5.5: Pharmacogenomics & Genetic Polymorphism

Introduction to Pharmacogenomics

Pharmacogenomics is the study of how an individual's genetic makeup influences their response to drugs. Genetic polymorphisms—variations in DNA sequences that occur in more than 1% of the population—can significantly alter drug pharmacokinetics (absorption, distribution, metabolism, excretion) or pharmacodynamics (receptor binding, immunological sensitivity). The primary focus of clinical pharmacogenomics is to predict therapeutic failure or avoid severe adverse drug reactions (ADRs) by genotyping patients prior to drug exposure.

Key areas of genetic variation include:

1. Phase I Metabolising Enzymes: Cytochrome P450 (CYP) enzymes (e.g., CYP2D6, CYP2C19).
2. Phase II Conjugation Enzymes: Thiopurine methyltransferase (TPMT).
3. Human Leukocyte Antigen (HLA) System: Immune-related genes associated with severe cutaneous reactions (e.g., HLA-B5701, HLA-B1502).

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CYP2D6 Polymorphisms

CYP2D6 is a highly polymorphic Phase I enzyme responsible for the metabolism of approximately 25% of all clinically used drugs. Based on their CYP2D6 genotype, individuals are classified into four main phenotypes:

• Poor Metabolisers (PM): Lack functional CYP2D6 enzymes.
• Intermediate Metabolisers (IM): Have reduced enzyme activity.
• Normal Metabolisers (NM / Extensive): Have normal enzyme activity.
• Ultra-rapid Metabolisers (URM): Have multiple copies of active CYP2D6 genes, leading to excessive enzyme activity.

Clinical Example 1: Codeine

Codeine is a weak opioid and is actually a prodrug with low intrinsic affinity for opioid receptors. It must undergo O-demethylation by CYP2D6 to be converted into its active metabolite, morphine, which provides the analgesic effect.

• Ultra-rapid Metabolisers (URMs): URMs rapidly convert codeine to morphine, resulting in unexpectedly high, toxic concentrations of morphine in the blood. This can lead to life-threatening respiratory depression, sedation, and death, even at standard therapeutic doses.
  • TGA Warnings / Contraindications: Due to cases of fatal respiratory depression (especially in infants whose mothers were codeine-prescribed URMs), the Therapeutic Goods Administration (TGA) in Australia contraindicates codeine in:
    1. Children under 12 years of age for any indication.
    2. Children under 18 years of age undergoing tonsillectomy or adenoidectomy for obstructive sleep apnoea.
    3. Breastfeeding mothers, as morphine is excreted into breast milk and can cause fatal toxicity in the breastfed infant.
• Poor Metabolisers (PMs): PMs (approximately 5–10% of Caucasians) cannot convert codeine to morphine. Consequently, codeine provides zero analgesic efficacy for these patients.

Clinical Example 2: Tamoxifen

Tamoxifen is a selective estrogen receptor modulator (SERM) used in the treatment and prevention of estrogen receptor-positive breast cancer. It is a prodrug that is metabolised by CYP2D6 to its active metabolite, endoxifen. Endoxifen has a 100-fold higher binding affinity for the estrogen receptor and is 30- to 100-fold more potent than parent tamoxifen.

• Clinical Consequence of PM Phenotype: Patients who are CYP2D6 poor metabolisers, or who have reduced activity, have significantly lower endoxifen concentrations. Clinical studies show these patients have a higher rate of breast cancer recurrence and shorter disease-free survival.
• The "Phenocopying" Trap: Co-administering strong CYP2D6 inhibitors (e.g., SSRIs like fluoxetine or paroxetine, or the antidepressant bupropion) with tamoxifen will block the CYP2D6 enzyme. This pharmacologically converts a normal metaboliser into a functional poor metaboliser (phenocopying), reducing tamoxifen efficacy.
• Management: If an antidepressant is required in a patient taking tamoxifen, select agents with minimal CYP2D6 inhibition, such as venlafaxine, citalopram, or escitalopram.

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CYP2C19 Polymorphisms

CYP2C19 is another highly polymorphic CYP enzyme. It plays a pivotal role in activating and inactivating various drugs.

Clinical Example: Clopidogrel

Clopidogrel is an antiplatelet prodrug widely used to prevent thrombotic events in patients with acute coronary syndrome (ACS) or those undergoing percutaneous coronary intervention (PCI) with stent placement.

• Activation Pathway: Clopidogrel requires a two-step hepatic oxidation process to be converted into its active thiol metabolite. The rate-limiting step is mediated primarily by CYP2C19. The active metabolite then irreversibly binds to platelet P2Y12 adenosine diphosphate (ADP) receptors, inhibiting platelet aggregation.
• Poor Metabolisers (PMs): Patients carrying two loss-of-function alleles (typically CYP2C19*2 or CYP2C19*3) produce minimal active metabolite.
  • Clinical Consequence: These patients exhibit significantly reduced platelet inhibition and a substantially higher risk of major adverse cardiovascular events (MACE), including stent thrombosis, myocardial infarction, and cardiovascular death.
• Clinical Action: Genotyping is increasingly used in cardiology. According to CPIC (Clinical Pharmacogenetics Implementation Consortium) and Australian guidelines, for patients identified as CYP2C19 intermediate or poor metabolisers, clopidogrel should be avoided.
  • Alternatives: Select antiplatelet agents that do not require CYP2C19 activation, such as ticagrelor (a direct-acting cyclopentyltriazolopyrimidine) or prasugrel (a prodrug activated by different CYP enzymes and not significantly affected by CYP2C19 polymorphisms). Note that prasugrel is contraindicated in patients with a history of stroke or transient ischaemic attack (TIA).

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HLA-B*5701 Polymorphism

The Human Leukocyte Antigen (HLA) gene complex encodes cell-surface proteins responsible for regulation of the immune system. Certain HLA alleles are strongly associated with severe, idiosyncratic drug hypersensitivity reactions.

Clinical Example: Abacavir

Abacavir is a nucleoside reverse transcriptase inhibitor (NRTI) used in the treatment of HIV-1 infection.

• Abacavir Hypersensitivity Syndrome (AHS): A potentially life-threatening systemic reaction. Symptoms typically appear within the first 6 weeks of therapy and include fever, rash, gastrointestinal distress (nausea, vomiting, diarrhoea, abdominal pain), constitutional symptoms (lethargy, malaise), and respiratory symptoms (dyspnea, cough).
  • The Danger: Re-exposure to abacavir after a suspected hypersensitivity reaction can trigger immediate, severe hypotension, cardiovascular collapse, and death.
• Genetic Association: There is an absolute association between AHS and the HLA-B*5701 allele. Abacavir binds directly to the antigen-binding cleft of the HLA-B*5701 protein, altering its shape and causing it to present self-proteins as foreign. This triggers a massive CD8+ T-cell-mediated immune response.
• Clinical Action in Australia: Screening for the HLA-B*5701 allele is mandatory for all patients in Australia prior to starting abacavir-containing therapy (e.g., Kivexa, Triumeq).
  • If Positive: Abacavir is absolutely contraindicated. The patient must be recorded as having an allergy to abacavir, and alternative antiretroviral regimens must be selected.
  • If Negative: The risk of AHS is extremely low (less than 1%), making it safe to start. However, patients should still be monitored, as rare non-HLA-B*5701-mediated hypersensitivity can occur.

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HLA-B*1502 Polymorphism

Another critical HLA association is between the HLA-B*1502 allele and severe cutaneous adverse reactions (SCARs).

Clinical Example: Carbamazepine

Carbamazepine is an anticonvulsant and mood stabiliser used for epilepsy, bipolar disorder, and trigeminal neuralgia.

• Stevens-Johnson Syndrome (SJS) & Toxic Epidermal Necrolysis (TEN): Life-threatening dermatological emergencies characterized by epidermal detachment, mucosal blistering, and systemic inflammatory response.
• Genetic Association: The HLA-B*1502 allele is strongly linked to carbamazepine-induced SJS/TEN. This allele is highly prevalent in populations of Asian ancestry (up to 10–15% in Han Chinese, Thai, Filipino, Malaysian, and Vietnamese populations, compared to < 1% in Caucasians).
• Australian / TGA Guidelines:
  • Prior to initiating carbamazepine, patients of ancestry where HLA-B*1502 is prevalent must be screened for the allele.
  • If Positive: Carbamazepine must not be started.
  • Cross-Reactivity Warning: There is significant genetic cross-reactivity. HLA-B*1502-positive patients are also at increased risk of SJS/TEN with other aromatic anticonvulsants, specifically phenytoin and oxcarbazepine. Therefore, these alternatives should also be avoided, and non-cross-reactive agents (e.g., sodium valproate, levetiracetam) should be chosen.

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Thiopurine Methyltransferase (TPMT) Polymorphisms

Thiopurine methyltransferase is a cytosolic enzyme that metabolises thiopurine drugs.

Clinical Example: Azathioprine & 6-Mercaptopurine

Azathioprine is a prodrug that is rapidly converted to 6-mercaptopurine (6-MP). 6-MP is used as an immunosuppressant in inflammatory bowel disease (Crohn's, ulcerative colitis), autoimmune conditions, and in leukaemia treatment.

Metabolic Pathway & TPMT Role

6-MP is metabolised by three competing pathways:

1. Xanthine Oxidase (XO): Converts 6-MP to inactive 8-hydroxymercaptopurine (inhibited by allopurinol).
2. Thiopurine Methyltransferase (TPMT): Converts 6-MP to inactive methyl-mercaptopurine (MeMP).
3. HGPRT Pathway: Converts 6-MP to active 6-thioguanine nucleotides (6-TGN). 6-TGNs are incorporated into DNA/RNA, causing cell death (the therapeutic mechanism).

                  Azathioprine
                       │
                       ▼
               6-Mercaptopurine (6-MP)
        ┌──────────────┼──────────────┐
        │              │              │
        ▼ (XO)         ▼ (TPMT)       ▼ (HGPRT)
   Inactive       MeMP (Inactive)  6-TGN (Active)
   Metabolite                     ┌───┴───┐
                                  ▼       ▼
                             Therapeutic Toxicity
                               Effect    (Myelosuppression)

TPMT Deficiency & Myelosuppression

Genetic variations in the TPMT gene lead to reduced or absent enzyme activity. The population is divided into:

• Normal / High Activity (approx. 89%): Standard metabolism.
• Intermediate Activity / Heterozygous (approx. 10%): Reduced metabolism.
• Low/Absent Activity / Homozygous Deficient (approx. 0.3%): Cannot metabolise 6-MP via TPMT.
• Clinical Consequence: In homozygous deficient patients, the lack of TPMT shunt pathway forces almost all 6-MP down the HGPRT pathway. This leads to an extreme accumulation of active 6-TGNs in the bone marrow, causing severe, life-threatening myelosuppression (leukopenia, pancytopenia) within weeks of initiating therapy.

Clinical Action in Australia

Prior to commencing azathioprine or 6-MP, baseline TPMT status must be determined. This can be done via:

• Genotype Testing: Identifying common non-functional alleles (*2, *3A, *3C).
• Phenotype Testing: Measuring actual TPMT enzyme activity in red blood cells (RBCs). Note: Phenotypic testing is inaccurate if the patient has received a red blood cell transfusion in the preceding 3 months.

Dosing Adjustments

• Homozygous Deficient (Low/Absent Activity): Avoid thiopurines entirely, or if essential, reduce the starting dose by 90% (e.g., dose 10% of normal, administered 3 times per week instead of daily) and monitor full blood counts (FBC) weekly.
• Heterozygous (Intermediate Activity): Start at a reduced dose (typically 30–50% of the standard dose) and titrate slowly based on tolerability and FBC.
• Normal/High Activity: Administer standard starting doses. Note: Patients with very high TPMT activity (often called "shunters") preferentially produce MeMP, which is hepatotoxic. They may experience therapeutic failure and hepatotoxicity.