March 26, 2008

Kanokporn Bhalang, DDS, MS, PhD

School of Dentistry, Chulalongkorn University

Pain : An unpleasant sensory and emotional experience associated with actual or potential tissue damage.

Experimental Pain : pain induced experimentally and assess quantitatively the responses by psychophysical, biomechanical, or physiological measures.

Temporomandibular Disorders (TMD) : collection of medical and dental conditions affecting the temporomandibular joint (TMJ) and/or the muscles of mastication, as well as contiguous tissue components.

Burning Mouth Syndrome (BMS) : a chronic pain syndrome that mainly affects middle-aged and older female patients. The term encompasses all forms of burning sensations in the oral cavity when the oral mucosa is clinically normal.

http://www.ornl.gov/hgmis

Explore howExplore how DNADNA impactsimpacts HEALTHHEALTH

Identify and understand

the differences in DNA sequence (A, T, C, G)

among human

populations

http://www.ornl.gov/hgmis

SimpleSimple• Single gene defect often causal

• DNA change - often significant protein

consequence

• Recognizable clinical phenotype

• Minor environmental impact

• Diagnosed by genetic testing

• Early onset

ComplexComplexComplexComplexComplexComplexComplexComplex• Multiple genes + environmental factors Multiple genes + environmental factors Multiple genes + environmental factors Multiple genes + environmental factors are causal. are causal. are causal. are causal.

• DNA change DNA change DNA change DNA change ---- slight change in function slight change in function slight change in function slight change in function of gene product.of gene product.of gene product.of gene product.

• Clinical phenotype usually highly Clinical phenotype usually highly Clinical phenotype usually highly Clinical phenotype usually highly

variable variable variable variable ---- over a continuum of over a continuum of over a continuum of over a continuum of expression from very mild to severe.expression from very mild to severe.expression from very mild to severe.expression from very mild to severe.

• Environmental impact Environmental impact Environmental impact Environmental impact ---- necessary for necessary for necessary for necessary for

disease manifestation. disease manifestation. disease manifestation. disease manifestation. • Genetic testing Genetic testing Genetic testing Genetic testing ---- Not diagnostic, may Not diagnostic, may Not diagnostic, may Not diagnostic, may

permit estimate of susceptibility or permit estimate of susceptibility or permit estimate of susceptibility or permit estimate of susceptibility or

prognosis .prognosis .prognosis .prognosis .• Adult onset, often chronic disease Adult onset, often chronic disease Adult onset, often chronic disease Adult onset, often chronic disease

state. state. state. state.

http://www.ornl.gov/hgmis

�� 99.9% of human genome are the same in all 99.9% of human genome are the same in all

people = people = 0.01% difference0.01% difference

�� approximately 1 SNP/ 1000 basesapproximately 1 SNP/ 1000 bases

�� 33--6 million6 million SNPs in the genomeSNPs in the genome

SNPs that are SNPs that are

located close to located close to

each other on a each other on a

chromosome tend chromosome tend

to be to be linkedlinked (go (go

together) through together) through

evolution.evolution.

International HapMap Consortium, Nature, 2003

BB

BB BB

CC

CCCC

International HapMap Consortium, Nature, 2003

� Animal model – knockout mice

� Human clinical pain phenotype

� Simple genetic diseases (rare)

� Congenital insensitive to pain (CIP)

� Congenital indifferent to pain (CIDP)

� Complex Genetic diseases

� Gene polymorphism also affects individual response

to phamacological agents.

Functional Class

Gene Condition Genetic Polymorphism

TransportersTransporters 55--HTTHTT TMDTMD 5HTTLPR5HTTLPR

Metabolic genesMetabolic genes COMTCOMT TMDTMD Val158Met and 3 Val158Met and 3

major haplotypesmajor haplotypes

ReceptorsReceptors ADRB2ADRB2 TMDTMD 3 major haplotypes3 major haplotypes

55--HTR2AHTR2A TMDTMD T102CT102C

CytokinesCytokines IL1B BMS C3954T

Diatchenko L, Nackley AG, Tchivileva IE, Shabalina SA, Maixner W. Genetic architecture of human pain perception. Trends Genet. 2007 Dec;23(12):605-13.

A multi-substrate enzyme catalyzing methylation of catecholamines.

Soluble COMTpresent in most tissuesmore effective in metabolizing

epinephrine*

Membrane bound COMTpresent only in the central nervous system†

more effective in metabolizing dopamine and norepinephrine.

* Mannisto PT and Kaakkola S. Pharmacol.Rev. 51 (1999) 593-628.† Hong J, Shu-Leong,H, Tao X, and Lap-Ping Y. Neuroreport, 9 (1998) 2861-2864.

COMT

Linkage disequilibrium analysis shows that the 6 SNPs cover 3 haploblocks.

� G/G (val/val)

� A/G (met/val)

� A/A (met/met)

rs6269 rs2097903 rs4633 rs4818 rs4680

val/met

rs165599

Soluble COMT transcript

Membrane-bound COMT transcript

Substitution of val by met reduces the

thermostability of COMT enzyme resulting in

reduction of enzyme activity.*

* Zubieta JK, et al. Science. 299 (2003) 1240-3.

rs6269 rs2097903 rs4633 rs4818 rs4680

Val/Met

rs165599

Soluble COMT transcript

Membrane-bound COMT transcript

Haploblock label Haploblock label %

#1 Low Pain G C G G 37.5%

Sensitivity (LPS)(LPS)

#2 Average Pain A T C A 47.3%Sensitivity (APS)(APS)

#3 High Pain A C C G 10.3%

Sensitivity (HPS)(HPS)

Other combinations 4.9%

Primary target for epinephrine.

Plays a critical role in mediating physiological and psychological responses to environmental stressors.

When transfected into human

embryonic kidney cells,

these genetic variants alter

production of COMT.

In Sprague-Dawley rats, pharmacological

inhibition of COMT alters thresholds of paw

withdrawal in response to mechanical and

thermal stimuli.

• 202 healthy females, followed for 3 years, to identify

newly developed cases of TMD.

• Collected blood samples at baseline.

• Assessed experimental pain sensitivity and

psychological traits at baseline.

Pain Threshold

The least intensity of a stimulus at which the subject

perceives pain.

Pain Tolerance

The greatest level of pain the subject can tolerate.

Hand held algometer

applied to four sites:1. Temporalis muscles 2. Masseter muscles3. TMJ 4. Ventral surfaces of

wrists.

Recording of force (kg) at

which subject reported

pain threshold

Total of 4 measures

Pressure Pain Pressure Pain

Threshold Threshold

Measurement on Measurement on

Masseter MuscleMasseter Muscle

Pressure Pain Pressure Pain

Threshold Threshold

Measurement on Measurement on

Temporalis MuscleTemporalis Muscle

Thermode applied heat increasing at 0.5°C/sec to three sites:right masseter muscleright hairy forearmthe dorsal surface of the

right foot

Recording of temperature at which subject reported pain threshold and pain tolerancetotal of 3 x 2 = 6 measures

Single Heat StimulusSingle Heat Stimulus

� Arm rendered hypoxic with a blood pressure cuff.

� Handgrip dynamometer squeezed for 20 repetitions.

� Recording of elapsed time until the subject reported pain threshold and pain tolerance. � total of 2 measures

Ischemic Pain Ischemic Pain

MeasurementMeasurement

Dynamometer Dynamometer

for Handgrip for Handgrip

ExerciseExercise

� Verbal ratings of fifteen 53 °C heat stimuli

� 1.5 second duration of each pulse

� 3 second interval between pulses

� applied to the right hand

� Ratings on visual analog scale:

� 0 = No perception

100 = worst imaginable pain

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150

20

40

60

80

100

Subject 112

Stimulus

Vis

ual

an

alo

g p

ain

rati

ng

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150

20

40

60

80

100

Stimulus

Mean

VA

S p

ain

rati

ng

(±± ±±

se)

Incoming Incoming Incoming Incoming

Pain Pain Pain Pain

signalsignalsignalsignal

NKNK--1 1

receptorreceptor

AMPA AMPA

receptorreceptor

NMDA NMDA

receptorreceptor

Ca++Ca++

NOS

LLLL----argininearginineargininearginine

NONONONO

Nociceptor Nociceptor Nociceptor Nociceptor Nociceptor Nociceptor Nociceptor Nociceptor

TerminalTerminalTerminalTerminalTerminalTerminalTerminalTerminal

Postsynaptic Postsynaptic Postsynaptic Postsynaptic Postsynaptic Postsynaptic Postsynaptic Postsynaptic

Dorsal Horn Dorsal Horn Dorsal Horn Dorsal Horn Dorsal Horn Dorsal Horn Dorsal Horn Dorsal Horn

Neuron Neuron Neuron Neuron Neuron Neuron Neuron Neuron

Substance P

Glutamate

Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain.Trends Neurosci. 2001 Aug;24(8):450-5.

Individual Z Score Individual Z Score

CalculationCalculation

A subject 's heat pain tolerance (Arm) = A subject 's heat pain tolerance (Arm) = 48.548.5 CC

Z score = (XZ score = (Xii -- X) = 48.5 X) = 48.5 -- 45.6 = 45.6 = 0.950.95

SD 2.57SD 2.57

Combined Z Score Combined Z Score

for a Subjectfor a Subject

C fiber Pain Threshold (Arm/Cheek/Foot)C fiber Pain Threshold (Arm/Cheek/Foot)++

C fiber Pain Tolerance (Arm/Cheek/Foot)C fiber Pain Tolerance (Arm/Cheek/Foot)++

Pressure Pain Threshold Pressure Pain Threshold (Wrist/Temporalis/Masster/TMJ)(Wrist/Temporalis/Masster/TMJ)

++Ischemic Pain Threshold Ischemic Pain Threshold

++Ischemic Pain Tolerance Ischemic Pain Tolerance

++WindupWindup

Combined ZCombined Z--Scores of All Pain MeasuresScores of All Pain Measures

For a SubjectFor a Subject

--2020 --1010 00 1010 2020 303000

1010

2020

3030

4040

5050

Integral ZIntegral ZIntegral ZIntegral ZIntegral ZIntegral ZIntegral ZIntegral Z--------score score score score score score score score

(all pain measures)(all pain measures)(all pain measures)(all pain measures)(all pain measures)(all pain measures)(all pain measures)(all pain measures)

Number of subjects

Number of subjects

Number of subjects

Number of subjects

Number of subjects

Number of subjects

Number of subjects

Number of subjects

More pain More pain More pain More pain More pain More pain More pain More pain

sensitivesensitivesensitivesensitivesensitivesensitivesensitivesensitiveLess pain Less pain Less pain Less pain Less pain Less pain Less pain Less pain

sensitivesensitivesensitivesensitivesensitivesensitivesensitivesensitive

n=202 subjectsn=202 subjectsn=202 subjectsn=202 subjectsn=202 subjectsn=202 subjectsn=202 subjectsn=202 subjects

mean=0.0, mean=0.0, mean=0.0, mean=0.0, mean=0.0, mean=0.0, mean=0.0, mean=0.0,

sd=10.6sd=10.6sd=10.6sd=10.6sd=10.6sd=10.6sd=10.6sd=10.6

median= median= median= median= median= median= median= median= --------1.21.21.21.21.21.21.21.2

skewness= 0.3skewness= 0.3skewness= 0.3skewness= 0.3skewness= 0.3skewness= 0.3skewness= 0.3skewness= 0.3kurtosis= kurtosis= kurtosis= kurtosis= kurtosis= kurtosis= kurtosis= kurtosis= --------0.1.0.1.0.1.0.1.0.1.0.1.0.1.0.1.

Muscle and Joint Muscle and Joint

PalpationPalpation

TemporalisTemporalis

MasseterMasseter

DigastricDigastric

SCMSCM

TrapeziusTrapezius

TMJTMJ

Intraoral SitesIntraoral Sites

��Lateral PterygoidLateral Pterygoid

��Tendons of TemporalisTendons of TemporalisEach participant was asked to rate the pain asEach participant was asked to rate the pain as

‘‘nonenone’’ = 0, = 0, ‘‘mildmild’’ = 1, = 1, ‘‘moderatemoderate’’ = 2, or = 2, or ‘‘severesevere’’ = 3. = 3.

I: Myofascial pain

Self reported muscle pain + 3 or more muscle

tender sites

II: Arthralgia

Self reported joint pain + TMJ tenderness

*Val158met polymorphism is associated with CNS-dependent temporal integration of pain, while COMT haplotypes are not.

-6

-3

0

3

6

9

12

Pain sensitivity

Pain sensitivity

Pain sensitivity

Pain sensitivity

Pain sensitivity

Pain sensitivity

Pain sensitivity

Pain sensitivity

(mean z

(mean z

(mean z

(mean z

(mean z

(mean z

(mean z

(mean z-- ---- -- score

score

score

score

score

score

score

score ±± ±±±± ±±se)

se)

se)

se)

se)

se)

se)

se)

APS/APS/

HPSHPS

APS/APS/

APSAPSHPS/HPS/

LPSLPSLPS/LPS/

APSAPSLPS/LPS/

LPSLPS

Haplotype combinationsHaplotype combinationsHaplotype combinationsHaplotype combinationsHaplotype combinationsHaplotype combinationsHaplotype combinationsHaplotype combinations

Less pain Less pain Less pain Less pain sensitivesensitivesensitivesensitive

More pain More pain More pain More pain sensitivesensitivesensitivesensitive

Individuals who produced reduced levels of COMT

(HPS and APS haplotypes) may have an increased

risk of developing persistent pain conditions.

The presence of even a single LPS haplotype diminishes, by as much as 2.32.3 times, the risk of

developing myogenous TMD.

In females, APS and HPS variants of the gene encoding COMT are associated withincreased responsiveness to noxious stimuli

2.3-fold elevation in risk of TMD onset

Two major functional variants of COMT provides different profiles of association with pain perception. COMT haplotype with summed pain sensitivity

Codon158 with windup

� Reduced COMT activity results in elevated levels of

catecholamines which promote the production of persistent

pain states via the stimulation of beta2 adrenergic receptors in

the central and peripheral nervous system.

SomatizationSomatization - psychological

distress arising from perception of bodily dysfunction.

PILL - Pennebaker Inventory for Limbic Languidness

STAI – State Trait Anxiety Inventory POMS – Profile of Mood States

BSI – Brief Symptom Inventory

Individuals who caried one haplotype coding for low Individuals who caried one haplotype coding for low

and one haplotype coding for high ADRB2 and one haplotype coding for high ADRB2

expression were 10 times less likely to develop expression were 10 times less likely to develop

TMDTMD

Functional Class

Gene Condition Genetic Polymorphism

TransportersTransporters 55--HTTHTT TMDTMD 5HTTLPR5HTTLPR

Metabolic genesMetabolic genes COMTCOMT TMDTMD Val158Met and 3 Val158Met and 3

major haplotypesmajor haplotypes

ReceptorsReceptors ADRB2ADRB2 TMDTMD 3 major haplotypes3 major haplotypes

55--HTR2AHTR2A TMDTMD T102CT102C

CytokinesCytokines IL1B BMS C3954T

Diatchenko L, Nackley AG, Tchivileva IE, Shabalina SA, Maixner W. Genetic architecture of human pain perception. Trends Genet. 2007 Dec;23(12):605-13.

� 5HTTLPR – 5 hydroxytryptamine (serotonin) transporter gene

promoter polymorphism

� The short form of this variant, designated s, is associated

with low transcriptional efficiency of the 5-HTT gene

promoter when compared with the longer forms, designated

l and xl.

•A significant association between the longer alleles (l and xl) of 5HTTLPR and the TMD.

•Genetic factors that involve the serotonergic system may play a role in the

pathogenesis of TMD.

Ojima K, Watanabe N, Narita N, Narita M. Temporomandibular disorder is associated with a serotonin transporter gene polymorphism in the Japanese population. Biopsychosoc Med. 2007 Jan 10;1:3.

� 5HTR2A : 5-hydroxytryptamine (serotonin) receptor 2A

� T102C polymorphism

� The C/C genotype was over represented in the patients

whereas T/T genotype was over represented in the controls

(P < 0.05).

� Suggests a possible role of the serotonergic system in this

disease, particularly at the receptor level.

Mutlu N, Erdal ME, Herken H, Oz G, Bayazit YA.T102C polymorphism of the 5-HT2A receptor gene may be associated with temporomandibular dysfunction.Oral Dis. 2004 Nov;10(6):349-52.

••C3954T polymorphismC3954T polymorphism

••Homozygous individuals for the T allele produce higher amount oHomozygous individuals for the T allele produce higher amount of ILf IL--1 1

than individuals displaying the C/C genotype.than individuals displaying the C/C genotype.

••Suggested that ILSuggested that IL--1 plays an important role in pain sensibility.1 plays an important role in pain sensibility.

Guimarães AL, de Sá AR, Victoria JM, de Fátima Correia-Silva J, Gomez MV, Gomez RS. Interleukin-1beta and serotonin transporter gene polymorphisms in burning mouth syndrome patients. J Pain. 2006 Sep;7(9):654-8.

� Until affordable sequencing methods arrive for everyday

use, the identification of intermediate phenotypes (such

as pain sensitivity) can serve as a good substitution.

� When the time comes, we can tailor individual therapies

for common orofacial pain conditions.

� Dr. William Maixner

� Dr. Luda Diatchenko

� Dr. Gary Slade

� Dr. Ray Sone Hovijitra