the association between single nucleotide polymorphisms...

THEASSOCIATIONBETWEENSINGLENUCLEOTIDEPOLYMORPHISMSANDPAINSENSITVITYINPATIENTSWITHTHEHYPERMOBILETYPEOFEHLERS-DANLOSSYNDROMETheinfluenceoftheOPRM1(rs1799971andrs563649),SLC6A4(5HTTLPRandrs3813034)andCOMT(rs4680andrs4818)genes.

VandorpeLeonardVanRansbeeckBenPromotor:Dr.LiesRombautCopromotor:Prof.Dr.PatrickCaldersAdissertationsubmittedtoGhentUniversityinpartialfulfillmentoftherequirementsforthedegreeofMasterofScienceinRehabilitationSciencesandPhysiotherapyAcademicyear:2016–2017

Acknowledgement

Thisstudywouldnothavebeenpossiblewithoutthehelpofseveralpeople.Therefore,wewouldlike

toacknowledgeeveryonewhomadeacontributiontothiswork.

Firstofall,wewould like to thankourpromotor,dr.LiesRombaut, forall thehelpandadviceshe

provided us throughout the course of this study. Secondly, we thank our co-promotor professor

PatrickCaldersforprovidinguswithusefulinformationandsupport.

We would also like to thank drs. Iris Coppieters for teaching us how to perform the pain

measurementsanddrs.IngeDeWandeleforprovidinguswithdataofthepatients.

AlotofgratitudegoestoprofessorPaulCouckeandhisteamattheCenterforMedicalGeneticsforall

the informationaboutgenetics theygaveusand theexecutionof theDNAanalysis. Without their

contributionregardinggenotypingofthepatients,thisstudywouldnothavebeenpossible.

Lastbutnotleast,aspecialthanksgoesouttoourfamilyandfriends,fortheirunconditionalsupport

duringthecourseofourfive-yearstudiesattheuniversity.

5

IndexListoffiguresandtables……………………………………….…………….…….…………………….…………...………….….7

1. Figures………………...……………………………………….……………………………………….………………….7

2. Tables.…………………………………………………………….……………………………………………………….7

Listofabbreviations………………………………………………………………….………………..…………………..………….8

Crosssectionalstudy…………….…………………………………………….……………….…………..………..………………..10

1. Abstract………………………………………………………………………………………………………………....... 10

English……………………………………………………………………..………………….…………….………. 10

Nederlands…………………………………………………….………………………………………..………….11

2. Introduction………………………………….………………………..…………………….……………….……..….. 12

3. Methods…..……………………………….…………………………..……………………………………..………..… 17

3.1Studydesign……………………………………………………………………………..…..…..….…….…17

3.2Studypopulation……………………………………………..…………………………………………….17

3.3Studyprocedure……………………………………………..………………………..…………………... 17

3.3.1Phenotyping……………………….…………………………..………………...….19

3.3.2Genotyping…………………………...………………………….....………….……22

3.4Statisticalanalysis……………………………………………………..…………………………........… 23

4. Results…………………………………………………………………..…………...………………….…………..……. 24

4.1Distributionofgenotypeandallelefrequencies………..…………………………….....…... 24

4.2Subjectcharacteristics………………………………………………..…………..…………........….… 25

4.3Self-reportedpainquestionnaires....…...…………………………………………….……...……..30

4.4Associationofgenotypeandpressurepainsensitivity..………………….……………….. 33

5. Discussion……………………………………………………………..……………………………..………………..…40

5.1Summaryofstudyfindings………………….…..……………..………………..…………….…….…40

5.2PressurepainsensitivityinOPRM1…………………………………….…………….………….…40

5.3PressurepainsensitivityinSLC6A4………………………………………………………….…….41

5.4PressurepainsensitivityinCOMT.……………..………………………….….……….….….…….42

5.5Subjectcharacteristicsandquestionnaires……………………………………………….……. 43

5.6Strengthsandlimitations…………………………………………………………………………….…43

5.7Suggestionsforfutureresearch…………………………………..……………………………..….. 44

6. Conclusion………..……………………………………………..………………………………….………………..….. 46

7. References………..……………………………………………..……………………………….……………….………47

6

Abstractinlekentaal…………………………………………………………………………………….……………………………52

Ethicalcommittee………………………………………………………..………………………….………………...….……………53

Addendum:Valorisation……………………………………………………………………………………………………………. 54

7

Listoffiguresandtables

1. FiguresFigure1:Timelineofprotocol

Figure2:ExampleofDNAsequencing

2. Tables Table1a:Distributionofgenotypefrequencies

Table1b:Distributionofallelefrequencies

Table2a:Demographicsofsubjects(OPRM1,rs1799971)

Table2b:Demographicsofsubjects(OPRM1,rs563649)

Table2c:Demographicsofsubjects(SLC6A4,rs3813034)

Table2d:Demographicsofsubjects(SLC6A4,5HTTLPR)

Table2e:Demographicsofsubjects(COMT,rs4818)

Table2f:Demographicsofsubjects(COMT,rs4680)

Table2g:Generaluseofmedicationofsubjects

Table3a:Questionnairesofcarrierswithdifferentgenotypes(OPRM1,rs1799971)

Table3b:Questionnairesofcarrierswithdifferentgenotypes(OPRM1,rs563649)

Table3c:Questionnairesofcarrierswithdifferentgenotypes(SLC6A4,rs3813034)

Table3d:Questionnairesofcarrierswithdifferentgenotypes(SLC6A4,5HTTLPR)

Table3e:Questionnairesofcarrierswithdifferentgenotypes(COMT,rs4818)

Table3f:Questionnairesofcarrierswithdifferentgenotypes(COMT,rs4680)

Table4a:Painscoresofcarrierswithdifferentgenotypes(OPRM1,rs4818)

Table4b:Painscoresofcarrierswithdifferentgenotypes(OPRM1,rs563649)

Table4c:Painscoresofcarrierswithdifferentgenotypes(SLC6A4,rs3813034)

Table4d:Painscoresofcarrierswithdifferentgenotypes(SLC6A4,5HTTLPR)

Table4e:Painscoresofcarrierswithdifferentgenotypes(COMT,rs4818)

Table4f:Painscoresofcarrierswithdifferentgenotypes(COMT,rs4680)

8

Listofabbreviations

A Adenine

ACE Angiotensin-converting-enzyme

AP Actionpotential

BMI Bodymassindex

C Cytosine

CIS Checklistindividualstrength

COMT Catechol-Omethyltransferase

CPM Conditionedpainmodulation

CWP Chronicwidespreadpain

DNIC Diffusenoxiousinhibitorycontrol

EDS Ehlers-Danlossyndrome

EIA Exercise-inducedanalgesia

FM Fibromyalgia

G Guanine

HADS HospitalAnxietyandDepressionQuestionnaire

HIT Hot-waterimmersiontest

HR Heartrate

HT Hypermobiletype

IPAQ Internationalpainactivityquestionnaire

Kg Kilogram

Kgf Kilogramforce

M Musculus

MET MetabolicEquivalentsofTask

Met Methionine

N.a. Notapplicable

NRS Numericalratingscale

NSAID Non-steroidalanti-inflammatorydrugs

OPRM1 Mu-opioidreceptorgene

PCR Polymerasechainreaction

PCS Paincatastrophizingscale

9

PPT Pressurepainthreshold

PVAQ PainVigilanceandAwarenessQuestionnaire

S Second(s)

SD Standarddeviation

SLC6A4 Serotonintransportergene

SNP SingleNucleotidePolymorphism

SPSS StatisticalPackageforSocialSciences

T Thymine

TS Temporalsummation

Val Valine

W Watt

WHO WorldHealthOrganization

10

1.AbstractEnglishBackground:Recentresearchindicatesapossibleassociationbetweenthepresenceofspecificsingle

nucleotidepolymorphismsandpainsensitivityinsomechronicpainpopulations.

Objectives:InvestigatingwhetheranassociationbetweenpolymorphismsintheOPRM1(rs1799971

andrs563649),SLC6A4(5HTTLPRandrs3813034)andCOMT(rs4680andrs4818)genesandpain

sensitivitycanbefoundinpatientswiththehypermobiletypeofEhlers-Danlossyndrome(EDS-HT).

Studydesign:Cross-sectionalstudy.

Method:Thestudyincluded11adultwomen,agedbetween18and55,sufferingfromEDS-HT.The

testprocedureconsistedoftwoparts:phenotypingandgenotyping.Thephenotypingpartcomprised

aclinicalexamination,self-reportedquestionnaires(PCS,PVAQ,IPAC,HADSandCIS),anamnesisand

measurements of pain sensitivity by pressure pain thresholds, temporal summation, conditioned

painmodulation and exercise induced analgesia.Ablood sample of eachparticipatingpatientwas

used for DNA analysis. Groups of patients were made for each SNP based on “wild type” vs.

“heterozygous+mutanttype”.Painsensitivitywascomparedbetweenthegenotypes.

Results: Two statistically significant differences were found in comparing pressure pain scores

between thegenotypesofOPRM1rs1799971. IngenotypesofCOMTrs4680, twodifferenceswere

found as well, indicating an association between these SNP’s and pain sensitivity. No significant

differences could be found inOPRM1 rs563649, SLC6A4 rs3813034, SLC6A45HTTLPR andCOMT

rs4680. No differences in pain cognitions were found except for the PCS questionnaire, at which

subjectswiththewildtypeofCOMTrs4680reportedasignificantlylowerscore.

Conclusion:Ourfindingssuggestthatanassociationbetweensomegenotypesandpainsensitivityin

EDS-HTpatientscanbemade.ThisisapplicableforOPRM1rs1799971andCOMTrs4818inrelation

topressurepain thresholds. ForOPRM1rs563649, SLC6A4 (5HTTLPRand rs3813034)andCOMT

rs4680, no such associations were found. However, due to influencing factors such as the small

populationgroup,thesestatementscannotbegeneralizedforallEDS-HTpatients.

Keywords: Ehlers-Danlos syndrome hypermobile type, OPRM1, COMT, SLC6A4, pain sensitivity,

pressurepainthreshold

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NederlandsAchtergrond: Recent onderzoek toont een mogelijke associatie aan tussen de aanwezigheid van

specifiekesinglenucleotidepolymorfismen(SNP)enpijngevoeligheidinverschillendepopulaties.

Doelstelling: Onderzoeken of er al dan niet een associatie tussen de OPRM1 (rs1799971 en

rs563649), SLC6A4 (5HTTLPR en rs3813034) en COMT (rs4680 en rs4818) genen en

pijngevoeligheidkanaangetoondwordenbijpatiëntenmethethypermobieletypevanEDS

Onderzoeksdesign:Cross-sectionelestudie.

Methode:Indezestudiewerden11volwassenvrouwengeïncludeerdmetleeftijdtussende18en55

jaar,dielijdenaanEDS-HT.Detestprocedurebestonduittweedelen:fenotyperingengenotypering.

De fenotyperingbestonduiteenklinischonderzoek,vragenlijsten(PCS,PVAQ, IPAC,HADSenCIS),

anamnese en meting van de pijngevoeligheid aan de hand van drukpijndrempels, temporele

summatie, geconditioneerde pijnmodulatie en inspanning-geïnduceerde analgesie. Een bloedstaal

vanelkepatiëntwerdgebruiktvoorDNA-analyse.GroepenperSNPwerdengemaaktopbasisvan

“wildtype”vs.“heterozygoot+mutanttype”.Pijngevoeligheidwerdvergelekentussendegroepen.

Resultaten:Erwerdentweestatistischsignificanteverschillengevondenbijhetvergelijkenvandruk

pijn scores tussen de genotypes van OPRM1 rs1799971. Tussen de genotypes van COMT rs4680

werden ook twee verschillen gevonden wat een associatie tussen deze SNP’s en pijngevoeligheid

aantoont. VoorOPRM1 rs563649, SLC6A4 rs3813034, SLC6A45HTTLPRenCOMT rs4680konden

geen significante verschillen aangetoondworden tussen genotype en pijngevoeligheid. Er werden

geenverschillengevondenomtrentpijncognitiesbehalvevoordePCSvragenlijst,waarbijpatiënten

methetwildetypevanCOMTrs4680eensignificantlagerescorevertoonden.

Conclusie:Onzebevindingen suggererendat eenassociatie tussengenotypeenpijngevoeligheid in

EDS-HTpatiëntengemaaktkanworden.DitgeldtvoorOPRM1rs1799971enCOMTrs4818inrelatie

totdrukpijndrempels.VoorOPRM1rs563649,SLC6A4 (5HTTLPRen rs3813034)enCOMTrs4680

werden geen dergelijke associaties gevonden. Desondanks kunnen onze bevindingen door

beïnvloedendefactoren,zoalsdekleinepopulatiegroep,nietgegeneraliseerdwordenvooralleEDS-

HTpatiënten.

Kernwoorden: Ehlers-Danlos syndroom hypermobiele type, OPRM1, COMT, SLC6A4, pijn

gevoeligheid,drukpijndrempel

12

2.IntroductionPAINPain is an unpleasant, sensory and emotional experience in the body that occurs as a result of

possibletissuedamageordisturbanceofthecellularmetabolism(1).

The damaged or disturbed cells release mediators that decrease the excitation threshold of the

nociceptors.Apainfulstimulusactivatesthesenociceptors.ThissignalistransmittedthroughtheA-

delta- and C-fibers up to the dorsal root ganglion. There, it enters the spinal cord and travels up

through the spinothalamic tract to themedulla.This tract is responsible forpainand temperature

sensation.Thesignaltravelsfromthemedullaupintothethalamuswhereitsynapseswithanother

neuron that travels to the somatosensory area and the postcentral gyrus.When the signal arrives

here, a pain sensation can be experienced. The descending pathways have their origin in the

hypothalamusandthesomatosensorycortex.Afterdescendingtothemedulla,thethalamicneurons

synapseontheascendingpathwaysinthespinalcordandmedullawheretheyinhibitnervesignals

thatareascending,causinganalgesia.

TYPESOFPAIN

Pain can occur in many different forms. Each type of pain has its own specific mechanisms and

symptoms. Nociceptive pain is pain caused by active or threatening tissue damage that leads to

activationofperipheralnociceptors.Theprimary functionof thiskindofpain isremovinga threat

(1).Thetissuedamagecanbesomaticorvisceral.Thenociceptionhasfourprocesses:transduction

(thestimulationoftheperipheralnociceptors),transmission(transferringthestimulusthroughthe

spinal cord to the brain), perception (recognizing of the pain in the brain) and modulation (the

feedbackofthebraintothetissuewherethepainstimulusoccurs).

Neuropathicpainisapaincausedbyinjurytothesomatosensorysystem(2).Itisatingling,burning

and electrical pain and clearly distinguishable fromnociceptive pain (3,4). The primary function of

painislost(1).Neuropathiescanbecausedbystretchorpressureonnervestructures.Withregard

to heritable connective tissue disorders, frequent (sub) luxations due to capsule-ligamentary

insufficiency can cause stretch on the nerves,which normally should be caught by the connective

tissue(5).Duetoadefectintheconnectivetissue,thesurroundingtissuesareweaker,resultingina

highercompressionofanerve(6,7).

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Lastly, there is dysfunctional pain. Dysfunctional pain may present as a result of nerve system

plasticity.Alsointhisformofpain,theprimaryfunctionofpainislost(1).Plasticitycaninducetwo

possible pathways: habituation (extinction of stimuli) or sensitization (enhancement of stimuli).

Prolongedorrepeatedexposuretopainstimulimaydevelopaprocessofcentralsensitization(3,4,8).

Anincreasedsensitivityoftheneuronsinthecentralnervoussystemresultsinspontaneousactivity,

increasedsensitivitytodifferentstimuli(pressure,light,cold,heat,etc.)andalargerprojectionarea.

This can lead to the concept of primary hyperalgesia (increased response to painful stimuli),

allodynia (a pain response to non-painful stimuli), referred pain (pain sensation even on

asymptomatic places) and increased temporal summation (build-up pain with repeated stimuli)

(9,10).Sensitizationprovidesanevidenced-basedexplanationfor"inexplicablepain"(11,12).

PAINMODULATINGSYSTEMS

Fortunately, thehumanbodyhas foundaway tomodulatepain.Theendogenouspainmodulating

system consists of twomajor subsystems, namely facilitation and inhibition. Dysfunctional pain is

characterizedbyreducedactivityofthepaininhibitionpathwaysoranabnormallyincreasedactivity

ofthepainfacilitatingpathways(13,14,15,16).

An increased facilitationcanbeprovokedwhenapatient isrepeatedlyexposedtopainstimuli, for

example in case of repetitive subluxations. Wind-up is the phenomenon in which dorsal horn

neurons in the spinal cord become more sensitive after repeated stimuli. In case of persisting

nociceptivestimuli,thedorsalhornremainsactivated.Thefollowingstimulidonothavetoreachthe

thresholdtobepassedontothenextnervecell.

Nexttothis,therearedescendingpathwaysfromdeeplylocatedbrainnuclei,whichmayalsocause

facilitation.Centersinthefrontalcortexwouldhaveastronginfluenceondifferentcoresinthebrain

stem(1,16).Thesemaycause"cognitive-emotionalsensitization".Cognitionsandemotionsaffectthe

painexperience.

Pain inhibition can occur through different ways. Primarily, there is the gate control mechanism

(non-painful stimuli inhibit the painful stimuli). Gate control is the inhibition of A-fibers that give

nociceptivestimulationbyinputofC-fibersbyinhibitinginterneurons(1,17).Furthermore,thereare

descendingpain inhibitorypathwaysandmechanisms (1,17) that areable to lowerapain stimulus.

Thepainloweringinfluencestartsfromdifferentareasofthebrainandisbroughttotheperiphery

throughspecificnervesinthespinalcord.

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GENETICFACTORS

Pain isoneof themostcommonandhamperingsymptomsindifferentpathologicalsituations. It is

not only a sensory perception but also an emotional one. This makes pain a very personal and

subjectivesensationthatcanbeinfluencedthroughvariablefactorssuchasindividualexperiences,

familialhistory,fearofpain,fatigue,moodandmanymore.Besidesthesepsychosocialfactors,pain

canalsobeinfluencedbyphysicalfactorsofwhichage,genderandethnicityarethemostimportant

determinants.Next to those influencing factors, recent research suggests that somegenetic factors

may also contribute to individual differences in pain sensitivity and the development of pain

complaints. Several genes have already been described in the literature in association to pain

sensitivitysuchascatechol-O-methyltransferase(COMT),serotonintransportergene(SLC6A4)and

mu-opioidreceptorgene(OPRM1).

Catechol-O-methyl transferase (COMT) is an enzyme that degrades catecholamines (such as

adrenaline, noradrenaline and dopamine) (18). These neurotransmitters are involved in the

modulationofpainamongotherphysiologicalprocesses.COMThas functionalpolymorphismsthat

contribute to the interindividual variability in human pain phenotypes such as pain sensitivity,

severity,chronicityandresponsetomedication.TwocommonpolymorphismsoftheCOMTgeneare

rs4680 and rs4818 (18). It has been shown that these polymorphisms can have an effect on the

dopaminepathwayswithpossibledifferences inthedopaminelevels. If thedopaminelevel ishigh,

the pain sensitivity will be reduced (19). COMT is found to be less expressed or less frequent in

patientswithchronicpain(18,19).

The serotonin transporter gene (SLC6A4) is part of the solute carrier family. This protein is

responsible for the transport of serotonin from the synaptic cleft to the presynaptic neuron (20).

Serotoninhasan influenceon thememory, sleep,emotionsandpainstimuliamongstother things.

This gene is found on chromosome 17. The most frequent genetic variations of the serotonin

transportergenearethe5HTTLPRandrs3813034polymorphisms.Thesechangescanplayapartin

theprocessingofpainstimuli.SLC6A4isresponsiblefortheproductionofserotonin.Serotoninhas

aninhibitinginfluenceonthedopaminelevelsresultinginlowerpainthresholds(20).

Themu-opioidreceptor(OPRM1)isoneofthethreeopioidreceptorsfoundinthehumanbody.The

substrates thathave an impact on these receptors are enkephalines andbeta-endorphins (22). The

mostcommonsinglenucleotidepolymorphismsofthemu-opioidreceptorgenearers1799971and

rs563649. This gene is located on chromosome 6. Stimulation of themu-opioid receptor leads to

15

activation of theG protein causing an increased efflux through the potassium (K+) channels and a

decreasedinfluxthroughthecalcium(Ca2+)andsodium(Na+)channels.Thisleadstoamorenegative

membranepotentialthatmakesrepolarizationmoredifficultandanAPhardertoachieve.Asaresult

thepainstimuluswillbehardertotransmit.Thereisalsoadecreasedreleaseofneurotransmitters

andthusareductionof thepainsensation. Inthecurrent literature,however,noassociationshave

been found between expression or frequency of this gene and pain sensitivity in patients with

chronicpain(22,23,24).

Insummary,littleresearchregardingtheassociationsbetweenpolymorphismsofcertaingenesand

chronic pain patients has been conducted. A few chronic pain patients groups have already been

studiedsuchasFMpatients,lowerbackpainpatientsandcancerpainpatients(19,21,22,28).However,a

lotofotherpatientpopulationscharacterizedbychronicpainhavenotyetbeenstudiedconcerning

the associations between pain sensitivity and genetic polymorphisms, including the Ehlers-Danlos

syndrome,hypermobiletype.

THEEHLERS-DANLOSSYNDROME

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of hereditary connective tissue

disorders characterized by articular hypermobility, skin extensibility, and tissue fragility (29,30).

Abnormalities in collagen and other connective tissues and proteins, based on genetic mutations,

leadtoanincorrectprocessofproteinsynthesis intheproductionofconnectivetissues.Thishasa

significantimpactontheentirebodybecauseconnectivetissuecanbefoundeverywhere;inbones,

ligaments,skin,bloodvessels,etc.Assuch,itreflectsinageneralfragilityofmanybodysystems.

Veryrecently,newdiagnosticcriteriaforEDShavebeenpublished.However,becauseinthisstudy

patientswereselectedbasedonthe‘old’criteria,thoseareexplainedbelow.Sixdifferenttypeshave

beenestablishedbytheVillefranchecriteria(30):theclassictype,thevasculartype,thehypermobile

type, kyfoscoliosis, arthrochalasis and dermatoparaxis. These types are classified according to the

nature,severity,inheritance,geneticdefectandclinicalcharacteristicsofthedisease.

Withafrequencybetween1:5.000and1:10.000,themostoccurringtypeofEDSisthehypermobile

type (EDS-HT). According to the Villefranche criteria, the two major criteria for this type are

generalized jointmobility and a hyperextensible, smooth, velvety skin. Theminor criteria include

recurring joint dislocations, chronic joint/limb pain and a positive family history (30). Other

symptoms typical for EDS-HT (among many others) are inflammation, bursitis, muscle tears,

16

decreasedproprioception,lossofsensitivity,tenderpoints,subluxations,etc.(29,30).Thistypeoccurs

morefrequentlyinwomen(29).

PAININEDSPATIENTS

Chronic pain is one of the most persistent and tampering symptoms patients with EDS-HT

experience. Originally, it was thought that pain in EDSwas only nociceptive pain caused by joint

injuryandluxations(8).However,thepainisoftennotproportionaltothepresenttissuedamageand

doesnotreacttosimplepainmedicationsuchasparacetamol.Neuropathicpainalsooftenoccursin

EDS patients. Nerve conduction studies are usually non-diagnostic and skin biopsy may reveal

absenceorreductionofsmallnervefibers(31).Neuropathicpainmaybecausedduetodirectnerve

impingement.Dysfunctionalpainmaybeinducedduetolongexposuretopainstimuli.Plasticityof

thenervesystemcancausecentralsensitization,whichmaymanifestitselfaspainhypersensitivity,

enhancedtemporalsummation,etc.(32).Thesemaybeargumentsforthepresenceofotherformsof

pain such as neuropathic pain or pain mechanisms such as central sensitization (25,26,27). The

pathophysiology of pain in EDS is possibly more complex than expected and musculoskeletal

involvementdoesnotadequatelyexplainthefullrangeofpainexperiencedbythesepatients(25).

Ahereditarydiseaseisnotcurable.Painmanagementisanimportantbutverydifficultfactorinthe

treatmentofEDS-HT.Themainobjectiveofthetreatment istoreducethepainthroughsurgeryor

conservative therapy including medication, physiotherapy, orthopaedic devices etc. although the

outcome of surgery and physiotherapy is often disappointing (27). EDS patients consume a large

amountofmedication,NSAID’sandopioids,accordingtotheirpainseverity.Bothofthesecarrythe

risk of serious side effects like constipation or gastrointestinal bleeding. Consumption of

antidepressants,sedativesandalsonon-pharmacologicaltreatmentsdonotofferafullsolutionand

eachhavetheirownsideeffects(27).Inthefuture,treatmentofchronicpainmightbeadaptedbased

on the genetic profile of the patient. Patients with different polymorphisms in certain genes, and

consequently with a variation in pain sensitivity, might be able to receive a different, specific

treatmentfittingtotheirgeneticprofile.

In fact, little is known about the associations between pain sensitivity and the genetic profiles in

patients with EDS-HT. Therefore, the purpose of this study was to investigate the associations

between pain sensitivity, measured by pressure pain thresholds and NRS pain scores, and

polymorphisms in COMT (rs4680 and rs4818), SLC6A4 (5HTTLPR and rs3813034) and OPRM1

(rs1799971andrs563649)inpatientsdiagnosedwithEDS-HT.

17

3.Methods3.1.StudydesignAcross-sectionalstudywasperformed.

3.2.StudypopulationThestudyincluded11adultwomen,agedbetween18and55,withthehypermobiletypeofEhlers-

Danlossyndrome(EDS-HT).Onlywomenwereincludedbecause90%ofEDS-HTpatientsarewomen

and toexclude the influenceofgenderdifferenceonpain sensitivity in the results.Patientshad to

fulfill bothmajor (skin involvement and generalized joint hypermobility) and two ormore of the

minor(recurringjointdislocations,positivefamilyhistoryandchroniclimb/jointpain)Villefranche

criteriaforEDS-HT(30)tobeincluded.

Exclusion criteria were: (1) surgery in the last year, (2) severe depression, (3) epilepsy, (4)

metabolic/neurological/cardiovascular or inflammatory diseases, (5) unstable pain medication

duringthelastmonth,(6)acutebackorlimbinjuryand(7)pregnancyorgivenbirthinthepastyear.

Subjectswerenotfamilyrelatedtoruleoutageneticorconsanguinityrelationship.

3.3.StudyprocedureThe subjects had to fulfill the in- and exclusion criteriamentioned above andhad to bewilling to

participateinthestudyafterinformationofthestudywasprovided.Ifso,awritteninformedconsent

wassigned.Thetestprocedureconsistedoftwoparts:phenotypingandgenotyping.

Thephenotypingpartincludedananamnesis,clinicalexamination,self-reportedquestionnaires,and

measurementsofpainsensitivity.Theanamnesis,painmeasurements,clinicalexamination,andself-

reportedquestionnaireswereconductedby twostudentsanda supervisor.Theevaluationofpain

sensitivityinvolvedameasurementofbaselinepressurepainthresholds(PPT),temporalsummation

(TS),conditionedpainmodulation(CPM)andexercise-inducedanalgesia(EIA).Foreachpatient,all

measurements were performed on the same day and in the same order. The genotyping part

consistedofDNA-analysis.AbloodsampleofeachparticipatingsubjectwasstoredattheConnective

tissue lab at the Medical Research Building of the Ghent University Hospital and available for

analysis.TheprotocolwasapprovedbytheEthicalCommitteeoftheGhentUniversityHospital.

18

Figure1:Timelineofprotocol

3.3.1Phenotyping

A.ClinicalexaminationandanamnesisDuring the clinical examination theweightand lengthof thepatientswasmeasured. If thepatient

reportedpain, itwas localized and its intensitydeterminedusing aNRS-score. The skin elasticity

was also measured (34) and a beighton score was determined (35). Medical and clinical relevant

informationsuchasmedicationuse,recentinjuries,etc.werequestionedintheanamnesis.

B.QuestionnairesFive self-reported questionnaires (dutch versions) were used, concerning psychosocial factors,

experiencesofpain,qualityoflifeandphysicalactivity:PCS,PVAQ,IPAC,HADSandCIS.

a.ThePainCatastrophizingScale(PCS)

The PCS (36) is a 13-item, 5-point rating scale (end points: ‘0’ not at all and ‘4’ all the time) that

assessesthedegreeofcatastrophiccognitionsapatientexperiencedwhileinpain.Itgeneratesatotal

cumulativescorefrom3individualsubscalesofthePCS(‘0’to ‘52’).Higherscoresindicatedhigher

ratingsofpaincatastrophizing.Thesesubscalesindicatetherateof(1)rumination,(2)magnification

and (3) helplessness, contributing to an overall factor of pain catastrophizing. There are cut-off

values where the 75th percentile (total score of 30) represents a clinically relevant level of

catastrophizing.ThePCShasbeenshowntohaveadequatetoexcellentinternalconsistency(36).

19

b.ThePainVigilanceandAwarenessQuestionnaire(PVAQ)

The PVAQ (37) evaluates attention or pre-occupation to pain, awareness and observation of pain

sensationsandconsistsof16itemsratedonasix-pointscale(‘0’never,‘1’rarely,‘2’sometimes,‘3’

often,‘4’mostlyand‘5’always).Thecumulativescorerangesfrom‘0’to‘80’(greatestpainvigilance).

ThePVAQshowedgoodinternalconsistencyandfairtest-retestreliability(37).

c.TheInternationalPhysicalActivityQuestionnaire;shortversion(IPAQ)

TheshortformoftheIPAQ(38)evaluatesphysicalactivity indaily lifebymeasuringfrequencyand

durationoffourspecifictypesofactivity(sitting,walking,moderate-intensityactivitiesandvigorous-

intensity activities) during the last 7 days. Each physical activity was expressed as an amount of

MetabolicEquivalentsofTask(MET=amountofoxygenaparticipantconsumesduringactivity)(38).

d.HospitalAnxietyandDepressionScale(HADS)

The HADS (39) measures anxiety and depression without involvement of physical complaints. It

consists of 14 itemsdivided in a depression and an anxiety scale both consisting of 7 items. Both

scalescanalsobeusedseparately.Eachitemisscoredonascaleof0-3,whichleadstoatotalscoreof

0-21peritem.Higherscorescorrelatewithmorecomplaints.Threegroupscanbeformedaccording

to the results: no depression/anxiety disorder (0-7), possible depression/anxiety disorder (8-10),

suspecteddepression/anxietydisorder(11-21).

e.ChecklistIndividualStrength(CIS)

TheCIS (40)measures the fatigue intensity of thepatient andbehavioral aspects related to this. It

consistsof20questionsregardingthe feelingof thepatientoverthepast twoweeks.Thesubscale

fatigueintensity,whichwasusedinthisstudy,consistsof8questions,eachratedonascorefrom1-

7.Atotalscorebetween8and56indicatestheleveloffatigueofthepatient.Higherscoresindicatea

higherleveloffatigue.

20

C.Measurementofpainsensitivity

Painsensitivitywasdeterminedby fourmeasurements(PPT,TS,CPMandEIA).Thepatientswere

askedtodeprivethemselvesoftheuseofcaffeine,alcohol,nicotine,high-intensityactivities(running,

etc.)during24hoursbeforetesting.Thetestsubjectswereaskednottotakepainmedicationonthe

dayofthestudy(exceptforparacetamolandNSAIDs).Medicationwithinfluenceonheartrateand

blood pressure (e.g. beta blockers) was stopped 24 hours before the examination (if clinically

justified).Thediscontinuationofthemedicationoccurredinconsiderationwiththephysician.

a. Pressurepainthreshold(PPT)

Pressurepain thresholdsweremeasuredwith a digital algometer (Wagner FDX®).The algometer

was held vertically on the skin and pressure was gradually increased (1kgf/s), until the subject

indicated thatanormalpressuresensechanged intoanunpleasant feeling.Thismoment indicated

the PPT. The real PPT amounted to the mean of two consecutive trials on the same location,

separated by a 30s interval. The locations of this testing were M. Trapezius (shoulder) and M.

Quadriceps (thigh), all at the dominant side of the body. The testing of M. Trapezius (pars

descendens)wasperformedinthemiddleofastraightlinebetweenthelateralsideoftheacromion

andprocessusspinosusofC7.ThetestingofM.Quadricepswasperformedinthemiddleofastraight

linebetween theSIAI and the topof thepatella.During testing the subjectsneeded to sit still and

comfortablewithfeetflatonthefloorandtolookstraightahead.Outcomemeasurementswerethe

meanpressurepainthresholdontheM.Trapezius(‘PPTT’)andthemeanpressurepainthresholdon

theM.Quadriceps(‘PPTQ’).

b. Temporalsummation(TS)

A break of two minutes was held between the last PPT measurement and the start of the TS

measurement,toavoidpossiblesummation.Tenconsecutivestimuliweregiventoexaminethepain

facilitation system. First, pressure was raised (with 2 kgf/s) to the mean pressure measured at

baselinePPT.Themeanpressurewasheldfor1sand1sofrestwasheldaftereachstimulus.During

thistestthesubjectswereaskedtoratethelevelofpainwithanNRS-score(0-10)atthefirst,fifth

andtenthstimulus.TheTSwasmeasuredconsecutivelyonM.TrapeziusandM.Quadriceps.Insome

patients,theM.TrapeziuswastestedfirstandtheM.Quadricepsafter.Inotherpatients,thisorder

21

was reversed. This selection was chosen randomly. Outcome measurements were the difference

between NRS-score at first and tenth stimulus of the M. Trapezius (‘TS T 10-1’) and the M.

Quadriceps(‘TSQ10-1’).

c. Conditionedpainmodulation(CPM)

Theendogenouspaininhibitionsystem,alsoknownasdiffusenoxiousinhibitorycontrol(DNIC)was

measuredwithahotwaterimmersiontest(HIT).Thisisaconditionedstimulusthatevokesalong-

termpainsensationandisthereforealsocalledaconditionedpainmodulation.HITgeneratesafocal

typeofpain,whichcancontributetoaconsistentactivationofinhibitorypathways.DuringHITthe

subjectswereasked toput the left lowerarmduring twominutes inahot-waterbath (46°C).The

subjectneededtokeephishandopenandcouldnotmakeafist.Theywerenotallowedtomovetheir

handinthewaterduringthesetwominutes.Acutepainstimuliwereappliedthrough10repetitive

pressure stimuliwith thealgometer.Theacutepressure stimuliwere started20 secondsafter the

lowerarmwasplacedinthehot-waterbath.Afterthese20seconds,10painstimuliweregivenon

theM.TrapeziusandM.Quadricepsatapressureequal to thebaselinePPT,withoutremovingthe

lowerarmoutof thewater.Again, thepatientwasasked to rate theexperiencedpainwithaNRS

scoreforthefirst, fifthandtenthstimulus.TheorderoftestingonM.QuadricepsandM.Trapezius

wasagainrandomizedbetweenpatients.Theoutcomemeasurementwasthedifferencebetweenthe

firstNRS-scoreduringimmersionandthefirstNRS-scorebeforeimmersiononM.Trapezius(‘CPMT

during-before’)andM.Quadriceps(‘CPMQduring-before’)

d. Aerobicpowerindextestforexercise-inducedanalgesia(EIA)

Theaerobicpower indextestconsistedofasubmaximalexercisetest tomeasureendogenouspain

inhibition or exercise-induced analgesia (EIA). The subjects sat on an electromagnetic bicycle and

needed to cycle continuously at 60-70 rounds perminute.Heart ratewasmonitored by a Polar®

heart rate meter. Before starting, heart rate in rest was written down and subjects needed to

acclimatize twominutes at a cycling position. The test started at 25W and thewattage increased

every minute with 25W until the submaximal exercise level of the subject was reached. The

submaximalexerciselevelwasdefinedas75%ofage-relatedmaximalheartrate(220-ageinyears).

After everyminute theHRwaswritten downuntil a submaximal levelwas reached. The testwas

completedwithacooling-downexercise(cyclingoneminuteat25W).Immediatelyafterthetest,the

22

PPT was measured again at M. Quadriceps and M. Trapezius. This PPT was compared with the

originalbaselinePPT.OutcomemeasurementswerethedifferencebetweenmeanbaselinePPTand

PPTafterexerciseonM.Quadriceps(‘EIAQafter-before’)andonM.Trapezius(‘EIATafter-before’).

3.3.2Genotyping

TheanalysisofDNAvariations (SNP’s)wasdone in threesteps.Firstly,DNAwasextracted froma

blood sample of all participating subjects. Starting with 5 ml blood, the white blood cells were

isolatedthroughcentrifugationfollowedbylysis,whichresultedinthereleaseofproteins,celldebris

and the DNA from the nucleus. Next, ethanolwas added, which precipitated the DNA specifically.

Consequently,DNAwassolvedinanaqueoussolutionanddilutedtoastandardconcentration.

ThesecondstepwastheamplificationofDNAfragmentsinourgenomethatcontainedtheSNP’sthat

needed to be characterized. The amplification of these DNA fragments was performed by the

PolymeraseChainReaction(PCR)usinggenespecificprimersflankingeachSNPofinterest.Foreach

PCRanalysis,areactionmixturewasmadecontainingDNA,primers,aheatstableTaqpolymerase,

the four freenucleotides (A, C,G andT) and the reactionbuffer.The reactionwasperformed in a

thermocyclermachinewithstandardcyclesettings.

Lastly, theDNAsequencingof theamplifiedPCRproductwasperformedinordertodeterminethe

exact sequenceof the SNP.The reaction,which is called a “Sanger SequencingReaction”,was also

performed in a thermocyclermachine. Consequently, theDNA sequence product generated by the

thermocycler is further processed whereby the DNA sequence itself is determined by capillary

electrophoresesontheAppliedBiosystems3730xlDNAanalyzer.Theresultingfilesareopenedwith

aprogramcalledFinchTVtoanalyzetheDNAsequences.

Figure2:ExampleofdigitalDNA-sequencing

23

3.4.Statisticalanalysis

Toanalyzethedata,StatisticalPackageforSocialSciences(SPSS)version24.0wasused.Descriptive

statisticsareshownasmean±standarddeviation(SD)forcontinuousdataandaspercentagesand

absolutefrequenciesforcategoricaldata.

Hardy-WeinbergequilibriumwasverifiedforobservedgenotypefrequenciesofCOMT(rs4680and

rs4818), OPRM1 (rs179971 and rs563649) and SLC6A4 (5HTTLPR and rs3813034) to detect

deviationsfromtheexpectedgenotypedistribution(Chi2-test).

Two groups were formed to compare the genotypes within one SNP. Each SNP consists of three

genotypes:twohomozygousandoneheterozygoustype.Thehomozygoustypescanbedividedina

“wildtype”anda“mutanttype”.ThewildtypeofaSNPisthehomozygousgenotypethatcomprises

theallelethatoccursmostfrequentlyinnature.Themutanttypeisthehomozygousgenotypewith

theallele thatoccurs less frequently.Themutant typeandheterozygoustypewereput together in

one groupbecause their respective groupswere too small to compare alone.This compiled group

wascomparedwiththewildtype.

Because the data was not distributed normally, a Mann-Whitney U test was used to compare

continuousdataof thewild type-groupandmutantandheterozygous-group.P-values<0,05were

regardedasstatisticallysignificantandneededfurtherattention.

24

4.Results4.1.DistributionofgenotypeandallelefrequenciesThedistributionofthegenotypesandallelefrequenciesofthesixSNP’scanbefoundintables1aand

1b.Basedontheirgenotypes,wedividedthesubjects intwogroups.Thefirstgroupcontainedthe

subjectswiththehomozygouswildtypeoftheSNPandtheothergroupcontainedtheheterozygous

andhomozygousmutanttypesoftheSNP.The‘wildtype’ofagenotypeisthehomozygoustypethat

occursmostfrequentlyinnature.Theoppositeiscalledthe‘mutanttype’.Thenumbersofsubjects

withaspecificgenotypeperSNPandtheirrespectivepercentageonthetotalnumbercanbefoundin

table 1a. The distribution of the different alleles in each SNP can be found in table 1b. Hardy

Weinberg analysis of the genotype data showed no disequilibrium for OPRM1 rs1799971

(Chi2=0,27424), OPRM1 rs563649 (Chi2=0,02494), SLC6A4 5HTTLPR (Chi2=2,39556), SLC6A4

rs3813034(Chi2=2,89941),COMTrs4680(Chi2=0,81818)andCOMTrs4818(Chi2=1,10008).

Table1a:Distributionofgenotypefrequencies Genotypefrequencies

Wildtype Heterozygous+mutanttype

OPRM1rs1799971

AA,n(%) AG+GG,n(%)8(72,7%) 3(27,3%)

OPRM1rs563649

GG,n(%) AG+AA,n(%)

10(90,9%) 1(9,1%)

SLC6A4rs3813034

AA,n(%) AC+CC,n(%)4(36,4%) 7(63,6%)

SLC6A45HTTLPR

LL,n(%) SL+SS,n(%)3(27,3%) 7(63,6%)

COMTrs4818

CC,n(%) CG+GG,n(%)2(18,2%) 9(81,8%)

COMTrs4680

GG,n(%) AG+AA,n(%)3(27,3%) 8(72,7%)

25

Table1b:Distributionofallelefrequencies Allelefrequencies

Wildallele Mutantallele

OPRM1rs1799971

A,n(%) G,n(%)19(86,4%) 3(13,6%)

OPRM1rs563649

G,n(%) A,n(%)21(95,5%) 1(4,5%)

SLC6A4rs3813034

A,n(%) C,n(%)15(68,2%) 7(31,8%)

SLC6A45HTTLPR

L,n(%) S,n(%)13(65%) 7(35%)

COMTrs4818

C,n(%) G,n(%)11(50%) 11(50%)

COMTrs4680

A,n(%) G,n(%)9(40,9%) 13(59,1%)

LegendA,C,G,T:AllelesL:Longalleleofthe5HTTLPRpolymorphismS:Shortalleleofthe5HTTLPRpolymorphism

4.2.Subjectcharacteristics

Atotalof11femalesubjectsagedbetween18-55years(mean:38±9,2)wereincludedinthisstudy.

Thedemographic characteristicsof the subjects foreachSNPare shown in tables2aup to2f.The

demographicsincludeage(years),weight(kg),length(m),BMI(kg/m2),skinelasticity(cm)andthe

beightonscore(/9)ofeachpatient.

Afewstatisticallysignificantdifferenceswerefound.InSLC6A4,rs3813034(table2c),asignificant

difference (p = 0,021) was found, concerning the length between the two groups. The wild type

groupwasfoundtobesignificantlytallerthanthemutanttypegroup. InCOMTrs4818(table2e),

another statistically significant difference (p=0,044) was found, concerning the age between the

differentgenotypes.Thewildtypecontainedsignificantlyyoungersubjectscomparedtothemutant

type. Thirdly, a significant difference (p=0,036), concerning the beighton score of the two groups,

26

wasfoundinCOMTrs4680(table2f).Subjectswithawildgenotypescoredsignificantlyloweronthe

beightontestthanthemutantgenotype.

Thegeneraluseofmedicationofthesubjectsisshownintable2g.Itisnotablethatalargediversity

of medication was used. Especially analgesics (54,5%), gastrointestinal medication (45,5 %) and

antidepressants (36,4 %) were used frequently. The analgesics comprise the use of paracetamol

(27,2%),NSAID (27,3%)andopioids from the firstWHOstep (45,5%).Only twopatients (18,2%)

tookmedicationfromWHOstep2andnobodyfromWHOstep3.Merelythreesubjects(27,3%)did

nottakemedicationofanysort.

Table2a:Demographicsofsubjects(OPRM1,rs1799971)

Totalgroup(n=11)

AA(n=8)

AG+GG(n=3)

Non-parametricP-value

Age(years) 38±9,2 35±5,4 46±13,0 0,259

Weight(kg) 68,5±9,07* 66,6±5,46* 71,7±14,30 0,456

Length(cm) 1,69±0,057* 1,69±0,07* 1,69±0,05 0,881

BMI(kg/m2) 23,9±3,18* 23,2±1,60* 25,0±5,20 0,451

Skinelasticity(cm) 1,49±0,635 1,51±0,564 1,43±0,945 0,837

Beightonscore(/9) 6±2,4 6±1,9 7±4,0 0,675

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtest*:Datafrom3patientsismissing

27

Table2b:Demographicsofsubjects(OPRM1,rs563649)

Table2c:Demographicsofsubjects(SLC6A4,rs3813034)

Totalgroup(n=11)

GG(n=10)

AG+AA(n=1)


Age(years) 38±9,2 38±9,6 37±0 0,634

Weight(kg) 68,5±9,07* 68,5±9,07* n.a. n.a.

Length(cm) 1,69±0,057* 1,69±0,057* n.a. n.a.

BMI(kg/m2) 23,9±3,18* 23,9±3,18* n.a. n.a.

Skinelasticity(cm) 1,49±0,635 1,45±0,635 1,9±0 0,338

Beightonscore(/9) 6±2,4 6±2,4 9±0 0,255

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtest*:Datafrom3patientsismissingn.a.:Notapplicable;alldatafromthisgroupismissing

Totalgroup(n=11)

AA(n=4)

AC+CC(n=7)


Age(years) 38±9,2 45±10,5 34±5,8 0,071

Weight(kg) 68,5±9,07* 70,0±11,55 67,0±7,26* 0,773

Length(cm) 1,69±0,057* 1,73±0,057 1,65±0,017* 0,021

BMI(kg/m2) 23,9±3,18* 23,3±3,78 24,5±2,89* 0,559


Beightonscore(/9) 6±2,4 6±2,9 7±2,2 0,627


28

Table2d:Demographicsofsubjects(SLC6A4,5HTTLPR)

Table2e:Demographicsofsubjects(COMT,rs4818)

Totalgroup(n=10)

LL(n=3)

SL+SS(n=7)


Age(years) 38±9,5 39±3,8 38±11,4 0,491

Weight(kg) 68,5±9,07* 65,0±0* 69,0±9,68 0,513

Length(cm) 1,69±0,057* 1,65±0* 1,70±0,059 0,275

BMI(kg/m2) 23,9±3,18* 24,0±0* 23,9±3,44 1,000


Beightonscore(/9) 7±2,5 8±2,3 6±2,6 0,409


Totalgroup(n=11)

CC(n=2)

CG+GG(n=9)


Age(years) 38±9,2 28±5,7 40±8,4 0,044

Weight(kg) 68,5±9,07* 64,0±8,49 70,0±9,49* 0,505

Length(cm) 1,69±0,057* 1,67±0,007 1,70±0,065* 0,505

BMI(kg/m2) 23,9±3,18* 23,0±2,83 24,2±3,49* 0,736

Skinelasticity(cm) 1,49±0,635 1,50±0 1,49±0,710 1,000

Beightonscore(/9) 6±2,4 6±2,1 7±2,5 0,467


29

Table2f:Demographicsofsubjects(COMT,rs4680)

Table2g:Generaluseofmedicationofsubjects

Totalgroup(n=11)

GG(n=3)

AG+AA(n=8)


Age(years) 38±9,2 42±8,5 36±9,4 0,305

Weight(kg) 68,5±9,07* 74,5±13,44* 66,5±7,74* 0,505

Length(cm) 1,69±0,057* 1,69±0,06* 1,69±0,062* 1,000

BMI(kg/m2) 23,9±3,18* 26,0±2,82* 23,2±3,19* 0,311


Beightonscore(/9) 6±2,4 4±1,7 7±1,9 0,036

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtest*:Datafrom3patientsismissing(GG;n=2,AG+AA;n=6)

Totalgroup(n=11)n(%)

Useofmedication 8(72,7%)

Analgesics• Paracetamol• NSAID(ibuprofen,naproxen,diclofenac)• Opioids(tramadol,codeine,morphine,fentanyl,oxycodone)• Other

6(54,5%)3(27,3%)3(27,3%)1(9,1%)4(36,4%)

Antidepressants(amitriptyline,duloxetine,trazodone) 4(36,4%)

Neuroleptics(gabapentin,pregabalin) 1(9,1%)

Sedatives(benzodiazepine) 2(18,2%)

Cardiovascularmedication(betablocker,diuretics,ACE-inhibitors) 2(18,2%)

Pulmonarymedication 0(0%)

Gastrointestinalmedication 5(45,5%)

Other(homeopathicremedies,nutritionalsupplements,cholesterolloweringmedication) 6(54,5%)

30

4.3.Self-reportedpaincognitions,centralsensitization,physicalactivityandqualityof

lifethroughpainquestionnaires

Five different questionnaireswere filled in by each subject: CIS, IPAQ, HADS, PCS and PVAQ. The

resultsarerepresentedintables3aupto3f.Meanscoresandstandarddeviationforeverytestwere

determinedforeachgroupofeachSNP.

Only one statistically significant difference was found. In COMT rs4680 (table 3f), a significant

difference (p=0,040) could be found, concerning the PCS questionnaire. Subjectswith thewildGG

genotypeofrs4680reportedasignificantlylowerscoreonthistestthansubjectswiththemutantAA

orAGgenotype.

Table3a:Questionnairesofcarrierswithdifferentgenotypes(OPRM1,rs1799971)

Totalgroup(n=11)

AA(n=8)

AG+GG(n=3)


CIS(totalscore/56) 39±12,5 37±12,3 45±13,5 0,259

IPAQ(MET) 2267±4470,5* 2445±5025,0* 1554±1357,7 0,419

HADS(totalscore/42) 13±8,7** 11±9,1** 19±4,4 0,120

PCS(totalscore/52) 22±14,7** 22±13,8** 23±19,6 1,000

PVAQ(totalscore/80) 39±18,4** 38±18,9** 41±21,2 0,796

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtest*:Datafrom1patientismissing**:Datafrom2patientsismissingMET:MetabolicEquivalentofTask(1MET=1,162W/Kg)Totalscore:Totalscoreonthepossiblemaximumontherespectivequestionnaires

31

Table3b:Questionnairesofcarrierswithdifferentgenotypes(OPRM1,rs563649)

Table3c:Questionnairesofcarrierswithdifferentgenotypes(SLC6A4,rs3813034)

Totalgroup(n=11)

GG(n=10)

AG+AA(n=1)


CIS(totalscore/56) 39±12,5 40±13,1 35±0 1,000

IPAQ(MET) 2267±4470,5* 896±1150,5* 14610±0 0,106

HADS(totalscore/42) 13±8,7** 13±8,7 n.a. n.a.

PCS(totalscore/52) 22±14,7** 22±14,7 n.a. n.a.

PVAQ(totalscore/80) 39±18,4** 39±18,4 n.a. n.a.

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtest*:Datafrom1patientismissing**:Datafrom2patientsismissingn.a.:Notapplicable;alldatafromthisgroupismissingMET:MetabolicEquivalentofTask(1MET=1,162W/Kg)Totalscore:Totalscoreonthepossiblemaximumontherespectivequestionnaires

Totalgroup(n=11)

AA(n=4)

AC+CC(n=7)


CIS(totalscore/56) 39±12,5 46±11,8 35±12,0 0,154

IPAQ(MET) 2267±4470,5* 1122±1459,5* 2758±5322,7 0,814

HADS(totalscore/42) 13±8,7** 14±7,8 13±10,2** 0,623

PCS(totalscore/52) 22±14,7** 20±17,0 24±14,3** 0,325

PVAQ(totalscore/80) 39±18,4** 34±21,6 43±16,9** 0,327


32

Table3d:Questionnairesofcarrierswithdifferentgenotypes(SLC6A4,5HTTLPR)

Table3e:Questionnairesofcarrierswithdifferentgenotypes(COMT,rs4818)

Totalgroup(n=10)

LL(n=3)

SL+SS(n=7)


CIS(totalscore/56) 41±12,4 29±5,2 45±11,3 0,051

IPAQ(MET) 2316±4738,8* 4870±8435,1 1040±1265,0* 0,787

HADS(totalscore/42) 15±7,8** 14±0** 15±8,5 0,826

PCS(totalscore/52) 23±15,1** 25±0** 23±16,3 0,510

PVAQ(totalscore/80) 40±19,4** 39±0** 40±21,0 0,827


Totalgroup(n=11)

CC(n=2)

CG+GG(n=9)


CIS(totalscore/56) 39±12,5 52±5,7 36±11,9 0,193

IPAQ(MET) 2267±4470,5* 179±252,4 2789±4912,1* 0,346

HADS(totalscore/42) 13±8,7** 16±15,6 13±7,5** 0,769

PCS(totalscore/52) 22±14,7** 32±22,6 19±12,6** 0,240

PVAQ(totalscore/80) 39±18,4** 59±13,8 33±15,8** 0,079


33

Table3f:Questionnairesofcarrierswithdifferentgenotypes(COMT,rs4680)

4.4.Associationofgenotypeandpressurepainsensitivity

Differentpainmeasurementswereexecutedonthepatients.Theirscoreswerecollectedintables4a

upto4f.InOPRM1rs1799971(table4a),twostatisticallysignificantdifferenceswerefound.Firstly,

thewildAAgenotypegroupshowedasignificantlylowerbaselinepainpressurethresholdontheM.

TrapeziusthanthemutantAG+GGgenotypegroup(p=0,025).Inaddition,thedifferenceofthePPT

on theM. Trapezius after and before EIA is significantly lower in the AA group compared to the

AG+GGgroup(p=0,024).

TwostatisticallysignificantdifferenceswerefoundinCOMTrs4818(table4e).ThewildCCgenotype

group showed a significant higher difference betweenNRS score at first and tenth stimulus onM.

TrapeziuscomparedtothemutantCG+GGtype(p=0,031).Furthermore,thewildtypealsoshoweda

significanthigherdifferencebetweenNRSscoresatfirststimulionM.Trapeziusbeforeandduring

hotwaterimmersioncomparedtothemutanttype(p=0,033).

Nosignificantdifferencesbetweengenotypesregardingpressurepainsensitivitycouldbe foundin

OPRM1rs563649(table4b),SLC6A4rs3813034(table4c),SLC6A45HTTLPR(table4d)andCOMT

rs4680(table4f).

Totalgroup(n=11)

GG(n=3)

AG+AA(n=8)


CIS(totalscore/56) 39±12,5 34±13,0 41±12,6 0,259

IPAQ(MET) 2267±4470,5* 911±1288,7* 2606±4980,1 0,686

HADS(totalscore/42) 13±8,7** 9±11,3** 15±8,4** 0,557

PCS(totalscore/52) 22±14,7** 10±3,5** 26±14,7** 0,040

PVAQ(totalscore/80) 39±18,4** 33±0,7** 41±20,8** 0,770

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtest*:Datafrom1patientismissing**:Datafrom2patientsismissing(onefromeachgroup)MET:MetabolicEquivalentofTask(1MET=1,162W/Kg)Totalscore:Totalscoreonthepossiblemaximumontherespectivequestionnaires

34

Table4a:Painscoresofcarrierswithdifferentgenotypes(OPRM1,rs1799971)

Totalgroup(n=11)

AA(n=8)

AG+GG(n=3)


BaselinePPT

Q(kgf)T(kgf)

4,24±2,5902,49±1,172

3,53±2,6621,98±0,906

6,15±1,1103,86±0,400

0,1530,025

TS Q10-1T10-1

2±2,62±2,3

2±2,53±2,3

1±3,01±2,0

0,5340,351

CPM Qduring-beforeTduring-before

-1±2,22±2,6

-1±2,03±2,6

0±3,01±2,1

0,5360,182

EIA Qafter-before(kgf)Tafter-before(kgf)

0,21±0,7500,47±1,334

0,20±0,842-0,07±0,443

0,23±0,5751,90±2,002

0,7590,024

LegendMean±standarddeviationNon-parametricP-value:MannWhitneyUtestkgf:kilogram-force(1kgf=9,80665N)PPTQ:meanpressurepainthresholdM.QuadricepsPPTT:meanpressurepainthresholdM.TrapeziusTSQ10-1:differencebetweenNRSscoreatfirstandtenthstimulusonM.QuadricepsTST10-1:differencebetweenNRSscoreatfirstandtenthstimulusonM.TrapeziusCPMQduring-before:differencebetweenNRSscoresatfirststimulionM.QuadricepsbeforeandduringhotwaterimmersionCPMTduring-before:differencebetweenNRSscoresatfirststimulionM.TrapeziusbeforeandduringhotwaterimmersionEIAQafter-before:differencebetweenmeanPPTandPPTonM.QuadricepsafterexerciseEIATafter-before:differencebetweenmeanPPTandPPTonM.Trapeziusafterexercise

35

Table4b:Painscoresofcarrierswithdifferentgenotypes(OPRM1,rs563649)

Totalgroup(n=11)

GG(n=10)

AG+AA(n=1)


BaselinePPT

Q(kgf)T(kgf)

4,24±2,5902,49±1,172

4,25±2,7302,50±1,235

4,15±02,43±0

1,0000,752

TS Q10-1T10-1

2±2,62±2,3

2±2,32±2,1

-2±0-1±0

0,1480,148


-1±2,22±2,6

0±1,83±2,5

-5±0-1±0

0,1100,152


0,21±0,7500,47±1,334

0,17±0,7770,44±1,401

0,63±00,79±0

0,5260,342


36

Table4c:Painscoresofcarrierswithdifferentgenotypes(SLC6A4,rs3813034)

Totalgroup(n=11)

AA(n=4)

AC+CC(n=7)


BaselinePPT

Q(kgf)T(kgf)

4,24±2,5902,49±1,172

3,98±2,7442,65±1,654

4,39±2,7102,40±0,946

0,7050,850

TS Q10-1T10-1

2±2,62±2,3

1±2,41±1,7

2±2,73±2,5

0,3870,387


-1±2,22±2,6

-1±1,51±2,2

-1±2,63±2,6

0,7020,216


0,21±0,7500,47±1,334

0,12±0,5130,38±0,693

0,26±0,8930,52±1,648

0,2560,449


37

Table4d:Painscoresofcarrierswithdifferentgenotypes(SLC6A4,5HTTLPR)

Totalgroup(n=10)

LL(n=3)

SL+SS(n=7)


BaselinePPT

Q(kgf)T(kgf)

4,42±2,6612,51±1,233

6,42±2,0572,84±0,839

3,56±2,5202,38±1,404

0,1380,569

TS Q10-1T10-1

2±2,62±2,4

0±2,01±1,5

2±2,63±2,5

0,2040,166


-1±2,32±2,6

-1±3,51±1,5

0±1,93±2,9

0,5640,302


0,23±0,7880,60±1,334

0,30±1,4440,22±0,520

0,20±0,4820,76±1,57

0,7320,819


38

Table4e:Painscoresofcarrierswithdifferentgenotypes(COMT,rs4818)

Totalgroup(n=11)

CC(n=2)

CG+GG(n=9)


BaselinePPT

Q(kgf)T(kgf)

4,24±2,5902,49±1,172

1,50±0,5661,31±0,074

4,86±2,4572,75±1,137

0,0990,157

TS Q10-1T10-1

2±2,62±2,3

4±2,86±2,1

1±2,41±1,5

0,1880,031


-1±2,22±2,6

-1±1,16±1,4

-1±2,41±1,9

0,4740,033


0,21±0,7500,47±1,334

0,47±0,622-0,16±0,106

0,15±0,7970,61±1,450

0,2380,288


39

Table4f:Painscoresofcarrierswithdifferentgenotypes(COMT,rs4680)

Totalgroup(n=11)

GG(n=3)

AG+AA(n=8)


BaselinePPT

Q(kgf)T(kgf)

4,24±2,5902,49±1,172

5,60±2,6892,85±1,012

3,74±2,5372,36±1,263

0,2210,540

TS Q10-1T10-1

2±2,62±2,3

1±3,11±1,7

2±2,43±2,4

0,3510,351


-1±2,22±2,6

-2±1,22±2,1

0±2,43±2,8

0,1480,681


0,21±0,7500,47±1,334

-0,49±0,689-0,28±0,497

0,47±0,6150,75±1,462

0,0520,184


40

5.Discussion5.1Summaryofstudyfindings

The purpose of this study was to detect an association between polymorphisms in the OPRM1

(rs1799971 and rs563649), SLC6A4 (5HTTLPR and rs3813034) and COMT (rs4680 and rs4818)

genes and pressure pain sensitivity in patients with the hypermobile type of Ehlers-Danlos

syndrome.Thedistributionof thegenotypeswas inequilibrium.A fewassociationsregardingpain

measurementswerefoundinOPRM1rs1799971andCOMTrs4818.ThebaselinePPTandEIAonthe

M. Trapezius were significantly lower in rs1799971 (OPRM1). In rs4818 (COMT), two significant

differenceswere foundconcerning theTSandCPM.Nosignificantassociationsbetweengenotypes

regarding pressure pain sensitivity in OPRM1 rs563649, SLC6A4 (5HTTLPR and rs3813034) and

COMTrs4680couldbedetected.

5.2PressurepainsensitivityinOPRM1

In current literature, there is little evidence forassociationsbetween theexpressionof rs1799971

andpainsensitivityinpatientswithchronicpain,suchasfibromyalgia.Thisstatementcorresponds

with findings inpreviousstudiesthathavebeenconducted(22,23,24). Additionally, inonestudy, the

frequencyofthers17999171polymorphismwaslowerinpatientswithfibromyalgia,butitdidnot

have a significant effect on pressure pain and fibromyalgia severity (24). Studies regarding

associations between the expression of rs1799971 and pain sensitivity in healthy populations

showeddifferentoutcomes(41,42).Findingsinthesestudiesstatethatthepainsensitivityofsubjects

fromtherareallelegrouphasbeendecreasedduetoagreaterbindingaffinityinthecellstructureof

thevariantreceptorforbeta-endorphin(41).Otherfunctionalpolymorphismscouldbeabletocause

changesinpainsensitivity,butmightnotbediscoveredyet(41).ConcerningEDSpatients,wesuspect

ourresultsofmeasurementstobein linewithpreviousfindingsinotherchronicpainpopulations,

suchasfibromyalgia-(24)andCWPpatients(23).

Regarding baseline PPT, current results show a significant difference in baseline PPT on the M.

TrapeziusbetweentheAAgroupandtheAG+GGgroup(p=0,025).SubjectswiththehomozygousAA

group showed significantly higher pain sensitivity compared to the combined AG+GG group. This

interfereswithresultsfoundinotherarticles(22,23,24).Previousstudiesconcerningfibromyalgiaand

CWP patients indicated that there was no significant association between this SNP and pain

sensitivity,butresultsofthisstudysuggestthereisapossibleassociationbetweentheexpressionof

41

rs1799971 in EDS patients and pressure pain thresholds. Subsequently, another significant

differencebetweenthegenotypegroupswasfoundregardingPPTonM.Trapeziusafterandbefore

EIA (p=0,024). The AA group showed significantly lower scores in comparison with the AG+GG

group.InEIA,theinfluenceofexerciseonpainsensitivityismeasured.PPT’sareexpectedtoincrease

due to activation of the mu-opioid receptors that trigger the release of beta-endorphins, causing

analgesia. In this study, patients from theAG+GG group showed adequate raises in PPT after EIA.

However,fiveoutofeightpatientsfromthewildtypegroup(AA)showedadecreaseofthePPTon

the M. Trapezius. This suggests that EDS patients from this group have possible dysfunctional

responsesoftheinhibitorypainpathwaysandaberrationsincentralpainmodulationtoexercise(43).

Measurement of the PPT’s on the M. Quadriceps before and after EIA showed no significant

differencesinbothwildandmutantgroups.

AnothercommonSNPofOPRM1isrs563649.NoassociationsbetweenthisSNPandpainsensitivity

were found in current literature (23,44). In this study, the findings of previous studies could be

confirmed.TherewerenosignificantdifferencesbetweenthewildGGgroupandthemutantAG+AA

group. However, the mutant group consisted of only one patient. Comparisons between the two

groupsarethereforelimitedandconclusionscannotbemade.

5.3PressurepainsensitivityinSLC6A4

The associations between heat pain perceptions and 5HTTLPR in adults with chronic pain were

examined in previous studies. One study stated that the intermediate 5HTTLPR-expressing group

(SL) was associated with greater heat pain thresholds compared to the high expressing group

(LL)(20). Another study compared thermal pain thresholds in healthy controls to fibromyalgia

patients.Painratingswerehigherinpatientswithfibromyalgia(21). Inthisresearch,pressurepain

thresholdsweremeasuredinsteadofheatpainthresholds,butasimilaroutcomecouldbeexpected.

However, contrary to previous findings, no significant associationswere found between 5HTTLPR

and pressure pain sensitivity in patients with EDS. Conform to findings in previous studies, only

intermediate and high expressing groups of 5HTTLPRwere discussed or used. Themutant group

consisted only of intermediate expressing genotypes (SL) and no low expressing genotypes (LL)

werepresent.ThiscouldbeanindicationfortheimportanceoftheS-alleleinpatientswithchronic

pain.

42

Only one article discussed the associations between pain thresholds and rs3813034 in healthy

adults.ThearticlestatedthattherewerenosignificantdifferencesbetweenthegenotypesofthisSNP

andpainthresholdsafterCPM.Similarly, inthisresearchnoassociationswerefoundbetweenpain

expressioninrs3813034andpressurepainthresholdsandCPM.Again,themutantgroupconsisted

ofonlyonesubjectwithanintermediateexpressinggenotype(AC)andnolowexpressinggenotypes

(CC)occurredinthisstudy.

5.4PressurepainsensitivityinCOMT

The most studied SNP in COMT is rs4680, also referred to as Val158Met. At codon 158 on

chromosome 22, a substitution between valine and methionine has been made, leading to three

different genotypes. The Val/Val genotype (GG) is the wild type of this SNP, while the Met/Met

genotype(AA)isthemutanttype.ThewildtypeischaracterizedbyhighCOMTenzymeactivitywith

thereforelowerdopaminelevels,leadingtohigherpainthresholds.Themutanttypeofrs4680shows

lower COMT enzyme activity resulting in higher dopamine levels and therefore higher pain

sensitivity.Thistrendhasbeenconfirmedinseveralstudiesindifferentpopulationsofpatientswith

chronicpain.TheMet/Metvariantoccursmorefrequentlyinthesepatientsandresultsinlowerpain

thresholds.(18,45,46,47,48).ThisleadstothehypothesisthatamongEDS-HTpatients,themajoritymight

have theMet/Met genotype (AA). However, this hypothesis could not be confirmed based on the

resultsofourstudy.OnlyoneoutofelevenpatientshadtheAAgenotype.Sevenotherpatientshad

theAGgenotype,whichcorrespondswithan intermediateactivityof theCOMTenzyme.MeanPPT

scoresofpatientsfromtheAA+AGgroupwerelowerthenthetotalgroupaverage,meaningthatthe

hypothesis that the variant Met-allele corresponds with lower pain thresholds still stands. This

differencewashowevernotsignificant.Nosignificantdifferencescouldbefoundatallbetweenthe

twogroups.Therefore,definiteconclusionsabouttheMet/MetandVal/Valvariantscannotbemade

intheEDShypermobilepopulation.

In current literature,most results indicateanassociationbetween theexpressionofCOMTrs4818

andpain sensitivity.TheGGgenotype is themutant typeand ismore stronglyassociatedwithFM

andalowerpainthreshold.Inthisstudy,threeEDSpatientswiththisgenotypewereincluded.Seven

ofthetotalofelevenpatientshadtheheterozygoustype(CG).Ahypothesiscanbeformedthatthe

GGandCGgenotypeareassociatedwithlowerpainthresholds(48).However,inthisstudysignificant

resultswerefoundindicatinghigherpainsensitivityinpatientswiththewildCCgenotype.Pressure

43

painmeasuredontheM.TrapeziuswassignificantlyhigherduringTS(p=0,031).Subsequentlythe

pressure pain threshold in CPM was also higher compared to the mutant type (p=0,033).

Respectively,forboththefacilitatingpainpathwaysandtheinhibitingpathwaysthepainsensitivity

washigherinthewildtypegroup.

5.5Subjectcharacteristicsandquestionnaires

ForeverySNP,age,weight,height,BMI,skinelasticityandabeightonscorewerecomparedbetween

the genotype groups. Only three statistically significant differences could be found between the

genotypes. These three differences occurred in three different SNP’s and were based on three

differentcharacteristics.

Concerning the pain questionnaires, only one significant difference was found. This difference

occurredbetweentherareandwild-allelegroupsofrs4680oftheCOMTgene.Itwasthetotalscore

onthePCSquestionnairethatdifferedsignificantly(p=0,040).TheGGgroup,thegenotypeassociated

with lower pain sensitivity, showed significantly lower scores on this questionnaire. Lower scores

correspondwithlowerratingsofpaincatastrophizing.Althoughasignificantresultispresenthere,

the filling in of the questionnaires remains very subjective. Peoplewho have the same score on a

questionnairemay actually feel or reactdifferently in real life. The results of thesequestionnaires

shouldthereforebeinterpretedcritically.Thisresultwaspreviouslyfoundinotherarticles(45).Itis

said thatFMpatientswith theMet/Metgenotype (AA) reportedworsescores forallpsychological

variables.Thisisinagreementwiththefindingsinthisstudy.

5.6StrengthsandlimitationsTheresultsmustbeviewedwithinthestrengthsandlimitationsofthestudy.Thisisthefirststudy

investigatingtheassociationbetweenpaininEDS-HTpatientsandgeneticfactors.

Firstly,thepatientsaskedtofillinquestionnairestodeterminetheseverityoftheirpainsymptoms,

presenceof central sensitization and to analyzepsychological factors.ThePCS,PVAQ, IPAQ,HADS

andCISwereusedforthis.Mostofthesepainquestionnairesshowgoodinternalconsistencyandfair

togoodtest-retestreliability(37,38).However, inmostofthecases,datawasmissingforoneortwo

patients out of almost every questionnaire. For OPRM1 rs563649, no data for theHADS, PCS and

44

PVAQ was retrieved of the heterozygous + homozygous mutant type group. Only for the CIS

questionnaire,alldatawascomplete.

Secondly, the study protocol of this study, including measurements of baseline pressure pain

thresholds, TS, CPM and EIA, was extensive and the tests were all standardized. Different pain

systemswereobservedandeverypainmeasurementwaslinkedtoPPT.Still,itispossiblethatother

painpathwaysintervenedwiththeresultssuchasthermalpain,ischemicpainorelectricpain.Ithas

beenproventhatthesepainsystemsmayhaveaninfluenceonpainsensitivity(50).Otherinfluencing

factorsalsomighthavebeenuseful likeheartrate,bloodpressureorphase inthemenstrualcycle.

Patientswere asked to deprive themselves of caffeine, nicotine, certainmedications and intensive

physicalexercisebutitcannotbeassuredifallpatientsrespectedtheserequirements.

The biggest limitation of this study was the small population. Only 11 EDS-HT patients could be

included. For one patient no genotyping could be performed regarding SLC6A4 5HHTLPR due to

unusablesequencing,soforthisgeneonly10EDSpatientswereobserved.Allincludedpatientswere

female,meaningthatneithergeneralstatements,neitheracomparisonwithmalepatientscouldbe

made.Ontheotherhand,themajorityofthepatientswithEDS-HTarewomen.Becauseofthesmall

patient group, it is hard to make general conclusions whether or not significant data could be

extended to the majority of EDS-HT patients or if it was influenced by chance or other patient

dependentfactors.Sincethiswasthefirststudyinvestigatingthismatter,nocomparisonswithother

studiesregardingthesamesubjectcanbemade.

5.7Suggestionsforfutureresearch

It isclearthat thissubjectstillneedsmuchattentionandexploration inthe future.This is the first

studyregardingtheassociationbetweenpainsensitivityandEDSpatients.Painsensitivityandpain

patternsaredifficulttocomprehendinthesetypesofpatientsandshouldthereforebeinvestigated

extensivelyinthefuture.Afewpossiblesuggestionsforfurtherresearchcanbemadebasedonthe

courseofthisstudy.

Firstofall, this studydiscusses the influenceonpainsensitivityof theOPRM1,SLC6A4andCOMT

geneswithtwoknownpolymorphismseach.Thereareundoubtedlyother,unknownpolymorphisms

thatstillhavetobefoundthatalsohaveapossibleassociationwithpainsensitivity(41).Additionally,

45

interactionsbetweengenesandtheirvariantSNP’sshouldalsobetakenintoaccount(33).Evenother

genesthanCOMT,OPRM1andSLC6A4shouldbeverifiedforpossibleassociationstopainsensitivity

orEDS.

Inthisstudy,patientshavebeencomparedbasedontheirgenotypes.Thisclassificationappearsto

bepopularandhasbeenused inmanypreviousstudies(18,45,48).Nevertheless,otherclassifications

shouldbe considered.They canprovidedifferentoutcomesbetweenpatient groupsandprovidea

different viewon thematter. For example,dividingEDSpatients according to the severityof their

symptoms could give a various perspective on their genotypes and pain experiences. Many

classificationsarepossibleandshouldbeconsidered.

ThepatientsofthisstudywereallwomensufferingfromthehypermobiletypeoftheEhlers-Danlos

syndrome.TobeabletoformconclusionsaboutEDSpatientsandtheirgenotypesorpainsensations,

itwouldbebettertocomparewithahealthypopulation.Iftheirresultsdiffersignificantlyfromthose

of the EDS patients, it can be more clear where the problem or abnormality is located. Another

interesting comparison isbetweena female andmalepopulation.Again, this canprovidedifferent

insightsinthediseaseanditscharacteristics.

Lastly, an interesting aspect for further research is the investigation of the clinical importance of

polymorphisms, especially those of COMT, SLC6A4 and OPRM1, regarding the effect on analgesic

responsesandopioidaddictionorabuseinchronicpatients.

46

6.ConclusionInconclusion,ourfindingssuggestthatanassociationbetweengenotypeandpainsensitivityinEDS-

HTpatientscanbemade.This isapplicableforOPRM1rs1799971andCOMTrs4818inrelationto

pressure pain threshold. For OPRM1 rs563649, SLC6A4 (5HTTLPR and rs3813034) and COMT

rs4680, no such associations were found. However, due to influencing factors such as the small

populationgroup,thesestatementscannotbegeneralizedforallEDS-HTpatients.

The exact mechanism and role of different genotypes of OPRM1, SLC6A4 and COMT in pain

sensitivityisnotyetverified.Theassociationsofthesegenesandpainsensitivityshouldbefurther

examined inhealthysubjectsandotherpatientgroups. Thesameapplies tostudieswithdifferent

painmodalitiesandothergeneticinfluencingfactors.

47

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52

Abstractinlekentaal

Achtergrond: Er is reeds onderzoek gebeurd dat aantoont dat er een mogelijk verband zou zijn

tussenafwijkingeninbepaaldegenenenpijngevoeligheidbijverschillendemensen.

Doelstelling:Wij gaan onderzoeken of er een associatie kan aangetoond worden tussen bepaalde

afwijkingen in genen (OPRM1 rs1799971 en rs563649, SLC6A4 5HTTLPR en rs3813034, COMT

rs4680enrs4818)enpijngevoeligheidbijpatiëntenmethethypermobiele typevanEhlers-Danlos

syndroom.

Methode:Indezestudiezaten11volwassenvrouwendielijdenaanhethypermobieletypevanhet

Ehlers-Danlossyndroom(EDS-HT).Detestprocedurebestonduithetinvullenvanvijfpsychologische

vragenlijsten en uit vier verschillende pijntestingen. Deze testen bestonden uit het meten van de

drukpijndrempelsinverschillendesituaties.Daarnawerdvanelkepatiënteenbloedstaalafgenomen

en hieruit werd het DNA geanalyseerd om te kijken of ze bepaalde afwijkingen in de genen

vertoonden.

Resultaten:Voortweevandezesgenendieweonderzochthebbenvondenweeenmogelijkverband

in relatie totpijngevoeligheidenvoordevier anderegenenvondenwegeenassociaties.Vooréén

genvondenweeenmogelijkverbandmeteenpsychologischevragenlijst.

Conclusie: Volgens deze studie is er bewijs dat er een verband zou zijn tussen twee van de

onderzochtegenenenpijngevoeligheidinEDS-HTpatiënten.Voordevieranderegenenwerdengeen

associaties gevonden. Toch kunnen we deze conclusies niet doortrekken voor alle EDS patiënten

omdatereenaantalbeperkendefactorenzijnzoalseentekleinetestgroep.

53

Ethicalcommittee

54

Addendum:ValorisationPotentieeltotvalorisatievanhetuitgevoerdeonderzoek Het is nietmogelijk omonsonderzoek reeds te valoriserenomdatdit nogmaarhet fundamentele

onderzoekisenhetzichdusnogmaarindeopstartfasebevindt.Er isnogverderonderzoeknodig

omklinischerichtlijnenoptestellenenteimplementeren.

Wij geven ons onderzoek een score van 6/10 omdat alle testen gestandaardiseerd verliepen en

enkele significantewaarnemingengevondenwerden.Desondankskunnendezenietmet zekerheid

wordenvastgesteldomwillevaninterveniërendefactoreneneenkleinepopulatie.Aangezienditde

eerstestudiewasomtrenteenassociatietussenpijngevoeligheidengeneticainEDSpatiëntenishet

onmogelijkomreedseenfinaleuitkomstomtrentditonderwerptebereiken.

Stakeholdersdieeenconcreteopportuniteitbijonsonderzoekkunnenhebbenzijnartsen,kinesisten,

EDS-patiëntenenpersonendieonderzoekdoennaarpijnengenetica.

the association between single nucleotide polymorphisms...

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