Department of Psychiatry, Osaka University Graduate School of Medicine, Osaka, Japan
Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Osaka, Japan
Correspondence: Ryota Hashimoto, MD, PhD, Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, D3, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. Email: firstname.lastname@example.org
Progressive cognitive decline has been an important issue in the treatment and care of patients with schizophrenia. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of catecholamine, including dopamine and noradrenaline. In this report, we examined a possible association of a genetic variant in the TH promoter region.
Association of a genetic variant in the TH promoter region, C-824T (rs10770141), with intellectual ability in 132 patients with schizophrenia and 282 healthy subjects was examined. The transcriptional activity of the plasmids harboring the TH promoter region with either C or T nucleotide at −824 was assayed using a luciferase gene as a reporter.
We found significant effects of the genotype on the full-scale IQ, verbal IQ, and performance IQ, in patients with schizophrenia. IQ was lower in individuals with the C/C genotype than those with T carriers. The plasmid with the T allele at −824 showed higher transcriptional activity than that with the C allele in a transient transfection experiment using a luciferase gene as a reporter, implying that the T carriers may have higher TH activities and retain higher levels of catecholamines in the brain.
The present data suggest that the biosynthesis of catecholamine by the action of TH should be deeply involved in decreased intellectual ability in patients with schizophrenia. This is the first report, as far as we know, showing a correlation between TH expression and IQ in humans.
Schizophrenia is a common and complex psychiatric disorder with strong genetic components. The neuropsychological profile of schizophrenia is typically characterized by a generalized cognitive deficit, including prominent, specific deficits in memory and learning, executive functions, attention, and processing speed.[1-3] There is increasing evidence indicating that cognitive dysfunction represents an independent symptom dimension associated with functional impairment and reduced quality of life in patients with schizophrenia.
Tyrosine hydroxylase (TH) is a rate-limiting enzyme for biosynthesis of dopamine and noradrenaline. Alteration in the TH expression can affect the levels of dopamine and noradrenaline in the brain, and the dopaminergic neurons are a primary target for treatment of schizophrenia. To date, many studies have investigated possible involvement of TH in the cause of schizophrenia, whereas most of the association studies failed to find significant genetic association between the TH gene and schizophrenia.[5-7]
Rao et al. investigated common human genetic variants governing transcription of the TH gene. They found a C-824T (rs10770141) variant in the proximal promoter of the TH gene associated with urinary catecholamine excretion and with blood pressure response to stress. The paper prompted us to explore a possible association of the TH variant with neuropsychiatric disorders and higher-order brain functions. IQ is a standardized measure of human intellectual capacity that takes into account a wide range of cognitive skills. The intellectual ability of patients with schizophrenia is lower than healthy subjects. Intellectual dysfunction in unaffected relatives of schizophrenia patients is very similar to but somewhat less pronounced than that in patients with schizophrenia. The estimated heritability of IQ is high in the general population (69–85%) and individuals with familial schizophrenia (64–74%).[11, 12] These findings suggest that intellectual dysfunction is a promising intermediate phenotype for schizophrenia.
In the present study, we examined the relevance of the C-824T variant of the TH gene on intellectual ability shown to be associated with genetic liability in schizophrenia. We found that T carriers of the C-824T variant showed higher IQ than individuals with C/C genotype. We also examined the effect of the C-824T polymorphism in the promoter region on the transcriptional activity of the TH gene using a reporter assay, and showed that the transcriptional activity of the T allele was higher than that of the C allele. Our data suggest that a catecholamine-biosynthesizing ability mediated by TH should be one of the factors influencing intellectual function in patients with schizophrenia.
The present study was conducted with 132 patients with schizophrenia (56.1% men [74 men and 58 women]; mean age ± SD, 37.2 ± 12.2 years) and 282 healthy subjects (41.8% men [118 men and 164 women]; mean age ± SD, 37.1 ± 12.2 years). All subjects were biologically unrelated within the second-degree of relationship and were of Japanese descent.[13, 14] Subjects were excluded if they had neurological or medical conditions that could potentially affect the central nervous system, such as atypical headache, previous head trauma with loss of consciousness, chronic lung disease, kidney disease, chronic hepatic disease, thyroid disease, active cancer, cerebrovascular disease, epilepsy, seizures, substance-related disorders or mental retardation. Cases were recruited from the Osaka University Hospital. Each patient with schizophrenia had been diagnosed by at least two trained psychiatrists according to the criteria of the DSM-IV based on the Structured Clinical Interview for DSM-IV (SCID). Controls were recruited through local advertisements at Osaka University. Psychiatrically, medically and neurologically healthy controls were evaluated using the non-patient version of the SCID to exclude individuals who had current or past contact with psychiatric services or received psychiatric medication. The mean age did not differ significantly between cases and controls (P = 0.90), while the female ratio, years of education and estimated premorbid IQ were significantly lower in the patients with schizophrenia than the controls (P < 0.05) (Table S1). Written informed consent was obtained from all subjects after the procedures had been fully explained. This study was conducted in accordance with the World Medical Association's Declaration of Helsinki and approved by the Research Ethics Committees of Osaka University and of Tokyo Institute of Technology.
SNP selection and SNP genotyping
We selected rs10770141 (C-824T) in the TH gene because this polymorphism has been associated with urinary catecholamine excretion and with blood pressure response to cold stress, as described in the Introduction. Venous blood was collected from the subjects, and genomic DNA was extracted from whole blood according to standard procedures. The polymorphism was genotyped using the TaqMan 5′-exonuclease allelic discrimination assay (Assay ID: C_31456586_10, Applied Biosystems, Foster City, CA, USA) as previously described.[15, 16] Detailed information on the PCR conditions is available upon request. No deviation from the Hardy–Weinberg equilibrium (HWE) was detected in the examined SNP in the patients or controls (P > 0.74) (patients with schizophrenia: C/C: 119; C/T: 13; T/T: 0; healthy subjects: C/C: 242; C/T: 39; T/T: 1). Because of the small number of subjects homozygous for the T allele, the T/T and C/T genotype groups were combined and treated as the T carriers for this study.
Measurement of intellectual abilities and assessment of current symptoms of schizophrenia
To assess intellectual abilities, we used the full-scale IQ, which is divided into performance IQ and verbal IQ, of the Japanese version of the Wechsler Adult Intelligence Scale-third edition (WAIS-III). The subjects were assessed by trained clinical psychologists to obtain full-scale, performance and verbal IQ scores on the WAIS-III. Current symptoms of schizophrenia were evaluated using the Positive and Negative Syndrome Scale (PANSS).
Based on the assumption that most of the demographic variables, such as age and education years, were not fitted to a normality distribution with the Kolmogorov–Smirnov test (P < 0.05), differences in clinical characteristics between patients and controls or between genotypes were analyzed using the non-parametric Mann–Whitney U-test for continuous variables and χ2-tests for categorical variables using pasw Statistics 18.0 (spss, Tokyo, Japan). Deviation from the HWE was tested separately in test cases and controls using χ2-tests for goodness of fit using SNPAlyze V5.1.1 Pro (dynacom, Yokohama, Japan).
The effects of diagnosis, TH genotype and their interaction on intellectual abilities were analyzed by two-way analyses of covariance (ancova). Diagnosis and genotype status were included in the analysis as independent variables. Full-scale, performance and verbal IQ scores were included as dependent variables. As intellectual abilities may be influenced by sex and years of education, these variables were corrected for as covariates. We did not include age as a covariate because IQ scores were already corrected for age. The Bonferroni correction was applied for multiple testing on two indices (verbal and performance IQ) of the WAIS-III to avoid type I error. The data of transient transfection experiments were analyzed by unpaired Student's t-tests. The significance level for all statistical tests was set at two-tailed P < 0.05.
Dual-luciferase assay of TH promoter variants
Approximately 2.5 kb of TH promoter region containing exon 1 and a part of intron 1 (-2169/+358; Base pair positions are numbered with respect to the ATG initiation codon) was amplified from human genomic DNA that had homozygous C allele of the C-824T polymorphism. Then the fragment was cloned into firefly luciferase vector pGVB2 (TOYOBO, Osaka, Japan). To construct a reporter plasmid containing −824T allele, a mutation was introduced to the amplified fragment at the position −824 using KOD-Plus Mutagenesis Kit (TOYOBO) and the mutant was then cloned into pGV-B2. Sequence analysis was performed to verify the nucleotide sequence other than C-824T between these two reporter plasmids.
Transient transfection to rat pheochromocytoma cell line, PC12, was performed using Lipofectamine 2000 (Invitrogen). Cells were plated into 24-well plates at approximately 80% confluence 1 day before transfection. PC12 cells in each well were transfected with 1.6 μg firefly or plasmid and 1.5 ng Renilla luciferase expression plasmid with a thymidine kinase promoter pRL-TK (Promega, Madison, WI, USA) as an internal control. Luciferase activities were measured 24 h after transfection using the Dual luciferase reporter assay system (Promega). For nerve growth factor (NGF) or forskolin (FSK) treatment, 10 μM NGF or FSK was added to the medium 6 h prior to the measurements.
Impact of genetic variants of the TH gene on intellectual abilities
When the two genotypes of rs10770141 (T carriers and C allele homozygotes) were compared, we found no differences in demographic variables (Table S1). First, we investigated the effects of diagnosis, TH genotype and their interaction on full-scale IQ (Table 1). We found significant effects of diagnosis (F1,408 = 56.8, P = 3.14 × 10−13, corrected P = 6.28 × 10−13), genotype of rs10770141 (F1,408 = 5.8, P = 0.017, corrected P = 0.034) and their interaction (F1,408 = 6.4, P = 0.012, corrected P = 0.024) on full-scale IQ. As expected, patients with schizophrenia showed significantly lower IQ than healthy subjects in all analyses of the present study. Because we found a significant diagnosis-genotype interaction on full-scale IQ, we separately examined the effect of genotype on full-scale IQ in patients with schizophrenia and healthy subjects. There was a significant effect of the genotype on full-scale IQ in patients with schizophrenia (F1,128 = 5.3, P = 0.023, corrected P = 0.046), whereas there was no significant effect in healthy subjects (F1,278 = 0.04, P = 0.85). Full-scale IQ was lower in patients with the C/C genotype of rs10770141 than those with the T/C genotype.
Table 1. Impact of genetic variant in the TH gene on intellectual function
Schizophrenia (n = 132)
Control (n = 282)
T/C (n = 13)
C/C (n = 119)
T carrier (n = 40)
C/C (n = 242)
*P < 0.05.
Means ± SD are shown. To control for confounding factors, the effects of diagnosis, TH genotype and their interaction on IQ were analyzed by two-way ancova, with sex and years of education as covariates.
Second, we investigated the effects of diagnosis, genotype and their interaction on verbal and performance IQ, respectively (Table 1). We found significant effects of diagnosis (F1,408 = 24.4, P = 1.14 × 10−6, corrected P = 2.28 × 10−6) and diagnosis-genotype interaction (F1,408 = 7.4, P = 0.0067, corrected P = 0.013) on verbal IQ and effects of diagnosis (F1,408 = 79.4, P = 1.67 × 10−17, corrected P = 3.34 × 10−17) and genotype (F1,408 = 7.4, P = 0.0068, corrected P = 0.014) on performance IQ. There was no significant effect of genotype on verbal IQ or diagnosis–genotype interaction on performance IQ (P > 0.05). When we separately examined the effect of the genotype on verbal IQ in patients with schizophrenia and healthy subjects, there was a marginally significant effect of the genotype on verbal IQ only in patients with schizophrenia (F1,128 = =5.0, P = 0.027, corrected P = 0.054), but not in healthy subjects (F1,278 = 1.3, P = 0.26). Verbal IQ was lower only in patients with the C/C genotype than those with the T/C genotype. On the other hand, performance IQ was lower in patients and controls with the C/C genotype than those with T carriers.
To find whether there was potential clinical impact on our outcomes, we additionally investigated the genotype effect on IQ, including chlorpromazine equivalent of total antipsychotics (mg/day) as covariate for the ancova. The genotype effect on full-scale and verbal IQ in patients with schizophrenia did not change even after controlling for the factor (P < 0.05), suggesting that the genotype may influence the full-scale and verbal IQ in patients with schizophrenia, despite potential impact of antipsychotic drugs on our outcomes.
Effect of the TH variant C-824T on the transcriptional activity
To examine whether the C-824T variant can affect the transcriptional activity of the TH gene, dual-luciferase reporter assay was performed using PC12 cells, which is derived from rat pheochromocytoma. We transfected the luciferase reporter plasmid harboring either C or T allele at −824. The plasmid possessing T allele at −824 was more active than one possessing C allele (Fig. 1, left two columns; each column n = 3, t = −17.48, d.f. = 4, P < 0.0001). The greater activities in the TH promoter with T than C allele at −824 were also observed when the cells were stimulated by NGF (Fig. 1, middle two columns; each column n = 3, t = −8.90, d.f. = 4, P = 0.0009) or FSK (Fig. 1, right two columns; each column n = 3, t = −15.7, d.f. = 4, P < 0.0001). Both NGF and FSK are known to increase the expression of TH in PC12 cells. Whereas activities of both promoters were increased by the treatments, the ratios of luciferase activity of T to C allele were not altered compared to those of cells with no stimulation. This suggests that the intracellular signals mediated by NGF and FSK to induce TH expression may not contribute to the effect of C-824T polymorphism.
In the present study, we found that T-carriers of the C-824T polymorphism in the TH gene showed higher IQ levels in patients with schizophrenia. Because the reporter plasmid with the T allele showed higher reporter activities, the T-carriers may have higher TH activities and increased levels of noradrenaline. Indeed, urinary noradrenaline excretion was reported to be higher in individuals with the T allele. Because the noradrenaline system was implicated in attention and arousal in both rodents and primates,[19, 20] it would be plausible that altered noradrenaline levels in the brain might affect IQ in humans.
While intellectual and neurocognitive deficits are characteristic features of schizophrenia, the degree of cognitive decline varies among the patients. To understand the genetic basis of cognitive decline in schizophrenia, a genome-wide association study (GWAS) was recently performed. The strongest association was observed at rs7157599 on chromosome 14, a missense polymorphism (Asn8Ser) in the delta4 desaturase gene (DEGS2) (P = 5.4 × 10−7). The most significant 10 markers include the SNP in the CUB and Sushi multiple domains-1 (CSMD1) gene, which has been identified as a new risk gene for schizophrenia by GWAS, the SNP in the carboxypeptidase X gene on 10q26.13, and the SNP near the bone morphogenetic protein 3. We further investigated the effects of the C-824T polymorphism in the TH gene on cognitive decline in patients participating in the present study. In the patients, there was no significant effect of the polymorphism on cognitive decline (P = 0.073). Additionally, no significant association with other polymorphisms near the TH gene was observed in the GWAS study, suggesting that the cognitive decline in patients with schizophrenia may be affected by distinct factors from intellectual function.
The C-824T polymorphism resides in the 5′-promoter region of the TH gene. Because TH is the rate-limiting enzyme for the biosynthesis of catecholamines, including dopamine and noradrenaline, the transcriptional activity of the TH gene is tightly regulated. Rao et al. found that common allelic variants, including C-824T and A-581G within the TH promoter region, exerted a heritable effect on autonomic control of the circulation, and changes in catecholamine secretion and the blood pressure response to environmental stress. Zhang et al. identified the transcriptional factors binding to the region, including the position −824 of the TH promoter. They clarified that variation at C-824T in the TH promoter altered the binding of some transcriptional factors, such as myocyte enhancer factor-2 (MEF2), sex-determining region Y (SRY), and forkhead box D1 (FOXD1).
Because the close correlation between age-related cognitive decline and the decrease in catecholamines and their receptors is well documented, the polymorphisms and SNP in genes relating to catecholamine metabolism and the receptors are candidates for genetic basis of individual difference in cognitive function. Several studies showed association of IQ with a polymorphism of the brain-derived neurotrophic factor gene, and weak association with a polymorphism of the catechol-O-methyltransferase gene. However, there was no report on implication of the TH gene with IQ. In this study, we for the first time found an association of C-824T SNP in the TH promoter region with full-scale IQ, verbal IQ, and performance IQ in patients with schizophrenia. Several lines of evidence suggest that the noradrenergic system has marked influence on the prefrontal cortex and on cognitive functioning in patients with schizophrenia. Therefore, the higher TH activity and the resulting increased level of noradrenaline may protect cognitive decline of the T-carriers of C-824T in patients with schizophrenia.
The genetic association between the TH polymorphism and IQ was observed only in patients with schizophrenia, but not in healthy individuals. Recent imaging studies showed that neuronal activities of prefrontal cortex were deteriorated in the brains of patients with schizophrenia, and the decreased neuronal activity in prefrontal cortex would be involved in neurocognitive deficits in schizophrenia. It would be plausible that the increases in the catecholamine content might be more effective to stimulate the deteriorated neuronal activities of prefrontal cortex in patients with schizophrenia compared with healthy individuals. Collectively, our data would support the idea that enhancement of the noradrenergic neuronal activity using a noradrenaline-specific reuptake inhibitor may have therapeutic effects on the neurocognitive deficits in patients with schizophrenia.
There are several limitations to interpreting our results. It is difficult to select healthy controls for IQ. For example, the controls were responders to local advertisements, but the patients were recruited as part of routine clinical diagnosis and treatment. Our significant results may be derived from sample selection bias. There were no common SNP with strong effects on IQ; however, multiple genetic variants with small effects influence IQ. A false positive association could not be excluded from our study because our results were based on a relatively small number of 13 patients carrying a T allele and 119 patients with C/C genotype. A future replication study using larger sample sizes is needed to confirm our findings. It is difficult to determine whether our results are directly linked to the TH polymorphism, to other polymorphisms in linkage disequilibrium with this variant, or to interaction between this genetic variant and other polymorphisms. An extensive search for other functional variants at this locus is needed to determine whether the C-824T polymorphism is the most strongly associated variant for IQ and TH gene expression.
In conclusion, we found that a functional genetic variant (C-824T) in the TH promoter region was associated with intellectual ability in patients with schizophrenia. The intellectual ability of the T carriers of the TH gene was higher in comparison with that of the C/C genotype. The plasmid with the T allele at −824 showed higher transcriptional activity than that with the C allele, implying that the T carriers may have higher TH activities and retain higher levels of catecholamines in the brain. These results may contribute to our understanding of the pathophysiology of schizophrenia.
We would like to thank all of the individuals who participated in this study. This work was supported by research grants from the Japanese Ministry of Health, Labor and Welfare (H22-seishin-ippan-001); KAKENHI, 22390225-Grant-in-Aid for Scientific Research (B), 23659565-Grant-in-Aid for Challenging Exploratory Research and Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); CREST of JST; and the Japan Foundation for Neuroscience and Mental Health. All authors declare that they have no conflict of interest.