Joseph Trakalo

Identification of candidate genes for psychosis in rat models, and possible association between schizophrenia and the 14-3-3η gene

Although the genetic contribution to schizophrenia is substantial, positive findings in whole-genome linkage scans have not been consistently replicated. We analyzed gene expression in various rat conditions to identify novel candidate genes for schizophrenia. Suppression subtraction hybridization (SSH), with polyA mRNA from temporal and frontal cortex of rats, was used to identify differentially expressed genes. Expression of mRNA was compared between adult Lewis and Fischer 344 (F344) rats, adult and postnatal day 6 (d6) F344, and adult F344 treated with haloperidol or control vehicle. These groups were chosen because each highlights a particular aspect of schizophrenia: differences in strain vulnerability to behavioral analogs of psychosis; factors that may relate to disease onset in relation to CNS development; and improvement of symptoms by haloperidol. The 14-3-3 gene family, as represented by 14-3-3γ and 14-3-3ζ isoforms in the SSH study, and SNAP-25 were among the candidate genes. Genetic association between schizophrenia and the 14-3-3η gene, positioned close to a genomic locus implicated in schizophrenia, and SNAP-25 genes was analyzed in 168 schizophrenia probands and their families. These findings address three different genes in the 14-3-3 family. We find a significant association with schizophrenia for two polymorphisms in the 14-3-3η gene: a 7 bp variable number of tandem repeats in the 5′ noncoding region (P=0.036, 1 df), and a 3′ untranslated region SNP (753G/A) that is an RFLP visualized with Ava II (P=0.028). There was no significant genetic association with SNAP-25. The candidate genes identified may be of functional importance in the etiology, pathophysiology or treatment response of schizophrenia or psychotic symptoms. This is to our knowledge the first report of a significant association between the 14-3-3η-chain gene and schizophrenia in a family-based sample, strengthening prior association reports in case–control studies and microarray gene expression studies.

Association analyses of the DAOA/G30 and D-amino-acid oxidase genes in schizophrenia: further evidence for a role in schizophrenia.

A number of linkage studies have previously implicated the region of chromosome 13q34 in schizophrenia. Chumakov and colleagues (2002) identified a gene complex called G72 (now termed d-amino acid oxidase activator: DAOA)/G30 in this region and performed association analyses of the DAOA/G30 as well as the d-amino-acid oxidase (DAAO) gene with schizophrenia. DAAO oxidizes d-serine, a potent activator of the N-methyl-d-aspartate (NMDA) type glutamate receptor in the human brain whereas the DAOA protein is considered an activator of DAAO. The interaction of these two genes has thus been implicated in the NMDA receptor regulation pathway in schizophrenia. To date, several studies have shown a relatively consistent positive association between schizophrenia and DAOA/G30, but not with DAAO. The aim of our study was to further evaluate the contributions of these genes to the susceptibility to schizophrenia using two different sample sets. Our sample consisted of 168 matched case-control pairs as well as a family-based sample (n=113) for transmission disequilibrium test. Significant associations between the DAOA/G30 M-7 and M-18 polymorphisms and schizophrenia were observed in our case-control sample whereas no associations were observed for DAAO markers. We also observed significant or suggestive transmission disequilibrium for DAOA/G30 M-7, M-23, and M-24 to probands with schizophrenia in our family-based sample. Subsequent analysis of haplotypes made up of four DAOA/G30 markers, one marker selected from each of two linkage disequilibrium blocks that were observed in our sample as well as both ends (M-7 and M-25), were also associated with schizophrenia. Our data provide further evidence that the DAOA/G30 locus may play a role in the pathophysiology of schizophrenia. Although no direct link to genetic polymorphism in these genes and NMDA receptor function has been revealed, the present findings support previous reports implicating DAOA/G30 as susceptibility genes for schizophrenia. Further research is warranted to determine the functional variation underlying these findings and to relate this to the pathophysiology of schizophrenia.

Human p53 tumor suppressor gene (TP53) and schizophrenia: Case–control and family studies

The human p53 tumor suppressor gene (TP53) is considered as a candidate susceptibility gene for schizophrenia because of its functions in neurodevelopment. To test for an association between TP53 and schizophrenia, both the case-control study and the transmission disequilibrium test (TDT) were performed on genotype data from eight polymorphisms in TP53. Our samples included 286 Toronto schizophrenia cases and 264 controls, and 163 Portuguese nuclear families. In the Toronto case-control study significant differences of allele frequencies of the CAA Ins/Del (p=0.027) and the 16bp Ins/Del (p=0.022) were detected. In TDT analysis we found significant differences for transmission of the CAA Ins/Del (p=0.017) in Portuguese schizophrenia families. Haplotype analysis also showed a significant association between TP53 and schizophrenia. These results provide further evidence that TP53 may play a role in the pathogenesis of schizophrenia.

Linkage disequilibrium between dopamine D1 receptor gene (DRD1) and bipolar disorder

BACKGROUND Based on the dopamine hypothesis, the dopamine D1 receptor gene (DRD1) is considered to be a good candidate gene for bipolar disorder (BP). METHODS In our study, three polymorphisms of the DRD1 gene, -800T/C, -48A/G, and 1403T/C, were analyzed in 286 BP trios. Both the transmission disequilibrium test (TDT) and haplotype TDT were performed on the genotype data to test for the presence of linkage disequilibrium between DRD1 and bipolar disorder. With the extended transmission disequilibrium test (ETDT), we also calculated the maternal transmission and paternal transmission for each allele. RESULTS Although no association was found for each individual polymorphism, there is a significant association between DRD1 and BP for haplotype TDT analysis (chi(2) = 16.068, df = 3, p =.0011). CONCLUSIONS These results indicate that DRD1 may play a role in the etiology of bipolar disorder.

Genetic and post-mortem mRNA analysis of the 14-3-3 genes that encode phosphoserine/threonine-binding regulatory proteins in schizophrenia and bipolar disorder

BACKGROUND Previous work with animal models of psychosis, human genetic studies, and human post-mortem gene expression studies implicate the 14-3-3 family of genes in schizophrenia. The 14-3-3 genes code for a family of proteins that bind to and regulate other proteins, and they modulate neurodevelopment, cell-division, signal transduction and gene transcription. OBJECTIVE To explore the role of five 14-3-3 isoforms (beta, gamma, epsilon, zeta, and eta) in schizophrenia by: (1) comparing mRNA levels in post-mortem brain from schizophrenic, bipolar and control subjects and (2) assessing genetic association with schizophrenia in both case-control and nuclear family samples. METHODS Quantitative PCR (q-PCR) was used to determine relative mRNA levels in dorsolateral prefrontal cortex (Brodmanns area 46) samples donated by the Stanley Medical Research Institute (SMRI). Selected SNPs were genotyped in all five isoforms for association analysis in both family and case-control samples. RESULTS No significant differences in 14-3-3 mRNA expression levels between the diagnostic groups were found. A significant genetic association with schizophrenia was found for the 14-3-3zeta isoform in a subset of nuclear families of British ancestry (TDT: chi(2)=7.2; df=1; p=0.0073), in the case-control sample overall (p=0.011), and in a subset of the case-control sample. CONCLUSION The results, in combination with other published evidence, suggest that further work is necessary to clarify what role the 14-3-3 genes may play in the etiology and pathogenesis of schizophrenia.

Investigation of polymorphism in the MDR1 gene and antidepressant-induced mania

ABSTRACTThe involvement of the multi-drug-resistant 1 P-glycoprotein gene (MDR1 P-gp) in the transport of antidepressants across the blood–brain barrier makes it a good candidate for the prediction of antidepressant response and side effects. We investigated the role of the MDR1 P-gp gene in predicting the induction of mania in bipolar patients (BP) treated with proserotonergic drugs. Participants met the DSM-IV criteria for BP or BPII and had at least one depressive episode treated with proserotonergic antidepressants. The first group (n=26) included patients with at least one DSM-IV manic/hypomanic episode developed during antidepressant treatment; the second group (N=29) included patients with no antidepressant-induced switches. The common polymorphism of the MDR1 was genotyped for both groups and comparison was made with respect to the presence/absence of induced mania between the two groups. No association between antidepressant-induced mania and the MDR1 alleles or genotypes was found (χ2=1.85, 2 df, P=0.39; χ2=0.13, 1 df, P=0.72).

Association between schizophrenia and the syntaxin 1A gene

BACKGROUND Both microarray and candidate molecule studies have demonstrated that protein and mRNA expression of syntaxin and other genes involved in synaptic function are altered in the cerebral cortex of patients with schizophrenia. METHODS Genetic association between polymorphic markers in the syntaxin 1A gene and schizophrenia was assessed in a matched case-control sample of 192 pairs, and in an independent sample of 238 nuclear families. RESULTS In the family-based sample, a significant genetic association was found between schizophrenia and one of the four single nucleotide polymorphisms (SNPs) tested: an intron 7 SNP (transmission disequilibrium test [TDT] chi(2) = 5.898; df = 1; p =.015, family-based association test [FBAT] z = 2.280, p =.023). When the results for the TDT and case-control analyses were combined, the association was stronger (n = 430; z(c) = 2.859; p =.004). Haplotype analysis supported the association with several significant values that appear to be driven by the intron 7 SNP. CONCLUSIONS The results should be treated with caution until replicated, but this is the first report of a genetic association between syntaxin 1A and schizophrenia.

N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia: TDT and case-control analyses.

The N‐methyl‐d‐aspartate glutamate receptors (NMDAR) act in the CNS as regulators of the release of neurotransmitters such as dopamine, noradrenaline, acetylcholine, and GABA. It has been suggested that a weakened glutamatergic tone increases the risk of sensory overload and of exaggerated responses in the monoaminergic system, which is consistent with the symptomatology of schizophrenia. We studied two silent polymorphisms in GRIN1. GRIN1/1 is a G/C substitution localized on the 5′ untranslated region; GRIN1/10 is an A/G substitution localized in exon 6 of GRIN1. Minor allele frequencies in our sample were calculated to be 0.05 and 0.2 respectively. We genotyped 86 nuclear families and 91 ethnically matched case‐control pairs. Both samples were collected from the Toronto area. We tested the hypothesis that GRIN1 polymorphisms were associated with schizophrenia using the transmission disequilibrium test (TDT) and comparing allele frequencies between cases and controls. The results are as follows: GRIN1/1: χ2 = 2.19, P = 0.14; GRIN1/10: χ2 = 1.5, P = 0.22. For the case‐control sample: GRIN1/1: χ2 = 0.013, P = 0.908; GRIN1/10: χ2 = 0.544, P = 0.461. No significant results were obtained. Haplotype analyses showed a borderline significant result for the 2,1 haplotype (χ2 = 3.86, P‐value = 0.049). An analysis of variance (ANOVA) to evaluate the association between genetic makeup and age at onset was performed, with no significant results: GRIN1/1, F[df = 2] = 0.42, P‐value = 0.659; GRIN1/10, F[df = 2] = 0.16, P‐value = 0.853. We are currently collecting additional samples to increase the power of the analyses.

Genetic association analysis of the glutathione peroxidase (GPX1) gene polymorphism (Pro197Leu) with tardive dyskinesia.

A possible involvement of oxidative stress in the pathophysiology of tardive dyskinesia (TD) has previously been proposed (reviewed in [Andreassen, O.A., Jorgensen, H.A., 2000. Neurotoxicity associated with neuroleptic-induced oral dyskinesias in rats. Implications for tardive dyskinesia? Progress in Neurobiology 61, 525-541.]). Long-term administration of neuroleptics alters dopaminergic turnover, which results in increased formation of reactive oxygen species (ROS). This is hypothesized to lead to TD through neuronal toxicity as a consequence of oxidative stress. In the present study, the relationship between TD and a possible functional polymorphism of the human glutathione peroxidase (GPX1) gene (an important antioxidant enzyme) was studied in 68 chronic treatment-refractory patients with schizophrenia. A proline (Pro) to leucine (Leu) substitution at codon 197 (Pro197Leu) in the GPX1 gene was genotyped. No significant difference in total Abnormal Involuntary Movements Scale (AIMS) scores was observed among patients in the three genotype groups. Moreover, no significant differences in genotype or allele frequencies were observed between subjects with and without TD. Our results suggest that the GPX1 gene polymorphism does not confer increased susceptibility to TD, although further studies are warranted before a conclusion can be drawn.

Family-Based Association Study of the Serotonin-2A Receptor Gene (5-HT2A) and Bipolar Disorder

Objectives: The serotonin 2A receptor gene (5-HT2A) is of great interest for research in neuropsychiatric disorders based on the observation that various neuroleptic agents and antidepressants bind with relatively high affinity at 5-HT2A receptors, and the fact that the receptor density in platelets tends to increase in depression. To test for the presence of association between 5-HT2A and bipolar disorder (BP), we studied a large number of triad families having probands affected with DSM-IV bipolar I (BPI), bipolar II (BPII) or schizoaffective disorder, bipolar type.Methods: Two polymorphisms of 5-HT2A, 102T/C, and His452Tyr were analyzed in the 274 bipolar triad families. Both the transmission disequilibrium test (TDT) and haplotype TDT were performed on the genotype data. We also calculated the maternal transmission and paternal transmission for each allele and compared the mean ages of onset across probands grouped by genotype at each of the two markers.Results: No significant transmission disequilibrium between the alleles of 5-HT2A and BP was found. Separate studies of the sub-phenotypes also failed to demonstrate significant association. However, we found a trend towards transmission disequilibrium with the haplotype 102C.His452 (p=0.0504). This trend may become more significant with a larger sample size.Significance: At present, results of this study suggest that the 5-HT2A is unlikely to play a major role in the genetic susceptibility to BP. Future studies will be directed towards increasing sample size, focusing on subtypes of BP or biochemical measures as phenotypes, and investigating other polymorphisms of 5-HT2A to provide more information at the DNA level.