To examine the cannabinoid hypothesis for pathogenesis of schizophrenia, we examined two kinds of polymorphisms of the CNR1 gene, which encodes human CB1 receptor, a subclass of central cannabinoid receptors, in schizophrenics and age-matched controls in the Japanese population. Allelic and genotypic distributions of polymorphism 1359G/A at codon 453 in the coding region and AAT triplet repeats in the 3′ flanking region in the Japanese population were quite different from those in Caucasians. Although the polymorphism 1359G/A was not associated with schizophrenia, the triplet repeat polymorphism of the CNR1 gene was significantly associated with schizophrenia, especially the hebephrenic subtype (P = 0.0028). Hebephrenic schizophrenia showed significantly increased rate of the 9 repeat allele (P = 0.032, OR = 2.30, 95% CI (1.91–2.69)), and decreased rate of the 17 repeat allele (P = 0.011, OR = 0.208, 95% CI (0.098–0.439)). The present findings indicated that certain alleles or genotypes of the CNR1 gene may confer a susceptibility of schizophrenia, especially of the hebephrenic type.
The cannabinoid hypothesis for the pathogenesis of schizophrenia is supposed to be based on the clinical facts that abuse of cannabis could precipitate the psychotic state, with hallucinations and delusions resembling schizophrenia1,2,3,4 and worsen positive symptoms of schizophrenia,5,6 even under regular medication of antipsychotics.7 Cannabinoid consumption could result in poor outcome and liability to relapse for schizophrenics.8,9,10 In addition, heavy cannabis users may develop an amotivational syndrome, reminiscent of some of the negative symptoms of schizophrenia.11 A Swedish cohort study showed that cannabis use before 18 years of age raises the incidence rate of schizophrenia six-fold.12 Another study showed that administration of delta-9-hydrocannabinol to normal volunteers induced cognitive impairment of three dimensions resembling closely that of schizophrenic patients.13 The hallucinogenic action of cannabis and marijuana mediated the central cannabinoid receptor, G protein-coupled receptor CB1, which was discovered in 1988.14 CB1 receptors were expressed abundantly throughout the brain, especially in substantia nigra, globus pallidus, hippocampus and cerebellum.15,16 CB1 receptors are encoded by the CNR1 gene (MIM114610), which was cloned by Matsuda et al in 1992.17 CB1 is located at 6q14–q15, which was included in a schizophrenia susceptibility locus, 6q13–q26, revealed by Cao et al18 using two independent series of pedigrees, which was designated by Schizophrenia 5 (SCZ5, OMIM 603175). Recently, two polymorphisms, AAT repeats microsatellite in the 3′ flanking region and 1359 G/A polymorphism at codon 453 in the coding exon of the CNR1 gene, were reported.19,20 To examine the cannabinoid hypothesis for schizophrenia, we examined these two polymorphisms in the CNR1 gene of schizophrenia in the Japanese population.
Genotypic distribution and allelic frequency of 1359 A/G and AAT repeat polymorphisms are summarized in Tables 1 and 2, respectively. Distributions of the alleles of the two kinds of polymorphisms of the CNR1 gene in both groups were within the values expected from Hardy–Weinberg equilibrium. GG, GA and AA of the 1359 G/A polymorphism genotype in controls were 94.2%, 4.4% and 1.5%, respectively. The genotypic distributions were not significantly different between controls and schizophrenia (G = 0.69, df = 1, P = 0.41), or among controls, hebephrenic and paranoid type schizophrenia (G = 2.39, df = 2, P = 0.30). The allelic frequency of the G allele and the A allele in controls was 96.4% and 3.7%, respectively. The allelic frequencies were not significantly different between controls and schizophrenia (G = 0.01, df = 1, P = 0.90), or among controls, hebephrenic and paranoid type schizophrenia (G = 1.70, df = 2, P = 0.43).
Allelic frequencies of AAT repeats of the CNR1 gene were shown in Table 2. Nine kinds of allele, (AAT)9, (AAT)10, (AAT)12–(AAT)18 repeat alleles were found. The most frequent allele of Japanese controls was (AAT)15 allele (34.8%), followed by (AAT)16 allele (28.7%), (AAT)14 allele (16.9%) and (AAT)17 allele (7.1 %). Alleles of (AAT)9 and (AAT)12 repeat were relatively rare and those of (AAT)10 and (AAT)18 repeat were few. The allelic distributions were significantly different between controls and schizophrenia (z = 1.995, P = 0.046). As to subcategories of schizophrenia, hebephrenic type, but not paranoid type schizophrenia were significantly different from controls (among the three groups, H = 10.17, P = 0.006; hebephrenic vs controls, z = 2.99, P = 0.0028; paranoid vs controls, z = 0.24, P = 0.81). Schizophrenia in all and hebephrenic type schizophrenia also showed a significantly increased rate of (AAT)10 allele (schizophrenia in all, G = 4.58, df = 1, P = 0.032; hebephrenia, G = 4.39, df = 1, P = 0.036), and significantly decreased rate of (AAT)18 allele (schizophrenia in all, G = 3.85, df = 1, P = 0.049, hebephrenia, G = 6.41, df = 1, P = 0.011). The odds ratios of hebephrenia for the (AAT)10 allele and the (AAT)18 allele were 2.30 (95% CI (1.91–2.69)) and 0.208 (95% CI (0.098–0.439)), respectively.
In the present study, we found that genotypic and allelic distributions of 1359A/G and AAT repeat polymorphism of the CNR1 gene in Japanese controls were quite different from those in the Caucasian population. Thus, Gadzicki et al19 reported that G and A allele frequencies of 1359G/A polymorphism were 76% and 24%, respectively in the German Caucasian population. Heterozygosities of this polymorphism in the Japanese and Caucasian population were 0.069 and 0.365. As to the AAT repeat polymorphism, Comings et al21 reported that the most frequent allele of the polymorphism in non-Hispanic Caucasians living in the USA was (AAT)13 repeat, followed by (AAT)17, (AAT)16 and (AAT)15. The allele of (AAT)13 repeats was as rare as 1.4% in the Japanese population. The allelic distribution of AAT repeats in the Japanese population was similar but not consistent with that in the Han Chinese population reported by Li et al.22 These results indicated that genotypic and allelic distributions of the two polymorphisms of the CNR1 gene could differ greatly among different races.
We have found that AAT repeats but not the 1359G/A polymorphism of the CNR1 gene were significantly associated with schizophrenia for the first time. As to subcategories of schizophrenia, hebephrenic type showed a significant association with AAT repeats polymorphism whereas the paranoid type did not. Tsai et al (2000)23 reported no association between AAT repeats of the CNR1 gene and Chinese schizophrenics. Certain reasons for this inconsistency are unknown, however, it is possible that racial difference, in the Japanese and Chinese population, may affect it. Alternatively, the composition of schizophrenic patients used in their study may considerably differ from that of the present study, although they did not show the ratio of each subtype of schizophrenia. If the paranoid type of schizophrenics was predominant in their subjects, a significant association may be ignored, because the present study showed that the paranoid type of schizophrenia did not associate with the CNR1 gene.
The hebephrenic type schizophrenia, which has been shown to be associated with the CNR1 gene in the present study, is characterized by predominant negative symptoms such as blunted affect, disorganized thought and deterioration of personality. Such symptomatic features of hebephrenic schizophrenia bear resemblance to chronic cannabinoid psychoses. Although acute use of cannabis can induce diverse types of psychotic state, such as panic reaction, confusional state, paranoid state and mania,24,25,26 chronic cannabis users often develop an ‘amotivational syndrome’27,28,29,30 typified by a diminution of ambition, productivity and motivation, which are often observed in hebephrenic schizophrenics. In addition, cannabinoid use affects cognitive function such as information processing and planning tasks.31 Such cognitive dysfunction is prominent in chronic schizophrenics like hebephrenics. Therefore, the endogenous cannabinoid system may be activated in the brain of patients with schizophrenia, especially with the hebephrenic type. A knockout study showed that central actions of cannabinoids are mediated via CB1 receptors encoded by the CNR1 gene.32 The CB1 receptor also mediates actions of endogenous cannabimimetic ligands, endocannabinoids like annadamide.33 It is possible that neurotransduction of CB1 receptors may be enhanced in the schizophrenic brain. A recent postmortem study supported this possibility which showed that receptor density of CB1 receptors in dorsolateral prefrontal cortex was increased in schizophrenics compared with controls.34 The present study has revealed an association of the CNR1 gene polymorphism with hebephrenic schizophrenia. The finding would support genetically the hypothesis that changes in the endogenous cannabinoid system of the brain may be involved in the pathogenesis of schizophrenia. The AAT repeat polymorphism locates in downstream about 16 kb of the CNR1 coding region. If the polymorphism affects transcription efficiency of the CNR1 gene as an enhancer, however, it is not still known, the present finding is very significant for the cannabinoid hypothesis of schizophrenia. Alternatively, the present finding may indicate an association with schizophrenia of other genes located close to the AAT repeat polymorphism of the CNR1 gene, although no genes or EST were registered in GenBank in the 3 prime region of the CNR1 gene to date. To confirm the significance of the study for the cannabinoid hypothesis of schizophrenia, the relationship of an allele containing a certain number of AAT repeats of the CNR1 gene and the transcription rate of the CNR1 gene should be specified. In addition, the present significant findings should be replicated by genomic control methods35 or family based control methods to avoid false positive results due to population stratification.
The subjects were 121 patients with schizophrenia (F20, 74 males and 48 females, age 44.7 ± 13.2 years) meeting ICD-10-DCR criteria who were outpatients or inpatients of psychiatric hospitals, and 148 age-matched normal controls (70 males and 78 females, age 44.5 ± 16.0 years) who had no known history of psychiatric disease in their families. Diagnosis was made by trained psychiatrists by interview. As to the subcategory of schizophrenia, hebephrenic (F20.1), paranoid (F20.0), catatonic (F20.2), and undifferentiated type (F20.3) accounted for 63, 55, two, and one patients, respectively. All subjects were Japanese, born and living in the middle western area of Japan. No subject abused any illicit drugs, such as cannabis and methamphetamine. This study was performed after approval by the ethics committee of Okayama University Medical School, Zikei Hospital and Takaoka Hospital, and all subjects provided written informed consent for the use of their DNA samples for this research.
Genomic DNA was extracted from peripheral leukocytes by the standard phenol/CHCl3 method. The region contained the 1359 G/A polymorphism amplified by PCR, using a mismatch primer set according to Gadzicki et al19 to create Msp I recognition sites (5′-GAAAGCTGCATCAAGAGCCC-3′, 5′-TTTTCCTGTG CTGCCAGGG-3′). PCR was performed in a final volume of 15 μl with 10% dimethyl sulfoxide and 1 unit of Supertaq (Sawady Co, Japan) in the reaction mixture. PCR conditions were as follows: 95°C for 5 min; 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, and 72°C for 5 min. The PCR products were analyzed on 3% agarose gel after digestion with Msp I. The region containing the AAT triplet repeat polymorphism was amplified according to Dawson et al20 (5′-GCTGCTTCTGTTAACCCTGC-3′, 5′-TACATCTCCG TGTGATGTTCC-3′). For analysis of polymorphic AAT repeats, the forward primer was 5′-end-labelled with Texas Red, and PCR products and the Texas red-labelled size standard were electrophoretically run on 6% polyacrylamide gel using an SQ5500 DNA sequencer (Hitachi Co, Japan) and each length was calculated using Fragyls 2 (Hitachi Co, Japan) computer software. Repeat numbers of AAT of the CNR1 gene were confirmed by direct sequencing using PCR samples from subjects homozygous for the repeat polymorphism of (AAT)9, (AAT)14, (AAT)16 and (AAT)17. All genotyping was carried out in a blinded fashion with control and patient samples mixed randomly. Statistical analysis between control and schizophrenia for the 1359 G/A polymorphism and each repeat allele of the AAT repeat polymorphism was done using the log-likelihood ratio test, and that for the overall allele of repeat polymorphism was done using the Mann–Whitney test. When comparison was made among three groups, control, hebephrenic type and paranoid type, the F test using the log-likelihood ratio test or the Kruskal–Wallis test was examined at first. If it was significant, each pair was examined as above mentioned.
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The authors are grateful to Dr Donn R Muhleman (Department of Medical Genetics, City of Hope, National Medical Center and Beckman Research Institute, USA) for providing sequence information on the CNR1 triplet repeats, and to Zikei Institute of Psychiatry (Okayama, Japan) for support in part by a grant.
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Ujike, H., Takaki, M., Nakata, K. et al. CNR1, central cannabinoid receptor gene, associated with susceptibility to hebephrenic schizophrenia. Mol Psychiatry 7, 515–518 (2002). https://doi.org/10.1038/sj.mp.4001029
- CNR1 gene
- cannabinoid receptor
- hebephrenic type
- association study
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