Pharmacogenetics - PGx

Diagnosis

Indications for Testing

  • Provide pre-therapeutic guidance for drug and dose selection
  • Provide post-therapeutic monitoring
    • Evaluate the cause of post-therapeutic adverse drug reactions or therapeutic failure
    • Optimize dose with pharmacokinetic or pharmacodynamic monitoring
  • Drug-gene associations (also see FDA-approved drugs with pharmacogenomic information in their labels)

Laboratory Testing

  • Germ-line genetic tests (whole blood)
    • 5-Fluorouracil and DPYD, TYMS
    • Antidepressants and CYP2D6, CYP2C19
    • Clopidogrel and CYP2C19
      • American Heart Association and American College of Cardiology recommend testing for patients at moderate to high risk of cardiovascular events who are treated with clopidogrel
    • Codeine and CYP2D6
    • Irinotecan and UGT1A1
    • Tamoxifen and CYP2D6
      • Most important therapeutic agent for treatment of estrogen receptor (ER)-positive breast cancer for the past three decades
      • Metabolites are ~100-fold stronger than parent compound
      • CYP2D6 is responsible for activity
    • Warfarin and CYP2C9, VKORC1
      • Narrow therapeutic index (causal agent for 10% of all adverse drug reactions)
      • Recommended by FDA
    • Interferon-α and ILB28
      • Inosine triphosphatase (ITPA)
    • HCV therapy and interleukin 28 B (IL28B), inosine triphosphatase (ITPA)
    • Abacavir and HLA-B*5701
      • 5-8% of Caucasians develop hypersensitivity reactions within 6 weeks of therapy
      • FDA considers test mandatory prior to therapy
    • Carbamazepine or lamotrigine and HLA-B*1502
    • Thiopurines and thiopurine methyltransferase (TPMT)
      • TMPT inactivates toxic metabolites of thiopurine prodrugs
      • Heterozygous individuals (7-15% of Caucasians) have intermediate TPMT activity
      • ~1/300 individuals are homozygous with low to negligible detoxification activity
      • Genetic and phenotype testing is available and may aid in dosage management
  • Monoclonal antibody therapy and molecular testing
    • Cetuximab or panitumumab and KRAS/NRAS
    • Trastuzumab and HER2/neu
    • Tyrosine kinase inhibitors, KIT and EGFR testing
  • Phenotype testing
    • Azathioprine/6-mercaptopurine and TPMT activity
    • Succinylcholine and pseudocholinesterase activity
  • Pharmacokinetic testing
    • Therapeutic drug monitoring (TDM) is available for many drugs and drug metabolites and can be used to assess phenotype of a patient post-therapeutically, as well as to optimize dose
      • Examples include tricyclic antidepressants and codeine

Screening

  • Pre-therapeutic drug selection/avoidance
    • Predict risk of toxicity and likelihood of response that is dose-independent
  • Pre-therapeutic dose selection
    • Predict risk of toxicity and likelihood of response that is dose-dependent
    • Select dose (eg, dose-escalate a patient that is predicted to have poor response or reduce dose for a patient that is predicted to be very sensitive to a drug)
    • Predict pharmacokinetics of a drug in order to optimize dosing frequency as well as determine best time to evaluate response to therapy (ie, estimate time to achieve steady state)
  • Familial testing may be appropriate
    • Testing options should be discussed with laboratory or genetic counselor

Monitoring

  • Post-therapeutic evaluation of adverse drug reactions or failure to respond
    • Dependent upon the following
      • Clinical factors
      • Clinical scenario (eg, whether a reaction is likely to be related to the drug and/or dose administered)
      • Compliance
      • Drug
      • Drug formulation

Clinical Background

Genetic variations associated with drug response or drug disposition may predispose a patient to risk of drug-related toxicity or lack of therapeutic benefit and are referred to as pharmacogenetic variants. Pharmacogenetics can explain and predict variations in both pharmacokinetic and pharmacodynamic processes.

As such, pre-therapeutic pharmacogenetic testing to identify people who have inherited clinically significant variants may guide drug and dose selection to promote personalized therapeutics. Pharmacogenetic testing may be designed to detect human germ-line variants, somatic variants (eg, tumor tissue), or genomic variants of an infectious organism (eg, virus). The goals of pharmacogenetic testing are to reduce the high number of nonresponders (averaging 30-60% of patients) and to reduce adverse drug reactions.

Pharmacogenetics Definitions

  • Definitions of common pharmacogenetics terms

    Definitions of Pharmacogenetics Terms

    Allele

    Alternate form of a gene located on a specific chromosome

    Chromosome

    Linear bodies in the cell nucleus containing most or all genes of the organism

    Deletion

    Absence of a section of genetic material from a gene or absence of one or more entire genes from a chromosome

    Duplication

    Part of a chromosome in which the genetic material, including an entire gene or genes, is repeated; process of forming a duplication

    Expression

    Detectable effect of a gene, usually manifested by the amount and/or type of protein

    Gene

    Functional unit of heredity occupying a specific locus on a chromosome; capable of reproducing itself exactly at each cell division; directs formation of an enzyme or other protein

    Genotype

    Genetic constitution of an individual gene; may reflect a single nucleotide polymorphism, mutation, or series of variants

    Haplotype

    Group of genetic variants from one or more genes (eg, of the major histocompatibility complex) located on a single chromosome that are closely enough linked to usually be inherited as a unit or characteristic pattern

    Heterozygote (HET)

    Two genes at corresponding loci on homologous chromosomes different for one or more loci; two different copies

    Homozygote

    Two genes at corresponding loci on homologous chromosomes identical for one or more loci; two identical copies

    Insertion

    Section of genetic material inserted into an existing gene sequence

    Linkage

    Relationship between genes on the same chromosome that causes them to be inherited together

    Metabolizers

    Poor metabolizer – lacking or near total lacking capacity to metabolize a substrate through a specific pathway
    Intermediate metabolizer – less than normal capacity to metabolize a substrate through a specific pathway
    Ultrarapid metabolizer – enhanced capacity to metabolize a substrate through a specific pathway
    Extensive metabolizer – normal population-based capacity to metabolize a substrate through a specific pathway

    Mutant (MUT)

    Change in hereditary material involving either a physical change in chromosome relations or a biochemical change in the codons that make up genes that are associated with a phenotype

    Phenotype

    Observable properties of an organism that are produced by the interaction of the genotype and the environment

    Single nucleotide polymorphism

    Naturally occurring substitution of a single nucleotide at a given location in genome of an organism, often resulting in phenotypic variability

    Variant

    Exhibiting variation or diversity, either genotypically or phenotypically

    Wild type

    Phenotype, genotype, or gene that predominates in a natural population of organisms or strain of organisms in contrast to that of mutant forms

Pathophysiology

  • Strengths and limitations of pharmacogenetic results are based on the following
    • Actual result (heterozygote vs mutant)
    • Allele frequency
    • Assay content
    • Clinically accepted guidelines
    • Drug choices available
    • Genotype-phenotype relationship
    • Methodology
    • Other factors that impact phenotype
      • Comedications
      • Clinical status
      • Age/sex
      • Alternate metabolic pathways
      • Other genes

Clinical Presentation

  • Pharmacogenetic variations
  • Adverse drug reactions with pharmacogenetic associations

    Examples of Adverse Drug Reactions With Pharmacogenetic Associations

    Drug

    Pharmacogenetic variation(s)

    Effect

    Warfarin

    CYP2C9, VKORC1

    Bleeding

    Azathioprine/6-Mercaptopurine/6-Thioguanine

    Thiopurine methyltransferase (TPMT)

    Neutropenia due to increased myelosuppression

    Irinotecan

    UGT1A1

    Severe diarrhea or neutropenia due to decreased metabolism

    Abacavir

    HLA-B*5701

    Hypersensitivity reaction (rash, fever, gastrointestinal symptoms) in Caucasian populations with mutation

    Carbamazepine

    HLA-B*1502

    Hypersensitivity reaction (rash, fever, gastrointestinal symptoms) in Asian populations with mutation

    Codeine, antidepressants, several other drugs

    CYP2D6

    Drug-specific

    RibavirinInosine triphosphatase (ITPA)Hemolytic anemia
    Therapeutic failures

    Examples of Therapeutic Failures

    Drug

    Pharmacogenetic variation(s)

    Effect

    Antiestrogens (tamoxifen), alpha-blockers, analgesics, anticonvulsives, antidepressants (amitriptyline), antidiabetics, antihypertensives, antipsychotics, beta blockers, norepinephrine reuptake inhibitors

    CYP2D6

    Drug specific

    Warfarin

    CYP2C9, VKORC1

    Bleeding or thrombosis

    Clopidogrel

    CYP2C19

    New arterial blood clots

    Interferon

    IL28B

    Disease progression

    Cetuximab panitumumab

    KRAS/NRAS

    Lack of therapeutic benefit

    Prescribed only in cases where mutation is lacking

    Tyrosine kinase inhibitorsBCR/ABL, c-Kit

    Presence necessary for therapeutic effect

    Characteristic mutations imply resistance to some tyrosine kinase inhibitors

    TrastuzumabHER2Presence of over expression necessary for therapeutic effort

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
5-Fluorouracil (5-FU) Toxicity and Chemotherapeutic Response, 7 Mutations 2007228
Method: Polymerase Chain Reaction/Single Nucleotide Extensions/Fragment Analysis
Predicts toxicity and responsiveness of tumor to 5-fluorouracil (5-FU) therapy

Only targeted mutations in the DPYD and TYMS genes will be detected by this panel

Rare diagnostic errors may occur due to primer-site mutations

Genetic and/or nongenetic factors that are not detected by this assay may affect

  • 5-FU drug metabolism
  • Efficacy
  • Risk for toxicity

Genotyping does not replace the need for therapeutic drug monitoring or clinical observation

Lack of detection of the targeted DPYD and TYMS mutations does not rule out

  • Risk for 5-FU toxicity
  • Degree of responsiveness to 5-FU
 
HLA-B*5701 Associated Variant Genotyping for Abacavir Sensitivity 2002429
Method: Polymerase Chain Reaction/High Resolution Melt Analysis

Identify individuals at risk for abacavir sulfate hypersensitivity reaction (ABC HSR) prior to therapeutic treatment

Screening prior to reinitiation of treatment in individuals who have previously tolerated abacavir but whose HLA-B*5701 status is unknown

Clinical sensitivity/specificity – for immunologically confirmed ABC HSR, 98% and 99%, respectively

Analytical sensitivity/specificity – 99% for prediction of HLA-B*5701 carrier status by SNP rs2395029 genotyping

Diagnostic errors can occur due to rare sequence variations

Rare recombination events between HCP5 SNP rs2395029 and HLA-B*5701 may occur

Nongenetic factors that may affect drug sensitivity are not identified

Testing for a genetic variant associated with ABC HSR does not replace the need for therapeutic drug or other clinical evaluation

 
Warfarin Sensitivity (CYP2C9 and VKORC1 ) 3 Mutations 0051370
Method: Polymerase Chain Reaction/DNA Hybridization/Electrochemical Detection

Aids in warfarin dosage planning

Most useful for

  • Warfarin-naïve individuals
  • Individuals with personal or family history of difficulty with anticoagulation
  • Individuals who are therapy adherent but difficult to treat
  • Individuals who might be placed on warfarin prophylactically after surgery (eg, those requiring <21 mg or >49 mg per week to maintain therapeutic INR)

Does not provide individualized dosing recommendations

Clinical sensitivity – 90% of CYP2C9 and VKORC1 mutations causing warfarin sensitivity in Caucasians are detected; the detection rate in other ethnicities is reduced

Other mutations that may impact warfarin sensitivity or resistance in these genes or other genes will not be detected

Rare mutations within primer or probe regions may interfere with the assay

Refer to Warfarin Genotyping Plus test if individualized warfarin dosing recommendations are needed

Warfarin Genotyping Plus 2004358
Method: Polymerase Chain Reaction/DNA Hybridization/Electrochemical Detection

Preferred test if individualized warfarin dosage planning is required

Individualized dosing recommendations are provided when specific individual parameters are submitted

   
Cytochrome P450 2C9 (CYP2C9) 2 Variants 0051103
Method: Polymerase Chain Reaction/DNA Hybridization/Electrochemical Detection

Pre-therapeutic testing to identify individuals who should avoid or have different dosing of medications metabolized by CYP2C9 such as

  • Anticoagulants (warfarin)
  • Anticonvulsants
  • Hypoglycemics
  • Some antidepressants
  • Cyclophosphamide
  • Nonsteroidal anti-inflammatory drugs (NSAIDs)

Screen individuals with personal or family history of adverse drug events or therapy failure when exposed to CYP2C9-metabolized drugs

Clinical sensitivity – >90% for Caucasians; unknown for other ethnicities

Variants tested

  • Decreased function
    • *2 (c.430C>T)
  • Nonfunctional
    • *3 (c.1075A>C)

CYP2C9 mutations other than *2 and *3 are not evaluated

Mutations in other genes associated with drug metabolism or drug response will not be detected; presence of other mutations (eg, VKORC1) may further impact drug metabolism

Drug metabolism may be affected by nongenetic factors

Mutation detection is not a substitute for therapeutic drug or clinical monitoring

Diagnostic errors can occur due to rare sequence variations

Refer to Warfarin Genotyping Plus test if individualized warfarin dosing recommendations are needed

Cytochrome P450 2D6 (CYP2D6) 14 Variants and Gene Duplication 0051232
Method: Polymerase Chain Reaction/Primer Extension

Pre-therapeutic testing to identify individuals who should avoid or have different dosing of medications metabolized by CYP2D6 such as tamoxifen

Screening for individuals with personal or family history of adverse drug event or therapy failure when exposed to CYP2D6-metabolized drugs

Clinical sensitivity – >95% for Caucasians; unknown for other ethnicities

Variants tested

  • Functional
    • *2 (2850C>T)
    • *2A (-1584C>G; 2850C>T)
  • Decreased function
    • *9 (2613-5delAGA)
    • *10 (100C>T)
    • *17 (1023C>T)
    • *29 (1659G>A)
    • *41 (2988G>A)
  • Nonfunctional
    • *3 (2549delA)
    • *4 (1846G>A)
    • *5 (gene deletion)
    • *6 (1707delT)
    • *7 (2935A>C)
    • *8 (1758G>T)
    • *12 (124G>A)
    • *14 (1758G>A)
  • Increased function
    • Duplicated functional alleles

Only the targeted CYP2D6 mutations will be detected; mutations in other genes will not be detected

Phase and copy number of detected CYP2D6 mutations may not be determined

Mutations in other genes associated with drug metabolism or drug response will not be detected

Drug metabolism may be affected by nongenetic factors

Mutation detection is not a substitute for therapeutic drug or clinical monitoring

Diagnostic errors can occur due to rare sequence variations

 
Cytochrome P450 2C19 (CYP2C19) 9 Variants 0051104
Method: Polymerase Chain Reaction/Primer Extension

Pretherapeutic testing to identify individuals who should avoid or have different dosing of medications metabolized by CYP2C19 such as

  • Antithrombotics (eg, clopidogrel/Plavix)
  • Amitriptyline (Elavil)
  • Escitalopram (Lexapro)
  • Tamoxifen (Nolvadex)

Screening for individuals with personal or family history of adverse drug event or therapy failure when exposed to CYP2C19-metabolized drugs

Clinical sensitivity – ~99% for Asians, ~87% for Caucasians

Variants tested

  • Decreased function
    • *9 (c.431G>A)
    • *10 (c.680C>T)
  • Nonfunctional
    • *2 (c.681G>A)
    • *3 (c.636G>A)
    • *4 (c.1A>G)
    • *6 (c.395G>A)
    • *7 (c.819+2T>A)
    • *8 (c.358T>C)
  • Increased function
    • *17 (c.806C>T; increased gene transcription)

Additional CYP2C19 mutations in this or other genes will not be detected

Mutations in other genes associated with drug metabolism or drug response will not be detected

Drug metabolism may be affected by nongenetic factors

Mutation detection is not a substitute for therapeutic drug or clinical monitoring

Diagnostic errors can occur due to rare sequence variations

Genotype results should be interpreted in the context of the individual clinical situation

 
KRAS Mutation Detection 0040248
Method: Polymerase Chain Reaction/Pyrosequencing

Order to assess resistance to anti-EGFR therapy (cetuximab and panitumumab)

40% of wild-type, KRAS-type tumors will not respond to anti-EGFR monoclonal antibodies

 
KRAS Mutation Detection with Reflex to BRAF Codon 600 Mutation Detection 2001932
Method: Polymerase Chain Reaction/Pyrosequencing

Order to assess response to EGFR-directed therapy

Detects mutations in codons 12, 13, and 61; if KRAS is not detected, BRAF codon 600 Mutation Detection will be performed

   
NRAS Mutation Detection by Pyrosequencing 2003123
Method: Polymerase Chain Reaction/Pyrosequencing

Screen individuals with rare colorectal cancers (CRCs) who may respond to anti-EGFR therapies

Screen individuals with melanoma who may respond to therapy targeted at downstream genes in the MAPK signaling pathway

Limit of detection – 10% mutant alleles

Oncogenic mutations outside of codons 12, 13, and 61 will not be detected

Presence or absence of mutations does not guarantee a positive response to anti-EGFR therapies

 
UDP Glucuronosyltransferase 1A1 (UGT1A1) Genotyping 0051332
Method: Polymerase Chain Reaction/Fragment Analysis

Use for dosage planning for individuals

  • Who will receive high-dose irinotecan (doses >150 mg/m2)
  • With a personal or family history of sensitivity to irinotecan
  • Who have experienced neutropenia while receiving irinotecan

Confirm suspected diagnosis of Gilbert syndrome

Clinical significance of (TA)5 and (TA)8 is not well-established

Other variants such as those associated with Crigler-Najjar syndrome will not be detected

(TA)5 and (TA)8 are not detected

Other factors that contribute to irinotecan toxicity will not be detected

 
Pseudocholinesterase, Dibucaine Inhibition 0020159
Method: Quantitative Enzymatic

Order to detect increased sensitivity in individuals who experience prolonged paralysis following succinylcholine or mivacurium administration

   
Mercaptopurine and Metabolites Quantitative, Blood 2011006
Method: Quantitative High Performance Liquid Chromatography-Tandem Mass Spectrometry 

Compliance monitoring

   
Thiopurine Methyltransferase, RBC 0092066
Method: Enzymatic/Quantitative Liquid Chromatography-Tandem Mass Spectrometry

Detect risk for severe myelosuppression with standard dosing of thiopurine drugs

Individualize dosing of thiopurine drugs

Does not replace clinical monitoring

Genotype cannot be inferred from TPMT activity (phenotype)

TPMT inhibitors may contribute to false-low test results

TPMT activity should be assessed prior to treatment with thiopurine drugs

Blood transfusion within 30 days will reflect donor status

 
EGFR Mutation Detection by Pyrosequencing 2002440
Method: Polymerase Chain Reaction/Pyrosequencing

Order to assess response to gefitinib therapy for non-small cell lung cancer

Detects mutations in EGFR exons 18, 19, 20, and 21

   
TPMT Genotype 2002573
Method: Qualitative Polymerase Chain Reaction

Aids in detecting individuals at high risk for myelosuppression when exposed to standard dose of thiopurines

Consider if red blood cell (RBC) TPMT testing is not practical, as in the following

  • Current treatment with thiopurine drugs (results will be falsely low)
  • Blood transfusion within the last 30-60 days
  • Treatment within the last 48 hours with naproxen, ibuprofen, ketoprofen, furosemide, sulfasalazine, mesalamine, olsalazine, mefenamic acid, thiazide diuretics, and benzoic acid inhibitors

TPMT polymorphism explains only 60% of the thiopurine hematotoxicity but none of the thiopurine induced liver injury

Continued monitoring on hematologic and liver parameters is necessary

 
Thiopurine Metabolites 2002575
Method: Qualitative High Performance Liquid Chromatography

Aids in therapeutic monitoring in individuals treated with thiopurines

   
Interleukin 28 B (IL28B)-Associated Variants, 2 SNPs 2004680
Method: Polymerase Chain Reaction/High Resolution Melt Analysis

Predict response to peginterferon (PEG-IFNα) and ribavirin (RBV) therapy  for chronic genotype 1 hepatitis C virus  (HCV-1) infection

SNPs other than those targeted will not be detected

Usefulness of the IL28B-associated SNPs for predicting therapy response for HCV genotypes other than HCV-1 is unknown; lack of favorable genetic factors should not be used to deny therapy

Mutations in genes and nongenetic factors that may affect response to HCV therapy are not detected

Diagnostic errors can occur due to rare sequence variation

 
Inosine Triphosphatase (ITPA) and Interleukin 28 B (IL28B)-Associated Variants, 4 SNPs 2006344
Method: Polymerase Chain Reaction/Single Nucleotide Extensions

Predict response to PEG-IFNα/RBV therapy for chronic HCV-1 infection

Aid in dose planning for chronic HCV-1 infection

Predict risk of RBV treatment-related anemia

SNPs other than those targeted will not be detected

Usefulness of the IL28B-associated SNPs for predicting therapy response for HCV genotypes other than HCV-1 is unknown; lack of favorable genetic factors should not be used to deny therapy

Mutations in genes and nongenetic factors that may affect response to HCV therapy are not detected

Diagnostic errors can occur due to rare sequence variations

 
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Warfarin, Urine 0091230
Method: High Performance Liquid Chromatography
Warfarin Quantitative, Serum or Plasma 0090805
Method: Quantitative High Performance Liquid Chromatography
Pseudocholinesterase, Total 0020167
Method: Quantitative Enzymatic