Abstract
Context:
Schizophrenia has been associated with disorder of the dopamine system, which is downregulated by projections of the serotonin pathway. Dopamine and serotonin levels are regulated by a system of transporters and enzymes. In this research, dopamine transporter polymorphism (DAT-VNTR), serotonin transporter polymorphism (5-HTTLPR), monoamine oxidase A (MAOA-uVNTR), and catechol-o-methyl transferase (COMT Val158Met) polymorphisms have been investigated.
Aims:
The aim of this study was to asses frequencies of these polymorphisms in the healthy control group and patients and to asses association with schizophrenia.
Settings and Design:
Three hundred and fourteen healthy volunteers and 306 schizophrenia patients were included. Schizophrenia was diagnosed by Diagnostic and Statistical Manual-IV of the American Psychiatric Association, and mini international neuropsychiatric interview questionnaire was used for screening of healthy population.
Materials and Methods:
Genotyping was performed using polymerase chain reaction (PCR) reaction followed by gel electrophoresis and PCR-restriction fragment length polymorphism.
Statistical Analysis:
Categorical data were analyzed using the Chi-square test, age between subgroups was compared using the Mann–Whitney test, and all polymorphisms were tested for Hardy–Weinberg equilibrium. Logistic regression analysis was used to set the prediction model of schizophrenia.
Results:
Difference in genotype distribution was observed for COMT Val158Met in female and DAT-VNTR polymorphism in overall sample P = 0.021 and P = 0.028, respectively. Statistically significant association of MAOA-uVNTR and schizophrenia was observed after adjustment for anamnestic predictors of disease. P = 0.010, 80.45% participants were correctly classified.
Conclusion:
Our results suggest an association of MAOA-uVNTR polymorphism with schizophrenia. The difference in the distribution of COMT Val158Met and DAT-VNTR polymorphism support the involvement of dopamine system components in the pathogenesis of schizophrenia.
Key words: Catechol-o-methyl transferase, dopamine transporter, monoamine oxidase, schizophrenia, serotonin transporter
INTRODUCTION
Schizophrenia is a severe psychological disease that affects around 1% of the general population.[1] Its etiology is still unclear, but there are several hypotheses that are trying to explain the biological background of schizophrenia. The most accepted hypothesis involves neurotransmitter dopamine and dopamine pathway. It has been changed and upgraded through time, and the latest research in this field has associated schizophrenia with prefrontal hypodopaminergia and subcortical hyperdopaminergia.[2] The neurotransmitter dopamine is involved in the regulation of motoric functions acting through the nigrostriatal pathway. Cognition, motivation, and reward are regulated through the mesolimbic and mesocortical pathways.[3] Projections of serotonergic neurons start from dorsal raphe nuclei and spread to the cortex and striatal region. Overlapping of these systems allows their interaction and downregulation of dopamine by serotonin.[4]
Considering the function and interaction of serotonin and dopamine systems, many studies have tried to find an association of genes involved in the regulation of these two systems and psychiatric disorders. Dopamine transporter is responsible for dopamine reuptake regulating dopamine levels. Polymorphisms of dopamine transporter gene SLC6A3 have been associated with altered transcriptional activity of dopamine transporter and thus have been investigated and associated with attention deficit hyperactivity disorder, schizophrenia, and Parkinson's disease.[5,6]
Another regulator of dopamine levels is catechol-o-methyl transferase (COMT) which has been considered as a treatment target of cognitive symptoms in schizophrenia. Because of its function in the prefrontal cortex, COMT became a gene candidate in genetic association studies for schizophrenia.[7] Variants of COMT gene have been associated with cortical function including cognitive symptoms and psychosis.[8]
Serotonin regulates psychological functions such as anxiety, emotions, and aggression which is why regulation of this neurotransmitter became subject of interest in many psychiatric studies. Serotonin function is regulated by serotonin transporter which is responsible for serotonin reuptake from the synaptic cleft.[9,10] Serotonin transporter transcription is regulated by SLC6A4 gene, and extensively studied functional polymorphism (serotonin transporter-linked polymorphic region [5-HTTLPR]) of this gene has been associated with depression, anxiety traits, and poor response to serotonin-selective reuptake inhibitor therapy.[11,12] However, some studies have failed to find any association of 5-HTTLPR polymorphism with depression.[9]
Monoamine oxidase A (MAOA) is an enzyme included in the degradation of biogenic amines such as serotonin, norepinephrine, and dopamine.[13] Its deficiency has been associated with aggressive behavior and mental retardation in males.[14] Furthermore, the association of MAOA gene with aggression and schizophrenia has been extensively investigated.[15]
Despite accepted dopamine hypothesis and interaction of serotonin and dopamine system, genetic association studies involving gene variants of these two systems have shown contradictory results.
In this research, we hypothesized that polymorphisms of dopamine transporter (DAT-VNTR), catechol-o-methyl transferase (COMT Val158Met), 5-HTTLPR, and monoamine oxidase-A (MAOA-uVNTR) might be associated with schizophrenia. The aim of this study was to assess the frequencies of these polymorphisms in healthy volunteers and patients with schizophrenia, to compare them, and to investigate their association with the disease.
MATERIALS AND METHODS
This study was conducted at the Department of clinical chemistry of the Sestre milosrdnice University Hospital Center in collaboration with the Clinic for psychiatry of the same institution. During a period of 6 years (June 2010–January 2017), 314 healthy volunteers and 306 patients with schizophrenia were included in this study. The sample size was estimated using available statistical tool for sample size estimation in genetic association studies by Gordon et al.[16,17] The sample size was calculated for all tested polymorphisms, and the highest required number was obtained for 5-HTTLPR. For this polymorphism, expected frequencies for controls (wild type allele = 0.48; variant allele = 0.52) and cases (wild type allele = 0.40; variant allele = 0.60) were obtained from the literature. The calculated number was 302 cases and 302 controls. Schizophrenia was diagnosed based on the Diagnostic and Statistical Manual-IV of the American Psychiatric Association by professional personnel. To avoid therapy effect, only individuals with the first episode of schizophrenia or individuals who were not using therapy were included in this study.
Healthy control group consisted of volunteers and blood donors. On inclusion, their health condition was examined by a clinician. Individuals with mental illness in anamnestic data, drug or alcohol abuse, or the presence of other chronic diseases were excluded from the study. To exclude potential current psychological disorders, the mini international neuropsychiatric interview questionnaire was used. The questionnaire is accepted as a diagnostic screening tool for outpatient evaluation.[18] Other demographic data such as marital status, education, and employment were collected for patients and control groups during their initial assessment by an interview with the physician. Faculty degree was treated as higher level of education compared to high school level (or lower).
This study was approved by the institution's ethical committee. All participants gave their written informed consent. Blood was sampled into K3EDTA tubes. Deoxyribonucleic acid was isolated using high pure PCR template preparation kit (Roche, Basel, Switzerland) and stored at +4°C until analysis. DAT-VNTR, MAOA-uVNTR, and 5-HTTLPR polymorphisms were genotyped using PCR followed by agarose gel electrophoresis. COMT Val158Met polymorphism was detected by restriction fragment length polymorphism method after PCR amplification. Primer and reaction conditions are presented in Table 1.
Table 1.
Polymerase chain reaction and electrophoresis conditions
| Polymorphism | Primer | PCR reaction | Electrophoresis |
|---|---|---|---|
| 5-HTTLPR | HTT419F: 5’-ATG CCA GCA CCT AAC CCC TAA TGT-3’HTT419R: 5’-GGA CCG CAA GGT GGG CGG GA-3’ | Reaction mix: Total volume 20 µL: H2O, 10XPCR buffer, 25 mM Mg2+, 25 mM dNTP, 5 µM primers, 6 U/µL fast start high fidelity polymerase Initial denaturation - 94°C/2 min Extension - 38 cycles: 94°C/45 s, 63°C/40 s, 72°C/60 s Final elongation - 72°C/10 min |
3.7% NuSieve 3:1 agarose gel containing 27 µL EtBr (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) Electrophoresis conditions: 110 V/45 min |
| MAOA-uVNTR | MAOAF: 5’-ACA GCC TGA CCG TGG AGA AG-3’MAOAR: 5’-GAA CGG ACG CTC CAT TCG GA -3’ | Reaction mix: Total volume 30 µL: H2O, 10XPCR buffer, 25 mM Mg2+, 25 mM dNTP, 5 µM primers, 5 U/µL Taq polymerase Initial denaturation - 95°C/2 min Extension - 37 cycles: 95°C/45 s, 70°C/30 s, 72°C/40 Final elongation - 72°C/10 min |
1.8% NuSieve GP agarose gel containing 27 µL EtBr (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) Electrophoresis conditions: 110 V/45 min |
| COMT Val158Met | COMTf: 5’-CTC ATC ACC ATC GAG ATC AA-3’COMTr: 5’-CAG TGA ACG TGG TGT GAA CAC-3’ | Reaction mix: Total volume 30 µL: H2O, 10XPCR buffer with 15 mM Mg2+, 25 mM dNTP, 5 µM primers, 5 U/µL Taq polymerase Initial denaturation - 94°C/2 min Extension - 35 cycles: 94°C/30 s, 60°C/30 s, 72°C/120 s Final elongation - 72°C/10 min RFLP: Incubation with NlaIII restriction enzyme during night |
PCR check: Commercial gel: PCR-check kit 110 V/15 min RFLP fragment detection: Spreadex EL 300 110 V/45 min. Gel (Elchrom Scientific AG, Cham, Switzerland). Staining with SYBR Gold (Invitrogen, Carslbad, CA USA) |
| DAT-VNTR | DAT1F: 5’-TGT GGT GTA GGG AAC GGC CT GAG-3’DAT1R: 5’-CTT CCT GGA GGT CAC GGC TCA AGG-3’ | Reaction mix: Total volume 30 µL: H2O, 10XPCR buffer with 15 mM Mg2+, 25 mM dNTP, 5 µM primers, 5 U/µL Taq polymerase, DMSO Initial denaturation - 95°C/2 min Extension - 35 cycles :95°C/60 s, 68°C/60 s, 72°C/60 s Final elongation - 72°C/10 min |
1.8% NuSieve 3:1 agarose gel containing 27 µL EtBr Electrophoresis conditions: 110 V/45 min |
5-HTTLPR – Serotonin-transporter-linked polymorphic region; MAOA-uVNTR – Monoamine oxidase A upstream variable number tandem repeats; COMT Val158Met – Catechol-o-methyl-transferase; DAT-VNTR – Dopamine transporter variable number tandem repeats; Mg2+ – Magnesium ions; dNTP – Deoxyribonucleotide triphosphate; DMSO – Dimethyl sulfoxide; EtBr – Ethidium bromide; RFLP – Restriction fragment length polymorphism; PCR – Polymerase chain reaction
Data are presented as numbers and percentages. Age is presented as median and range. All polymorphisms were tested for Hardy–Weinberg equilibrium. Genotype, allele frequencies, and other categorical data were compared using the Chi-square test. If the conditions for the Chi-square test were not met (small sample size), z-test (comparison of proportions) was performed. Age between subgroups was compared using the Mann–Whitney test. The level of significance was set at P < 0.05.
Anamnestic data in combination with genotypes were analyzed using logistic regression analysis for prediction of negative outcome – schizophrenia. Variables included in the logistic regression analysis were all four polymorphisms and variables statistically significant in previous data analysis: gender, marital status, education, and employment [Table 2]. Logistic regression analysis was performed in two steps. First, using a univariate model to avoid dropping out significant variables due to low statistical power caused by the inclusion of too many variables at the same time and for that reason, relaxed P value was used (P < 0.25) according to Sperandei.[19] Statistically significant variables from the univariate model (gender, marital status, education, employment, and MAO-uVNTR polymorphism) were included in multivariate analysis. The level of significance using a multivariate model was set at P < 0.05; odds ratio (OR) and 95% confidence intervals (95% CIs) were used as measures of association of tested variables and schizophrenia. Statistical analysis was performed using MedCalc statistical software (v12.7.2.0, Ostend, Belgium).
Table 2.
Demographic characteristics of participants
| Healthy control (n=314) | Schizophrenia (n=306) | P | |
|---|---|---|---|
| Age (years) | 36 (18-68) | 38 (18-84) | 0.281* |
| Male gender, n (%) | 164 (52.2) | 222 (72.5) | <0.001** |
| Married, n (%) | 135 (62.2) | 81 (29.8) | <0.001** |
| Faculty degree, n (%) | 138 (63.3) | 52 (18.3) | <0.001** |
| Employed, n (%) | 187 (86.2) | 86 (31.7) | <0.001** |
Age is presented as median (range). *Mann-Whitney test; **Chi-square test
RESULTS
Anamnestic data in schizophrenia patients and healthy controls are presented in Table 2. Schizophrenic patients were mostly males, not married, with a lower education degree, and mostly unemployed (P < 0.001).
All tested polymorphisms were in Hardy–Weinberg equilibrium: MAOA-uVNTR P = 0.986, DAT-VNTR P = 0.937, 5-HTTLPR P = 0.878, and COMT Val158Met P = 0.187.
Genotype and allele frequencies are presented in Table 3.
Table 3.
Allele and genotype frequencies in a healthy control and schizophrenia patients
| Healthy control |
Schizophrenia |
P |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| All, n (%) | Male, n (%) | Female, n (%) | All, n (%) | Male, n (%) | Female, n (%) | All | Male | Female | |
| MAOA | |||||||||
| High/high | 178 (58.9) | 114 (74.0) | 64 (43.2) | 166 (55.9) | 131 (61.6) | 35 (43.2) | 0.010 | 0.010 | 0.242 |
| High/low | 58 (19.2) | / | 58 (39.2) | 38 (12.8) | / | 38 (46.9) | |||
| Low/low | 66 (21.9) | 40 (26.0) | 26 (17.6) | 93 (31.3) | 85 (39.4) | 8 (9.9) | |||
| High | 302 (67) | 114 (74) | 186 (63) | 200 (53) | 131 (61.6) | 102 (67) | <0.001 | 0.010 | 0.941 |
| Low | 149 (33) | 40 (26) | 110 (37) | 178 (47) | 85 (39.4) | 60 (33) | |||
| 5-HTTLPR | |||||||||
| LL | 105 (33.4) | 57 (35.4) | 48 (32.0) | 122 (39.9) | 88 (40.0) | 34 (39.5) | 0.147 | 0.107 | 0.495 |
| LS | 152 (48.4) | 81 (50.3) | 71 (47.3) | 125 (40.8) | 88 (40.0) | 37 (43.0) | |||
| SS | 57 (18.2) | 23 (14.4) | 31 (20.7) | 59 (19.3) | 44 (20.0) | 15 (17.4) | |||
| L | 364 (58) | 196 (61) | 168 (56) | 367 (60) | 264 (60) | 105 (61) | 0.509 | 0.867 | 0.331 |
| S | 264 (42) | 126 (39) | 132 (44) | 245 (40) | 176 (40) | 67 (39) | |||
| COMT | |||||||||
| Val/Val | 63 (20.0) | 36 (22.4) | 27 (18) | 75 (24.8) | 50 (22.7) | 25 (29.1) | 0.237 | 0.992 | 0.021 |
| Val/Met | 168 (53.5) | 81 (50.3) | 85 (56.7) | 144 (47.1) | 111 (50.5) | 33 (38.4) | |||
| Met/Met | 83 (26.4) | 44 (27.3) | 38 (25.3) | 87 (28.4) | 59 (26.8) | 38 (44.2) | |||
| Val | 295 (47) | 155 (48) | 144 (46) | 294 (48) | 211 (48) | 83 (43) | 0.750 | 0.981 | 0.345 |
| Met | 333 (53) | 167 (52) | 156 (53) | 318 (52) | 229 (52) | 109 (57) | |||
| DAT | |||||||||
| 10/10 | 150 (47.8) | 78 (48.4) | 72 (48.0) | 154 (50.3) | 114 (51.8) | 40 (46.5) | 0.587 | 0.581 | 0.931 |
| 10/9 | 131 (41.7) | 68 (42.2) | 63 (42.0) | 133 (43.5) | 94 (42.7) | 39 (45.3) | 0.709 | 0.994 | 0.722 |
| 9/9 | 28 (8.9) | 13 (8.1) | 14 (9.3) | 13 (4.2) | 9 (4.1) | 4 (4.8) | 0.028 | 0.338 | 0.318 |
| Other genotypes | 5 (1.6) | 2 (1.2) | 1 (0.7) | 6 (1.9) | 3 (1.4) | 3 (3.5) | 0.983 | 0.775 | 0.284 |
| 10 | 431 (70) | 224 (70) | 207 (69) | 441 (74) | 322 (74) | 119 (72) | 0.163 | 0.290 | 0.692 |
| 9 | 187 (30) | 94 (30) | 91 (31) | 159 (26) | 112 (26) | 47 (28) | |||
COMT – Catechol-o-methyl-transferase; DAT – Dopamine transporter; VNTR – Variable number tandem repeats; 5-HTTLPR – Serotonin-transporter-linked polymorphic region; MAOA – Monoamine oxidase A
Significant differences in frequencies of MAOA-uVNTR polymorphism have been observed between healthy volunteers and patients with schizophrenia (P = 0.010), as well as male healthy volunteers and schizophrenic males (P = 0.010). Low activity alleles were more frequent in the schizophrenia subgroup. This difference was not observed in the female population investigated (P = 0.134). The Val158Met genotype of COMT polymorphism was found more frequent in females of the control group (P = 0.021). This difference was not observed in healthy males (P = 0.992). 9/9 genotype of DAT-VNTR polymorphism was more frequent in the healthy group than in patients with schizophrenia (P = 0.028), but no difference was found between groups when divided by gender. There was no statistically significant difference in the distribution of 5-HTTLPR polymorphism.
Results of univariate and multivariate logistic regression are presented in Table 4.
Table 4.
Logistic regression analysis results
| Variable | Univariate analysis |
Multivariate analysis |
||
|---|---|---|---|---|
| OR (95%CI) | P | OR (95%CI) | P | |
| Gender (male) | 2.38 (1.71-3.33) | <0.001 | 1.05 (0.64-1.72) | 0.849 |
| Marital status (not married) | 3.88 (2.66-5.67) | <0.001 | 2.35 (1.48-3.73) | <0.001 |
| Education (high school or lower) | 7.19 (4.78-10.83) | <0.001 | 3.93 (2.45-6.30) | <0.001 |
| Employment (unemployed) | 13.27 (8.35-21.07) | <0.001 | 8.59 (5.17-14.29) | <0.001 |
| COMT | 0.96 (0.76-1.97) | 0.699 | ||
| DAT-VNTR | 0.87 (0.68-1.12) | 0.278 | ||
| 5-HTTLPR | 0.92 (0.74-1.15) | 0.469 | ||
| MAOA-uVNTR | 1.17 (0.98-1.41) | 0.083 | 1.41 (1.08-1.84) | 0.010 |
Correct classification of schizophrenia: 80.45%. Dependent variable: Schizophrenia; Independent variables: Gender, marital status, education, employment, COMT, DAT-VNTR, 5-HTTLPR, MAOA-uVNTR. Relaxed level of significance for univariate analysis was set at 0.25. Independent variables which were statistically significant (gender, marital status, education, employment, and MAOA-uVNTR) were included in multivariate analysis. COMT – Catechol-o-methyl-transferase; DAT-VNTR – Dopamine transporter variable number tandem repeats; 5-HTTLPR – Serotonin-transporter-linked polymorphic region; MAOA-uVNTR – Monoamine oxidase A upstream variable number tandem repeats; OR – Odds ratio; CI – Confidence interval
Statistically significant variables using the univariate model were gender, marital status, education, employment, and MAOA-uVNTR polymorphism. The univariate model revealed that the male gender, single marital status, high school level (or lower) of education, and unemployment were predictors for schizophrenia. 5-HTTLPR, COMT Val158Met, and DAT-VNTR ORs were not statistically significant in the univariant model and were not included in the multivariate model. However, MAOA-uVNTR polymorphism OR (95% CI) was 1.17 (0.98–1.41) and P = 0.083, which met criteria for inclusion in the multivariate model. The multivariate logistic regression showed that marital status, education, and employment status remained statistically significant, as well as MAOA-uVNTR (OR [95% CI] = 1.41 [1.08–1.84]; P = 0.01). Using this model, we managed to correctly classify 80.45% participants.
DISCUSSION
In this study, we found that low activity allele of MAOA-uVNTR polymorphism is significantly associated with schizophrenia even after adjusting for all significant anamnestic predictors of disease (male gender, marital status, education, and employment status). COMT Val158Met showed a difference in the genotype distribution of female participants between healthy control and schizophrenia patients. DAT-VNTR polymorphism showed a difference in genotype distribution between healthy and control; however, as for 5-HTTLPR polymorphism, overall association with schizophrenia was not observed. Using the proposed regression model, 80% participants could be correctly classified as schizophrenia patients.
Variable number tandem repeats (u-VNTR) polymorphism located upstream from the coding region of the MAOA gene was described. Its sequence comprises 30 base pairs (bp) repeated in 3, 3.5, 4, or 5 copies. The most frequent alleles found in Caucasians, according to Sabol et al., are the 4-repeat (64.8%) followed by a 3-repeat allele (33.1%).[13] Alleles with 3.5 or 5 copies are quite rare, comprising only 2.1%. The investigated allele frequencies in Croatian healthy population are in agreement with these findings. Namely, the frequencies of the 4-repeat, 3-repeat, and rare alleles in the healthy population investigated were 66%, 31%, and 3%, respectively. Furthermore, Sabol et al. assessed the allele transcriptional activity and discovered that 3.5- and 4-repeats are optimal allele lengths for transcriptional activity, while lower transcriptional activity was associated with 3- and 5-repeat alleles.[12] Conversely, Deckert et al. found that 5 and 4 repeats alleles of MAOA-uVNTR showed higher transcriptional activity, suggesting that longer alleles have higher transcriptional activity.[20] Since both studies agreed on the activity of 3- and 4-repeat alleles, we decided to include only participants with 3-(low activity) and 4-repeats (high activity) in our study. We found that the low activity allele (3-repeat) of MAOA-uVNTR was more frequent in male schizophrenic patients. Since MAOA gene is located on the short arm of chromosome X (Xp11.3) only female carriers can be heterozygotes. It is indicative that the presence of one high activity allele could have a protective effect which is possible only in females. Purves-Tyson et al. measured MAOA enzyme activity in postmortem brain tissue of schizophrenic patients and discovered higher MAOA activity. Their results could be explained by higher serotonin degradation with consequently reduced dopamine inhibition through 5-HT2 receptors in the substantia nigra.[21] Furthermore, higher MAOA activity in patients with schizophrenia could be explained by the possible effect of prolonged antipsychotic treatment. MAOA-uVNTR polymorphism has previously been investigated in schizophrenia research and was found related to schizophrenia. However, data are inconsistent, and a meta-analysis showed no association of this polymorphism with schizophrenia.[22]
Schizophrenia is a complex disorder characterized by a spectrum of symptoms mostly categorized as negative positive and general. It has been reported that negative symptoms have been associated with MAO-uVNTR polymorphism.[23] Furthermore, low allele activity was associated with the development of aggressive behavior in adults who were exposed to maltreatment during childhood, suggesting a modifying role of MAOA-uVNTR on brain development.[24] Further studies are needed to clarify whether the association of MAOA-uVNTR polymorphism is associated with schizophrenia or this association is merely a reflection of certain symptoms.
Dopamine projections are followed by projections of the serotonin pathway, which starts in the dorsal raphe nuclei and follows the dopamine pathway in substantia nigra, striatal, and cortical region where it inhibits dopamine action.[25,26] The serotonin transporter is the main regulator of serotonin action, and its 5-HTTLPR polymorphism has been associated with mental disorders such as schizophrenia and depression.[27,28,29] Serotonin transporter gene SLC6A4 is located on chromosome 17q11.2. A functional polymorphism of the 5-HTTLPR region consists of a 44 bp insertion/deletion sequence. Long variant (L) is associated with 2-fold higher expression compared to short allele (S).[30] In our study, no differences were found in frequencies of 5-HTTLPR genotype between schizophrenia patients and control.
Dopamine transporter gene, i.e. SLC6A3 is located on chromosome 5p15.33 with the VNTR polymorphism located in the untranslated region. This polymorphism consists of 40 bp sequence, repeated 3–11 times. Although the distribution of alleles varies between different populations, the most frequent alleles are the 10- and 9-repeats. 10 repeat allele is the most frequent in Caucasians (72%), followed by 9-repeat allele (28%), which is associated with lower transcriptional activity.[31,32] Imaging brain studies have shown that the density of dopamine transporter is higher in the striatum, where it regulates dopamine action to the greatest extent. In contrast, its density is lower in the prefrontal cortex where dopamine action is mainly regulated by COMT. A nine-repeat allele of dopamine transporter has been associated with lower transcriptional activity and therefore lower dopamine reuptake.[33,34] In our study, we found that the 9/9 genotype was less frequent in schizophrenia patients in comparison with healthy control (4.2 vs. 8.9%). Considering the dopamine hypothesis of schizophrenia which suggests subcortical hyperdopaminergia, we expected a higher frequency of 9/9 genotype in patients with schizophrenia. Our findings can be explained by conflicting results of studies on the transcriptional activity of DAT-VNTR polymorphism. For example, analysis on postmortem brain tissue has shown higher mRNA expression of 10/10 genotype in patients with schizophrenia but the lower expression in healthy controls. In this research, mRNA expression has been considered to correlate with gene expression; however, the density of dopamine transporter was not measured so the effect of external factors or drug abuse cannot be excluded. Research conducted on the cell culture model showed a higher expression of a 10-repeat allele.[35] In contrast, single-photon emission computed tomography (SPECT) analysis has shown that 10-repeat allele expression is lower compared to the 9-repeat allele. Limitation of SPECT analysis is low resolution because radioactive labeling does not specifically bind to dopamine transporter but also binds to serotonin transporter.[36,37] Our study suggests an association of DAT-VNTR polymorphism with schizophrenia; however, the exact mechanism of action and the possible effect of other regulatory elements should be investigated.
Another regulator of dopamine concentration is COMT which has been associated with schizophrenia.[7] This enzyme is present in two isoforms, longer, membrane-bound (MB) and shorter, soluble, S-isoform. COMT gene is located on chromosome 22q11.21. G > A substitution at position 158 (Val158Met) (rs4680) results with reduced activity of MB COMT isoform.[38,39] In our study, we found a statistically significant difference in COMT Val158Met polymorphism frequency between healthy and schizophrenic patients, but only in female participants. Dopamine function in the prefrontal cortex in association with COMT Val158Met seems to depend also on external factors. It has been investigated that optimal prefrontal cortex function is associated with a narrow dopamine level. Although COMT is important for regulation of dopamine level, the effect of Val158Met polymorphism is more complicated than high or low enzymatic activity. It has been suggested that a combination of environment and genetic factors like COMT Val158Met variant can contribute dopamine level.[7] Although promising, association studies of COMT Val158Met polymorphism and schizophrenia are inconsistent possibly due to the complex effect of COMT Val158Met polymorphism on dopamine levels. In our study, the association of COMT Val158Met polymorphism in a female population could be explained by molecular heterosis due to the relatively high frequency of heterozygous carriers.[40]
Distribution of gender in our study differed significantly between the schizophrenia group and healthy control which might affect our findings. Therefore, we performed logistic regression analysis to adjust for the influence of different variables on schizophrenia as an outcome. Participants who were not married, had a lower level of education, and were unemployed, had significantly higher odds to develop schizophrenia. It should be noted that our study is case–control by its design, and our results present participants in one moment of their lives. Abovementioned education level, marriage status, and unemployment are data collected at the time of participant recruitment and should be considered as indicators of poor cognitive abilities and poor establishing of interpersonal relationships and social functioning.
Limitation of our study is that we did not asses environment factors such as traumatic life events which might contribute to schizophrenia development.
In contrast to a traditional concept where one genetic variant is considered beneficial compared to another, the concept of genetic plasticity seems to be a more appropriate approach. In general, gene association studies are trying to find a direct association between certain genes and disease (in this case, schizophrenia). The direct association is difficult to find because psychiatric disorders depend also on environmental factors and some genes might be beneficial in combination to certain environmental factors.[41]
CONCLUSION
According to our study, MAOA-uVNTR seems to be a significant predictor of schizophrenia in combination with marriage status, education, and employment. Our findings support the complex and multifactorial nature of schizophrenia.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
- 1.Kahn RS, Sommer IE, Murray RM, Meyer-Lindenberg A, Weinberger DR, Cannon TD, et al. Schizophrenia. Nat Rev Dis Primers. 2015;1:15067. doi: 10.1038/nrdp.2015.67. [DOI] [PubMed] [Google Scholar]
- 2.Howes OD, Kapur S. The dopamine hypothesis of schizophrenia: Version III – The final common pathway. Schizophr Bull. 2009;35:549–62. doi: 10.1093/schbul/sbp006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Le Moal M, Simon H. Mesocorticolimbic dopaminergic network: Functional and regulatory roles. Physiol Rev. 1991;71:155–234. doi: 10.1152/physrev.1991.71.1.155. [DOI] [PubMed] [Google Scholar]
- 4.Jacobs BL, Azmitia EC. Structure and function of the brain serotonin system. Physiol Rev. 1992;72:165–229. doi: 10.1152/physrev.1992.72.1.165. [DOI] [PubMed] [Google Scholar]
- 5.Yang B, Chan RC, Jing J, Li T, Sham P, Chen RY, et al. Ameta-analysis of association studies between the 10-repeat allele of a VNTR polymorphism in the 3'-UTR of dopamine transporter gene and attention deficit hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2007;144B:541–50. doi: 10.1002/ajmg.b.30453. [DOI] [PubMed] [Google Scholar]
- 6.Lee CS, Samii A, Sossi V, Ruth TJ, Schulzer M, Holden JE, et al. In vivo positron emission tomographic evidence for compensatory changes in presynaptic dopaminergic nerve terminals in Parkinson's disease. Ann Neurol. 2000;47:493–503. [PubMed] [Google Scholar]
- 7.Tunbridge EM, Harrison PJ, Weinberger DR. Catechol-o-methyltransferase, cognition, and psychosis: Val158Met and beyond. Biol Psychiatry. 2006;60:141–51. doi: 10.1016/j.biopsych.2005.10.024. [DOI] [PubMed] [Google Scholar]
- 8.Williams HJ, Owen MJ, O'Donovan MC. Is COMT a susceptibility gene for schizophrenia? Schizophr Bull. 2007;33:635–41. doi: 10.1093/schbul/sbm019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kobiella A, Reimold M, Ulshöfer DE, Ikonomidou VN, Vollmert C, Vollstädt-Klein S, et al. How the serotonin transporter 5-HTTLPR polymorphism influences amygdala function: The roles of in vivo serotonin transporter expression and amygdala structure. Transl Psychiatry. 2011;1:e37. doi: 10.1038/tp.2011.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Culej J, Štefanović M, Ćelap I, Nikolac N, Karlović D. Serotonin transporter polymorphism (5-HTTLPR) in croatian population. Mol Biol Rep. 2015;42:553–8. doi: 10.1007/s11033-014-3800-7. [DOI] [PubMed] [Google Scholar]
- 11.Domínguez-López S, Howell R, Gobbi G. Characterization of serotonin neurotransmission in knockout mice: Implications for major depression. Rev Neurosci. 2012;23:429–43. doi: 10.1515/revneuro-2012-0044. [DOI] [PubMed] [Google Scholar]
- 12.Hariri AR, Mattay VS, Tessitore A, Kolachana B, Fera F, Goldman D, et al. Serotonin transporter genetic variation and the response of the human amygdala. Science. 2002;297:400–3. doi: 10.1126/science.1071829. [DOI] [PubMed] [Google Scholar]
- 13.Sabol SZ, Hu S, Hamer D. A functional polymorphism in the monoamine oxidase A gene promoter. Hum Genet. 1998;103:273–9. doi: 10.1007/s004390050816. [DOI] [PubMed] [Google Scholar]
- 14.Brunner HG, Nelen M, Breakefield XO, Ropers HH, van Oost BA. Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A. Science. 1993;262:578–80. doi: 10.1126/science.8211186. [DOI] [PubMed] [Google Scholar]
- 15.Schlüter T, Winz O, Henkel K, Eggermann T, Mohammadkhani-Shali S, Dietrich C, et al. MAOA-VNTR polymorphism modulates context-dependent dopamine release and aggressive behavior in males. Neuroimage. 2016;125:378–85. doi: 10.1016/j.neuroimage.2015.10.031. [DOI] [PubMed] [Google Scholar]
- 16.Gordon D, Finch SJ, Nothnagel M, Ott J. Power and sample size calculations for case-control genetic association tests when errors are present: Application to single nucleotide polymorphisms. Hum Hered. 2002;54:22–33. doi: 10.1159/000066696. [DOI] [PubMed] [Google Scholar]
- 17.Gordon D, Levenstien MA, Finch SJ, Ott J. Errors and linkage disequilibrium interact multiplicatively when computing sample sizes for genetic case-control association studies. Pac Symp Biocomput. 2003;8:490–501. doi: 10.1142/9789812776303_0046. [DOI] [PubMed] [Google Scholar]
- 18.Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, et al. The mini-international neuropsychiatric interview (M.I.N.I.): The development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry. 1998;59(Suppl 20):22–33. [PubMed] [Google Scholar]
- 19.Sperandei S. Understanding logistic regression analysis. Biochem Med (Zagreb) 2014;24:12–8. doi: 10.11613/BM.2014.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Deckert J, Catalano M, Syagailo YV, Bosi M, Okladnova O, Di Bella D, et al. Excess of high activity monoamine oxidase A gene promoter alleles in female patients with panic disorder. Hum Mol Genet. 1999;8:621–4. doi: 10.1093/hmg/8.4.621. [DOI] [PubMed] [Google Scholar]
- 21.Purves-Tyson TD, Owens SJ, Rothmond DA, Halliday GM, Double KL, Stevens J, et al. Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain. Transl Psychiatry. 2017;7:e1003. doi: 10.1038/tp.2016.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Li D, He L. Meta-study on association between the monoamine oxidase A gene (MAOA) and schizophrenia. Am J Med Genet B Neuropsychiatr Genet. 2008;147B:174–8. doi: 10.1002/ajmg.b.30570. [DOI] [PubMed] [Google Scholar]
- 23.Camarena B, Fresán A, Aguilar A, Escamilla R, Saracco R, Palacios J, et al. Monoamine oxidase a and B gene polymorphisms and negative and positive symptoms in schizophrenia. ISRN Psychiatry. 2012;2012:852949. doi: 10.5402/2012/852949. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Caspi A, McClay J, Moffitt TE, Mill J, Martin J, Craig IW, et al. Role of genotype in the cycle of violence in maltreated children. Science. 2002;297:851–4. doi: 10.1126/science.1072290. [DOI] [PubMed] [Google Scholar]
- 25.Nedergaard S, Bolam JP, Greenfield SA. Facilitation of a dendritic calcium conductance by 5-hydroxytryptamine in the substantia nigra. Nature. 1988;333:174–7. doi: 10.1038/333174a0. [DOI] [PubMed] [Google Scholar]
- 26.Williams J, Davies JA. The involvement of 5-hydroxytryptamine in the release of dendritic dopamine from slices of rat substantia nigra. J Pharm Pharmacol. 1983;35:734–7. doi: 10.1111/j.2042-7158.1983.tb02880.x. [DOI] [PubMed] [Google Scholar]
- 27.Hu XZ, Lipsky RH, Zhu G, Akhtar LA, Taubman J, Greenberg BD, et al. Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder. Am J Hum Genet. 2006;78:815–26. doi: 10.1086/503850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Kaiser R, Tremblay PB, Schmider J, Henneken M, Dettling M, Müller-Oerlinghausen B, et al. Serotonin transporter polymorphisms: No association with response to antipsychotic treatment, but associations with the schizoparanoid and residual subtypes of schizophrenia. Mol Psychiatry. 2001;6:179–85. doi: 10.1038/sj.mp.4000821. [DOI] [PubMed] [Google Scholar]
- 29.Kenna GA, Roder-Hanna N, Leggio L, Zywiak WH, Clifford J, Edwards S, et al. Association of the 5-HTT gene-linked promoter region (5-HTTLPR) polymorphism with psychiatric disorders: Review of psychopathology and pharmacotherapy. Pharmgenomics Pers Med. 2012;5:19–35. doi: 10.2147/PGPM.S23462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Heils A, Teufel A, Petri S, Stöber G, Riederer P, Bengel D, et al. Allelic variation of human serotonin transporter gene expression. J Neurochem. 1996;66:2621–4. doi: 10.1046/j.1471-4159.1996.66062621.x. [DOI] [PubMed] [Google Scholar]
- 31.Prata DP, Mechelli A, Fu CH, Picchioni M, Toulopoulou T, Bramon E, et al. Epistasis between the DAT 3' UTR VNTR and the COMT val158Met SNP on cortical function in healthy subjects and patients with schizophrenia. Proc Natl Acad Sci U S A. 2009;106:13600–5. doi: 10.1073/pnas.0903007106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Kang AM, Palmatier MA, Kidd KK. Global variation of a 40-bp VNTR in the 3'-untranslated region of the dopamine transporter gene (SLC6A3) Biol Psychiatry. 1999;46:151–60. doi: 10.1016/s0006-3223(99)00101-8. [DOI] [PubMed] [Google Scholar]
- 33.Mill J, Asherson P, Browes C, D'Souza U, Craig I. Expression of the dopamine transporter gene is regulated by the 3' UTR VNTR: Evidence from brain and lymphocytes using quantitative RT-PCR. Am J Med Genet. 2002;114:975–9. doi: 10.1002/ajmg.b.10948. [DOI] [PubMed] [Google Scholar]
- 34.Heinz A, Goldman D, Jones DW, Palmour R, Hommer D, Gorey JG, et al. Genotype influences in vivo dopamine transporter availability in human striatum. Neuropsychopharmacology. 2000;22:133–9. doi: 10.1016/S0893-133X(99)00099-8. [DOI] [PubMed] [Google Scholar]
- 35.Fuke S, Suo S, Takahashi N, Koike H, Sasagawa N, Ishiura S, et al. The VNTR polymorphism of the human dopamine transporter (DAT1) gene affects gene expression. Pharmacogenomics J. 2001;1:152–6. doi: 10.1038/sj.tpj.6500026. [DOI] [PubMed] [Google Scholar]
- 36.Jacobsen LK, Staley JK, Zoghbi SS, Seibyl JP, Kosten TR, Innis RB, et al. Prediction of dopamine transporter binding availability by genotype: A preliminary report. Am J Psychiatry. 2000;157:1700–3. doi: 10.1176/appi.ajp.157.10.1700. [DOI] [PubMed] [Google Scholar]
- 37.van Dyck CH, Malison RT, Jacobsen LK, Seibyl JP, Staley JK, Laruelle M, et al. Increased dopamine transporter availability associated with the 9-repeat allele of the SLC6A3 gene. J Nucl Med. 2005;46:745–51. [PubMed] [Google Scholar]
- 38.Chen J, Lipska BK, Halim N, Ma QD, Matsumoto M, Melhem S, et al. Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): Effects on mRNA, protein, and enzyme activity in postmortem human brain. Am J Hum Genet. 2004;75:807–21. doi: 10.1086/425589. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Sagud M, Mück-Seler D, Mihaljević-Peles A, Vuksan-Cusa B, Zivković M, Jakovljević M, et al. Catechol-O-methyl transferase and schizophrenia. Psychiatr Danub. 2010;22:270–4. [PubMed] [Google Scholar]
- 40.Comings DE, MacMurray JP. Molecular heterosis: A review. Mol Genet Metab. 2000;71:19–31. doi: 10.1006/mgme.2000.3015. [DOI] [PubMed] [Google Scholar]
- 41.Belsky J, Jonassaint C, Pluess M, Stanton M, Brummett B, Williams R, et al. Vulnerability genes or plasticity genes? Mol Psychiatry. 2009;14:746–54. doi: 10.1038/mp.2009.44. [DOI] [PMC free article] [PubMed] [Google Scholar]