1

Unraveling Persistent Pain Conditions

American College of Rheumatology

Chicago

November 9, 2011

William Maixner DDS, PhD

Center for Neurosensory Disorders

University of North Carolina

Disclosure: Algynomics Inc. Cofounder and equity shareholder

ENV

IRO

NM

ENTA

L CO

NTR

IBU

TION

High Psychological Distress

High State of Pain Amplification

Anxiety

Depression

Stress Response

Impaired Pain

Regulatory Systems

Pro-inflammatory

State Blood Pressure

Na+, K+-ATPase

Serotonin transporter

BDNF

12q11.2

Cannabinoid receptors

MAO

11q23

Adrenergic receptors NMDA POMC

COMT

Interleukins

5q31-32 22q11.21

Opioid receptors Prodynorphin DREAM NGF

IKK NET

Somatization

Tissue Injury

CREB1

Serotonin receptor GR

Dopamine receptors

Mood

GAD65 CACNA1A

Acute and Persistent Pain Conditions

6q24-q25 1p13.1 5q31-q32 9q34.3 Xp11.23

Hypothesized Risk Determinants

Diatchenko et al, Pain 123: 226-30, 2006

Heritability

Clinical Pain

• Low back pain 30-68%

• Fibromyalgia 51%

• Neck pain 34-58%

• Knee pain 44%

• Osteoarthritis 30-46%

• Pelvic pain 41%

• Headache

– Males 39-44%

– Females 49-58%

Experimental Pain

• Punctuate hyperalgesia 55%

• Heat pain threshold 53%

• Pain during burn induction 34%

• Itch after histamine iontophoresis 35%

• Pain during acid iontophoresis 31%

• Pain during ATP iontophoresis 22%

• Brush evoked allodynia area 25%

• Cold pressor 60%

• Contact heat 26%

• Pressure threshold 10%

Functional Class Gene Condition Transporters SLC6A3, or DAT1 PTSD

SLC6A4, or 5-HTT, or SERT FMS, CFS, D-IBS, migraine, TMJD

ABCB1, or MDR1 Efficacy of µ-opioid analgesia

Metabolic genes, enzymes, and transcription

regulators COMT TMJD, analgesia, migraine, FMS

CYP2D6 Opioid analgesia

GCH1 LBP

MTHFR MA

ACE MA

SPTLC1, or SPT1 HSAN I

HSN2 HSAN II

IKBKAP, or IKAP HSAN III

NGFB HSAN V

Receptors NTRK1, or TRKA HSAN IV

ADRA2A, ADRA2C C-IBS

ADRB2 TMJD

DRD2 MA, PTSD

DRD4 MO, FMS

MC1R Opioid analgesia

OPRM1, or MOR µ-opioid analgesia

5-HTR2A MA,TMJD

Cytokines IL1A, IL1B, and IL1RN LBP

IL1RN VVS

IL1B VVS, BMS

IL6 LBP

IL10 IBS

TNF IBS

LTA MO

Ion channels ATP1A2 BM, FHM type 2

KCNS1 LBP, Neuropathic

CACNA1A MA, FHM type 1

CACNA2D3 LBP

SCN1A FHM type 3

SCN9A IL6, or IFNB2 PE, PEPD, CAIP, CIDP Diatchenko et al.. Trends in Genetics 23:605-13, 2007 and Kim et al. Journal of Pain 10:663-693, 2009

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Orofacial Pain: Prospective Evaluation and Risk Assessment

William Maixner, Program Director Site PIs Richard Ohrbach, Univ. at Buffalo Joel Greenspan, Univ. of Maryland Roger Fillingim, Univ. of Florida Core Directors Gary Slade, Epidemiology Core Luda Diatchenko, Genomics & Bioinformatics Core Charles Knott, Data Coordinating Center External Advisory Committee Gary Macfarlane, Chair

OPPERA Study Design • 5-year prospective cohort study of first-onset TMD

• 5-year prospective cohort study of first-onset TMD

– 3,276 initially-TMD-free adults aged 18-44 years were recruited by community-wide advertisement in FL, MD, NY and NC

– Baseline interviews, questionnaires, physical examination, quantitative sensory testing and blood sample collection

– Follow-up questionnaires are sent once every three months to identify potential incident cases

– Re-examination of potential cases to verify TMD case status based on research diagnostic criteria

• Three additional, nested studies

– 3,276 initially-TMD-free adults aged 18-44 years were recruited

ENV

IRO

NM

ENTA

L CO

NTR

IBU

TION

High Psychological Distress

High State of Pain Amplification

Anxiety

Depression

Stress Response

Impaired Pain

Regulatory Systems

Pro-inflammatory

State Blood Pressure

Na+, K+-ATPase

Serotonin transporter

BDNF

12q11.2

Cannabinoid receptors

MAO

11q23

Adrenergic receptors NMDA POMC

COMT

Interleukins

5q31-32 22q11.21

Opioid receptors Prodynorphin DREAM NGF

IKK NET

Somatization

Tissue Injury

CREB1

Serotonin receptor GR

Dopamine receptors

Mood

GAD65 CACNA1A

6q24-q25 1p13.1 5q31-q32 9q34.3 Xp11.23

Generalized Persistent Pain Conditions Psychological Constructs

Psychological Risk

Somatic Awareness

Stress

Coping

Mood/Affect

Global Psychological

Function

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Adjusted Standardized Odds Ratios for Psychosocial Variables

Sta

ndar

diz

ed O

dds

Rat

io

Standardized odds ratios were adjusted for study site, sex, and race/ethnicity

ENV

IRO

NM

ENTA

L CO

NTR

IBU

TION

High Psychological

Distress

High State of Pain Amplification

Anxiety

Depression

Stress Response

Impaired Pain

Regulatory Systems

Pro-inflammatory

State Blood Pressure

Na+, K+-ATPase

Serotonin transporter

BDNF

12q11.2

Cannabinoid receptors

MAO

11q23

Adrenergic receptors NMDA POMC

COMT

Interleukins

5q31-32 22q11.21

Opioid receptors Prodynorphin DREAM NGF

IKK NET

Somatization

Tissue Injury

CREB1

Serotonin receptor GR

Dopamine receptors

Mood

GAD65 CACNA1A

6q24-q25 1p13.1 5q31-q32 9q34.3 Xp11.23

Generalized Persistent Pain Conditions

Pain Sensitivity Constructs • Pressure Pain Thresholds (PPT): tested at multiple sites bilaterally

Temporalis, Masseter, TMJ, Trapezius, Lateral Epicondyle

• Cutaneous Mechanical Pain Threshold and Suprathreshold Ratings: tested on digit dorsum (also ratings of aftersensation)

• Heat Pain Threshold, Tolerance, and Suprathreshold Ratings: Tested on ventral forearm (also ratings of aftersensation)

Odds Ratios (adjusted for sex, age, race/ethnicity, and study site) for Pain Sensitivity Measures Distinguishing TMJD Cases from Controls

N.B.: For threshold and tolerance measures, the original metric was reverse-coded, so the odds ratio

represents the relative increase in odds of having TMJD with greater pain sensitivity for all measures.

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ENV

IRO

NM

ENTA

L CO

NTR

IBU

TION

High Psychological

Distress

High State of Pain Amplification

Anxiety

Depression

Stress Response

Impaired Pain

Regulatory Systems

Pro-inflammatory

State Blood Pressure

Na+, K+-ATPase

Serotonin transporter

BDNF

12q11.2

Cannabinoid receptors

MAO

11q23

Adrenergic receptors NMDA POMC

COMT

Interleukins

5q31-32 22q11.21

Opioid receptors Prodynorphin DREAM NGF

IKK NET

Somatization

Tissue Injury

CREB1

Serotonin receptor GR

Dopamine receptors

Mood

GAD65 CACNA1A

6q24-q25 1p13.1 5q31-q32 9q34.3 Xp11.23

Generalized Persistent Pain Conditions Association Methods

Candidate Gene Genome-Wide Association

Hypothesis driven (“confirmatory”) Hypothesis neutral (“exploratory”)

Relatively inexpensive per sample Expensive per sample (but price decreasing)

Relatively expensive per SNP Very inexpensive per SNP

Good power in moderately sized studies Requires very large sample sizes

Results are easily interpreted Results may require much work to interpret

Limited to few genes at a time “Genome-wide” coverage

Pain Research Panel

Assessment of 3295 SNPs from 350 genes implicated in key pathways that regulate the perception of pain • Affymetrix ParAllele microarray platform using Molecular Inversion Probe (MIP) technology • 3 domains relevant to hypothesized risk pathways • Genes coding for proteins that mediate or modify the therapeutic effects of pharmacological agents used to treat pain • SNP choice selective for putatively functional loci • LD coverage of entire gene at r2 > 0.8 • 160 ancestry-informative markers

Nociceptive

transmission

Inflammation

Mood and affect

Association Tests

Logistic Regression model: y = βØ + β1(allele dosage) + β2(sex) + β3-6(5 sites) + β7-8(2 race eigenvectors) + e

CHR SNP GENE Call Rate MAF (W) MAF (B) OR Joint_P OPPERA_P UNC_P

5 rs2963155 NR3C1 99.88% 0.22 0.24 0.62 5.22E-05 0.0098 0.0014

13 rs9316233 HTR2A 99.94% 0.19 0.36 0.64 0.00039 0.049 0.00023

5 rs3756612 CAMK4 100.0% 0.18 0.05 1.51 0.00064 0.011 0.024

7 rs7800170 CHRM2 99.95% 0.48 0.37 0.72 0.00087 0.040 0.098

7 rs728273 IFRD1 99.56% 0.41 0.69 1.38 0.0010 0.0029 0.026

349 TMD cases vs 1612 controls

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0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 1 2 3 4

observed

expected

λ=1.00

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Association Tests

Logistic Regression model: y = βØ + β1(allele dosage) + β2(sex) + β3-6(5 sites) + β7-8(2 race eigenvectors) + e

348 combined TMD cases vs 1612 controls: Tier 1

λ=1.00

CHR SNP GENE Call Rate MAF (W) MAF (B) OR P

1 rs3024496 IL10 100.0% 0.52 0.36 0.76 0.0059

4 rs7696139 ADRA2C 99.64% 0.22 0.60 0.74 0.0072

20 rs1556832 ADRA1D 100.0% 0.53 0.23 1.29 0.0082

1 rs1800896 IL10 99.95% 0.53 0.27 0.77 0.0086

1 rs2236857 OPRD1 99.85% 0.27 0.33 1.32 0.0087

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2 2.5

-lo

g(p

-val

ue)

expected -log(p-value)

AD

RA

2C

IL1

0

AD

RA

1D

OP

RD

1

CO

MT

Putative Genetic Polymorphims Associated with TMD Case Status

Gene Protein Function

NR3C1 Glucocorticoid receptor gene HPA Axis Function and inflammation

HTR2A Serotonin 2A receptor Pain transmission , TMD, CWP

CAMK4 Calcium/calmodulin-dependent protein kinase 4 gene

Pain transmission and opioid analgesia

CHRM2 Muscarinic cholinergic receptor 2 Mood and inflammation

IFRD1 Interferon-related developmental regulator 1 Induced by NFG, neutrophil function

GRK5 G protein-coupled receptor kinase 5 Regulation of G protein-coupled receptors including ADRB2

COMT Catecholamine-O-transferase Pain transmission, TMD and opioid function

ADRA2C Alpha-2C Pain transmission

OPRD Delta opioid receptor Pain transmission

IL10 Interleukin 10 Inflammation and Pain

GRIN2A Ionotropic N-methyl-D-aspartate (NMDA) receptor 2A

LTP, Pain transmission

Association Tests

Logistic Regression model: y = βØ + β1(allele dosage) + β2-4(4 sites) + β5-6(2 race eigenvectors) + e

CHR SNP GENE Call Rate MAF (W) MAF (B) OR P

4 rs1563826 EREG 100.0% 0.21 0.51 0.41 3.66E-05

14 rs10498313 PRKD1 97.48% 0.21 0.15 1.89 0.00011

1 rs2236857 OPRD1 99.93% 0.27 0.32 1.83 0.0019

5 rs2963155 NR3C1 99.98% 0.22 0.24 0.52 0.0021

7 rs1140475 EGFR 100.0% 0.12 0.08 2.08 0.0023

127 TMD cases and 231 “supercontrols”

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0.5

1

1.5

2

2.5

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3.5

4

4.5

5

0 1 2 3 4

observed

expected

λ=1.00

Future Directions

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Need to Stratify Heterogeneous Populations into Homogenous Subgroups

• Not all pain patients are created equal – several pathways to pain and suffering

• Need to assess intermediate phenotypes associated with causal pathways

• Statistical approaches that identify relatively homogenous patient subgroups or clusters based on intermediate phenotypes and clinical signs and symptoms. – Principal Component Analysis to determine latent constructs – Clustering of latent constructs – Machine Learning

• Integrate the information associated with many polymorphisms - each

expressing a relatively small effect on the phenotype or cluster of interest.

An Example of Stratification and Drug Target Identification

Descriptive Statistics from Questionnaires Age (years)

Sex (% female)

Race (% white)

Fibromyalgia Impact Questionnaire (FIQ) Total Score

Fibromyalgia Health Assessment Questionnaire (HAQ)

Disability In Dressing And Grooming

Disability In Arising

Disability In Eating

Disability In Walking

Disability In Hygiene

Disability In Reach

Disability In Grip

Disability In Daily Activities

Multidimensional Assessment of Fatigue (MAF)

Severity Of Fatigue

Distress Of Fatigue

Interference With Daily Life

Frequency Of Fatigue In Last Week

Medical Outcomes Study – Sleep Scale (MOS-Sleep)

Sleep Disturbance

Sleep Adequacy

Sleep Somnolence

Hours Of Sleep

Short Form McGill Pain Questionnaire (McGill)

Affective Component Of Pain

Sensory Component Of Pain

Pain In Past Week (VAS)

Sheehan Disability Scale (SDS)

Social Life Disruption

Family/Home Disruption

Daily Diary Measures

Mean Pain (VAS)

Mean Sleep Quality (VAS)

Short Form 36 (SF36)

General Health

Physical Function

Role Physical

Bodily Pain

Vitality

Social Function

Role Emotional

Mental Health

Hospital Anxiety and Depression Scale (HADS)

• Overlap of factors, with single factor predominating in each cluster • Multiple factors represent challenge for therapy selection

Factor and Cluster Analysis

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SNP Associations of FM Cases vs Controls

The PCA Component Associated with Pain Research

Panel SNPs as Quantitative Traits in a Linear Regression, Incorporating Age as a Covariate

Clusters Contrasted Against Healthy Controls to

Detect SNPs Associate with Subtypes of FM

New biomarkers

and drug targets

Knowledge

Base

MedScan®

Text

ResNet Database

Pain Panel SNP Data

Creating Biological Pathways Using SNP Data from

Pain Research Panel

1.25 million events of

regulation between

compounds, proteins,

and cellular processes 161 pathways

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Biological Pathways Associated with Clusters Cluster 1 (n=67)

High Pain Cluster 3 (n=66)

Low Pain Cluster 4 (n=49) Non- Fatigued

Cluster 5 (n=24) Effective Sleepers

PATHWAY P PATHWAY P PATHWAY P PATHWAY P

NTRK -> FOXO/MYCN

0.007 CCR1 -> STAT 0.042 T-cell receptor ->

CREBBP 0.018

EGFR/ERBB ->

STAT 0.007

NGFR -> MEF 0.015

TGFBR ->

ATF/GADD/MAX/T

P53

0.029

Skeletal

Myogenesis

Control

0.013

TLR3 -> NF-kB 0.043 TGFBR ->

MEF/MYOD/MYOG 0.029

EGFR/ERBB2 ->

CTNNB 0.016

NTRK -> AP-

1/CREB/ELK-

SRF/MYC/SMAD3

/TP53

0.048 CCR5 -> TP53 0.044 Gonadotrope Cell

Activation 0.028

NGFR -> NF-kB 0.056 CCR2/5 -> STAT 0.079

AngiotensinR ->

STAT 0.031

TGFBR ->

CREB/ELK-SRF 0.082 EGFR -> CTNND 0.038

EGFR -> ZNF259 0.038

UNIFYING THEMES:

NTRK & NGFR CCR1 TGFBR & CCR2/5 EGFR

OPPERA TMD Pathways Data (Extreme Phenotypes)

Pathway

# related

pathways P Values

EGFR -> signaling pathways 7 0.00129

GFR ->signaling pathways 3 0.00516

TGFBR -> signaling pathways 3 0.01026

AdenosineR -> AP-1 signaling 0.01361

FibronectinR -> AP-1/ELK-SRF/SREBF signaling 0.01423

DopamineR2 -> AP-1/CREB/ELK-SRF signaling 0.01727

NeurotensinR -> ELK-SRF/AP-1/EGR signaling 0.01904

VasopressinR2 -> CREB/ELK-SRF/AP-1/EGR signaling 0.02339

EndothelinRa -> AP-1/CREB signaling 0.03353

ICAM1 -> AP-1/CREB/ELK-SRF signaling 0.03353

TLR -> AP-1 signaling 0.04296

NGFR -> AP-1/CEBPB/CREB/ELK-SRF/TP53 signaling 0.04310

T-cell receptor -> AP-1 signaling 0.04467

EctodysplasinR -> AP-1 signaling 0.04467

VEGFR -> ATF/CREB/ELK-SRF signaling 0.04533

CCR5 -> TP53 signaling 0.04889

Diatchenko

Pathway Analysis – Human Pain Sensitivity EGF Pathways and Pain

• EREG

– Epiregulin is a mitogenic peptide that binds to EGFR

– Active in multiple cell types, including fibroblasts, macrophages, keratinocytes

• EGFR

– Anti-ErbB antibody treatment reduces opioid reqirements in cancer treatment

– EGFR regulates DOR and MOR via tyrosyl phosphorylation and activation of GRK2 (Chen et al, Mol Biol Cell 2008)

– EGFR inhibition improves sensory and functional recovery after SCI in rats (Erschbamer et al, J of Neuroscience, 2007)

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EGFR Receptor Antagonist Produces Analgesia in Multiple Pain Assays Assessed in Mice

Data provided by Jeff Mogil – McGill University

EGFR Agonists Produce Hyperalgesia – Mouse Formalin Model

Data provided by Jeff Mogil – McGill University

NTRK -> AP-1 pathways – gene SNPs contributions

dark blue – SNPs contributing to pathway P<0.05 light blue – SNPs contributing to pathways 0.05>P<0.1

Different Genes – Common Cluster

FM Patient 1 FM Patient 2

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A Future for Human Pain Genetic Biomarkers

• Discovery of biological pathways underlying traits and diseases

• Drug discovery – target identification

• Diagnostic and prognostic markers enabling individualized decisions regarding efficacy and risk of :

• Pharmacotherapies

• Behavioral therapies

• Invasive procedures

• Eric Bair • Kanokporn Bhalang • Luda Diatchenko • Greg Essick • Richard Gracely • Mark Hollins • Kevin Kahn • Pei Feng Lim • Dylan Maixner • Sam McLean • Andrea Nackley-Neely • Asgier Sigurdsson • Gary Slade • Shad Smith • Kati Thieme • Inna Tchivileva • William Whitehead • Denniz Zolnoun

• Bruce Weir, University of Washington • Roger Fillingim, University of Florida

(Gainesville) , • Dmitry Shagin - Institute of Bioorganic

Chemistry (Moscow, Russia) • Sergei Makarov – Attagene Inc.

Center for Neurosensory Disorders National Institutes of Health

• David Goldman - NIAAA

• Inna Belfer - NIDCR/NIAAA • Mitchell Max - NIDCR • Ke Xu - NIAAA • Sveta Shabalina – NCBI • Dmitri Zaykin - NIEHS

Collaborators

Supported by:

DE017018, DE016155, DE007333, DE00366, NS45685, AR/AI-44564, AR-30701, AR/AI-4403, AA000301, G192BR-C4

UNC School of Dentistry

Comprehensive Center for Inflammatory Disorders

Thurston Arthritis Center

Attagene Inc.

Algynomics Inc.