Gerry Higgins PhD, MDAdjunct Research Professor, Computational Medicine and

Bioinformatics, University of Michigan Medical School;Vice President, Pharmacogenomic Science, Assurex Health, Inc.

PSYCHIATRIC PHARMACOGENOMICS:

Clinical Translation & Novel Variant Discovery

Conflict of interest statement:

Dr. Gerald A. Higgins is an employee of AssureRx Health, Inc. (Mason, OH) and holds options in the company. He serves as an Adjunct Research Professor at the University of Michigan Medical School. He states that he has no financial interest in the response to this presentation.

CLINICAL TRANSLATION

The Impact of Psychiatric Disease→More than 350 million people are affected by

depression;→It is the number 1 cause of disability, U.S. and

worldwide;→Almost 2/3 of those who commit suicide have

depression; →34% of female patients diagnosed with bipolar

disorder attempt suicide, 19% of male bipolar patients attempt suicide;

→Majority of perpetrators of mass shootings in the U.S. since 1970 displayed psychiatric symptoms including paranoia, delusions, and depression, and 74% were on psychiatric medications; but

→In the new 2016 NIH budget, $5.6 B is earmarked for cancer research, only $404 M for depression research and $259 M for research on schizophrenia.

Challenges in Psychiatric Pharmacogenomics→ No usable objective diagnostic measures in clinical

practice, must rely on surrogate phenotypes and rating scales;

→Comorbid psychiatric conditions and comorbid non-psychiatric conditions confound differential diagnosis;

→Most psychiatric medications were developed in the 1950’s, including lithium, chlorpromazine and imipramine;

→There has been virtually no CNS drug development since 2000, with the exception of ketamine-like antidepressants;

→79 M Americans were prescribed psychiatric medications in 2014 - 25% of the U.S. population;

→Psychiatric medications exhibit unacceptably high levels of adverse events – 90,000 annual visits to the ER in the U.S. – 50% of all prescription drug-related hospitalizations.

David Mrazek, Psychiatric Pharmacogenomics & Assurex Health, Inc.

CYP2C19

CYP2D6

Use with Caution

CYP1A2

SLC6A4

5HTR2A

Genotype Phenotype Interpretive Drug Sorting

Medication Binning

Effect of Composite

Phenotype on Each Drug

Use as Directed

Use with Caution and More Frequent

Monitoring

Metabolizer Status

Metabolizer Status

Metabolizer Status

Transporter Activity

Receptor Activity

CYP2C9 Metabolizer Status

One reason why pharmacogenomic testing is adopted in psychiatry, but not in cardiology

(e.g., warfarin, clopidogrel)

Cardiologists:17% in solo practice in U.S.

Psychiatrists:85% in solo practice in U.S.

Another reason why pharmacogenomic testing is being adopted in psychiatry?

ANXIETY DISORDERS DEPRESSIVE DISORDERS COMORBID DISORDERS$0.00

$2,000.00

$4,000.00

$6,000.00

$8,000.00

$10,000.00

$12,000.00

$14,000.00

INCONGRUENT WITH GENESIGHT TEST RESULTS CONGRUENT WITH GENESIGHT TEST RESULTS

p <0.0001

$6874 $3579

p <0.007 p <0.0001

$4056

Winner, JG et al. Combinatorial pharmacogenomic guidance for psychiatric medications reduces overall pharmacy costs in a 1 year prospective evaluation. Current Med. Res. & Opinion. 2015. 1–11. doi:10.1185/03007995.2015.1063483

ANOVA one way test

Status – Clinical Translation, Assurex PGx testsTo date: Several published trials → Demonstration of clinical validity & utility, albeit with small numbers of patients;

Ongoing:Almost 30 clinical trials underway, many investigator-initiated, including 1 large RCT involving the National Network of Depression Centers (NNDC) and many other sites;

Reimbursement:Only genotype-based test in pharmacogenomics approved for reimbursement by the Centers for Medicare and Medicaid Services (CMS) nationwide;

Challenges:How to keep improving the science – success fosters competition;FDA regulation of LDTs is on the horizon.

PHARMACOGENOMIC VARIANT DISCOVERY

Novel Pharmacogenomic Variants & Pathways→Higgins GA, Allyn-Feuer A, Handelman S, Sadee W, Athey BD. The

epigenome, 4D nucleome and next-generation neuropsychiatric pharmacogenomics. Pharmacogenomics. 16(14), 1649–1669 (2015).

→Higgins GA, Allyn-Feuer A, Athey BD. Epigenomic mapping and effect sizes of noncoding variants associated with psychotropic drug response. Pharmacogenomics. 16(14), 1565–1583 (2015).

→Higgins GA, Allyn-Feuer A, Barbour E, Athey BD. A glutamatergic network mediates lithium response in bipolar disorder as defined by epigenome pathway analysis. Pharmacogenomics. 16(14), 1547–1563 (2015).

→Wiley JW, Higgins GA, Athey BD. Stress and glucocorticoid receptor transcriptional programming in time and space: Implications for the brain–gut axis. In press

→Higgins GA, Allyn-Feuer A, Barbour E, Austic E, Athey BD. Candidate pharmacodynamic variants for stratification of ketamine antidepressant response are associated with chromatin-mediated neuroplasticity. In preparation

→Higgins GA, Allyn-Feuer A, Barbour E, Athey BD. A novel antidepressant network in human brain mediates antidepressant response. In preparation

Collaborators

Brian D. Athey, Ph.D.

Ari Allyn-Feurer Edward Barbour

John W. Wiley, M.D. Wolfgang Sadee, Dr.rer.nat

The majority of assignable SNPs from GWAS in neuropsychiatric pharmacogenomics are located

within putative enhancers

Intron; 1161; 57%

Intergenic; 228; 11%

5'UTR; 324; 16%

3'UTR, 190, 9%Coding variant; 121; 6%

A. Location of lead SNPs and small indels reported in pharmacogenomic GWAS (from NHGRI)

Enhancer; 23Enhancer boundary, 1

Promoter boundary; 3

Promoter, 3

Transcribed; 3

B. SNPs that could be assigned using our bioinformatics pipeline & the Epigenome Roadmap

Effect sizes of pharmacogenomic SNPs are large

DILI: Drug-induced liver injury;SAE: Safety and adverse events;SSR: Selective serotoninergic reuptake inhibitors.

Used with permission from:Lilian Warren, Statistical Genetics,

GlaxoSmithKline.

Effect sizes of SNPs from neuropsychiatric GWAS are large

Higgins GA, Allyn-Feuer A, Athey BD. Epigenomic mapping and effect sizes of noncoding variants associated with psychotropic drug response. Pharmacogenomics. 16(14), 1565–1583 (2015).

Tagging SNPs for enhancers that cause drug-induced cutaneous injury

Addiction SNPs for enhancers

Analgesia SNPs

Lithium-response SNP that disrupts a promoter

Spatial associations elucidate adverse drug events in trans & cisA. The SNP rs4959235 within an intron of

SLC22A23 is significantly associated with Seroquel® (quetiapine)-induced prolonged QT interval in some patients [1]. It identifies a known enhancer.

B. In trans, this enhancer contacts the transcription start site of the NOS1AP gene.

C. Mutations in the NOS1AP gene, as well as altered expression, mediate drug-induced prolonged QT syndrome:

• Chang KC et al. Nitric oxide synthase 1 adaptor protein, an emerging new genetic marker for QT prolongation and sudden cardiac death. Acta Cardiol. Sin. 29:217–225. (2013)

• Jamshidi Y et al. Common variation in the NOS1AP gene is associated with drug-induced QT prolongation and ventricular arrhythmia. J. Amer. Coll. Cardiol. 60: 841-850. (2012)

• Kapoor A et al. QT Interval-International GWAS Consortium: An enhancer polymorphism at the cardiomyocyte intercalated disc protein NOS1AP locus is a major regulator of the QT interval. Amer. J. Human Genet. 94: 854-869. (2014)

• Tomás M, Napolitano C, De Giuli L et al. Priori Polymorphisms in the NOS1AP gene modulate QT interval duration and risk of arrhythmias in the long QT syndrome. J. Amer. Coll. Cardiol. 55: 2745-2752. (2010)

[1] Aberg K et al. Genome-wide association study of antipsychotic-induced QTc interval prolongation. Pharmacogenomics. 12:165–72. (2012).

Epigenome pathway analysis identifies a lithium-response network in human

brain

SNPs from 13 published studies on gene-SNP association and 2 GWAS of lithium response in bipolar disorder.

SNPs from 18 GWAS on bipolar disorder, other psychiatric disorders, and adverse events associated with lithium response.

19 SNPs after LD pruning, in non-coding domains of 10 genes

Set of 23,312 SNPs in LD (r2>0.8) with reported lead SNPs in same populations

Impute SNPs using genetic and epigenetic fine mapping

Lithium Response

802

217LithiumRespons

e SNPs

[1] Higgins GA, Allyn-Feuer A, Barbour E, Athey BD. A glutamatergic network mediates lithium response in bipolar disorder as defined by epigenome pathway analysis. Pharmacogenomics. 16(14), 1547–1563 (2015).

SNP Imputation and Bioinformatics Pipeline [1]

Gene set enrichment followed by ‘blinded’ pathway analysis using IPA®

shows that they are associated with a single CNS network (p = 1 x 10-26)

Subsequent publications by members of the NIH-funded Consortium on Lithium Genetics (ConLiGen)

In press, Nature

Same gene set and subset of the same SNPs, tested in a clinical population of lithium-responsive bipolar patients & controls.

SNP rs116323614 within an intron of the SESTD1 gene reached p < 2.47 x 10-08 in a GWAS of lithium-responders versus controls. SNP rs201013542 in high LD, is found in human brain.

“SESTD1: SEC14 and spectrin domains 1, a calcium channel regulator that regulates neurite elongation. Protein acts in the phosphoinositide signaling pathway, and interacts with RAC1 and GSK3B.” –TransFac Pro

Top pathway hit in Panther/ GO when entire gene set was entered:

Labeled as phosphoinositide signaling pathway by Panther/GO

PI3K

ATK1

PDK1

RAC1

SESTD1

Adverse events of treatment with lithium salts:Correlations with genes in this pathway

1.SBSN (Subsarin) & alopecia universalis: • Patel, Mansi, Shannon Harrison, and Rodney Sinclair. Drugs and hair

loss. (2013). Dermatologic Clin. 31(1), 67-73.• Lesiak, K., Bartlett, J. R., & Frieling, G. W. (2015). ‘Drug-Induced

Alopecia’. In Cutaneous Drug Eruptions (pp. 215-227). Springer London.

2.HTR1A (5-hydroxytryptamine (Serotonin) Receptor 1A) & weight gain:• Murphy E & McMahon FJ. Pharmacogenetics of antidepressants, mood

stabilizers, and antipsychotics in diverse human populations. Dis. Med. (2013). 16(87), 113-122.

• Wysokinski A & Kloszewska I. Mechanisms of increased appetite and weight gain induced by psychotropic medications. (2014). J. Advanced Clin. Pharmacol. 1.1, 12-33.

• Can A et al (2014). Molecular actions and clinical pharmacogenetics of lithium therapy. Pharmacol. Biochem. Behavior. 123, 3-16.

3. SLC1A2 (Solute Carrier Family 1 Member 2) & hand tremor:

• Their S et al. Polymorphisms in the glial glutamate transporter SLC1A2 are associated with essential tremor. Neurology. 79(3), 243-248 (2012);

• Brandler WM. Common variants in left/right asymmetry genes and pathways are associated with relative hand skill. PLoS Genetics. 9(9):e1003751 (2013);

• Their S et al. Polymorphisms in the glial glutamate transporter SLC1A2 are associated with essential tremor. Neurology. 79(3), 243-248 (2012).

4. CACNA1c (Subsarin) & Brugada Syndrome (lithium unmasking of long QT and sudden cardiac death)-

SNP rs1016388 located within an intron of the CACNA1C gene

is significantly associated with Brugada Syndrome

[1] Invitae® Brugada syndrome panel: https://www.invitae.com/en

SNP POSITION LOCATIONCHROMATIN

STATE

Left Ventricle

rs4756764 11: 13280971 Intron of ARNTL Enhancer

rs2314339 17: 40096958 Intron of NR1D1 Genic enhancer

rs12412727 10: 62113906 Intron of ANK3 Enhancer

rs1016388 12: 2322118 Intron of CACNA1C Enhancer

rs4354668 11:35440726 5’UTR, SLC1A2 Promoter- TSS

“Brugada syndrome is a disorder characterized by sudden death associated with one of several ECG patterns characterized by incomplete right bundle-branch block and ST-segment elevation. It can be drug-induced, with lithium accounting for the majority of the cases. Lithium “un-masks” an inherent heart defect, commonly in carriers of the intronic CACNA1C SNP rs1016388 [1].”

SNPs in bipolar risk genes SNPs in lithium response genes = Pharmacogenomic: Disease Coupling

GENE SIGNIFICANT LITHIUM HITS SIGNIFICANT BIPOLAR HITS

ANK3PMID:19448189; PMID:23378337; PMID: 24625017; PMID:25377609; PMID:14609504

PMID:18711365; PMID:20351715;PMID:21926972; PMID:22182935; PMID:23453885PMID:24618891

ARNTL PMID:22384149; PMID:24636202; PMID:24679394

PMID: 16528748; PMID: 18228528;PMID: 19839995; PMID: 24679394

CACNA1C

PMID: 25194313; PMID: 25445986;PMID: 25678966; PMID: 26476274

PMID: 18711365; PMID: 20351715; PMID: 21926972; PMID: 23453885; PMID: 24280982; PMID: 25056061

CANG2 PMID: 18408563 PMID: 18408563; PMID: 23038240

DLG4 PMID: 26297903 PMID: 21559497; PMID: 23759419;PMID: 22005598

GRIA2 PMID: 19448189; PMID: 24885933 PMID: 22122651; PMID: 25308386

GSK3B

PMID: 17628506; PMID: 19754466; PMID: 22990942; PMID: 24033914; PMID: 24677591 PMID: 24992082; PMID: 25345732; PMID: 26267417

PMID: 20492846; PMID:20668436; PMID: 14729229; PMID: 21886606 PMID: 21922001;PMID: 23449817; PMID: 25041379; PMID: 25435019

HTR1A PMID: 10893489; PMID: 10758249; PMID: 20159346; PMID: 24534415

PMID: 20594600; PMID: 22752684; PMID: 23926239

NR1D1 PMID: 19818381; PMID: 21781277; PMID: 24625017 PMID: 19267705; PMID: 18804497

SLC1A2

PMID: 23023733; PMID: 24354796 PMID: 24534415 PMID: 23023733; PMID: 25406999

Candidate pharmacodynamic variants for stratification of ketamine antidepressant response are associated with chromatin-

mediated neuroplasticity

Regulatory variants were examined in pharmacodynamic targets

Bioinformatics pipelineImpute SNPs from GWAS and gene association studies using epigenetic fine

mapping SNPs from clinical trials using knowledge –based discoverywww.clinical-trials.gov

Learning machine to detect regulatory variants [2]

Transcription factor annotation and Hi-C mapping [4]

Phylogenetic module complexity analysis to detect causal SNPs [6]Learning machine to detect splice

variants [5]

Examine GTEx data on normal RNA-Seq [3]

Map mRNA using Allen Brain Atlas [1]

OUTPUT

[1] Sunkin SM et al. Allen Brain Atlas: an integrated spatio-temporal portal for exploring the central nervous system. Nuc. Acids Res. 41, D996-D1008 (2013).

[2] Lee D et al. A method to predict the impact of regulatory variants from DNA sequence. Nature Genet. (2015) doi:10.1038/ng.3331

[3] Pirinen M et al. Assessing allele-specific expression across multiple tissues from RNA-seq read data. Bioinformatics. btv074 (2015).

[4] Higgins GA et al. Epigenomic mapping and effect sizes of noncoding variants associated with psychotropic drug response. Pharmacogenomics. 16(14), 1565–1583 (2015).

[5] Vaquero-Garcia J et al. A new view of transcriptome complexity and regulation through the lens of local splicing variations, preprint

[6] Claussnitzer M et al. Leveraging cross-species transcription factor binding site patterns: From diabetes risk loci to disease mechanisms. Cell. 156, 343–358 (2014). doi:10.1016/j.cell.2013.10.058

Microarray analysis suggests that several pharmacodynamic genes are targets

Hippocampal formation, parahippocampal gyrus and inferior temporal cortex:

Dorsolateral frontal cortex:

Microarray shows that CYP2B6 and CYP2D6 are expressed in brain as well

Putative Pharmacogenomic Variants that may Impact Transcription

Putative Pharmacogenomic Variants that may Impact Transcription

Promoter_BoundaryGRIN2B. rs3764028_G;SK-N-SH cells.

Gene Set Enrichment of Spatial Contacts by Hi-C

Others have found similar results

Bharadwaj R et al. Conserved higher order chromatin regulates NMDA receptor gene expression and cognition. Neuron. 84, 997–1008 (2014):

Thakurela S et al. Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity. Genome Res. 25, 1–16 (2015):

Summary – Novel Variant Discovery→ These are methodological approaches for discovery of

candidate pharmacoepigenomic variants – their function and clinical utility must be demonstrated in biological experiments and clinical studies;

→For the lithium response pathway we discovered using bioinformatics analysis, there are indications that others have found similar results using the same variants in the pathway, or closely related to the pathway, in a subsequent clinical study and a GWAS;

→ We believe that these strategies will uncover what some have called “missing hereditability” in pharmacogenomic response and adverse events, and how the epigenome impacts variation in gene regulation through presumptive environmental effects.

Is lithium response an inherited trait?

From: Song J et al. Genome-wide association study identifies SESTD1 as a novel risk gene for lithium responsive bipolar disorder. Mol. Psychiatry. 1–8 (October 2015). doi:10.1038/mp.2015.165

Lithium response in bipolar disorder is a genetic trait: PCA analysis of familial clusters

Excellent lithium-responders

Poor lithium response, but excellent response to lamotrigine

Poor lithium-responders, may respond with antipsychotics

Goldstein D et al. Principal component analysis of lithium and lamotrigine-responsive and treatment-resistant pedigrees in bipolar disorder reveals distinct pharmacogenomic population structure. Genomics. 106 (2015).

Lithium rescues loss of brain tissue loss in bipolar disorder

Amygdala and hippocampus: → 15 studies published between 2009 and 2015 to datePrefrontal cortex: → 6 studies published between 2009 and 2015 to dateAnterior caudate, striatum, thalamus and/or nucleus accumbens: → 4 studies published between 2009 and 2015 to dateCingulate cortex: → 3 studies published between 2009 and 2015 to dateInsular cortex: → 2 studies published between 2009 and 2015 to dateAngular gyrus: → 2 studies published between 2009 and 2015 to dateInferior temporal cortex: → 1 study published between 2009 and 2015 to dateOccipital cortex: → 1 study published between 2009 and 2015 to dateCerebellum: → 1 study published between 2009 and 2015 to date

From: Burmeister M, McInnis MG, Zöllner S. Psychiatric genetics: progress amid controversy. Nature Rev. Genet. 9, 527-540 (2008).

The 4D nucleome

“Most causal variants are located in the regulatory part of the genome, not in the exome.”

[1] Kellis M et al. PNAS. 111:6131–6138 (2014).[2] Albert, FW, Kruglyak L. Nature Reviews Genetics (2015).

PHARMACOEPIGENOMIC VARIANT DISCOVERY