An association study between 5-HTTLPR polymorphism, COMT polymorphism, and Tourette's syndrome

Psychiatry Research 97 Ž2000. 93᎐100

An association study between 5-HTTLPR polymorphism, COMT polymorphism, and Tourette’s syndrome Maria Cristina CavalliniU , Daniela Di Bella, Marco Catalano, Laura Bellodi Istituto Scientifico H San Raffaele, Department of Neuroscience, Uni¨ ersity of Milan Medical School, Via L. Prinetti, 29, 20127 Milan, Italy Received 1 November 1999; received in revised form 14 August 2000; accepted 10 September 2000

Abstract Several lines of evidence suggest that a genetic component underlies Tourette’s syndrome ŽTS.. We investigated both the role of the insertionrdeletion polymorphism in the promoter region of the serotonin transporter gene Ž5-HTTLPR. and that of the Val-158-Met substitution in the catechol-O-methyl-transferase ŽCOMT. gene in conferring susceptibility to TS. Fifty-two TS patients were recruited and compared with a control group of 63 healthy subjects. Neither a genotypic nor an allelic association was found; subdividing TS patients according to clinical variables, such as a co-diagnosis of obsessive᎐compulsive disorder ŽOCD. and a positive family history for obsessive compulsive disorder or tics, also failed to reveal a significant association. The lack of significance for 5-HTTLPR and COMT polymorphisms in conferring liability to TS does not exclude a role of different functional polymorphisms in genes coding for serotonergic or dopaminergic structures in the etiology of TS. In fact, TS is a complex disorder and these genes most likely have only a minor genetic effect in its etiology. 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serotonin; Catecholamine; Association studies; Obsessive᎐compulsive disorder; Spectrum disorders

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Corresponding author. Tel.: q39-02-2643-3315; fax: q39-02-2643-3265. E-mail address: [email protected] ŽM.C. Cavallini.. 0165-1781r00r$ - see front matter 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 1 7 8 1 Ž 0 0 . 0 0 2 2 0 - 1

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1. Introduction Tourette’s syndrome ŽTS. is an early onset disorder Žbefore 18 years., characterized by chronic motor tics and at least one vocal tic according to DSM-IV diagnostic criteria ŽAmerican Psychiatric Association, 1994.. In the general population the disorder is quite rare Ž1r10 000., even though it interferes with functioning, especially when associated with co-morbid conditions such as obsessive᎐compulsive disorder ŽOCD.. From an etiologic point of view, several studies have suggested that TS is a genetic condition with a Mendelian inheritance and incomplete penetrance ŽDevor, 1984; Pauls and Leckman, 1986; Eapen et al., 1993; Hasstedt et al., 1995). From a neuroanatomical point of view, dysfunction of subcortical structures, including the basal ganglia, has been hypothesized in the etiology of TS ŽBraun et al., 1993; Palumbo et al., 1997.. To date, neuroleptics are the main treatment for TS, suggesting that a dysfunction in dopaminergic pathways might be implicated in the development of the disorder. Dopamine receptor genes DRD1, DRD2, DRD3, DRD4 and DRD5 have been investigated with regard to their possible involvement in conferring susceptibility to TS ŽGelernter et al., 1990; Nothen et al., 1994; ¨ Brett et al., 1995; Barr et al., 1996, 1997; Comings et al., 1996., but their role in the etiopathogenesis of TS still remains unclear. Furthermore, the dysfunction could be localized at a different point of the dopaminergic pathway, such as, for example, at the level of catechol-O-methyltransferase ŽCOMT., an enzyme that inactivates adrenaline, noradrenaline, dopamine, and levodopa. In Parkinson’s disease ŽPD., COMT is responsible for much of the levodopa degradation in the periphery known to be associated with motor fluctuations and dyskinesia ŽMartinez-Martin and O’Brien, 1998.. Furthermore, it has been observed that fluctuations of levodopa in PD are associated with the exacerbation of tics ŽShale et al., 1986.. Recently, a functional G ª A transition has been described in the COMT gene, resulting in a Val-158-Met substitution. This polymorphism is associated with a three- to four-fold lower level of enzyme activity. Homozygotes for the low-activity

allele have lower levels of COMT with higher levels of L-dopa in the brain ŽLachman et al., 1996.. An excess of homozygotes for the low-activity allele of the COMT gene has been observed in men with clinical OCD ŽKarayiorgou et al., 1997, 1999., a condition frequently associated with TS. A recent study failed to find any linkage between this polymorphism and TS in five Canadian TS families ŽBarr et al., 1999.; however, it is not possible to exclude that the COMT polymorphism may have a minor effect in conferring liability to TS ŽGreenberg, 1993.. Nevertheless, a dysfunction of other neurotransmitter systems in TS cannot be ruled out. In fact, there is evidence pointing to a complex serotoninrdopamine interaction in several brain areas. Serotonergic projections from the dorsal raphe area have been shown to inhibit dopamine function both at the midbrain level, where they inhibit the firing of dopamine cells projecting from the substantia nigra, and in the striatum and cortex, where they inhibit the synaptic release of dopamine, and probably the synthesis of dopamine. Serotonergic agonists enhance the inhibition of the dopamine system, while lesions at the raphe nuclei, or the administration of a serotonergic antagonist, disinhibit dopamine transmission ŽKapur and Remington, 1996. Data from biochemical studies have shown generally lower values of serotonin and its metabolites in subcortical brain regions in TS patients ŽAnderson et al., 1992.. A reduced concentration of 5-hydroxyindolacetic acid Ž5-HIAA. in cerebrospinal fluid ŽCSF. in TS patients could reflect the inadequate modulation of dopaminergic activity by serotonergic neurons ŽCohen et al., 1978.. Data from pharmacological studies may also indicate a role for serotonin in TS. The use of serotonin re-uptake inhibitors ŽSRIs. in the treatment of TS ŽBajo et al., 1999. has proved to be effective in tic reduction in some TS children. The serotonin transporter Ž5-HTT. is a prime target for SRIs. A 44-bp deletionrinsertion functional polymorphism within the promoter region of the serotonin transporter gene Ž5-HTTLPR. leads to lower transcriptional rates of 5-HTT protein ŽLesch et al., 1996.. The aim of our study was, therefore, to assess

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through an association case᎐control study whether the COMT Val-158-Met and the 5-HTTLPR polymorphisms could play a role in conferring susceptibility to TS in an Italian population of TS patients.

2. Methods Fifty-two TS subjects, 30 males and 22 females, were recruited in our Department and included in the study. All patients were diagnosed according to DSM-IV full diagnostic criteria ŽAmerican Psychiatric Association, 1994.. Specific tics were scored using the Yale-Brown Tourette syndrome scale ŽLeckman et al., 1989.. Table 1 presents demographic and clinical features of the patients. A co-diagnosis of OCD was made in 53.84% of TS patients; OCD onset was antecedent to TS in 5% of cases, subsequent in 60% of cases, while in the remaining 35% it was not possible to clearly evaluate the respective onset of the two disorders. Only OCD co-morbidity was considered, and OC symptoms were further investigated using the Yale-Brown obsessive᎐compulsive scale ŽGoodman et al., 1989.. Information on family history of OCD and TSrtic disorders was collected from each proband using the family history method. No differences were found between male and female TS patients for age, age at onset, or positive family history for tics or OCD. Female TS patients had a higher frequency of OCD co-diagnosis Ž16r22. than male TS patients Ž12r30. Ž ␹ 2 s 5.47, d.f.s 1, Ps 0.019.. TS females with OCD had a slightly earlier onset of TS than TS females without OCD Ž7.68" 3.4 vs. 12 " 7.01 years., while

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for male OCDrTS patients we observed the inverse trend Ž10.3" 4.19 vs. 8.18" 4.53 years.. Sixty-three healthy control subjects, 24 males Žmean age s 35 years, S.D.s 10.91. and 39 females Žmean age s 25.89 years, S.D.s 3.71. were recruited from department personnel. Experienced psychiatrists ŽCMC, BL. personally evaluated control subjects. DSM-IV diagnoses in first-degree relatives represented the criteria for exclusion from the study. Informed consent was obtained from patients and control subjects, who were all unrelated and of Italian descent. 2.1. Laboratory procedures Genomic DNA was extracted from anti-coagulated thawed blood according to the method of Lahiri and Nurnberger Ž1991.. 2.1.1. COMT typing Two primers were used to amplify a 96-bp fragment of the COMT gene containing the NlaIII polymorphism in codon 158: forward primer 5⬘TCACCATCGAGATCAACCCC-3⬘ and reverse primer 5⬘-ACAACGGGTCAGGCATGCA-3⬘. A 25-␮l PCR reaction was performed according to standard PCR protocols. After initial denaturation at 95⬚C for 5 min, 35 cycles were carried out at 94⬚C for 1 min, 64⬚C for 1 min, and 72⬚C for 1 min. Then 10 ␮l of PCR reaction mixture was digested with 5U NlaIII ŽNew England Biolabs. in a 20-␮l reaction, according to the manufacturer’s specifications. Digested products were electrophoresed on 14% polyacrylamide gel, and stained with ethidium bromide, thus allowing differentiation of allelic variants Žvariant condition-

Table 1 Demographic and clinical characteristics of TS sample

Mean age " S.D. Mean age at onset " S.D. OCD co-diagnosis Family history for tic disorder Family history for OCD

30 male TS

22 female TS

52 total TS

31.63" 15.68 9 " 4.44 40% 66.66% 33.33%

27.09" 10.22 8.86" 4.88 73% 59.1% 50%

29.711" 13.71 8.93" 4.6 53.84% 63.46% 40.38%

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Table 2 COMT polymorphism association results U

COMT polymorphism

N

HrHa Ž%.

HrLb Ž%.

LrL Ž%.

L Ž%.

P-value

d.f.

Control subjects Tourette’s patients

63 52

18 Ž28. 17 Ž33.

30 Ž48. 24 Ž46.

15 Ž24. 11 Ž21.

47 44

Genotypic s 0.878 Allelic s 0.704

2 1

Male TS Male control subjects

30 24

8 Ž27. 7 Ž29.

15 Ž50. 13 Ž54.

7 Ž23. 4 Ž17.

48 48

Genotypic s 0.833 Allelic s 0.773

2 1

Female TS Female control subjects

22 39

9 Ž41. 11 Ž28.

9 Ž41. 17 Ž44.

4 Ž18. 11 Ž28.

39 50

Genotypic s 0.524 Allelic s 0.308

2 1

TS with OCD FHc TS without OCD FH

21 31

11 Ž52.3. 13 Ž41.9.

1 Ž4.7. 10 Ž32.2.

28.57 53.22

Genotypic s 0.052 Allelic s 0.041

2 1

9 Ž42.8. 8 Ž25.8.

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Chi-square statistics with P after Bonferroni correction for multiple tests s 0.016. H s high activity allele. b L s low activity allele. c FHs family history. a

ing a low activity: 64 bp q18 bp q13 bp, constant digestion band; variant conditioning a high activity: 83 bp q13 bp, constant band.. 2.1.2. 5-HTTLPR typing For PCR reactions, forward primer 5⬘GGCGTTGCCGCTCTGAATGC-3⬘ and reverse p rim e r 5 ⬘-G A G G G A C T G A G C T G G A CAACAAC-3⬘ were employed. A 25-␮l PCR reaction was performed according to the protocol previously described ŽDeckert et al., 1997.. PCR products were separated on 3% agarose gels supplemented with ethidium bromide allowing identification of the long Ž528 bp. and the short Ž484 bp. variants. 2.2. Statistical methods Comparison between groups was performed with student’s t-test and chi-square statistics Žtwo-tailed test. subprograms of the BDMP statistical package ŽDixon, 1990.. The control group was in Hardy᎐Weinberg equilibrium, both for COMT Ž ␹ 2 s 0.633, d.f.s 2, Ps 0.729. and 5-HTTLPR Ž ␹ 2 s 0.392, d.f.s 2, Ps 0.822. polymorphisms. With our sample size and a power Ž1 y ␤ . of 80%, the minimum detectable significant odds

ratio would be 3.1 for the COMT polymorphism and 3.05 for 5-HTTLPR ŽEPINFO, 1994.. To evaluate the association of different clinical variables with COMT and 5-HTTLPR polymorphisms, we performed multiple tests; a Bonferroni correction for multiple testing was therefore applied to the analyses Ž P level of significance s ␣r3 s 0.05r3s 0.016..

3. Results 3.1. COMT polymorphism (Table 2) No association was found between the COMT polymorphism and TS Žgenotypic: ␹ 2 s 0.261, d.f.s 2, Ps 0.878; allelic: ␹ 2 s 0.145, d.f.s 1, P s 0.704.. When the sample was analyzed according to sex distribution, no malerfemale difference in distribution of alleles and genotypes of COMT was found Žgenotypic: ␹ 2 s 2.355, d.f.s 2, Ps 0.308; allelic: ␹ 2 s 1.542, d.f.s 1, Ps 0.214.. Both male and female allelic and genotypic frequencies of TS patients are similar to those of the control group Žsee Table 2.. No significant associations emerged when the sample was subdivided according to the following

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factors: OCD co-diagnosis Žgenotypic: ␹ 2 s 2.679, d.f.s 2, Ps 0.261; allelic: ␹ 2 s 2.329, d.f.s 1, P s 0.127.; positive family history for tics Žgenotypic: ␹ 2 s 4.657, d.f.s 2, Ps 0.097; allelic: ␹ 2 s 3.119, d.f.s 1, Ps 0.077.; and positive family history for OCD Žgenotypic: ␹ 2 s 5.883, d.f.s 2, Ps 0.052, allelic: ␹ 2 s 4.173, d.f.s 1, Ps 0.041.. No differences in allele or genotype distribution were found when OCD co-morbidity or family history for tics or OCD was considered in the male sub-sample of patients. 3.2. 5-HTTLPR polymorphism (Table 3)

No association was found between the 5-HTTLPR polymorphism and TS Žgenotypic: ␹ 2 s 0.522, d.f.s 2, Ps 0.77; allelic: ␹ 2 s 0.008, d.f.s 1, Ps 0.929.. There was also no malerfemale difference in the distribution of alleles and genotypes of 5-HTTLPR in patients Žgenotypic: ␹ 2 s 1.548, d.f.s 2, Ps 0.461; allelic: ␹ 2 s 0.006, d.f.s 1, Ps 0.939.. Subdividing the sample according to OCD co-diagnosis or family history for tics or OCD also failed to reveal an association of TS with 5-HTTLPR ŽTable 3..

4. Discussion

Our data seem to exclude a role of both the COMT Val-158-Met polymorphism and the 5HTTLPR polymorphism in conferring susceptibil-

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ity to TS. Clinical and genetic variables, such as co-diagnosis of OCD and positive family history for tics, also did not reveal an association between TS and the two candidate genes. Even though positive findings in the literature ŽKarayiorgou et al., 1997, 1999. support a role for COMT in OCD development in male patients, no evidence of a specific association was found in our patients. Several types of data support the hypothesis that TS may be part of the OCD spectrum; serotonin alterations seem to be involved in OCD etiopathogenesis and we cannot exclude a priori a role of 5-HTT in the etiology of TS. The role of other dopaminergic and serotonergic candidate genes has not been completely ascertained in TS or OCD ŽCatalano et al., 1994; Novelli et al., 1994; Brett et al., 1995; Billett et al., 1997; Cavallini et al., 1998; Bengel et al., 1999.. Nevertheless, evidence suggests that dopaminergic blockers improve tics, in addition to the serotonergic agents effective in juvenile TS ŽBajo et al., 1999.. Furthermore, there is some evidence that, while neuroleptics are needed to improve TS with OC symptoms, the addition of SRI drugs leads to an improvement of obsessive symptoms in these patients. These apparently contradictory data could be explained by the fact that psychotropic drugs act through indirect pathways on different neurotransmitter systems Ži.e. serotonergic projections influence dopaminergic activity; Kapur and Remington, 1996.. It has been suggested that TS is a polygenic disorder and that genes involved in the metabolism of dopamine, serotonin, norepinephrine, and other neurotransmitters may only

Table 3 5-HTTLPR polymorphism association results U

5-HTTLPR polymorphism

N

LrLa Ž%.

LrSb Ž%.

SrS Ž%.

S Ž%.

P-value

d.f.

Control subjects Tourette’s patients

63 52

21 Ž33. 14 Ž26.9.

34 Ž53.9. 28 Ž53.8.

8 Ž12.7. 10 Ž19.2.

39 46.15

Genotypic s 0.77 Allelic s 0.929

2 1

U

Chi-square statistics with P after Bonferroni correction for multiple tests s 0.016. L s longrwild allele. b Ss shortrdeleted allele. a

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contribute between 1 and 10% of the phenotypic variance ŽComings, 1995a,b.. We therefore cannot exclude that our negative result is due to the power of our sample; we are able, in fact, to identify an odds ratio of at least 3. Case᎐control studies could also present some limitations, due to population stratification, for example. Familybased association methods ŽTerwilliger and Ott, 1992. could overcome this effect, particularly given the wide variation in the COMT allele among human populations ŽPalmatier et al., 1999.. To date, no differences have been found in the frequency of the low-activity allele between our control subjects and mixed European people ŽPalmatier et al., 1999. Žgenotypic: ␹ 2 s 2.698, d.f.s 2, Ps 0.259; allelic: ␹ 2 s 1.795, d.f.s 1, P s 0.18.. Despite certain advantages, the familybased sampling procedure is sensitive to other effects, like difficulties in collecting triads and the lack of power in many situations ŽRisch and Teng, 1998; Owen et al., 2000.. TS is a rare disorder, and the recruitment of cases with their parents may therefore be a burdensome task. Because TS is a complex phenotype, an innovative approach may be to study TS by including several clinical and phenotypic elements in analyses, such as drug therapy responses or symptomatological structure Ži.e. TS with OCD co-diagnosis vs. TS without OCD co-diagnosis.. A more detailed phenotypic description of TS may help identify subgroups of patients that are more genetically homogeneous, and therefore more suited for genetic dissection of this complex trait. For example, aggressive conduct is frequently found in TS. Dysfunction in serotonergic, and also catecholaminergic, mechanisms has been associated with aggressive behaviors: an increased serotonin activity has been invoked in aggressive subjects ŽMoffitt et al., 1998., and low COMT activity has been associated with violent behaviors in schizophrenic patients ŽLachman et al., 1998.. Although information about aggressive conduct is unfortunately not available for our TS sample, an intriguing development of a serotonergic or catecholaminergic genetic hypothesis in TS could be represented by a study of the relationship between the proposed polymorphisms with disrupted behaviors.

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