Original Article
This study was conducted to investigate the association of the Catechol-O-Methyl Transferase (COMT) polymorphism with Tourette's disorder (TD) in a Korean sample of families with TD probands. The relationship between the risk alleles and specific clinical features (tic severity, comorbidity, drug response) was also explored. Patients were recruited from the Tic Disorder clinic at the Child & Adolescent Psychiatric Division of Seoul National University Hospital and assessed through a 2 stage evaluation. Firstly, all of the patients and parents underwent a semistructured interview using the Korean version of the Kiddie-Schedule for Affeetive Disorders and Schizophrenia for School Age children, Present and Life time Version (K-SADS-PL). Secondly, all of the patients underwent a clinical interview and tic severity assessment with the Korean version of the Yale Global Tic Severity Scale (YGTSS). The subjects in the control group were recruited from the health promotion center of our hospital and were evaluated by means of the Symptom Checklist 90 (SCL-90) and Structured Clinical Interview for DSM-IV (SCID-IV). Through this process, a total of 42 children and dolescents with TD, their 84 parents and 86 control subjects were finally recruited. Genotyping for the Val158Met polymorphism of the COMT gene was done by a standardized method. After the collection of the genetic data of all of the patients, parents and control subjects, a case-control comparison and transmission dysequilibrium test were performed using SPSS version 11. Based on the case-control comparison, the frequencies of the L-allele and LL genotype were significantly higher in the TD group. However, no differences were found in the transmission disequilibrium test (TDT). No significant differences were found in the family history of tic, Attention Deficit Hyperactivity Disorder (ADHD), Obsessive-Compulsive Disorder (OCD), drug response or comorbid conditions among the TD patients with the three different genotypes. Although the results of this study should be interpreted cautiously, due to the small sample size and negative finding in the TDT test, this is the first report of a positive association between the functional polymorphism of the COMT gene and TD.
Correspondence: Soo Churl, Cho, MD, Department of Psychiatry, Clinical Research Institute, Institute of Human Behavioral Medicine, Seoul National University Hospital, 28 Yungun-dong, Chongro-gu, Seoul 110-744, Korea
Tel: +82-2-2072-2450, E-mail: soochurl@snu.ac.kr
Tourette's disorder (TD) is one of most importunate and troublesome disorders in childhood, affecting 0.05-0.1% of school age children. TD is characterized by recurrent motor and vocal tic, which involves involuntary, erratic and sudden movement/vocalization. Comorbidity with other psychiatric conditions, such as attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), other anxiety disorders and aggressive behavior, is frequently found in clinical populations1,2,3.
Although the etiology of TD is unknown, the involvement of genetic factors is strongly supported by diverse genetic epidemiologic studies4,5,6. Family and adoption studies have suggested that TD is a highly genetically heritable disorder7. Studies of twins reported that the concordance rate between monozygotic twins could be 100%, including in the case of chronic tic disorder8,9,10,11.
In the segregation analysis of TD families, the mode of transmission remains a highly controversial issue. Some researchers have insisted on the autosomal dominant pattern with a reduced penetrance rate5,12,13, while others persisted in their support of other models14, Nowadays, the multifactorial-polygenic model is the most widely accepted one15,16.
Recent biological studies have supported the hypothesis that the catecholamine system, including the dopamine/norepinephrine neural network, is implicated in the pathophysiology of TD and, consequently, catecholamine related candidate genes have been the initial candidates for the association studies of TD17,18,19.
Surprisingly, there have been very few genetic association studies concerning the role of the Catechol-O-Methyl Transferase (COMT) gene in TD. Only two studies dealt with the genetic association of COMT and TD. One of them was conducted by Calvallini et al20. in Italy using a case-control design. The authors of this study did not find any positive association between COMT (VAL-158-Met polymorphism) and TD. Barr et al21. tested for the linkage of the COMT allele (as in Calvallini et al's study) to TD in five multi-generational families. However, they found no significant evidence for such a linkage in their parametric linkage analysis.
To our knowledge, there have been no reports which involved both the family based transmission disequilibrium test (TDT) and a case control study for the role of the COMT gene in TD. The objective of this study was to investigate the association of the COMT polymorphism with TD in a Korean sample of families with TD probands. The relationship between the risk alleles and specific clinical features (tic severity, comorbidity, drug response) is also explored.
Methods and Materials
Subjects
Patients were recruited from the Tic Disorder clinic at the Child & Adolescent Psychiatric Division of Seoul National University Hospital in Seoul, Korea. A consensus diagnosis of TD with or without comorbidity was achieved through a two stage evaluation process.
First, an evaluation was conducted with a semistructured interview using the Kiddie-Schedule for Affective Disorders and Schizophrenia for School Age Children, Present and Lifetime Version (K-SADS-PL), modified to assess the DSM-IV criteria and applied to the parents by trained child psychiatric fellows and residents22. The inter-rater reliability for the TD diagnosis was 0.91. Second, a clinical evaluation for TD and comorbid conditions was conducted by child psychiatrists for the children who had previously been interviewed with the K-SADS-PL.
Third, the Yale Global Tic Severity Scale was completed by the parents. A cognitive evaluation was performed for all of the patients with the Korean version of the Weschler Intelligence Scale for Children and the Attention deficit Diagnostic System by a trained child psychologist to estimate their overall IQ and attentional problems.
The sociodemographic data, developmental data, family history and treatment history were systematically collected from the parents.
The parents were evaluated by means of the Symptom Checklist 90 (SCL-90) and clinical interview. For the control group, seventy eight subjects were recruited from the health promotion center in Seoul National University Hospital (SNUH). The controls were evaluated using the clinical interviews, SCL-90 and Structured Clinical Interview for DSM-IV (SCID-IV).
Through these processes, a total of 42 children and adolescents with TD, their 84 parents, and 86 control subjects were finally recruited.
This research was approved by the ethical committee of the clinical research institute of SNUH. Informed consent was obtained from all of the parents and control subjects
Genotyping
Genomic DNA was extracted from peripheral leukocytes in the blood samples obtained from both the patients and control subjects, using a standard phenol/chloroform method (Invitrogen Easy-DNATM Kit, Boehringer Mannheim, SanDiego, California, USA) according to the manufacturer's instructions.
The Val158Met polymorphism of the COMT gene (gene map locus: chromosome 22q11.2, G/A1947 polymorphism in the HSCOMT 2 gene, gene bank accession number z26491, dbSNP: rs165388) was tested from a the 217-bp polymerase chain reaction product amplified by using the oligonucleotide primers 5°-TCG TGG ACG CCG TGA TTC AGG-3° and 5°-AGG TCT GAC AAC GGG TCA GGC-3° (Bioneer, Seoul, Korea). The polymerase chain reaction product was digested by the restriction enzyme Nla III (New England Biolabs, Beverly, Mass.) for 6 hours at 37℃. After electrophoresis at 100 V for 35 minutes on 2% Metaphor agarose gels (FMC, Rockland, Maine) containing 0.5% ethidium bromide, the gels were photographed under ultraviolet light.
Restriction fragments with lengths of 114, 83, and 20 bps revealed the COMTH allele, while those with lengths of 96, 83, 20, and 18 bps revealed the COMTL allele.
Statistical analysis
The allele frequencies of the individual loci were estimated by counting. The allele frequencies among the TD probands, their parents and the control subjects were compared by the chi-square test using SPSS for Windows version 11.0.
The TDT was performed, however homozygote parent-proband pairs with the same genotype were exclud-ed, since the transmission status of the parental alleles could not be determined.
Using the student t-test and ANOVA performed by the SPSS software, the relationship between the clinical variables and specific alleles was evaluated.
Results
42 children and adolescents with TD, their 84 parents and 86 control subjects were assessed. Table 1 presents the demographic and clinical characteristics of the 42 TD probands. All of them were of Korean descent, which was assumed to be a very homogenous ethnic group. The comorbidities of the TD subjects were ADHD (30.9%), OCD (7.1%), other anxiety disorders (7.1%) and others (4.7%). The family loading of tic disorder was greater than 28% in the patients with TD. The mean IQ was 107.4, which is a high average level.
The control subjects were free from any psychiatric conditions and had no history of tic disorder and no family loading of chronic tic disorder or Tourette's disorder.
1. The allele frequencies for COMT in the TD probands and controls (Table 2)
The frequencies of the H allele of COMT in the TD probands and controls were 28 (33.3%) and 90 (52.3%), respectively. The frequencies of the L allele in the TD probands and controls were 56 (66.7%) and 82 (47.7%), respectively. The allele distributions in the patients with TD were significantly different from those in the controls. The frequency of the COMT gene allele (L) was significantly higher in the TD group than in the controls
X2=15.7, df=1, p<0.01)
2. The genotype frequencies for COMT in the TD probands and controls (Table 2)
The frequencies of the HH, HL and LL genotypes of the TD probands were 7 (16.7%), 14 (33.3%) and 21 (50%), respectively, while the corresponding frequencies of the controls were 16 (18.7%), 58 (67.4%) and 12 (13.9%), respectively. The frequency of the LL genotype of the COMT gene was significantly higher in the TD group than in the controls X2=16.6, df=1, p<0.01)
3. TDT result for COMT in TD families (Table 3)
Family trios with uninformative transmissions were excluded from the TDT test (e.g. if the genotypes of the trios were all homozygous with either the HH or LL genotype, then the trios were considered to be unusable for the TDT test). No difference was found between the transmitted to a risk factor (allele) and the not-transmitted to the same risk factor for the disease (TDT: X2=0.083, df=1, p>0.75)
4. COMT genotype and clinical characteristics of TD subjects (Table 4)
There was no significant difference in the family history of tic, ADHD, OCD, drug response or comorbid conditions among the TD patients with the three different genotypes.
Discussion
The results of this study suggest that there is a significant involvement of the COMT gene (Val158Met) in the TD susceptibility of Korean children and adolescents. An excess of the L allele (Met allele) in the probands was observed when they were compared to an ethnically matched control sample. In the family based TDT analysis, however, no evidence of preferential transmission of the L allele at the COMT gene was detected in the study subjects.
However, evidence of an association between the COMT L allele and TD was detected in the comparison of the TD subjects with the control group. This discrepancy in the results between the case-control comparison and TDT can be explained in various ways. One of the possible reasons is that the positive finding in the case-control comparison is due to population stratification23. False positive results resulting from population stratification is notorious in case control studies of genetic association, especially in the case of multi-ethnic subjects24. The Korean ethnic group, however, is generally considered to be sufficiently homogenous for concern about genetic stratification to be unwarranted. The probability of population stratification seems to be very low in Korean subjects.
Another possible reason for the negative result in the TDT study is that the study design has low statistical power. A previous meta-analysis study of 7 repeat alleles of DRD4 for ADHD showed that there was much stronger evidence of an association between ADHD and DRD4 in the case-control studies than in the family based studies25. Some researchers reported that the negative results in the family based analysis could be explained by the relatively low statistical power of the family based method25. In this study, because the genotypes of the trios were all homozygous with the HH or LL genotype, these family trios with uninformative transmissions were excluded from the TDT test. It was suggested that the non-replication of positive findings is to be expected in family based studies when the samples are not very large. Under the condition of a small sample size, the case control design is more reasonable for the investigation of genetic effects in complex genetic disorders such as TD.
The COMT gene is an enzyme that plays an essential role in the dopamine metabolism. The enzymatic activity of COMT in humans is substantially regulated by a common functional genetic polymorphism. This genetic polymorphism results in a 3- to 4- fold difference in COMT activity. The low activity variant of the enzyme contains a methionine residue at amino acid 158 of the membrane bound COMT protein, whereas the high activity variant has a valine residue at this site. Homozygosity for the low activity allele (LL genotype) is found in approximately 25% of Whites and 30% of Koreans26. Heterozygotes (LH type) Heterozygotes(LH type) have intermediate levels of COMT activity.
Previous research shows that this functional polymorphism in the COMT gene is associated with diverse mental disorders such as alcohol related disorder, panic disorder, obsessive compulsive disorder and certain types of major psychotic disorders in the adult group27,28,29.
There have been few genetic studies of behavioral and emotional disorders, especially Tourette's disorder. A few research groups studying TD patients from different ethnic backgrounds reported that there was an association between the COMT gene and Tourette's disorder. One research group in Italy using 52 patients with TD and 63 healthy control subjects found that there was a negative association between the COMT gene and TD20. Another genetic study in Canada involving five multigenerational families, failed to prove the existence of a linkage between TD and the COMT gene21. To the best of our knowledge, there have only been two genetic studies that dealt with the issue of direct association and linkage, making this the third genetic study, and the first such study to find a positive association between the COMT gene and Tourette's disorder.
However, in this study, none of the clinical variables such as the drug response, comorbidity or familial loading, were associated with the polymorphism. This implies that the COMT gene is only associated with the vulnerability to TD, and not with the drug response, familial loading or comorbidity.
The results of this study should be interpreted cautiously because of the small sample size and the negative finding in the TDT test. However, this is the first report of a positive association between the functional polymorphism of the COMT gene and TD.
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