucose-6-Phosphate-Dehydrogenase Deficiency

Glucose-6-phosphate-dehydrogenase deficiency (G6PDD, favism) is one of the most common genetic disorders, with an estimated 400 million affected individuals worldwide. It is inherited as an X-linked condition; mostly males are clinically affected. As with the hemoglobinopathies (sickle cell anemia and thalassemia), the incidence of G6PDD varies considerably by region due to a partial resistance to malaria in carriers. G6PD deficiency results in diminished production of reduced glutathione, which protects erythrocytes from oxidative stress. Affected patients have a reduced capacity to detoxify peroxides that, consequently, damage the erythrocyte membrane and cause hemolysis.

Hemizygous or homozygous individuals may develop severe hemolytic crises in reaction to various external factors such as oxidative stress triggered by infections, ingestion of fava beans (broad beans), or the administration of certain medications such as quinine, chloroquine, primaquine, sulfonamides, and aspirin. Once the diagnosis is made, crises may be prevented through avoidance of the triggering substance. All affected persons should wear a medical ID bracelet and carry a medical emergency card.

Acute Intermittent Porphyria

Acute intermittent porphyria is an autosomal dominant disorder caused by half-normal activity of porphobilinogen deaminase, the second enzyme of heme biosynthesis. Clinically, it is characterized by acute episodes of (abdominal) pain, psychiatric symptoms, and tachycardia. These attacks are triggered by various external factors, including drugs (e.g., cytochrome P-450 enzyme inducers such as barbiturates, pyrazolone, sulfonamides, and halothane), hormones (e.g., progesterone), nutritional factors (e.g., fasting and low cellular glucose), smoking, alcohol, and stress.

The diagnosis is typically made in early adulthood (peak age 30 years), and the incidence is estimated to be 1 in 10,000 individuals. Females are more frequently clinically affected than males. Penetrance is low; only 10% to 20% of individuals with a disease-causing mutation in the HMBS gene manifest with the disease.


Acute Intermittent Porphyria—A Dominant Metabolic Disorder

Most inborn errors of metabolism show autosomal recessive inheritance since, in most instances, the half-normal activity of an enzyme (caused by a heterozygous mutation) is sufficient for physiological function. In contrast, porphobilinogen deaminase (PBGD), a highly regulated enzyme that functions at the beginning of a biosynthetic pathway, exhibits autosomal dominant inheritance. There are two pathogenetic factors: on one hand, half-normal PBGD activity might be sufficient at baseline, but not when there is increased demand for heme biosynthesis, and on the other hand, there is high toxicity of the respective accumulating substrates (aminolevulinic acid and porphobilinogen).

A significant amount of the heme synthesized in the liver is not utilized for hemoglobin but is integrated into numerous enzymes, especially cytochrome P-450 oxidases. Heme synthesis is regulated primarily by its cellular concentration: low heme concentrations result in a strong activation of the first enzyme of this metabolic pathway, δ-aminolevulinic acid synthase. This explains why crises are triggered especially by drugs that induce cytochrome P-450 enzymes. These drugs deplete the cellular concentration of heme, resulting in a strong stimulation of heme biosynthesis that cannot be adequately contained due to the metabolic blockage.


Clinical Relevance

Clinical symptoms can be numerous and misleading. Episodes can manifest as nonspecific, often colicky, abdominal discomfort. Unfortunately, some patients undergo unnecessary abdominal surgery, as signs and symptoms of porphyria can mimic those of acute appendicitis. The neurological and psychiatric symptoms of this condition are protean, are nonspecific, and often pose a diagnostic challenge. These include polyneuropathy with paresis, mood changes, fatigue, and, in severe cases, seizures. Cardiovascular symptoms, such as hypertension or tachycardia, may also occur.

Diagnostics

In 50% of cases, the urine in an acute attack is red or red-brownish, which is enhanced by exposure to air and light. A marked increase in the urinary excretion of porphobilinogen confirms the diagnosis.

Therapy

In an acute crisis, it may be necessary for the patient to receive intensive medical care. Medication associated with causing the disease must be stopped immediately. Heme arginate (as a source of heme) and intravenous glucose infusion can revert activation of heme biosynthesis in the liver. Toxic metabolites may be removed by forced diuresis. The most important therapy, in the long term, is educating the patient about the avoidance of triggers. Affected persons should wear a medical ID bracelet and carry a medical emergency card.

Pharmacogenomics


Pharmacogenetics:
The study of the impact of single genetic variants on drug metabolism.

Pharmacogenomics:
The study of drug metabolism in relation to the whole genome or the individual’s overall genetic constitution.


Whenever a physician administers a medication to a patient, there is a degree of uncertainty as to how effective the treatment will be and if there will be any unwanted side effects. In most cases, the desired effect is achieved; sometimes the medication has no effect at all, or the effect is excessive or paradoxical. Usually, unexpected drug response is multifactorial rather than monogenic, since it does not result from a single rare mutation but rather from a multitude of functionally important genetic variants.

Pharmacogenomics strives for an individualized genetic profile of the patient that can predict the effect of a medication in the respective patient. Such an individualized, genetic-biochemical profile could, theoretically, make it possible to calculate, in advance, the adequate dose and application of a medication. It remains to be seen how rapidly and for which therapies such an approach can be realized.