Pharmacogenomics: SNPs and Haplotypes

• Pharmacogenomics is a branch of pharmacology using DNA sequence variation to inform drug development and testing.
• An application of pharmacogenomics is the correlation of individual genetic variations with drug responses.
• Genes affect response to drugs.

Review of Terminology

• Allele: a variant form of a gene and a pair of alleles from each parent represents the genotype of a specific gene.
• SNP (Single Nucleotide Polymorphism): a single DNA nucleotide difference occurring in > 1% of a population.
• SNPs occur once every 300 bps of DNA.
• SNPs may affect response to drugs, toxins, and risk of diseases.
• SNPs are generally not pathologic (unlike mutations) but can lead to alternative responses to drugs.
• Haplotype (haploid genotype): a set of DNA variations that tend to be inherited together.
• SNPs are placed in order and shown in the figure.
• Combination of alleles in a cluster on the same chromosome is inherited together.

International HapMap Project

• Goal: Discover genetic factors that contribute to disease susceptibility, protection against illness, and drug response.
• Data: SNP frequencies, genotypes, and haplotypes.
• Result: 12 million SNPs from 4 different groups of individuals including Yoruba in Nigeria, Utah residents of western European ancestry, Japanese, and Chinese participants.

Genome-Wide Association Studies (GWAS)

• GWAS compares genetic data (HapMap and genotype) of affected vs. unaffected individuals to determine which SNPs may play a role in human disease.
• GWAS have been used to identify polymorphism-dependent disease association and drug response variability.
• More frequent occurrence of SNPs or haplotypes may indicate a potential role in disease or drug sensitivity
• GWAS has identified many SNPs that play a role in drug efficacy and toxicity.

Three Ways Polymorphisms Can Affect Protein Function

• Regulatory polymorphism: Occurs in gene regulatory sequence not in protein-coding region, such as a transcription factor binding site.
• Structural RNA polymorphism: Alters mRNA processing and translation, such as a splice site mutation.
• Coding region polymorphism: Alters protein sequence and function, such as CYP450 enzymes.

Pharmacogenomic Studies

• ~130 FDA-approved drugs contain pharmacogenomic-related warnings on labels.
• Many warnings are related to SNPs that may increase the risk of serious adverse drug responses.
• Patients carrying the HLA-B*5701 allele are at a higher risk of a hypersensitivity reaction to abacavir.

Warfarin

• Warfarin inhibits vitamin K epoxide reductase (VKORC1) and formation of functional coagulation factors.
• It is the most widely prescribed oral anticoagulant in North America and a drug often responsible for emergency room visits.
• Optimized warfarin dosage is required to inhibit coagulation, but not so much to result in hemorrhage.
• Variability in warfarin responses is largely due to polymorphisms in two enzymes: CYP2C9 and VKORC1.
• CYP2C9 is a CYP450 enzyme that metabolizes and inactivates warfarin.
• VKORC1 is vitamin K epoxide reductase, which is partially- but not completely- inhibited by warfarin during therapy.
• CYP2C9*1 has 100% activity and inactivates warfarin.
• CYP2C9*2 has 12% activity.
• CYP2C9*3 has 5% activity.
• A G→A SNP in the VKORC1 promoter region (not coding region) decreases VKORC1 synthesis.
• Less warfarin is required to inhibit the lower amounts of VKORC1 in individuals with the G→A SNP.
• CYP2C92 and CYP2C93 polymorphisms lead to warfarin buildup.

Drug Sensitivity

• Conventional warfarin dosage taken by individuals with CYP2C92 or CYP2C93 polymorphisms results in elevated warfarin levels in circulation, causing excessive bleeding.
• A lower dose is recommended for patients with polymorphisms that decrease the activity of both enzymes.
• AmpliChip CYP450 is used to determine the dose of therapeutics metabolized by cytochrome P450 proteins CYP2D6 and CYP2C19.
• AmpliChip CYP450 analyzes polymorphisms in two CYP450 genes: CYP2D6 (20 of 70 variant alleles tested) and CYP2C19 (3 of 19 variant alleles tested).
• Drugs affected by AmpliChip testing include antidepressants, antipsychotics, antiarrhythmics, opiates, antiemetics, beta-adrenergic receptor blocker drugs, anticonvulsants, proton pump inhibitors, anticoagulants, benzodiazepines, and antimalarials.

AmpliChip CYP450 Protocal

• Obtain a DNA sample from patient (whole blood).
• PCR amplifies CYP2D6 and CYP2C19.
• DNA is fragmented and fluorophore labeling.
• Hybridization and labeling with streptavidin-tagged fluorophores on a microarray.
• Data analysis uses a hybridization pattern analyzed by an algorithm compiling genotype information (for selected common polymorphisms and gene duplications) to predict CYP2D6 and CYP2C19 enzymatic activity.
• Type of polymorphism, duplication, or deletion, determines the metabolizer phenotype with categories of ultrarapid, extensive (standard dose), intermediate, and poor.

Drug Response

• With active drugs, ultrarapid metabolizers have too rapid of a drug metabolism.
• Extensive metabolizers have the expected response to the standard dose and are considered the most common type of metabolizer.
• Intermediate metabolizers metabolize slower than normal rate.
• Poor metabolizers have a too slow or no drug metabolism.
• Clopidogrel (Plavix®) dosage is determined by AmpliChip results.
• Clopidogrel inhibits ADP-stimulated platelet activation and reduces risk of stroke.
• Effectiveness of clopidogrel depends on activation to an active metabolite by the cytochrome P450 (CYP) system, principally CYP2C19.
• Poor metabolizers treated with clopidogrel at recommended doses exhibit higher cardiovascular event rates following acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI) than patients with normal CYP2C19 function.
• Tests are mandated to identify a patient's CYP2C19 genotype, used as an aid in determining therapeutic strategy.
• Individuals taking conventional Clopidogrel dosages with CYP2C192 or CYP2C193 polymorphisms have reduced effectiveness and are at an increased risk of thrombosis and myocardial infarction at standard drug doses.

Abacavir

• HLAB polymorphism causes hypersensitivity to abacavir (Triumeq®, Ziagen®).
• Self-peptide antigens presented by HLAB*57:01 are perceived as non-self.
• Abacavir is used to treat HIV and the intended target is HIV reverse transcriptase.
• HLAB*57:01 also binds abacavir.
• HLAB is the Major histocompatibility complex class I, B; and the human version is called human leukocyte antigen (HLA).
• HLAB presents peptide antigens, including those from infectious agents, on the surface of every cell.
• Cells presenting non-self antigens bound to HLAB are targeted for destruction.
• HLA-B*57:01 polymorphism elicits a drug-induced autoimmune response.
• The CD8+ T cells recognize & destroy cells that present non-self peptides on HLA.
• Cells presenting self-peptides are not destroyed.
• HLA-B5701 binds abacavir, which presents self-peptides bound to abacavir-HLAB5701 complex.
• This complex is perceived by T cells as foreign, eliciting an auto-immune response to destroy the cell.

HLA-B Polymorphisms

• HLA-B polymorphisms have been associated with hypersensitivity reactions to abacavir* (HIV therapy).
• HLA-B polymorphisms have been associated with hepatotoxicity due to Flucloxacillin (antibiotic).
• HLA-B polymorphisms have been associated with Stevens-Johnson syndrome induced by Carbamazepine* (epileptic seizure and neuralgia treatment).
• Stevens-Johnson syndrome features severe, toxic necrolysis of the skin and mucous membranes and is most common in individuals of Asian descent.
• Genetic testing is mandated prior to prescribing abacavir and carbamazepine.

Cancer Treatment

• SNPs in the somatic (i.e., germline) genome may alter drug efficacy/toxicity.
• Tumor genomes are highly mutated, which may be advantageous for designing chemotherapeutics specifically for tumor proteins.
• With cancer treatment many other factors are considered.
• Several germline (somatic) polymorphisms affect cancer drug activity.
• Poor metabolizers, due to CYP2D polymorphisms, may not get effective doses of tamoxifen.
• Increased toxicity of irinotecan (colon cancer therapeutic) with UGT1A1 promoter region alterations decreases enzyme activity.
• Coding region TPMP SNPs can reduce activity of TPMT coding region
• Coding region TPMP SNPs causes toxicity to mercaptopurine and thioguanine (cancer drugs) by altering thiopurine S-methyltransferase (TPMT, cancer drug target)
• Patients with TPMT polymorphisms have an increased risk of toxicity with conventional doses of Imuran®.
• Less active TPMT results in more 6-TGN and toxicity.
• Gefitinib targets epidermal Growth Factor Receptor (EGFR) for the non-small cell lung cancer drug.
• Polymorphisms in tumor cells with polymorphisms in their EGFR kinase domain respond better to Gefitinib than healthy cells.
• Vemurafenib targets the signaling protein, BRAF, which is involved in the Ras pathway and present in 60% of melanomas.
• Specifically, the drug vemurafenib (V600E mutation in BRAF) targets at a specific point mutation in the BRAF protein (Val→Glu) in late-stage melanomas.
• Decreased activity of TPMT can cause bone marrow toxicity associated with thioguanine and mercaptopurine treatment (cancer chemotherapy).
• Enzyme activity test from blood cells is a physician-preferred method for thiopurine S-methyltransferase (TPMT) testing.
• Enzyme activity test from blood cells is a physician-preferred method for Glucose-6-phosphate dehydrogenase (G6PD) testing, which features over 140 SNPs and decreased G6PD activity can cause hemolysis from antibacterial and antimalarial drugs.