1. 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

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

3. 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

4. Drugs: 138 Entries: 155 ≥2 entries: 14 CYP2D6: 37 CYP2C19: 14
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5. SSRI PD and PK Pathways SLC6A4 HTR CYP2D6
Brain-derived neurotrophic factor
PharmGBK.org
Whirl-Carillo et al (2012) CPT 92:414-17
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6. 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
Zhou (2009) Clin Pharmacokinet 48: (Part 1) and 48: (Part 2)
Gaedigk et al (2008) CPT 83:
Llerena et al (2008) Pharmacogenomics 10:17-28 6

7. 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
The Human Cytochrome P450 (CYP) Allele Nomenclature Database at
Over 100 allelic variants and subvariants defined to date
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8. 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
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Gaedigk (2013) Int Rev Psych 25:534-53
Tandem arrangements 8

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

10. 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
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11. Whole genome sequencing
2D D7
2D6
NGS reads are short ( bp)
Algorithms align reads to reference sequence
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Gaedigk et al (2013) ISSX and manuscript in preparation

12. CYP2D6 challenge: accurate alignments
2D D7
2D6
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13. 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
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14. 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
and
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15. CPIC guidelines for CYP2D6
Update
Epub Jan
Published 2013
Guideline for CYP2D6 and CYP2C19 Genotypes and Dosing for SSRIs
early stages
Epub Jan
15/25

16. CYP2D6 and CYP2C19/TCA
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17. 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
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18. SLC6A4
Gly56Ala
Ile425Val
Promoter
Variable number of
GC-rich, 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
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19. 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 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 (
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20. 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
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21. 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 (
Mrazek (2010) Psychiatric Pharmacogenomics
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22. 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 (
Mrazek (2010) Psychiatric Pharmacogenomics
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23. 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
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24. The individual patient
To individualize patient care
History
Labs, imaging, etc
[Pharmaco]Genetics
Evidence-based pharmacology and drug therapy 24

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