O:57 Analysis of linkage disequilibrium with schizophrenia in three candidate genes: GRM-3, AKT-1 and NRG-1
S.G. Schwab∗, C. Plummer, A. Doyle, M. Albus, M. Borrmann-Hassenbach, W. Maier, D.B. Wildenauer
Aims/Background Association of DNA sequence variants with schizophrenia has been reported for the metabotropic glutamate receptor 3 (GRM3) the protein kinase B (AKT-1) and neuregulin 1 (NRG1). Whereas NRG1 is located in a chromosomal region with good support for linkage with schizophrenia, there has been only weak support for linkage to the chromosomal locus of GRM3 with schizophrenia and no support for linkage to the chromosomal region of the AKT1 locus.
Methods We genotyped SNPs for all three regions based on previous reports and attempted replication of association with schizophrenia using a family based design. Statistical analysis was performed using the program Famhap.
Results
AKT1: A P value of 0.0005 was obtained for rs4983559. This SNP is located approximately 17 kb upstream of the AKT1 gene and revealed overall the lowest P value obtained for all 9 SNPs analysed in the AKT1 region. GRM3: Five SNPs have been analysed. SNP rs2237562 was nominally associated with schizophrenia (P = 0.01). NRG1: We genotyped 13 SNPs located in the exon dense 3′ region of the gene, and one SNP and two microsatellite markers previously implicated in susceptibility to schizophrenia by Stefansson et al (AJHG, 2002, 72:83). Single marker analysis revealed association of schizophrenia with rs10503929 (p =.017). Haplotype analysis including 7 of the 16 markers revealed lowest P value of P =.000004 for a five-marker haplotype and a global P value corrected for multiple testing for these 7 markers of P = 0.00035.
Conclusions We conclude that DNA variation in all three candidate genes contribute to susceptibility for schizophrenia.
Presenter: Sibylle G Schwab, School of Psychiatry and Clinical Neurosciences, and Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, QEII MC, B-Block, Ground Floor, Hospital Ave, Nedlands, WA 6009
Tel: 08 9346 2711
Fax: 08 9346 1818
Email: sschwab@cyllene.uwa.edu.au
O:58 Evaluation of linkage disequilibrium in the TNF alpha gene region on chromosome 6p in families with schizophrenia
B. Morar∗, S.G. Schwab, M. Albus, W. Maier, B. Lerer, D. Wildenauer
Aims/Background The association of the TNFα−G308A promoter polymorphism with schizophrenia has complemented clinical findings of increased TNFα cytokine levels in schizophrenic patients, with some support for a functional consequence of the variant. Our studies of genetic causes of schizophrenia supported findings of linkage to the MHC region where the TNFα gene is located as well as association with the −G308A promoter polymorphism. While the common G-allele shows association in our sample, the A-allele has been reported to be associated by other groups. This suggests linkage disequilibrium (LD) rather than direct involvement in the disorder. To examine the association of other DNA variants in this area with the disorder and to define the LD pattern of the region, we analysed 36 SNPs in a ∼165 kb region around this polymorphism.
Methods The SNPs were typed in 204 families using restriction fragment length polymorphism analysis and allele specific PCR using fluorescent probes (Taqman and Amplifluor technology). FAMHAP was used to carry out tests of association and LD analysis using haplotypes constructed from the family data.
Results We identified three markers (including the −G308A polymorphism) and multiple haplotypes showing significant association (p < 0.05) with schizophrenia. Inter-marker LD analysis indicated that all three markers and a majority of associated haplotypes localise to one of two high LD regions.
Conclusions Data analysis in our family sample allows us to refine the region that may contain an allele conferring susceptibility to schizophrenia to 30 kb. However, in light of our nominally significant association results and the long range LD known to exist in the MHC, we cannot exclude that markers in the refined region may be in LD with a susceptibility allele elsewhere in the MHC.
Presenter: Bharti Morar, School of Psychiatry and Clinical Neurosciences, and Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, QEII MC, B-Block, Ground Floor, Hospital Ave, Nedlands, WA 6009
Tel: 08 9346 2711
Fax: 08 9346 1818
Email: bmorar@cyllene.uwa.edu.au
O:59 The presence of alternatively spliced variants and the relative expression of dysbindin in lymphoblast cell lines of schizophrenic patients
H.P Ludewick∗, S.G Schwab, M. Albus, J. Berry, W. Maier, M. Borrmann-Hassenbach, D.B. Wildenauer
Aims/Background Genetic factors contribute to schizophrenia and several regions in the genome have been shown to harbor susceptibility genes for schizophrenia. One such region, on chromosome 6p22 to which the dystrobrevin binding protein gene (DTNBP1, dysbindin) maps was shown by us and others to be associated with schizophrenia. The association was with SNPs in intronic region and no obvious functional SNPs identified. Dysbindin has been detected in the brain and may be involved in synapse formation and thus neuronal signalling. Multiple splice transcripts with introns have been identified for dysbindin. Due to the absence of an association with a coding SNP we investigated if susceptibility to schizophrenia may be due to altered gene expression of dysbindin.
Methods Total RNA extracted from lymphoblast cell-lines of affected individuals was amplified by RT-PCR and sequenced. Quantitative real time RT-PCR using Taqman probes spanning exon 5/6 and exon 9/10 of DTNBP1 respectively was used to quantify the expression of dysbindin. The relative expression of dysbindin was normalised with two housekeeping genes (GUSB and GAPDH). The ΔΔCt method was used for the analysis of the relative expression.
Results Studying the variety of dysbindin transcripts in lymphocytes we have been able to show that dysbindin was alternatively spliced. Using primers located in exon 1 and 6 of the gene, we have been able to identify a splice variant missing exon 2 and 3. In addition an isoform of dysbindin exhibiting an extended exon 9 has been identified.
Conclusion We obtained evidence for dysbindin splice variants being present in lymphocytes. These splice variants might be differentially regulated in schizophrenia.
Presenter: Herbert P Ludewick, School of Psychiatry and Clinical Neurosciences, and Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, QEII MC, B-Block, Ground Floor, Hospital Ave, Nedlands, WA 6009
Tel: 08 9346 2711
Fax: 08 9346 1818
Email: hlud@cyllene.uwa.edu.au
O:60 Lack of association of DRD2 gene TaqI polymorphism with schizophrenia in an Iranian population
J. Behravan∗, M. Hemayatkar, H. Toufani, E. Abdollahian
Biotechnology Laboratory, Biotechnology and Pharmaceutical Research Centers, Mashhad University of Medical Sciences, Mashhad, IRAN
Aims D2 dopamine receptor (DRD2) gene has been reported to be one of the most relevant candidate genes in schizophrenia. In this study we investigated the association between Taq IA and Taq IB dopamine D2 receptor polymorphisms and psychopathology of schizophrenia.
Methods The study subjects were 38 acutely exacerbated schizophrenic patients who were all Iranian descent. Control population consisted of 63 healthy individual within the same range of age as patients and were also of Iranian decent. The Taq IA and Taq IB genotypes, the A1 and A2 alleles and, the B1 and B2 were determined by Restriction Fragment Length Polymorphism (RFLP) of the amplified DNA fragments by Polymerase Chain Reaction (PCR).
Results For each polymorphism (A or B) the patients were divided into three genotype groups; i.e., the patients with alleles: A1/A1, A1/A2, A2/A2; B1/B1, B1/B2 and B2/B2. No significant association was found between Taq1A or Taq1B gene polymorphisms and schizophrenia in patients compared to controls. When study subjects were divided into male and female subgroups, the distribution of the A1A1 genotype differed in females (patients vs controls) but such difference was not observed in males. However, statistical analyses proved that the observed differences were not significant.
Conclusion Our findings show that in this population study, there was no genetic association between Taq1A and Taq1B gene polymorphisms and schizophrenia. Further clinical studies should be designed to confirm and further evaluate these findings.
O:61 Dysfunction of genes regulating membrane exocytosis in schizophrenia
J. Weidenhofera,b,c, N.A. Bowdena,b,c, R.J. Scott b,c,d, P.A. Tooney∗a,b,c
aNeuroscience Institute of Schizophrenia and Allied Disorders (NISAD), NSW Australia. bSchool of Biomedical Sciences, Faculty of Health, University of Newcastle, NSW 2308, Australia. cHunter Medical Research Institute, Newcastle, NSW 2308, Australia. dDivision of Genetics, HAPS, John Hunter Hospital, NSW 2308, Australia
Background The amygdala is implicated in the pathophysiology of schizophrenia, however the genes involved in the dysfunction of the amygdala in schizophrenia are yet to be identified.
Aim To study global gene expression in the amygdala in post-mortem tissue from schizophrenia and non-psychiatric control subjects.
Methods Gene expression in amygdalae from post-mortem pairs of schizophrenia and non-psychiatric control brains matched for age, gender, postmortem interval and brain hemisphere obtained from the NSW Tissue Resource Centre, was studied using oligonucleotide arrays consisting of 19000 gene transcripts (n = 7 pairs) and real-time PCR (n = 11 pairs). Western blotting of amygdala extracts was used to check protein levels. The effects of antipsychotic medication were addressed by culturing SH-SY5Y neuroblastoma cells in the presence of haloperidol or clozapine.
Results Genes involved in presynaptic function, myelination and cellular signalling were identified as being consistently dysregulated. In particular, the expression of three genes involved in the cytomatrix active zone, RIMS2 (p = 0.007, t = 3.42, df = 6), RIMS3 (p = 0.001, t = 4.87, df = 6) and piccolo (p = 0.014, t = 2.91, df = 6) were significantly up-regulated and in vitro culture of SH-SY5Y with haloperidol and clozapine indicate that this is unlikely to be due to treatment with antipsychotic drugs. Western blot analysis indicates that RIMS2 protein levels are also significantly increased (p = 0.047, t = 1.98, df = 6) in the amygdala in these subjects with schizophrenia.
Conclusions These results implicate dysregulation of membrane exocytosis via changes in the levels of proteins that are components of the cytomatrix active zone of synapses in the amygdala in the pathophysiology of schizophrenia.
O:62 Investigation of associations between Single Nucleotide Polymorphisms (SNP) of the Muscarinic1 Receptor (M1R) gene and cognitive deficits in schizophrenia
A. Khademy-Deljo∗1,2, S. Sundram1,2, E. Scarr1, C. Pantelis1, B. Dean1
1Mental Health Research Institute of Victoria.
2Northern Psychiatry Research Centre
Background Cognitive dysfunction is a major predictor of psychosocial impairment in schizophrenia, however its aetiology is unknown. The cholinergic system has been implicated in cognitive dysfunction and studies have shown a decrease in the density of the M1R in Brod-mann's Area 9 in post-mortem brain tissue from subjects with schizophrenia. Further a recent study demonstrated an association between a SNP in the M1R gene and impaired performance on a cognitive test, WCST, in people with schizophrenia.
Aims This NHMRC funded study will further investigate any potential association between M1R SNPs and cognitive performance in schizophrenia.
Method 250 patients with schizophrenia will be recruited from the Northern Area Mental Health Service. DNA will be extracted from whole blood. Subjects will complete the WCST, Controlled Oral Word Association Test, and Positive and Negative Syndrome Scale.
Results Have initiated and currently recruiting subjects. Early screening of the human M1R gene has identified 9 possible SNPs.
Conclusion If associations are demonstrated between M1R SNPs and impaired performance on tests of executive cognitive functioning then, this will, for the first time, potentially allow for screening of patients with schizophrenia. Those with this vulnerability could then have the opportunity to receive cognitive remediation therapies prior to the onset of impairment with the aim of delaying or preventing cognitive deterioration. It would also open avenues for the pharmacological targeting of the M1R for the development of new treatments aimed at precenting cognitive impairment.
O:63 Violent Behaviour Associated with a Novel ApoB Gene Mutation (apoB-29.4), which causes Hypocholesterolemia
P.F. Edgar∗1, A.J. Hooper2, N.R. Poa1, J.R. Burnett2
1Molecular Psychiatry Research Group, Department of Psychological Medicine, Christchurch School of Medicine, University of Otago, Christchurch, New Zealand.
2Department of Core Clinical Pathology and Biochemistry, PathWest Laboratory Medicine WA, Royal Perth Hospital, Perth, Australia and School of Medicine & Pharmacology, University of Western Australia, Perth, Australia
A 26-year-old male, the proband, presented with persecutory delusions and suicidal behavior. Five, out of ten, of his paternal male relatives in two prior generations, died by violent suicide and one of the five also committed a double homicide. The proband was found to be hypocholesterolemic and heterozygous for a novel, hypocholesterolemia causing, mutation of apolipoprotein B (apoB-29.4). His mother and paternal grandmother were normocholesterolaemic, whereas a surviving paternal uncle was hypocholesterolaemic and heterozygous for apoB-29.4. This indicated that the proband inherited the mutation from his paternal grandfather. Our results support an inheritable relationship between violent behaviour and hypocholesterolaemia.
O:64 Molecular mechanisms of Lithium Chloride: What can mice teach us about Bipolar Disorder?
A.F. Chetcuti∗, L.J. Adams, C.E. Duncan, P.B. Mitchell, P.R. Schofield
Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), 384 Victoria Street, Darlinghurst NSW 2010, AUSTRALIA
Aims/Background Bipolar disorder is a common psychiatric disorder that affects an estimated 0.8–1.6% of the population. Patients experience mood swings, manic episodes, syndromal major depression and have a substantial lifetime risk of suicide. Breakthroughs in the treatment of bipolar disorder have progressed slowly. Lithium chloride has been used successfully since the 1950s, yet the underlying mechanism of its action is still not known. The aim of this study was to identify genes in the mouse brain that are regulated by lithium chloride.
Methods Lithium chloride was administered by injecting mice daily for 1 week. Control mice received saline solution. Lithium serum concentrations were measured for treated mice and total RNA was extracted from whole mouse brains. The expression profile for 12,488 transcripts was determined by hybridising mouse brain cRNA probes to Affymetrix U74Av2 GeneChip microarrays. The gene expression profiles were analysed using Affymetrix Microarray Analysis Suite (MAS) software.
Results Data analysis was undertaken and differentially expressed transcripts were determined. Using 4-fold difference relative to control mice, 18 transcripts were downregulated and only 1 transcript was upregulated after lithium treatment. Altered gene expression was confirmed using real-time quantitative PCR.
Conclusions Animal models are being used as tools to further understand the biology and biochemistry of human mental disorders such as bipolar disorder. The identified genes may represent novel targets for the development of new compounds for the treatment of bipolar disorder.
