Genotypic and Haplotypic Association of Catechol-O-Methyltransferase rs4680 and rs4818 Gene Polymorphisms with Particular Clinical Symptoms in Schizophrenia

Catechol-O-methyl transferase (COMT) gene variants are involved in different neuropsychiatric disorders and cognitive impairments, associated with altered dopamine function. This study investigated the genotypic and haplotypic association of COMT rs4680 and rs4618 polymorphisms with the severity of cognitive and other clinical symptoms in 544 male and 385 female subjects with schizophrenia. COMT rs4818 G carriers were more frequent in male patients with mild abstract thinking difficulties, compared to CC homozygotes or C allele carriers. Male carriers of COMT rs4680 A allele had worse abstract thinking (N5) scores than GG carriers, whereas AA homozygotes were more frequent in male subjects with lower scores on the intensity of the somatic concern (G1) item, compared to G carriers. Male carriers of COMT rs4818–rs4680 GA haplotype had the highest scores on the G1 item (somatic concern), whereas GG haplotype carriers had the lowest scores on G2 (anxiety) and G6 (depression) items. COMT GG haplotype was less frequent in female patients with severe disturbance of volition (G13 item) compared to the group with mild symptoms, while CG haplotype was more frequent in female patients with severe then mild symptoms. These findings suggest the sex-specific genotypic and haplotypic association of COMT variants with a severity of cognitive and other clinical symptoms of schizophrenia.


Introduction
Schizophrenia, one of the most severe mental disorders, is associated with the imbalance between resilience and vulnerability in relation with life stressors [1][2][3]. Schizophrenia is characterized by positive, negative and cognitive symptoms, according to the DSM-5 criteria [4]. Cognitive symptoms include disturbances in executive function, working and verbal memory, language, learning, attention, processing speed, vigilance, and problem solving [4][5][6][7], leading to decreased social functioning and adjustment and employment reduced dopaminergic function, will be associated with pronounced cognitive decline and more severe clinical symptoms in schizophrenia, compared to presence of the A or C allele of the COMT rs4680 or rs4818 polymorphisms, respectively.

Subjects
The study included 929 subjects (544 male and 385 female participants) with schizophrenia, and some of them were included in our previous studies [28][29][30]. Patients were included in the study if they had schizophrenia for at least 5 years, and if they were 18-65 years old. Before participation, patients were informed in detail about the procedures, and after they had signed the written informed consent, the study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki from 1975). Both in-and out-patients participated. Exclusion criteria were intellectual disabilities, first-episode psychosis, substance abuse and/or dependence in the previous three months, and if they had any comorbid severe somatic or neurological disorder.
They were diagnosed using the Structured Clinical Interview for DSM-IV (SCID) [36], while the severity of clinical symptoms was evaluated using the gold standard, the Positive and Negative Syndrome Scale (PANSS) [32], and the presence of cognitive decline was determined with PANSS cognitive subscale, that includes items P2 (conceptual disorganization), N5 (abstract thinking), G10 (disorientation), and G11 (attention problems) [33,34]. Additionally, subjects were subdivided into those with mild and moderate to severe symptoms with cut off points: 4 for particular PANSS items, i.e., 16 for PANSS cognitive subscale, 28 for PANSS positive and negative subscale, and 64 for PANSS general subscale, as reported previously [37]. Average PANSS score was 103 (88; 124). All subjects were recruited from the University Hospitals from the Zagreb County (Department for Psychiatry and Psychological Medicine at the University Hospital Centre Zagreb and from Department for Biological Psychiatry and Psychogeriatrics, University Psychiatric Hospital Vrapce, Zagreb, Croatia). The study was conducted with the approval of the Ethics Committees of the University Hospital Center Zagreb and University Psychiatric Hospital Vrapce, Zagreb, Croatia.

Genotyping
All subjects were sampled during regular check-ups in the morning, and blood was processed on the same day. Genomic DNA was isolated from the peripheral blood using the salting-out method [38]. The COMT rs4818 (assay ID: C__2538750_10) and COMT rs4680 (assay ID: C__25746809_50) polymorphisms genotypes were determined using the primers and probes from Applied Biosystems as TaqMan ® Drug Metabolism Genotyping Assays (Applied Biosystems, Foster City, CA, USA) on ABI Prism 7300 Real time PCR System apparatus (Applied Biosystems, Foster City, CA, USA), according to the procedures described by Applied Biosystems. Thermal cycler conditions were 10 min at 95 • C followed by 50 cycles of denaturation at 92 • C for 15 s and elongation at 60 • C for 90 s. The 10 µL reaction volume contained around 30 ng of DNA. Besides the codominant model, which includes all three genotypes, dominant, and recessive models for both COMT polymorphisms, as well as the allelic model, were used.

Statistical Analyses
The data were analyzed using Prism version 7.00 (GraphPad Software, Inc., San Diego, CA, USA). Due to the deviation from a normal distribution (determined with Kolmogorov-Smirnov test), nonparametric tests were used for all analyses. Multiple linear regression was performed to analyze the effect of sex, age, and smoking on severity of cognitive decline (PANSS cognitive scores) and severity of other individual items of the PANSS in patients with schizophrenia. The Mann-Whitney U test was used for independent pair comparisons and Kruskal-Wallis ANOVA, with post hoc Dunn test was used for multiple comparisons. Box-plot diagrams were used for visual representation of significant results,  where the central box represented the interquartile range, the middle line represented  the median, the vertical line extended from the minimum to the maximum value, while  separate dots represented the outliers. Extreme values (more than 3 box-lengths outside of the box) were excluded from the analyses. Genotype, allele, and haplotype distribution among subjects with mild and severe symptoms of schizophrenia was calculated using the χ 2 -test, while the standardized residuals (R) were calculated to determine which parameter was the most significant contributor to the differences among groups [39].
The Hardy-Weinberg equilibrium for COMT rs4818 and rs4680 polymorphisms was calculated using χ 2 -test [40]. LD pairwise values for two COMT SNPs and haplotype frequencies were determined using the Haploview software v. 4.2 [41] and represented with D' value. Loci are considered to be in linkage if D' is >0.80. PLINK v. 1.07. software was used to assign best-estimate haplotype pairs to each individual using the expectationmaximization algorithm [42]. G*Power 3.1 Software (Germany) [43] was used to determine a priori sample size. For all two tailed analyses, the p-value (0.05/2 = 0.025) was corrected because two SNPs were compared and the results were considered significant if p < 0.025. Required sample size (with α = 0.025; power (1 − β) = 0.800; and a small effect size = 0.15) for Mann-Whitney test was N = 416, whereas for Kruskal-Wallis ANOVA (for 3 groups, the required sample size was N = 432; and for 4 groups, the required sample size was N = 492). As the study included N = 929 participants, it had adequate sample size and statistical power.

Demographic Data
The study included a total of 929 subjects (544 male and 385 female participants) with schizophrenia. Male subjects were significantly younger (p < 0.001), and had more severe total (p < 0.001), positive (p < 0.001), negative (p < 0.001), and general (p < 0.001) symptoms of schizophrenia, as well as higher PANSS cognitive scores (p < 0.001), than female subjects (Table 1). Moreover, male subjects were more frequently smokers (69.1%, R = 2.1; p < 0.001) than female subjects (51.7%). Multiple linear regression was performed to analyze the effect of sex, age, and smoking on severity of cognitive decline (PANSS cognitive scores) in patients with schizophrenia. Younger age (β = −0.211; p < 0.001) and male sex (β = −0.360; p < 0.001) were significant predictors of higher PANSS scores, while smoking (β = −0.006; p = 0.858) was not associated with PANSS cognitive scores (R 2 = 0.228; F = 63.398; p < 0.001). Since male subjects were significantly younger than female, and there were significant differences in PANSS scores between two sexes, all analyses were performed in male and female subjects separately.
Genotype and allele frequencies of the rs4818 and rs4680 polymorphisms located in the COMT gene have been determined in total 929 subjects. Minor allele frequency (MAF) and corresponding Hardy-Weinberg equilibrium significance value for each polymorphism, as well as linkage disequilibrium between COMT rs4818 and rs4680 polymorphisms have been determined using Haploview software v. 4.2. MAFs for rs4818 and rs4680 were 0.386 (HW p value = 0.859) and 0.498 (HW p value = 0.658), respectively. Haplotype analysis showed strong LD between COMT rs4818 and rs4680 polymorphisms (D' × 100 = 88; LOD > 2) ( Figure 1), hence haplotypes for COMT rs4818 and rs4680 block were determined for each subject using expectation-maximization algorithm. The most common haplotype was CA, which was present in 48.0% of subjects, followed by GG (36.3%) and CG (13.4%) haplotypes. The rarest haplotype was GA (2.2%). and corresponding Hardy-Weinberg equilibrium significance value for each polymorphism, as well as linkage disequilibrium between COMT rs4818 and rs4680 polymorphisms have been determined using Haploview software v. 4.2. MAFs for rs4818 and rs4680 were 0.386 (HW p value = 0.859) and 0.498 (HW p value = 0.658), respectively. Haplotype analysis showed strong LD between COMT rs4818 and rs4680 polymorphisms (D' × 100 = 88; LOD > 2) (Figure 1), hence haplotypes for COMT rs4818 and rs4680 block were determined for each subject using expectation-maximization algorithm. The most common haplotype was CA, which was present in 48.0% of subjects, followed by GG (36.3%) and CG (13.4%) haplotypes. The rarest haplotype was GA (2.2%).
There were no significant differences in the distribution of the COMT rs4818 and rs4680 genotypes, alleles, or haplotypes between male and female patients, as well as between smokers and non-smokers (Table 2).   There were no significant differences in the distribution of the COMT rs4818 and rs4680 genotypes, alleles, or haplotypes between male and female patients, as well as between smokers and non-smokers (Table 2). The severity of the PANSS positive, negative, and general psychopathology symptoms of schizophrenia, and cognitive decline evaluated with PANSS cognitive subscale, that includes items P2 (conceptual disorganization), N5 (abstract thinking), G10 (disorientation), and G11 (attention problems), as well as scores on particular items of PANSS scale, were evaluated in regard to COMT rs4818 and rs4680 polymorphisms and COMT rs4818-rs4680 haplotype in male and female subjects separately.
Polymorphism COMT rs4818 was not associated with any of the symptoms of schizophrenia in male or in female subjects when the total scores were evaluated (Tables 3 and 4). However, when subjects were divided according to the severity of symptoms into those with mild symptoms and those with moderate and severe symptoms (Table 5), G carriers were more often present (25.6%) in male subjects with mild difficulties in abstract thinking (item N5) compared to male CC carriers (17.2%) (R = 1.2; χ 2 = 5.210; p = 0.022), or male C allele carriers (20.2%) (R = 1.6; χ 2 = 5.042; p = 0.025). Additionally, nominal association of COMT rs4818 polymorphism and scores on G1 item (corresponding to the intensity of somatic concern) was observed, where G allele was under-represented (19.3%) in male subjects with mild symptoms (R = −1.4; χ 2 = 4.186; p = 0.041) compared to C allele carriers (24.7%); however, after correction for multiple testing, this association did not reach statistical significance.  Polymorphism COMT rs4680 was associated with N5 (abstract thinking) scores in male subjects, where A carriers had higher scores than GG carriers (U = 23575.5; p = 0.023 (Table 3, Figure 2a). As shown in Table 5, when subjects were divided according to the severity of symptoms, COMT rs4680 AA homozygotes were more frequent (18.1%) in subjects with lower scores on G1 (somatic concern) item (R = 1.7; χ 2 = 5.061; p = 0.024), compared to G carriers (23.7%). However, this association was not confirmed in female patients (Table 6).
Both COMT polymorphisms showed an association with the PANSS general scores in female patients when they were subdivided in mild and severe symptoms category (Table 6); however, very low number of subjects (<2) in certain groups could possibly lead to false significant results, and since they were not confirmed when total PANSS general scores were evaluated, they were considered artefact.
The carriers of the rarest COMT rs4818-rs4680 haplotype (GA) had the highest scores on G1 item (somatic concern), while GG haplotype had the lowest scores on G2 (anxiety) and G6 (depression) items, compared to other haplotypes in male subjects (Table 3, Figures 2b-d and 3b-d). Similarly, as shown in Table 6, in female subjects the GG haplotype was less frequent (31.8%; R = −1.4) in the group with severe disturbance of volition (G13 item) compared to the group with mild symptoms (39.8%), while CG haplotype was over-represented in group with severe symptoms (18.2%; R = 1.8), compared to those with mild symptoms (11.8%) (χ 2 = 9.356; p = 0.025). Table 5. The distribution of the genotypes and alleles of the COMT rs4818 and rs4680 polymorphisms, and their haplotype block, in male subjects with mild and severe symptoms of schizophrenia divided by scores on PANSS total, positive, negative, general psychopathology and cognitive subscales as well as on particular PANSS items.

Discussion
Our results have shown a significant genotypic and haplotypic association of COMT rs4680 and rs4818 gene polymorphisms with particular clinical symptoms evaluated by the individual PANSS subscales scores in male and female patients with schizophrenia. Our detailed analysis detected several associations between COMT rs4680 and rs4818 and the severity of particular individual PANSS items. In male patients with schizophrenia, the presence of the COMT rs4818 G allele was more often detected in those with mild "difficulties in abstract thinking" (N5), compared to CC homozygotes or C allele carriers. Regarding the other COMT SNP, in male patients, the presence of the COMT rs4680 A allele was associated with N5 scores compared to GG carriers, and the presence of the

Discussion
Our results have shown a significant genotypic and haplotypic association of COMT rs4680 and rs4818 gene polymorphisms with particular clinical symptoms evaluated by the individual PANSS subscales scores in male and female patients with schizophrenia. Our detailed analysis detected several associations between COMT rs4680 and rs4818 and the severity of particular individual PANSS items. In male patients with schizophrenia, the presence of the COMT rs4818 G allele was more often detected in those with mild "difficulties in abstract thinking" (N5), compared to CC homozygotes or C allele carriers. Regarding the other COMT SNP, in male patients, the presence of the COMT rs4680 A allele was associated with N5 scores compared to GG carriers, and the presence of the COMT rs4680 AA homozygous genotype was more frequently found participants with lower scores on the intensity of somatic concern (G1) item compared to G carriers. In females with schizophrenia, there were no significant associations between individual PANSS items (clinical symptoms of schizophrenia) and COMT rs4680 and rs4818 genetic variants. Concerning haplotype associations, male patients with schizophrenia who were COMT rs4818-rs4680 GA haplotype carriers had the highest scores on G1 item (somatic concern), whereas GG haplotype carriers had the lowest scores on G2 (anxiety) and G6 (depression) items. Female patients with schizophrenia had less frequent COMT GG haplotype associated with severe disturbance of volition (G13 item) compared to females with mild symptoms, while female patients with CG haplotype had more frequent severe then mild symptoms. All these associations were sex-specific.
In male patients with schizophrenia, lower N5 "Difficulties in abstract thinking" scores in COMT rs4680 GG carriers than in A carriers, and overrepresentation of COMT rs4818 G carriers compared to CC polymorphism in participants with less severe N5 scores, suggest an association between the presence higher COMT activity alleles and better preservation of abstract thinking. Actually, N5 was the only PANSS-negative item which severity was related to any of the two aforementioned COMT polymorphisms in males. Although abstract thinking belongs to the PANSS negative symptoms, according to a meta-analysis of the results of 45 factor analyses, it was not a part of the core negative items and was actually a symptom of disorganization/cognitive dimension [44]. The inability to engage in abstract thinking is a long-known typical symptom of schizophrenia [45]. It measures proverb interpretations and abstract similarities, by detecting the relationship between object, ideas, principles, and concepts. Its impairment is expressed as concrete form of cognition and speech, such as providing more concrete responses to proverbs [46]. Many studies have investigated abstract thinking in schizophrenia. Difficulty in abstract thinking was associated with the impaired decision making [47], distorted sensory experiences [48], and EEG findings of reduced alpha 1 spectral amplitude and lower Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery 5 neuro-cognitive composite score [49].
Interestingly, baseline higher severity of PANSS N5 item was, along with P2 and G9 items, among three baseline PANSS items which predicted treatment resistance [50]. In another study, the PANSS item "abstract thinking" accounted for an association between negative symptoms and cognition [51], and was also found, together with conceptual disorganization, hostility, and uncooperativeness, to represent important mediating roles across different communities of schizophrenia symptoms [52]. In first-episode patients, baseline N5 and N7 were lower in remitters after one year of treatment, than in nonremitters, while other negative items were not [53]. In addition, P5 "abstractive thinking" was the only PANSS-negative symptom to predict depressive trajectory [54].
While these studies highlight the distinct position of N5 item among other PANSS negative symptoms [50][51][52], to best of our knowledge, only one study investigated all 30 PANSS items in relation with COMT polymorphism. This study found no associations between any individual PANSS item in males, including N5, with the COMT rs4680 polymorphism [55]. However, this study included a lower number of patients, and recruited only individuals with youth-onset schizophrenia with the mean age of 32. In our study, male patients were 38 years old. In turn, the age of onset of schizophrenia influences cognitive functioning, given that patients with youth-onset schizophrenia had severe cognitive deficits, in contrast to individuals with late-onset schizophrenia who maintained relatively preserved cognition [56]. Another study has focused on COMT polymorphism and abstraction in patients with schizophrenia. Our results disagree with the findings of better performance on abstraction test in individuals with schizophrenia who had greater number of COMT rs4680 A alleles in a dose-response fashion, although this effect was small [57]. However, the aforementioned study had small sample size for genetic study, included ethnically heterogeneous outpatient population with the average illness duration of 24 years, and abstraction was measured by a subtest from The Delis-Kaplan Executive Function System [57]. While those studies [55,57] and the present trial were crosssectional, future studies are needed to establish the associations between COMT functional polymorphisms longitudinally, i.e., to explore if the relationships between COMT rs 4680 and rs4818 is detected in the acute phase change during successful treatment. Of note, treatment with antipsychotics decreases the scores on essentially all PANSS symptom, at least in non-treatment resistant patients [58], and it was depicted that abstract thinking in several diagnostic categories, including schizophrenia, improves over time [46].
The unique association of lesser severity of PANSS N5 component with the highactivity COMT rs4680 and rs4818 alleles in our study, indirectly suggests the involvement of lower prefrontal dopamine levels in better performance in abstract reasoning in males with schizophrenia. Namely, COMT genotypes at SNPs rs4680 and rs4818, but not rs737865 or rs165599, were associated with altered levels of S-COMT, but not MB-COMT, in the dorsolateral prefrontal cortex (DLPFC) of subjects who died from different causes, including suicide [59]. While it was reported that reduced activity in the left frontotemporal and the right orbitofrontal cortex could be a critical region for the deficit in abstract thinking in schizophrenia [60], low COMT activity in healthy men, resulting from the presence of the A allele of COMT rs4680 was associated with reductions in resting blood flow in frontal regions, compared to high COMT activity groups [61]. It may be speculated that males with lower-activity COMT polymorphisms in our study may have had changes in cortical neural activity and perfusion, which may have contributed to deficits in abstract thinking. Given the important role of abstract thinking in schizophrenia, it remains to be determined whether males with low-activity COMT alleles, who had worse N5 scores, differ in the degree of improvement of deficits in abstract thinking.
Both G2 "anxiety" and G6 "depression" items are a part of PANSS anxiety and depression dimension, according to the factor analysis [62]. Our findings suggest that high COMT activity related to low dopamine availability was associated with the lowest anxiety and depression dimension scores in males. In another study in Croatian population, COMT rs4680 was not associated with affective/depressive PANSS factors [63]. Our results do not agree with the findings of the higher levels of depression in patients with COMTrs4680 GG genotype, compared to A carriers, in patients with first-episode schizophrenia, whereby COMT rs4680 polymorphism moderated an association between the severity of depression and stressful life events [64]. That study had small sample size, and in both cited studies males and females were not separately analyzed [63,64]. Importantly, in patients with schizophrenia, COMT DNA methylation was inversely correlated with depressed subdomain of the PANSS, i.e., the higher the depressive symptoms, the lower the DNA methylation [65]. Future studies exploring the associations of COMT polymorphisms and depressive symptoms in schizophrenia need to consider childhood and life-time stressors, which may strongly influence this relationship.
The disturbance of volition (PANSS G13 item) refers to impaired will to move, and an altered timing of awareness of movement [66]. It belongs, along with G5 and several other items, to the disorganization factor [44]. In our female patients with schizophrenia, no associations between COMT genotypes or haplotypes and the severity of any PANSS sub-scores or individual items were observed. In line, no associations were found between PANSS item G13 and COMT rs4680 polymorphism in females, although female patients in that study had several other correlations [55]. In that cited study, females were about 19 years younger than our female patients, and had youth-onset schizophrenia [55]. However, when symptoms in the present study were categorized according to their severity, GG haplotype was less frequent in females with severe disturbance of volition, while the CG haplotype was over-represented in the group with severe symptoms, compared to those with mild symptoms. Disturbance of volition is commonly observed in individuals with schizophrenia. For example, it was the fifth-most-common PANSS single symptom in firstepisode schizophrenia, reported in 65.4% of participants [67]. Moreover, the higher severity of disturbance of volition was, along with uncooperativeness, predictive of improved therapeutic benefit of paliperidone [68], but also during the initial asenapine treatment [69]. Despite being recognized as a common symptom in schizophrenia, disturbance of volition has received less attention than other PANSS items, and we were unable to find any data on the relationship between N5 item and either dopamine or COMT variants, but targeting such interactions appear as an attractive goal for future studies.
Sex-specific correlations of COMT polymorphism with different parameters have been previously reported in both preclinical and clinical studies. In preclinical studies, the administration of COMT inhibitor tolcapone produced more prominent changes in dopamine metabolism in female than male rat brains [70], while ovariectomy decreased, and estradiol replacement restored COMT levels, respectively [71]. In healthy women, increases in estradiol during the menstrual cycle were associated with improved working memory performance for participants who were GG carriers, and an association was in the opposite direction for AA carriers [72]. Sex divergent effects of COMT rs4680 polymorphism have been shown previously on the effects on the relationship between executive dysfunction and psychotic-like experience in college students [22]. In addition, among healthy individuals with COMT rs4633-rs4680 T allele, males had higher COMT specific activity in serum than females [73]. Moreover, the higher number of associations in males in our study may also be related to their substantially higher severity as well as wider range of severities in PANSS total scores and all other scales in male patients.
While the associations were sex-specific, in general 1) positive psychotic symptoms were not associated with any COMT genotypes or haplotype combinations, and 2) the presence of mainly high-activity COMT genotypes and haplotype was correlated with lower psychopathology on some indicators of PANSS negative and general psychopathology symptoms. Such findings somehow contribute to the general view that COMT is the most important regulator of dopamine function in PFC, but has a minor role in striatum and nucleus accumbens [74], whereas positive symptoms of schizophrenia are connected with hyperactivity of dopaminergic neurotransmission in striatum. However, an association between high-activity COMT and lower intensity of some negative and general symptoms is surprising, seeing that negative and cognitive symptoms are thought to arise from hypodopaminergic functioning in the frontal lobe and additional mesolimbic structures [75,76]. However, such findings were not detected in all patients with schizophrenia, and, given the high variance in dopaminergic measures in this population [77], it remains to be determined what is the role of COMT polymorphism in the complex landscape of symptoms in schizophrenia.
In addition, polygenic risk score (PGS) was associated with the genetic liability for schizophrenia, but it is not yet applied in precision medicine at the individual level. For example, it predicted treatment resistance in one study [78], but was not associated with the onset of treatment-resistance in another study [79]. Moreover, PGS predicted lower antipsychotic response in first-episode patients [80], but was not associated with the variance in cognitive test scores [81] or poorer cognitive outcome in schizophrenia [82]. Maybe the biggest obstacle for establishing precision medicine in schizophrenia is the lack of laboratory tools to predict the outcome. Moreover, schizophrenia is not a single disorder, but rather a group of distinct disorders with some overlapping symptoms [83]. Our correlations of COMT variants, implicated in higher dopamine degradation with lower severity of particular symptoms, may be an interesting topic for future longitudinal studies. Such studies should address the predictive value of different COMT genotypes and haplotypes on the trajectories of symptoms. Of note, genetics itself will probably never be a single factor that determines the severity of the outcome of schizophrenia, due to the presence of many other factors unrelated to genes, such as non-compliance, stressful life events, and substance abuse. Nevertheless, we hypothesize that different COMT variants may contribute to our understanding on the complexity of psychopathology in schizophrenia and our results may provide a small contribution to the field.
Taking into consideration that all our associations were sex-specific, we also recommend separate analyses for males and females in schizophrenia, or controlling the results for patient's sex, especially in relation with studies addressing COMT variants. Our results add to the current knowledge on sexually dimorphic influence of COMT gene upon psychiatric phenotypes [84], that was also reported in healthy individuals [85]. In addition, it would be interesting to explore how different COMT genotypes and their haplotype combinations relate to psychopathology in different stages throughout the course of schizophrenia in male and female participants.
It is intriguing to put the COMT gene into evolutionary perspective. Due to its influence in the prefrontal cortex in complex cognitive functioning, and the role of COMT enzyme in modulating prefrontal dopamine levels, COMT functional polymorphisms may have impacted adaptation to new environments during human evolution. Of note, G allele of COMT rs4680 is the ancestral allele, while A allele is a result of mutation, which is considered advantageous for human evolution because A allele was unique to humans and related to higher intelligence [86] and better insight problem solving [85]. Schizophrenia is very rare in societies with hunter-gatherer lifestyles [83]. In turn, hunter-gatherers had lower frequency of A allele than farming or industrialized populations [86]. While it is unknown if COMT gene has any role in the evolutionary paradox in schizophrenia, we speculate that the relation between COMT polymorphisms and haplotype combinations (associated with higher enzymatic activity), with lower intensity of some symptoms in schizophrenia, may suggest distinct impact of the COMT genes on schizophrenia onset and schizophrenia severity.

Limitations and Strengths
This study was cross-sectional, and therefore the observed associations do not imply a causal relationship. The severity of several PANSS items/symptoms was associated with COMT genotypes and haplotypes in patients with schizophrenia.
Limitation is that only two polymorphisms were determined. However, it has been shown that COMT rs4680 is a functional polymorphism affecting COMT activity, protein abundance, and protein stability [17], while rs4680 and rs4818, but not rs737865 or rs165599 genotypes, were associated with altered levels of S-COMT, in the human dorsolateral prefrontal cortex (DLPFC) [59]. In a preclinical study, COMT knockout mice had 60% higher dopamine levels in PFC, but not in nucleus accumbens [87]. However, in a postmortem study, the mean abundance of COMT mRNA in DLPFC of psychiatric patients, including those with schizophrenia, did not differ by the COMT genotype [88]. Therefore, the relationship between COMT genetic variants and COMT activity in brain cortical areas is complex and incompletely understood. Many other factors may also influence COMT activity, such as COMT methylation level. For example, participants with schizophrenia who received atypical antipsychotics, most often risperidone, had lower incidence of COMT DNA methylation than patients on typical antipsychotics [65].
However, this study has strengths, such as large sample size and the ethnically homogenous Caucasian population, which is important given the pronounced differences in prevalence of COMT genotypes and haplotypes across different ethnic groups. Apart from genotype frequencies, psychopathology ratings may also differ across different ethnic groups. For example, patients from China scored 20-30% higher on 5 PANSS items, including disturbance of volition, whereas they had 20% lower scores on another 11 items, including abstract thinking, compared to American inpatients [89]. Moreover, while the vast majority of studies on COMT investigated the rs4680 genotype, the present study included COMT rs4680 and rs4818 polymorphisms, which is important because it was suggested that the COMT rs4818 polymorphism may be responsible for an even larger variation in the COMT activity than the rs4680 polymorphism [20], and that COMT haplo-types are associated with schizophrenia susceptibility [90]. In addition, the frequency of the COMT rs4680 and rs4818 genetic variants was controlled for the effect of smoking and sex.

Conclusions
Our findings demonstrated sex-specific associations of COMT high-activity rs4680 and rs4818 variants and haplotype combinations with mostly lower severity of several individual PANSS symptoms in patients with schizophrenia. Longitudinal studies are needed to establish whether those correlations remain significant during treatment and if different COMT genotypes and haplotypes have predictive value on trajectories of treatment.