Introduction to Pharmacogenetics and Genomics

Introduction to Pharmacogenetics and Pharmacogenomics

Conceptual Overview: Patients often respond differently to the exact same drug. While the majority of individuals respond within a standard therapeutic range, a significant proportion may experience outlier responses due to inherent biological differences.
Anecdotal Example (Paracetamol/Panadol): A common example of variability is the response to paracetamol ( $\text{Panadol}$ ). While it typically does not cause drowsiness, a small subset of the population (including the lecturer) feels extremely sleepy to the point of being unable to drive. This suggests a predisposition, potentially due to a genetic variant, that affects how the drug is metabolized or broken down.
Factors Influencing Drug Response: * Genetics: Inherited variants that dictate metabolic rates or target sensitivity. * Environment: Diet (e.g., orange juice improves iron absorption, while dairy products interfere with it). * Age: Metabolism naturally slows down as an individual ages, altering drug processing over time. * Co-confounders: The combination of environmental factors and genetic inheritance determines the individual response.

Definitions and Scale of Variation

Pharmacogenetics: Refers to the genetic basis of variation in drug response. It typically focuses on how a single specific gene—such as one encoding a transporter, ion channel, or receptor—influences drug outcomes.
Pharmacogenomics: A broader term used when multiple genes (the entire genome) influence the outcome of how a human responds to drugs.
Frequency of Variation: Humans are almost identical genetically, yet each individual possesses between $300$ and $1,000$ nucleotides that vary. These variations depend on ethnicity and geographical ancestry.
Impact of Variation: While many genetic variants are common and do not affect drug response (allowing for standard medications like NSAIDs, paracetamol, and aspirin), specific variants can strongly impact drug efficacy and safety.

Basic Mechanisms of Genetic Influence

The Pathway: Genetic variant in DNA $\rightarrow$ altered protein product $\rightarrow$ altered Pharmacodynamics (drug-target binding) or Pharmacokinetics (ADME: Absorption, Distribution, Metabolism, and Elimination).
Enzyme Activity Scenarios: * Reduced Enzyme Activity: Leads to slower drug breakdown. This results in higher drug concentrations in the blood, leading to potential toxicity. * Increased Enzyme Activity: Leads to accelerated metabolism. This causes drug levels to drop rapidly, resulting in a lack of therapeutic effect or benefit.

Evidence for Heritability in Drug Response

Twin Studies: Used to determine the genetic contribution by comparing identical vs. fraternal twins. * Example: $Succinylcholine$ metabolism. Identical twins showed nearly identical paralysis times after receiving the drug because both were deficient in the necessary breakdown enzyme. Fraternal twins showed much greater variability in response, indicating a strong genetic influence.
Family Studies: Identifying variants inherited over generations within a single family. * Example: $CYP2D6$ polymorphism. This enzyme affects the metabolism of certain antihypertensive drugs. Families may have a history of specific drug responses (e.g., inherited paracetamol-induced sleepiness) following Mendelian inheritance patterns.
Complexity of Traits: * Monogenic Traits: Driven by a single gene; results are often predictable and relate to specific metabolic pathways or side effects. * Polygenic/Multigenic Traits: Driven by multiple genes; require large sample sizes and significant statistical power to identify patterns of association.

Fundamentals of Genetic Architecture

Gene Components: * Promoter: DNA segment before the gene start that controls expression levels (on/off). * 5' Untranslated Region (5' UTR): Part of the mRNA that does not become protein but regulates how much protein is synthesized. * Exons: The blue "coding sequences" that are joined together to form mature messenger RNA (mRNA). * Introns: Gray non-coding parts located between exons; these are removed during processing. * 3' Untranslated Region (3' UTR): Affects the stability and lifespan of the mRNA within the cell. * Intergenic Region: DNA between two separate genes; may serve as a promoter for adjacent genes.
Alleles: Different versions of the same gene resulting from mutations. Humans inherit one copy from each parent (maternal and paternal).
Genotype vs. Phenotype: * Genotype: The genetic makeup (the alleles inherited). * Phenotype: The observable response to a drug.

Patterns of Inheritance and Polymorphism

Autosomal Dominant: One inherited variant allele is sufficient to alter drug response (e.g., $succinylcholine$ toxicity).
Autosomal Recessive: Two non-functional alleles are required for an altered response (e.g., codeine metabolism via $CYP2D6$ ).
Codominant: Both alleles contribute to the phenotype; the effect typically falls between dominant and recessive.
X-linked: Gene is located on the X chromosome. Because males have only one X chromosome, they are more significantly impacted as they cannot "silence" a defective copy like females can.
Polymorphism Types: * Single Nucleotide Polymorphism (SNP): A change in a single DNA base (e.g., $A \rightarrow G$ ). These occur every $100$ to $1,000$ base pairs. * Indels: Small sections of DNA added or removed. Rare in coding regions but can cause total loss or sudden gain of activity. * Copy Number Variations (CNV): Large sections or entire chromosomes are duplicated or deleted (e.g., $Trisomy\,21$ ).

Functional Impact of SNPs

Coding Region SNPs: * Non-synonymous: Changes the amino acid, potentially altering protein folding, causing a null function, or increasing activation. * Synonymous: The base change does not alter the amino acid, but may affect mRNA stability or post-translational modification. * Nonsense/Premature Stop: Results in a truncated, non-functional protein.
Non-coding Region SNPs: * Promoter: Can stop transcription or cause over-transcription (too little or too much protein). * 3' UTR: Changes mRNA stability. * Introns: Can alter splicing, leading to the inclusion or removal of specific exons and the creation of a new protein. * Example: A promoter variant in $UGT1A1$ leads to decreased enzyme activity.

Haplotypes and Linkage

Haplotype: A group of genetic variants inherited together on a single chromosome. Each person has two haplotypes (one maternal, one paternal).
Linkage Equilibrium: Variants are inherited independently and randomly.
Linkage Disequilibrium (LD): Variants occur together more frequently than expected by chance. This can vary by population and geography but can be broken by recombination over evolutionary time.

Ethnicity and Population Specifics

Cosmopolitan Polymorphisms: Found in all populations; likely older variants acquired during early evolution.
Population-Specific Polymorphisms: Specific to ethnic groups or geographical isolates. * High Altitude Adaptation: Variants in genes affecting red blood cells to maximize oxygen absorption. * Doxorubicin: Used for lung cancer in Caucasians but causes cardiotoxicity specifically in Indian populations. * Warfarin: Blood thinner dosing varies significantly. East Asians generally require a lower dose due to polymorphisms in metabolism genes that make them slower metabolizers. Europeans require intermediate doses, and Africans show a mixed response. * Clopidogrel (Antiplatelet): High frequency of poor metabolizers in East Asians increases the risk of heart attack if the drug is not activated properly. * Carbamazepine (Anti-epileptic): A specific $HLA$ subtype found in Southeast Asia (rare in Europeans) triggers immune hypersensitivity, leading to $Steven\,Johnson\,Syndrome$ (severe blisters/rashes).

Study Designs and Identifying Traits

Measuring Phenotypes: Enzyme activity, metabolite levels, physiological effects (e.g., blood pressure), or gene expression.
CYP2D6 Activity Test: Measured using the ratio of the drug $dextromethorphan$ to its metabolite in urine. A smaller ratio indicates fast metabolism; a higher ratio indicates a poor metabolizer.
The Quinidine Effect: Quinidine can make a person appear to be a poor metabolizer even if they don't have the genotype; this highlights the importance of distinguishing genetic from environmental factors.
Genotyping: DNA is typically sourced from white blood cells or buccal (cheek) swabs. It remains stable throughout life.
Hardy-Weinberg Equilibrium: Used to confirm that genotyping results are not due to sequencing errors or population bias.
Variant of Unknown Significance (VUS): A genetic variant where the functional impact on drug response is currently unknown.

Lab Validation of Genetic Variants

Sequence-based Methods: Checking if variants occur in highly conserved evolutionary regions.
Structure-based Methods: Computational modeling of protein stability and binding.
In-vitro Systems: Isolating a variant, introducing it into mammalian cells via plasmids, and measuring the phenotypic effect. * Example Outcome A: Increased $K_m$ , meaning the drug does not bind the receptor as well. * Example Outcome B: Decreased $V_{max}$ , meaning the rate of metabolism has slowed or the receptor is lost. * CYP3A5 Example: An intronic SNP leads to an early stop codon and a truncated, inactive enzyme, causing drug accumulation in the system.

Pharmacogenetic Phenotype Categories

Pharmacokinetic (PK) Alterations: Variants in enzymes and transporters (ADME). * CYP2D6: Metabolizes $15\,to\,25\%$ of all drugs, particularly antidepressants and antipsychotics. Poor metabolizers risk toxicity; ultra-rapid metabolizers clear the drug too fast for benefit. * Omeprazole: $CYP2C19$ variants can reduce its activation, failing to treat acid reflux.
Pharmacodynamic (PD) Alterations: Variants in the drug target itself (receptors, ion channels). * Target Mutations: Can cause direct disease or reduce enzyme activity (e.g., $TYMS$ polymorphism or serotonin receptors).
Disease-Modifying Polymorphisms: The variant doesn't change PK or PD but increases the baseline risk of an adverse event. * Thrombosis: Having $Factor\,V$ or prothrombin variants makes one prone to clots; taking prothrombotic drugs then amplifies this existing risk. * Arrhythmia: Polymorphisms in cardiac channels increase susceptibility to arrhythmia when taking macrolides or antihistamines.

Personalized Therapy in Cancer

Somatic Mutations: Unlike germline mutations (present in all cells), these are acquired only in tumor cells.
Targeted Therapies: * Lung Cancer: Mutations in $EGFR$ (epidermal growth factor receptor) allow treatment with $Gefitinib$ . * Breast Cancer: $HER2$ ( $ERBB2$ ) positive tumors are treated with the monoclonal antibody $Trastuzumab$ , which blocks growth signaling.
Double Layer of Genetics: Treatment must consider both tumor somatic mutations and the patient's germline polymorphisms (e.g., $TYMS$ variants affecting sensitivity and toxicity).

Clinical Implementation and Ethics

Standard of Care: Most drugs use generic doses optimized for the "average" population. Dose adjustments currently focus more on renal and liver function than genetics.
Barriers to Uptake: High cost of sequencing units and the need for tiered, reproducible clinical data.
Ethics: Concerns regarding genetic discrimination and the necessity of solid evidence before altering treatment based on variant composition.

Questions & Discussion

Question: Two patients have the same genotype; one has a normal response, one shows toxicity. Why? * Answer: Environmental factors like diet or age can alter the phenotype despite an identical genotype.
Question: Which variant has a major functional effect? * Answer: A nonsense mutation, as it incorporates a stop codon and leads to the loss of the protein.
Question: Why use the Hardy-Weinberg equation? * Answer: To confirm if genotype frequencies fit the expected population distribution and rule out sequencing errors.
Question: Definition of an allele? * Answer: Different versions of the same gene.
Question: Definition of Copy Number Variation? * Answer: The gain or loss of a whole gene or large DNA segment.
Question: What describes a pharmacogenetic phenotype? * Answer: Variability in drug absorption, metabolism, and transport (ADME).