Catechol-O-methyltransferase gene and obsessive–compulsive symptoms in patients with recent-onset schizophrenia: Preliminary results☆

2.1. Subjects 

All patients were admitted to the Adolescent Clinic of the Academic Medical Center in Amsterdam. After a complete description of the study to the subjects, written informed consent was obtained. The study was approved by the Medical Ethics Committee of the Academic Medical Center in Amsterdam. Patients who met DSM-IV (American Psychiatric Association, 1994) criteria for schizophrenia, schizoaffective disorder or schizophreniform disorder were included in the study. Exclusion criteria were organic brain syndrome, endocrine disorder, and substance-induced psychosis. Diagnosis at discharge was established according to DSM-IV criteria and was based on a semi-structured clinical interview by two independent psychiatrists as described previously (de Haan et al., 2002); diagnoses were based on all available clinical information including the semi-structured interview, examination of case records and information from relatives and mental health professionals. On admission, symptoms were rated with the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987); the extent of OCS was assessed with the Yale-Brown Obsessive–Compulsive Scale (Y-BOCS) (Goodman et al., 1989a, Goodman et al., 1989b). The Y-BOCS consists of 10 topics with scores ranging from 0 (no symptoms) to 4 (severe symptoms). Obsessions were defined as persistent, intrusive, unwanted, and repetitive thoughts not related to the patient's delusions. Compulsions were defined as repetitive goal-directed rituals clinically distinguishable from schizophrenic mannerisms or posturing.

2.2. DNA extraction and genetic analysis 

Blood samples were collected from all subjects for DNA isolation. Genomic deoxyribonucleic acid (DNA) was extracted using a filter-based method (QIAamp DNA Mini Kit, Qiagen Ltd, United Kingdom). Genotyping of the COMT Val158Met polymorphism (rs4680) was performed by primer extension and analyzed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as previously described (Sauer and Gut, 2002). All DNA samples were genotyped in duplicate to ensure reliability. In brief, a polymerase chain reaction (PCR) was performed to amplify the fragments of DNA containing the Val158Met polymorphism using the following primers: forward 5′-CAC CTG TGC TCA CCT CTC CT-3′ and reverse 5′-GGG TTT TCA GTG AAC GTG GT-3′. For the PCR the following reagents were used: 1× PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 5 ng forward primer, 5 ng reverse primer, 0.5 U HotFirePol DNA polymerase (Solis Biodyne, Estonia), 20 ng genomic DNA template, dH2O to a final reaction volume of 10 μl. PCR was carried out on a Biometra Thermocycler machine, using the following conditions: initial 15-min denaturing step at 95°, followed by 35 cycles of 95° for 1 min, 58° for 1 min, 72° for 1.5 min, and a final extension phase of 72° for 10 min. Five microliters of PCR product were treated with 0.2 U shrimp alkaline phosphatase (USB) on 37° for 45 min to dephosphorylate remaining dNTPs. Next, single base primer extension (PEX) using ddNPTs was performed to generate short, single-stranded allele-specific products. We used a biotinylated UV-cleavable PEX primer with the following sequence: 5′-GATGGTGGAT-L-TCGCTGGC-3′; the UV-cleavable site is indicated by L (BioTeZ, Berlin, Germany). For PEX the following reagents were used: 5 mM MgCl2, 0.4 μM PEX primer, 0.2 mM ddATP, 0.2 mM ddGTP, 2.5 U ThermiPol (Thermipol, Solis Biodyne, Estonia), dH2O to a final reaction volume of 10 μl. Primer extension was carried out on a Biometra Thermocycler machine, using the following conditions: initial 5-min denaturing step at 95°, followed by 70 cycles of 95° for 15 s, 40° for 30 s, 72° for 30 s. Dependent on the genotype, the primer was elongated with either a ddATP or a ddGTP. The primer extension product was purified using a 384-well streptavidin coated plate (Bruker Daltonics, Germany) according to the manufacturer's instructions. After exposure to UV light (366 nm, 8 W) for 10 min, the 3′ end was released and dissolved into 10 μl dH2O and subsequently spotted onto a MALDI plate (Bruker Daltonics, Germany). Time-of-flight MS was performed on a Bruker III Daltonics Mass Spectrometer using FlexControl v2.0 software. As the varying incorporated nucleotides differ in their mass, the allele-specific products can be determined in the mass spectrum. Data were analyzed using Genotools v2.0 software (Bruker Daltonics, Germany).

2.3. Statistics 

Analysis was performed using the Statistical Package for the Social Sciences (SPSS), version 12.0. The population was divided into three groups according to COMT genotype. We tested between-group differences in age, educational level and PANSS scores using analysis of variance (ANOVA). Correlations between Y-BOCS scores and PANSS scores were calculated using Pearson's Rho. Between-group differences in the use of medication were assessed using Fisher's exact test. Because data were not normally distributed, we used non-parametric testing (2-tailed) to determine between-group differences in Y-BOCS scores (Kruskal–Wallis and Mann–Whitney U). Several reports have been published of induction of OCS in patients with schizophrenia by olanzapine and clozapine (Morrison et al., 1998, de Haan et al., 1999, Mottard and de la Sablonniere, 1999). Therefore, we looked at the use of these antipsychotics in the three genotype groups using Fisher's exact test to test whether medication use could be a confounding factor. In addition, we analyzed differences in mean Y-BOCS scores between COMT genotype groups controlling for the use of antipsychotic medication with analysis of covariance (ANCOVA), entering medication as fixed factor in the model. Level of statistical significance was defined as P<0.05.

4.1. COMT and risk for OCD without schizophrenia 

It is still unclear whether the Val158Met polymorphism increases the risk of OCD: although three studies reported that the Met allele increased the risk of OCD (Karayiorgou et al., 1997, Karayiorgou et al., 1999, Alsobrook et al., 2002), five studies either failed to replicate this (Azzam and Mathews, 2003, Erdal et al., 2003, Meira-Lima et al., 2004) or found an association with either homozygosity (Schindler et al., 2000) or heterozygosity (Niehaus et al., 2001) at this locus. Therefore, similar to results from studies investigating the association between COMT and schizophrenia (Munafo et al., 2005), the association between the COMT Val158Met polymorphism and OCD is still inconclusive. However, three out of eight studies reported an association between OCD and the low-activity Met allele, whereas we found an association between the high-activity Val allele and more OCS in schizophrenia. It is important to emphasize that we have investigated effects of the COMT genotype on OCS in patients with schizophrenia, therefore making it difficult to compare our results with those from studies investigating the association between the COMT genotype and OCD without schizophrenia. Also, most studies looking at COMT and OCD investigated the risk of OCD in the presence of a specific COMT genotype, whereas we took a dimensional approach using Y-BOCS scores as a quantitative phenotype. One study that used a similar approach reported an association between the COMT high-activity Val allele and phobic anxiety in a large sample of over 1200 subjects (McGrath et al., 2004). In this respect, the Val allele could be considered a risk allele for anxiety symptoms.

4.2. Limitations and advances of the study 

This study has several limitations. Sample size is relatively small; therefore, results should be considered preliminary. However, two recent studies (Wahlstrom et al., 2007, McIntosh et al., 2007) reported on the effect of the COMT Val158Met polymorphism in samples of similar size (70 and 77 subjects, respectively). Thus, our sample size is small but not uncommon. A second limitation of our study is genetic heterogeneity. Since the effect of the COMT Val158Met genotype on the extent of OCS remained significant when we analyzed our data including male patients of Dutch ancestry only (N=54; Kruskal–Wallis: χ2=6.60, df=2, P=0.037), we think it is unlikely our results are false-positive. Also, since the COMT Val158Met polymorphism is functional, i.e. leads to an amino acid substitution known to affect the thermostability of the COMT protein (Chen et al., 2004), it is likely that effects on enzymatic activity are similar across ethnic populations. Moreover, Chen et al. (2004) established effects of the COMT Val158Met polymorphism on COMT protein stability using postmortem brain tissue from an ethnically mixed sample including white Caucasian and African American subjects: 66% of healthy controls and 52% of schizophrenia patients were African Americans. To our best knowledge, there is no evidence that ethnicity modulates functional effects of the COMT Val158Met polymorphism on COMT activity. However, the precise relationship between ethnicity and these functional effects has not yet been investigated; future research is needed to examine this. Previous studies reported that the Val allele is more frequent in non-Caucasian samples (Palmatier et al., 1999). Thus, ethnicity could confound our data if non-Caucasian subjects (who are likely to carry more Val alleles) had more OCS than Caucasian subjects. However, in our sample Y-BOCS scores did not differ between ethnic groups. Therefore, we consider ethnicity an unlikely confounder in our study. Nevertheless, since this is the first report of an association between the COMT Val158Met polymorphism and OCS in patients with schizophrenia, our findings need to be replicated in a large and genetically homogeneous sample.

Several reports have described the induction of OCS by clozapine and olanzapine (de Haan et al., 1999, Mottard and de la Sablonniere, 1999, Ongur and Goff, 2005). However, since no randomized controlled trials examining this have been performed yet, it is controversial whether these agents really induce OCS. In our study no difference in the use of olanzapine and clozapine between genotype groups was observed; therefore we do not think this to be a confounding factor. Furthermore, when we analyzed our data correcting for antipsychotic medication as a possible confounding factor, the effect of the COMT genotype on Y-BOCS scores seemed increased in comparison to the ‘uncorrected’ analysis (P<0.001 and P=0.012, respectively). In a recent report, clozapine was reported to induce OCS in some patients with schizophrenia, whereas it was associated with a decrease in existing OCS in others (de Haan et al., 2004); it is possible that these differences can be explained by genetic variation.

Our analyses included only male patients with schizophrenia; future studies should investigate whether COMT genotype has similar effects on OCS in female patients with schizophrenia. Differentiating between males and females is important, since previous studies reported gender-specific effects of the COMT genotype on the risk for OCD (Karayiorgou et al., 1997, Karayiorgou et al., 1999, Alsobrook et al., 2002). Furthermore, gender-specific effects of COMT have been reported in COMT-deficient mice (Gogos et al., 1998) and may result from the down-regulation of COMT expression by estrogens (Jiang et al., 2003).

4.3. Neurobiological mechanism 

Prefrontal dysfunction and impaired dopaminergic neurotransmission resulting in abnormal prefrontal regulation of striatal activity are mechanisms hypothesized to play a role in the pathophysiology of schizophrenia (Weinberger et al., 2001). Disturbances in dopaminergic systems in schizophrenia may not only lead to psychotic symptoms, but also to OCS since evidence for increased dopaminergic neurotransmission in the striatum in patients with OCD has been found (van der Wee et al., 2004, Denys et al., 2004). Disruption of frontostriatal circuits have been observed in both patients with schizophrenia and OCD. Structural MRI studies have identified specific parts of the frontostriatal system that are altered in OCD (Pujol et al., 2004). Furthermore, patients with OCD show decreased fronto-striatal responsiveness during planning and executive tasks (van den Heuvel et al., 2005), which is a finding similar to those in patients with schizophrenia (Morey et al., 2005). It is possible that by indirect downstream effects on dopamine regulation of striatum and basal ganglia, the COMT gene may modulate the risk for OCS and OCD in schizophrenia.

Apart from dopamine, COMT is also important for the degradation of noradrenalin: e.g. COMT-deficient mice show changes in the levels of dopamine and its metabolites, but also in noradrenalin levels (Gogos et al., 1998, Huotari et al., 2002). The noradrenergic system is involved in behavioral responses to stress, i.e. anxiety (Morilak et al., 2005), and a relative increase of noradrenalin levels by selective serotonin and norepinephrine reuptake inhibitors (SNRIs) reduces anxiety in depressed patients (Blier and Szabo, 2005). Similarly, people with the COMT Met/Met genotype (with relatively increased noradrenalin levels) may be less vulnerable to the development of anxiety disorders (Eley et al., 2003, Rothe et al., 2006). Thus, the mechanism by which COMT influences the severity of OCS in patients with schizophrenia may be via modulation of dopaminergic or noradrenergic systems; further research is needed to clarify which of these pathways is the most important one.

4.4. COMT: a possible pharmacogenetic target? 

Recently, pharmacological inhibitors of COMT, e.g. entacapone and tolcapone, have received increased attention in the treatment of patients with Parkinson's disease (Keating and Lyseng-Williamson, 2005). COMT inhibitors stabilize dopamine levels by reducing the breakdown of dopamine (Muller et al., 2006) and have been reported to ameliorate cognitive symptoms in patients with Parkinson's disease (Gasparini et al., 1997). Tolcapone, a COMT inhibitor acting both in the peripheral and in the central nervous system (Keating and Lyseng-Williamson, 2005), may have important therapeutic implications for patients with schizophrenia. However, further research is warranted to investigate the safety and efficacy of the use of COMT inhibitors in patients with schizophrenia.

In conclusion, our preliminary data suggest that the COMT high-activity Val allele is associated with more obsessive–compulsive symptoms in young males with schizophrenia. Therefore, our results further support the hypothesis that the COMT Val158Met polymorphism may be a modifier gene for the symptomatology of schizophrenia.