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Possible association of temporomandibular joint pain and dysfunction with a polymorphism in the serotonin transporter gene

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Possible association of temporomandibular joint pain and dysfunction with a polymorphism in the serotonin transporter gene
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  308 T he concept of a single temporomandibular joint(TMJ) syndrome or disorder has been replacedwith a series of diagnostic categories (such asmyofascial pain and dysfunction,internal derange-ment,arthritic disorders,and muscle hyperactivity dis-orders). Although many pain syndromes can affect theface or the head and neck region,the more prevalentconditions include TMJ disorders and atypical facialpain. 1,2 Previously,the myofascial pain dysfunctionsyndrome included all TMJ/masticator muscle pain, jaw dysfunction and joint-clicking conditions. 3,4 The pathophysiologic mechanisms underlying TMJdysfunction and pain have been linked to disturbance inserotonin (5-HT) metabolism and transmission. 5-8 Theactivity of the 5-HT is regulated by the gene known asthe 5-HT transporter  (5-HTT) gene that has 2 identifiedpolymorphic regions (Fig 1). The 5-HTT gene is locatedon chromosome 17q 11.2,and a functional 44-base pair(bp) deletion/insertion polymorphism has been identi-fied in the 5´-flanking promoter region of 5-HTT gene(5-HTT gene-linked polymorphic region [5-HTTLPR]),which can create a short (S) and a long (L) allele.The 5-HTTLPR polymorphism is situated in a guaninecytosine (GC)-rich region composed of 20- to 23-bp repeat-ing units. The S and L alleles have 14 and 16 repeat ele-ments,respectively. 9,10 The short variant,S,is associatedwith the reduced transcriptional efficiency of the 5-HTTgene promoter that results in lowered serotonin uptakeactivity,when compared with the long,L,variants. 9-11 Recently,another polymorphic region that conformed a 17-bp variable number of tandem repeats (VNTR) in the sec-ond intron of the 5-HTT gene was also described. 12,13 a Department of Psychiatry,Medical Faculty of Gaziantep University,Gaziantep,Turkey. b Department of Medical Biology and Genetics,Medical Faculty of Mersin Uni-versity,Mersin,Turkey. c Department of Oral and Maxillofacial Surgery,Faculty of Dental School,Sel-cuk University,Konya,Turkey. d Department of Medical Biology and Genetics,Medical Faculty of GaziantepUniversity,Gaziantep,Turkey. e Department of Orthodontics,Faculty of Dental School,Selcuk University,Konya,Turkey.Reprint requests to:Hasan Herken,MD,Department of Psychiatry,MedicalFaculty of Gaziantep University,Gaziantep,Turkey; e-mail,hasanherken@hot-mail.com.Submitted,September 2000; revised and accepted,December 2000.Copyright ©2001 by the American Association of Orthodontists.0889-5406/2001/$35.00 + 0 8/1/115307 doi:10.1067/mod.2001.115307 ORIGINAL ARTICLE Possible association of temporomandibular jointpain and dysfunction with a polymorphism inthe serotonin transporter gene Hasan Herken,MD, a Emin Erdal,PhD, b Necip Mutlu,DDS,PhD, c Ömer Barlas,RA, d Osman Cataloluk,PhD, d Fatih Oz,DDS, e and Enis Güray,DDS,PhD e Gaziantep,Mersin,and Konya,Turkey The purpose of this study was to evaluate the relationship between temporomandibular joint pain anddysfunction and serotonin transporter (5-HTT) gene polymorphism.Forty-eight patients withtemporomandibular joint pain and 111 healthy control subjects were examined.The results for the patients andcontrol subjects were not significantly different ( P  > .05).The analysis of genotype distribution (homozygousfor STin 2.10 genotypes of the variable-number tandem-repeat polymorphism) showed significant differencesbetween the patients and control subjects ( P  = .003).ST 2.10 allele was more frequent in the patients withtemporomandibular joint pain and dysfunction.In the control group, however, STin 2.12/12 genotype wassignificantly higher ( P  = .017).In the patients who were homozygous or heterozygous for variable-numbertandem-repeat variants of 5-HTT STin 2.12 copies, the average scores of somatization and anger weresignificantly higher than those who were homozygous for STin 2.10 variant ( P  < .05).The patients who werehomozygous for STin 2.10 genotype were also homozygous for “L”genotype ( P  = .019).However, this was notthe condition in the control subjects.This study does not provide evidence to support the involvement of 5-HTT gene-linked polymorphic region in temporomandibular joint pain and dysfunction.Our findings indicatedthat only the presence of the homozygous STin 2.10 genotype of variable-number tandem-repeat is likely toplay a substantial role in the genetic predisposition to temporomandibular joint pain and dysfunction and thatthe STin 2.12/12 genotype may have a protective role against temporomandibular joint pain and dysfunction.(Am J Orthod Dentofacial Orthop 2001;120:308-13)   American Journal of Orthodontics and Dentofacial Orthopedics  Herken et al 309 Volume 120  ,Number 3 The 5-HTT gene proved to be associated with anumber of conditions (such as autism,severe alco-holism,anxiety disorder,seasonal affective disorder,fibromyalgia,migraine,depression,schizophrenia,and bipolar affective disorder). 14-22 Although the 5-HTT has been proposed as a possible candidate for itsinvolvement in the pathogenesis of major psychosisand bipolar affective disorders, 11,17 the gene was notproved to be involved in the pathogenesis of mooddisorders. 23,24 Because of the presence of a possible pathophysio-logic role of the 5-HT in TMJ dysfunction and pain,wethought that the polymorphism in the 5-HTT gene,which determines the 5-HT activity,could also have arole in this disease state. For this reason,the aim in thisstudy was to clarify the association of the 5-HTT genewith TMJ pain disorder (TMJPD) by examining thepolymorphisms in the promoter region (5-HTTLPR)and in the second intron (VNTR). MATERIAL AND METHODS Forty-eight patients with TMJPD who were consec-utively admitted to the Department of Orthodontics &Oral and Maxillofacial Surgery Clinic,Faculty of Den-tal School,Selcuk University,Turkey,were included inthe study. The diagnoses of TMJPD were assignedaccording to our standard examination protocol thatwas modified from the previously described diagnosticcriteria 25 (Fig 2). The control group consisted of 111individuals who represented different social groups.The patients and healthy subjects who participated inthe study were volunteers.The age,gender,and duration of illness were docu-mented. Personnel interviews were conducted,andblood samples were obtained after physical examina-tion. Those candidates who had mental retardation,drug dependence,or somatic or neurologic illnesses(eg,hypothyroidism mimicking a depressive state,positive toxicologic findings) that could impair thepsychiatric evaluation were excluded. Persons whosefirst-degree relatives had endogenous psychoses oralcoholism were also excluded. Psychiatric evaluation The patients were evaluated with the SymptomChecklist 90 Revised (SCL-90-R), 26 Beck DepressionScale (BDS), 27 and State-Trait Anxiety Inventory I andII (STAI-I and -II). 28 Minnesota Multiphasic Personality Inventory,apersonality questionnaire used to identify personaltraits of patients, 29 was performed on the patients andhealthy controls.The SCL-90-R measures somatization,obsession,compulsion,depression,anxiety,sensitivity and hostil-ity between people,phobic and paranoid thinking,psy-choticism,and it includes a global severity index. Weused a Turkish version of the SCL-90-R.The BDS evaluates depressive symptoms and atti- Fig 1. Locations of polymorphic regions in serotonin transporter gene.  310  Herken et al  American Journal of Orthodontics and Dentofacial OrthopedicsSeptember 2001 tudes. A single summed score can range from 0 to 63;a higher score indicates greater depression. We used aTurkish version of the BDS.The STAI I-II is a 2 × 20-item inventory and givesscores that index the current anxiety level of the subject(“state”) and degree to which the subject is prone toexperience anxiety (“trait”). We used a Turkish versionof the STAI I-II. Molecular analysis DNA was extracted from patients and control sub- jects from whole blood by standard techniques. A func-tional 44-bp insertion/deletion polymorphism in thepromoter region of the SERT gene was typed by poly-merase chain reaction (PCR) amplification of DNAwith flaking primers 5´-CTTGTT GGGGATTCTCC-CGCCTGGCGTT- 3´ (forward) and 5´-TCGAGGCT-GAGCGTCTAGAGGG ACTGAGCTGG-3´ (reversed).PCR was performed with GC Rich PCR system (RocheMolecular Biochemical) in a 50 µ L reaction mixturethat contained 100 to 200 ng DNA,100 µ L deoxyri-bonucleoside triphosphate,20 pmol of each primer,and1.5 mmol/L MgCl 2 . DNA was denatured at 95°C for 3minutes and 35 cycles at 95°C for 1 minute for denatu-ration,1 minute at 60°C for annealing and 1 minute at72°C for extention,followed by 7 minutes at 72°C for final extention. Amplification products wereresolved by electrophoresis on 2% agarose gels next to a DNA molecular weight standard and visualizedwith ultraviolet ethidium bromide staining. Alleles were designated S (484 bp) and L (528 bp),as was previously described. 9 The 17-bp VNRT polymorphism in the second intronof the SERT gene was typed by PCR with primers 5´-TGGATTTCCTTCTCTCAGTGATTGG-3´ (forward)and 5´-TCATGTTCCTA GTCTTACGCCAGTG-3´(reversed). PCR was performed in a 50 µ L volume with20-100 ng DNA,100 µ m deoxyribonucleoside triphos-phate,20 pmol of each primer,1 mmol/L MgCl 2 ,20 µ mol/L Tris-HCl pH 8.6,50 µ mol/L KCl,0.2%(weight/vol) bovine serum albumin,and 1 U Taq poly-merase (MBI Fermentas). PCR conditions were 2 min-utes for initial denaturation at 94°C,40 cycles at 94°Cfor 1 minute for denaturation,1 minute at 57°C forannealing,and 2 minutes at 72°C for extension,fol-lowed by 10 minutes at 72°C for final extention. Ampli-fication products were resolved by electrophoresis on2% agarose gels next to a DNA molecular weight stan-dard and visualized with ultraviolet ethidium bromidestaining. Alleles were designated 390 bp (12 copy,STin2.12) 360 bp (10 copy,STin 2.10),or 345 bp (9 copy,STin 2.9) as was previously described. 30 Statistical analysis Statistical analyses were performed with SPSS forWindows (version 7.5; SPSS,Chicago,Ill). T  test,chi-squared test,and 1-way analysis of variance (ANOVA)tests were used for the statistical analyses of data. Aprobability value of less than .05 was considered sta-tistically significant. RESULTS There were 17 female and 31 male patients,with amean age of 21.5 ±3.87 years,in the study group. Inthe control group,there were 61 healthy female and 50healthy male volunteers,with a mean age of 22.12 ±4.08 years. The genders,ages,and psychiatric familyhistory of the patients and control subjects were notsignificantly different ( χ 2 , P > .05).The results of genetic analyses of the 5-HTT genepolymorphism are summarized in Table I. The 5-HTTLPR results of the patients and control subjects Fig 2. Standard clinical examination protocol.   American Journal of Orthodontics and Dentofacial Orthopedics  Herken et al 311 Volume 120  ,Number 3 were not significantly different ( χ 2 , P > .05). The analy-sis of genotype distribution,homozygous for STin 2.10genotypes of the VNTR polymorphism,showed signifi-cant differences between the patients and control sub- jects ( χ 2 ,11.95; degrees of freedom [df],2; P = .003).STin 2.10 allele was more frequent in the patients withTMJD. In the control group,however,STin 2.12/12genotype was significantly higher than in the patients( χ 2 ,5.70; df,1; P = .017). One patient who was found tohave STin 2.9 genotype was dropped from the study.According to the SCL 90-R test,the average of somatization subscale scores of the patients who werehomozygous for STin 2.12 variant was higher than forthose patients who were homozygous and heterozy-gous for STin 2.10 variant (1-way ANOVA,f = 3.76;df,2; P = .034).In the patients who were homozygous or heterozy-gous for VNTR variants of 5-HTT STin 2.12 copies,the average scores of somatization and anger were sig-nificantly higher than for those patients who werehomozygous for STin 2.10 variant (t,2.83; P = .008and t,2.12; P = .042,respectively). However,theirSTAI-I point averages were not significantly different(t,2.03; P = .05). There was no association with the 5-HTTLPR results and test scores,SCL 90-R,BDS,andSTAI-I and II (1-way ANOVA, P > .05).When the combined VNTR and 5-HTTLPR resultswere analyzed for the patient and control groups,it wasfound that the patients who were homozygous for STin2.10 genotype were also homozygous for L genotype(Fisher exact test, P = .019),(Table II). However,thiswas not the condition in the control subjects (Table III). DISCUSSION Several association studies with the 5-HTTLPR poly-morphism have shown a higher frequency of the S allelein the individuals with affective disorders. 10,11,17 The 5-HTTLPR was also proposed as a possible candidate genein major psychoses,because it modulates the 5-HTTgene at the transcriptional level,with the S allele corre-sponding to low 5-HT uptake activity. 10,11 The individu-als with either 1 or 2 copies of the S form had higher neu-roticism scores than did the individuals who werehomozygous for the L variant. Moreover,patients withseasonal affective disorder were less likely to have theL/L genotype and more likely to have the S allele,ascompared with control subjects. 31 In this study,there wasno relationship between the study and the control groupswith regard to S and L allele variants of 5-HTTLPR.A polymorphic region that contained a 17-bpVNTR in the second intron was found to be associatedwith bipolar disorders. 13 Liu et al, 17 and Gutierrez et al 32 found the frequency of allele 10 in bipolar affective dis-order and major depression with melancholia to be sig-nificantly higher than in the control group. On the otherhand,Rees et al 33 found a significant excess of 12 repeat Table I. Distribution for 5-HTTLPR and VNTR variants in patients and healthy control subjects 5-HTTLPRVNTR genotypePolymorphismS/SS/LL/LSTin 2.10/10STin 2.10/12STin 2.12/12 No. of patient * (%)11 (22.9)22 (45.8)15 (31.3)16 † (40.0)11 (27.5)13 (32.5)No. of control subjects ‡ (%)36 (32.4)42 (37.8)33 (29.7)16 (14.5)34 (30.9)60 (54.5)T OTALS (n)476448324573 S/S  ,Short/short; S/L ,short/long;  L/L ,long/long. * N = 48 patients. † χ 2 ,11.95; degrees of freedom,2; P = .003. ‡ N = 111 healthy control subjects.   Table II. Combined analysis of the frequency of geno-types of 5-STT gene in patients S/S +L/LTotalS/L (%)(%)(%) STin 2.10/106 (40.0)9 * (60.0)15 (100)STin 2.10/12 and 12/1219 (79.2)5 (20.8)24 (100)T OTALS 25 (64.1)14 (35.9)39 (100) S/S  ,Short/short; S/L ,short/long;  L/L ,long/long. * Fisher exact test, P = .019. Table III. Combined analysis of the frequency of geno-types of 5-HTT gene in control group S/S or S/L L/L Total (%)(%)(%) STin 2.10/1010 (62.5)6 (37.5)16 (100)STin 2.10/12 and 12/1265 (70.7)27 (29.3)92 (100)Totals75 (69.4)33 (30.6)108 (100) S/S  ,Short/short; S/L ,short/long;  L/L ,long/long.Fisher exact test, P = .56.  312  Herken et al  American Journal of Orthodontics and Dentofacial OrthopedicsSeptember 2001 alleles in bipolar patients. The functional polymor-phisms located in the VNTR variants in intron-2 of the5-HTT gene were found to be associated with TMJPD inpatients of Turkish srcin. The homozygous STin 2.10genotype of the VNTR was significantly higher in thepatients with TMJPD than in the control subjects.Although some functional effects of 44-bp inser-tion/deletion with reduced 5-HTT uptake that arecaused by lower expression have been well confirmed,there have been no in vitro data on the functional effectsof VNTR in intron 2 as yet. Both polymorphisms maybe working in an integrated fashion and may likewise beassociated with transcriptional activity in a synergisticfashion. For this reason,we analyzed the combinedresults for patients and control subjects separately(Table III). Even though the number of citations thatevaluated VNTR and 5-HTTLPR polymorphismstogether has not been enough to draw a conclusion,when the 2 polymorphisms were evaluated together,thepatients who were homozygous for the STin 2.10 geno-type were also found to have homozygous L genotype.It was shown that the frequency of the L genotypewas high in depressive suicide victims. 34 In the study,however,we found that patients with TMJPD had beenhomozygous for both STin 2.10 and L genotypes. Thisfinding should be taken into account in the further stud-ies of this kind. On 1 side,there was no relationshipbetween the psychiatric symptoms and 5-HTTLPR poly-morphism. On the other side,the patients who werehomozygous for STin 2.12 genotype were found to havesignificantly higher somatization scores than thosepatients with homozygous STin 1.10 genotype. Further-more the somatization and anger scores of patients withhomozygous or heterozygous STin 2.12 variant of VNTRwere significantly higher than for those patients with thehomozygous STin 2.10 variant. In the light of these find-ings,we propose that the VNTR polymorphism has animpact on the psychiatric symptoms of the patients.It was proposed that individuals who have the ten-dency of somatization also have the tendency of myofascial pain. 35 However,we found that the STin2.12/12 genotype,which is thought to have a higherprotective capacity,shows higher somatization ten-dency when compared with the STin 2.10/10 genotype.This suggests that there might be a weak relationshipbetween somatization and TMJPD and that genetic fac-tors may be involved in the susceptibility to TMJPD. CONCLUSION This study does not provide evidence to support theinvolvement of 5-HTTLPR in TMJPD. Our findingsindicated that only the presence of the homozygousSTin 2.10 genotype of VNTR is likely to play a sub-stantial role in the genetic predisposition to TMJPDand that the STin 2.12/12 genotype may have a protec-tive role against TMJPD. Further studies are requiredfor the confirmation of our results and to find thegenetic basis of TMJPD in other populations.We thank to Dr Yildirim A. Bayazit from theDepartment of Otolaryngology,Faculty of Medicine,Gaziantep University,for reviewing and revising themanuscript. REFERENCES 1.Laskin DM. Etiology of the pain-dysfunction syndrome. J AmDent Assoc 1969;79:147-53.2.Lerman MD. A unifying concept of the TMJ pain-dysfunctionsyndrome. J Am Dent Assoc 1973;86:833-41.3.Eversole LR,Stone CE,Matheson D,Kaplan H. Psychometricprofiles and facial pain. Oral Surg Oral Med Oral Pathol1985;60:269-74.4.Eversole LR,Machado L. 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