Variants in doublecortin- and calmodulin kinase like 1, a gene up-regulated by BDNF, are associated with memory and general cognitive abilities.

BACKGROUND: Human memory and general cognitive abilities are complex functions of high heritability and wide variability in the population. The brain-derived neurotrophic factor (BDNF) plays an important role in mammalian memory formation. METHODOLOGY / PRINCIPAL FINDING: Based on the identification of genes markedly up-regulated during BDNF-induced synaptic consolidation in the hippocampus, we selected genetic variants that were tested in three independent samples, from Norway and Scotland, of adult individuals examined for cognitive abilities. In all samples, we show that markers in the doublecortin- and calmodulin kinase like 1 (DCLK1) gene, are significantly associated with general cognition (IQ scores) and verbal memory function, resisting multiple testing. DCLK1 is a complex gene with multiple transcripts which vary in expression and function. We show that the short variants are all up-regulated after BDNF treatment in the rat hippocampus, and that they are expressed in the adult human brain (mostly in cortices and hippocampus). We demonstrate that several of the associated variants are located in potential alternative promoter- and cis-regulatory elements of the gene and that they affect BDNF-mediated expression of short DCLK1 transcripts in a reporter system. CONCLUSION: These data present DCLK1 as a functionally pertinent gene involved in human memory and cognitive functions.

Identification of genes co-upregulated with Arc during BDNF-induced long-term potentiation in adult rat dentate gyrus in vivo.

Brain-derived neurotrophic factor (BDNF) is a critical regulator of transcription-dependent adaptive neuronal responses, such as long-term potentiation (LTP). Brief infusion of BDNF into the dentate gyrus of adult anesthetized rats triggers stable LTP at medial perforant path-granule synapses that is transcription-dependent and requires induction of the immediate early gene Arc. Rather than acting alone, Arc is likely to be part of a larger BDNF-induced transcriptional program. Here, we used cDNA microarray expression profiling to search for genes co-upregulated with Arc 3 h after BDNF-LTP induction. Of nine cDNAs encoding for known genes and up-regulated more than four-fold, we selected five genes, Narp, neuritin, ADP-ribosylation factor-like protein-4 (ARL4L), TGF-beta-induced immediate early gene-1 (TIEG1) and CARP, for further validation. Real-time PCR confirmed robust up-regulation of these genes in an independent set of BDNF-LTP experiments, whereas infusion of the control protein cytochrome C had no effect. In situ hybridization histochemistry further revealed up-regulation of all five genes in somata of post-synaptic granule cells following both BDNF-LTP and high-frequency stimulation-induced LTP. While Arc synthesis is critical for local actin polymerization and stable LTP formation, several of the co-upregulated genes have known functions in excitatory synaptogenesis, axon guidance and glutamate receptor clustering. These results provide novel insight into gene expression responses underlying BDNF-induced synaptic consolidation in the adult brain in vivo.

Gene expression profiles of head and neck carcinomas from Sudanese and Norwegian patients reveal common biological pathways regardless of race and lifestyle.

PURPOSE: To explore possible range of gene expression profiles in head and neck squamous cell carcinomas (HNSCC) and pairwised normal controls from Sudanese (n = 72) and Norwegian (n = 45) patients using a 15K cDNA microarray and to correlate the findings with clinicopathologic variables. EXPERIMENTAL DESIGN: Samples from Sudan were grouped according to anatomic location/patients' habit of toombak (snuff) use, and 37 pools of 2 to 11 tumors matched to 37 pools of their normal controls from the same patients, respectively, were prepared. For Norway, eight pools of 3 to 11 tumors matched to eight pools of their normal controls from the same patients, respectively, were prepared according to anatomic location. Pools (n = 45) were hybridized to microarrays. For controls, 33 of the pools were hybridized against Human Reference RNA. Scanned array images were recorded, and data analysis was done in groups. For verification, results for selected genes were analyzed using quantitative real-time PCR/immunohistochemistry. RESULTS: We identified 136 genes from Sudan and 154 from Norway as differentially expressed between tumors and controls. Changes of the genes found were confirmed in >70% of the pools by hybridization against Reference RNA. Seventy-three genes and three main pathways (signal transduction, cell communication, and ligand-receptor interaction) were of relevance to the HNSCCs from both countries. Hierarchical clustering of the 73 genes identified subclasses of mixed tumors from the two populations, two independent subgroups for Norwegian tumors by their anatomic sites, and five subgroups for Sudanese tumors by their toombak habits. Quantitative real-time PCR/immunohistochemistry validated the microarray-based data. CONCLUSIONS: Differences in gene expression between tumor and nontumor tissues were identified in HNSCCs. Analysis of the two population groups revealed a common set of 73 genes within three main biological pathways. This indicates that the development of HNSCCs is mediated by similar biological pathways regardless of differences related to race, ethnicity, lifestyle, and/or exposure to environmental carcinogens. Of particular interest, however, was the valuable association of gene expression signature found with toombak use and anatomic site of the tumors.

Evaluation of five different cDNA labeling methods for microarrays using spike controls.

BACKGROUND: Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay--aminoallyl), two protocols with dendrimer technology (3DNA Array 50 and 3DNA submicro), and hapten-antibody enzymatic labeling (Micromax TSA). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5-50 microg. RESULTS: The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively). CONCLUSION: We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.

Pharmacogenetics of tardive dyskinesia: combined analysis of 780 patients supports association with dopamine D3 receptor gene Ser9Gly polymorphism.

Variability among individuals in their therapeutic response to psychotropic drugs and in susceptibility to adverse effects is considerable. Pharmacogenetics addresses the contribution of genetic factors to this variability. An important focus of interest in pharmacogenetics has been on candidate genes that play a role in susceptibility to the antipsychotic drug-induced adverse effect, tardive dyskinesia (TD). Four published studies have reported an association between a serine (ser) to glycine (gly) polymorphism in exon 1 of the dopamine D3 receptor gene (DRD3) and TD; three failed to replicate this finding and one found an insignificant trend. We examined the association in a pooled sample of 780 patients (317 with TD and 463 without TD) drawn from 6 research centers, who were divided into 8 groups based on their population origin. The analysis employed stepwise logistic regression so as to allow confounding effects of group, age, and gender to be taken into account. TD was significantly associated with DRD3 gly allele carrier status (x(2)=4.46, df 1, p =.04) and with DRD3 genotype (x(2)=6.62, df 2, p =.04) over and above the effect of group. Similar positive effects were observed when controlling for age and gender (x(2)=5.02, df 1, p =.02 for gly allele carrier status; x(2) = 7.51, df 2, p =.002 for genotype). Examining abnormal involuntary movement scores as a continuous variable, we found that patients homozygous for the gly allele had significantly higher scores than ser-gly heterozygotes (p =.006) or ser-ser homozygotes (p <.0001). We also performed a meta-analysis that included, besides the groups in the combined analysis, three other published studies on DRD3 and TD. The Mantel-Haenszel pooled odds ratio for DRD3 gly allele carrier status increasing susceptibility to TD was 1.33 (95% CI 1.04-1.70, p =.02); the cumulative pooled estimate showed an odds ratio of 1.52 (95% CI 1.08-1.68, p <.0001). These findings support a small but significant contribution of the DRD3 ser9gly polymorphism to TD susceptibility that is demonstrable over and above population effects and the effect of age and gender on the phenotype.

Homozygosity for the Gly-9 variant of the dopamine D3 receptor and risk for tardive dyskinesia in schizophrenic patients.

This study was undertaken to re-examine whether homozygosity for the Gly-9 variant (allele 2) of the dopamine D3 receptor gene (DRD3) is associated with increased risk for tardive dyskinesia (TD) in schizophrenic patients. Seventy-one antipsychotic-treated subjects with schizophrenia from Newcastle upon Tyne, UK, were genotyped for the presence of allele 1 (Ser-9) and allele 2 (Gly-9) of the dopamine D3 receptor (DRD3) Ser-9-Gly polymorphism. Among 32 patients with TD, 7 subjects (22 %) were homozygous for the Gly-9 variant (2-2 genotype), whereas 4 out of 39 patients (10 %) without TD had this genotype. The non-significant tendency in this sample towards an over-representation of allele 2 and the 2-2 genotype among schizophrenic patients with TD is in line with our initial report as well as recent studies by others, indicating that the Gly-9 allele of DRD3 may be a susceptibility factor for the development of TD in neuroleptic-treated individuals with schizophrenia. There are, however, some recent non-supportive reports, and since the trend in our present study failed to reach statistical significance, further studies on larger samples and future meta-analysis may be necessary to establish the role of the DRD3 in the pathogenesis of TD.