Gene Polymorphisms: How They Alter Enzymes, transporters and CNS Response Proteins Feb 21, 2014 Andrea Gaedigk, MS, PhD Professor, School of Medicine, University of Missouri-Kansas City and Children’s Mercy Hospital & Clinics, Division of Clinical Pharmacology & Therapeutic Innovation, Kansas City, MO American Society for Experimental NeuroTherapeutics | 16th Annual Meeting

Disclosure Member of the Clinical Pharmacology Implementation Consortium (CPIC) No financial COI American Society for Experimental NeuroTherapeutics | 16th Annual Meeting 2/25

Learning Objectives Understand genetic variation in pharmacogenes Understand the challenges of implementing pharmacogenetics into practice Examples for CYP2D6 (drug metabolism) SLC6A4 (serotonin transporter) HTR (serotonin receptor) American Society for Experimental NeuroTherapeutics | 16th Annual Meeting 3/25

Drugs: 138 Entries: 155 ≥2 entries: 14 CYP2D6: 37 CYP2C19: 14 www.fda.gov/drugs/scienceresearch/researchareas/pharmacogenetics 4/25

SSRI PD and PK Pathways SLC6A4 HTR CYP2D6 Brain-derived neurotrophic factor PharmGBK.org Whirl-Carillo et al (2012) CPT 92:414-17 5/25

Drugs metabolized by CYP2D6 Antiarrythmics encainide flecainide sparteine perhexiline propafenone mexiletine Antidepressants Antipsychotics amitriptyline aripiprazole clomipramine chlorpromazine desipramine duloxetin fluoxetine fluvoxamine haloperidol imipramine minaprine nortriptyline paroxetine perphenazine risperidone thioridazine venlafaxine zuclopenthixol Others alprenolol amphetamine atomoxetine bufuralol chlorpheniramine codeine debrisoquine dexfenfluramine dextromethorphan duloxetin lidocaine metoclopramide methoxyamphetamine ondansetron oxycodon perhexiline phenacetin phenformin promethazine tamoxifen tramadol ß-blockers carvedilol S-metoprolol nebivolol propafenone propranolol timolol <2% 3-12% <8% <8% ……many more http://medicine.iupui.edu/flockhart Zhou (2009) Clin Pharmacokinet 48:689-723 (Part 1) and 48:761-804 (Part 2) Gaedigk et al (2008) CPT 83:234-242 Llerena et al (2008) Pharmacogenomics 10:17-28 6

CYP2D6 Complex and highly polymorphic gene locus Single nucleotide polymorphisms (SNPs) Small deletions or insertions (indels) Large deletions (e.g. entire gene) Gene copy number variation (CNVs) Pseudogenes Gene rearrangements (hybrid genes, tandems) Allele frequencies across populations www.pharmgkb.org/download.action?filename=CYP2D6_Literature_Table_and_Legend.pdf The Human Cytochrome P450 (CYP) Allele Nomenclature Database at www.cypalleles.ki.se Over 100 allelic variants and subvariants defined to date 7/25

CYP2D6 copy number variation (CNVs) Loss of entire CYP2D6 gene Gene duplications and multiplications Duplication of functional and non-functional gene units Need to discriminate for accurate genotype determination 8/25 Gaedigk (2013) Int Rev Psych 25:534-53 Tandem arrangements 8

CYP2D6 copy number variation (CNVs) CYP2D7/6 gene hybrids Nonfunctional CYP2D7-derived T-insertion in exon 1 Tandem arrangements Functional or nonfunctional 9/25 Gaedigk (2013) Int Rev Psych 25:534-53 9

CYP2D6 allele/genotype assignment …is a default strategy *1 *2 *41 *10 *4 *56A *56B 100C>T 1846G>A 2850C>T 3201C>T 4180G>C 2988G>A 10/25

Whole genome sequencing 2D8 2D7 2D6 NGS reads are short (30-200 bp) Algorithms align reads to reference sequence 11/25 Gaedigk et al (2013) ISSX and manuscript in preparation

CYP2D6 challenge: accurate alignments 2D8 2D7 2D6 12/25

Predicting phenotype from genotype Challenges Assigning allele function/activity Assigning genotype function/activity predicting phenotype from genotype Clinically actionable? Current Drug Metabolism 2014 Epub Feb 2 13/25

CPIC Clinical Pharmacogenetics Implementation Consortium Incorporation of genomic data into routine clinical practice Established in 2009 Housed at PharmGKB.org/page/cpic Chaired by Mary Relling, St. Jude Children’s Research Hospital and PAAR4Kids >70 members of diverse backgrounds from almost 100 institutions To develop PGx-based dosing guidelines and updates Designed to help clinicians understand HOW available genetic test results should be used to optimize drug therapy, rather than WHETHER tests should be ordered 12 guidelines published (3 with updates) Relling & Klein (2011) CPT 89:464 www.pharmgkb.org/view/dosing-guidelines.do?source=CPIC and www.pharmgkb.org/page/cpicGeneDrugPairs 14/25

CPIC guidelines for CYP2D6 Update Epub Jan 23-2014 Published 2013 Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing for SSRIs early stages Epub Jan 30-2014 15/25

CYP2D6 and CYP2C19/TCA 16/25

SLC6A4 (SERT, 5-HTT, OCD1) SoLute Carrier family 6A member 4 Sodium:neurotransmitter symporter Monoamine transporter that transports serotonine from the synaptic cleft to the presynaptic neuron Terminates action of serotonin Polymorphisms in SLC6A4 extensively scrutinized for many psychiatric and neurologic conditions and disorders Target of many antidepressants/psychotics including SSRIs which reduce binding of serotonin to transporter Polymorphisms predictive of drug response? SLC6A4 not on FDA biomarker list Level 3 (low) evidence on PharmGKB 17/25

SLC6A4 Gly56Ala Ile425Val Promoter Variable number of GC-rich, 20-23 bp long Repeat elements. 43 bp insertion causes ‘long’ allele Short (14 repeats) The coding (pink) exons and noncoding and intronic areas (blue) of SLC6A4; note the 5HTT-LPR polymorphism is ~1.4 kb upstream and the intronic VNTR near exon 2. A polymorphism in intron 2 of SLC6A4 consists of a 17 bp VNTR, termed STin2 VNTR. Abbreviations: 5HTT-LPR, serotonin transporter protein linked polymorphic region; VNTR, variable number of tandem repeats.  The short variation leads to less transcription for SLC6A4, and it has been found that it can partly account for anxiety-related personality traits Long (16 repeats) Murphy et al (2008) Neuropharmacol 55:932-60 De Neve (2011) J Hum Genet 56:456-59 18/25

SLC6A4 Short allele Long allele May be useful for Dominant allele; results in decreased concentration of the transporter protein Poorer response to stressful events Patients may respond to SSRI therapy slowly, taking up to 12 weeks Long allele Homozygous subjects may demonstrate response to SSRI therapy in 3 to 4 weeks May be useful for Evaluating patients who have failed therapy with SSRIs Predicting response time to improvement with SSRIs Identifying patients who might respond favorably to a class of antidepressants other than SSRI Identifying patients who have diminished amounts of the serotonin transporter and, hence, an altered response to SSRI therapeutics Evaluating patients with treatment-resistant depression The coding (pink) exons and noncoding and intronic areas (blue) of SLC6A4; note the 5HTT-LPR polymorphism is ~1.4 kb upstream and the intronic VNTR near exon 2. A polymorphism in intron 2 of SLC6A4 consists of a 17 bp VNTR, termed STin2 VNTR. Abbreviations: 5HTT-LPR, serotonin transporter protein linked polymorphic region; VNTR, variable number of tandem repeats.  The short variation leads to less transcription for SLC6A4, and it has been found that it can partly account for anxiety-related personality traits Interpretive Handbook (Mayo Medical Laboratories (www.mayomedicallaboratories.com/) 19/25

HTR (5-HT Receptor) G protein-coupled receptor Mediate excitatory and inhibitory neurotransmission Activated by their natural ligand serotonin Modulate the release of many neurotransmitters and hormones Influence many biologics and neurological processes such as aggression, anxiety, mood, cognition, appetite, etc Target of may drugs including antidepressants, antipsychotics, antiemetics, hallucinogens, etc Seven HTR families HTR2B and 2C most relevant for SSRI response Polymorphisms have been associated with psychiatric disorders and drug response HTRs not on FDA biomarker list Level 3 (low) evidence on PharmGKB many SSRIs (but not fluoxetine, which is a 5-HT2C antagonist[7]) indirectly stimulate 5-HT2C activity by increasing levels of serotonin in the synapse although the delayed mood elevation that's usually typical of SSRIs is usually paralleled by the downregulation of the 5-HT2C receptors. An overactivity of 5-HT2C receptors may contribute to depressive and anxiety symptoms in a certain population of patients. Activation of 5-HT2C by serotonin is responsible for many of the negative side effects of SSRI and SNRI medications, such as sertraline, paroxetine, venlafaxine, and others. Some of the initial anxiety caused by SSRIs is due to excessive signalling at 5-HT2C. Over a period of 1–2 weeks, the receptor begins to downregulate, along with the downregulation of 5-HT2A, 5-HT1A, and other serotonin receptors. This downregulation parallels the onset of the clinical benefits of SSRIs. 5-HT2C receptors exhibit constitutive activity in vivo, and may retain the ability to influence neurotransmission in the absence of ligand occupancy. Thus, 5-HT2C receptors do not require binding by a ligand (serotonin) in order to exhibit influence on neurotransmission. 5-HT2C receptors mediate the release and increase of extracellular dopamine in response to many drugs 20/25

HTR2A -1438A>G 74C>A 1178G>A 1354C>T 1 2 3 Linked with -1438A>G G ↓ expression His452Tyr Thr25Asn -1438A>G Associated with SSRI response and side effects and antipsychotic response GG genotype responds better and has higher adverse drug reactions to SSRIs GG genotype associated with poorer response to clozapine and typical antipsychotics 74C>A Not been studied in vivo In vitro studies show a 30 fold decrease in aripiprazole agonist potency for the A allele; individuals with the A allele are expected to respond poorly to the drug 1178G>A Better response of the AA genotype to citalopram 1354C>T TT and CT genotypes are associated with poorer response to clozapine Interpretive Handbook (Mayo Medical Laboratories (www.mayomedicallaboratories.com/) Mrazek (2010) Psychiatric Pharmacogenomics 21/25

Variable promoter tandem repeat HTR2C 2 3 -759C>T 1 4 5 6 -997G>A -697G>C 69G>C Cys23Ser Variable promoter tandem repeat Located on the X chromosome Males are hemizygous Polymorphisms may affect males and females to a different extent Adverse effects of antipsychotics Weight gain Tardive dyskinesia -697G>C tag SNP Linkage with tandem repeat, -997G>A, -759C>T and 69G>A CT and TT genotypes are associated with less weight gain caused by antipsychotic therapy Interpretive Handbook (Mayo Medical Laboratories (www.mayomedicallaboratories.com/) Mrazek (2010) Psychiatric Pharmacogenomics 22/25

Impact on Clinical Care and Practice The majority of pharmacogenes (ADME genes) are highly polymorphic leading to a wide range of activity Pharmacogenetic testing can be highly valuable for the individual patient (precision medicine) Often controversial or void of literature regarding the association between genetic variation and drug response Genotype tests cover the most common gene variants Rare/novel/complex variants may not be captured Multitude of barriers that need to be overcome CPIC guidelines How to interpret a genotype Guidance for drug choice and dosage 23/25

The individual patient To individualize patient care History Labs, imaging, etc [Pharmaco]Genetics Evidence-based pharmacology and drug therapy 24

Tribute to David Mrazek, MD Pioneer in Psychiatric Pharmacogenomics 25/25