Ch trial, the latency to reach the platform was recorded. Three days after acquisition, defined by an average latency less than 10 seconds, the platform was removed and a 60 second probe trial was conducted. The data from the probe trial was analyzed with Ethovision software (Noldus, Netherlands), which also determined the path of the subject in the pool and the time spent in each quadrant.Statistical analysesAnalyses of mouse data used PASW statistics 18 (SPSS) and t tests (Excel). Analyses of variance and covariance (ANOVA and ANCOVA) used between subjects factors of genotype sex, and dose, age as a cofactor, and within-subjects factors where appropriate including time for locomotion and trial day for Morris water maze. Analyses of human association data and assessment of correlations between genotypes at nearby SNPs used PLINK (pngu.mgh.harvard. edu/ purcell/plink). Normalized gene expression levels were compared between human major vs minor allele homozygotes using t tests. Bonferroni corrections were performed using (http:// www.quantitativeskills.com/sisa/calculations/bonfer.php) with 0.6 and 0.8 correlations (r) between SNPs.Results Searches for common CSMD1 variation related to addiction and/or schizophreniaDatabases contained no common CSMD1 copy number/insertion/deletion variants, though rarer copy number variants that cover much of this large gene are documented. Only three missense variants (rs28455997, rs6558702 and rs11984691), which were distant from the most schizophrenia-associated SNP rs10503253, displayed minor allele frequencies > 0.03. There was nominally-significant association between CSMD1 PX-478 web genomic markers and levels of CSMD1 mRNA expression. We order PX105684 detected CSMD1 mRNA in RNA extracted from cerebral cortical specimens from each of the 181 individuals tested. Data from thirty nine genomic markers that displayed minor allele frequencies > 0.03 were analyzed. The CSMD1 intron 3 trinucleotide repeat that is annotated as rs71534387 and the intron 5 SNP rs10503223 displayed the most robust associations (Fig 1; p = 0.03/0.02, respectively; t tests). The Bonferroni-corrected thresholds for two sided t tests are p < 0.012 and p < 0.024 with r = 0.6 and r = 0.8, respectively; these nominally-significant associations thus did not definitively surpass Bonferroni correction for the numbers of markers tested. These two markers displayed D' values of 0.53 in individuals who donated the brain samples studied here. rs71534387 data comes from 104 individuals who were either hetero or homozygous for the 200 bp vs 206 bp alleles. These nominally-significant associations corresponded to 18/25 differences in levels of expression. Expression is lower in individuals with longer rs71534387 repeat lengths and in rs10503223 = G homozygotes. These differences in postmortem brain expression help to support heterozygous and/or homozygous csmd1 knockout mice as models for common CSMD1 allelic variation of relevance for addiction and, possibly, schizophrenia-related phenotypes. The schizophrenia-associated SNP rs10503253 itself did not provide the nominally-significant associations noted for the nearby rs71534387 trinucleotide repeat, however.PLOS ONE | DOI:10.1371/journal.pone.0120908 July 14,5 /CSMD1 Variants and AddictionFig 1. Differential expression of CSMD1 mRNA in cerebral cortical samples from individuals with different rs71534387 SSLP genotypes. Mean +/standard error of the mean (SEM) of relative expression of CSMD1 mRNA in cerebral cortic.Ch trial, the latency to reach the platform was recorded. Three days after acquisition, defined by an average latency less than 10 seconds, the platform was removed and a 60 second probe trial was conducted. The data from the probe trial was analyzed with Ethovision software (Noldus, Netherlands), which also determined the path of the subject in the pool and the time spent in each quadrant.Statistical analysesAnalyses of mouse data used PASW statistics 18 (SPSS) and t tests (Excel). Analyses of variance and covariance (ANOVA and ANCOVA) used between subjects factors of genotype sex, and dose, age as a cofactor, and within-subjects factors where appropriate including time for locomotion and trial day for Morris water maze. Analyses of human association data and assessment of correlations between genotypes at nearby SNPs used PLINK (pngu.mgh.harvard. edu/ purcell/plink). Normalized gene expression levels were compared between human major vs minor allele homozygotes using t tests. Bonferroni corrections were performed using (http:// www.quantitativeskills.com/sisa/calculations/bonfer.php) with 0.6 and 0.8 correlations (r) between SNPs.Results Searches for common CSMD1 variation related to addiction and/or schizophreniaDatabases contained no common CSMD1 copy number/insertion/deletion variants, though rarer copy number variants that cover much of this large gene are documented. Only three missense variants (rs28455997, rs6558702 and rs11984691), which were distant from the most schizophrenia-associated SNP rs10503253, displayed minor allele frequencies > 0.03. There was nominally-significant association between CSMD1 genomic markers and levels of CSMD1 mRNA expression. We detected CSMD1 mRNA in RNA extracted from cerebral cortical specimens from each of the 181 individuals tested. Data from thirty nine genomic markers that displayed minor allele frequencies > 0.03 were analyzed. The CSMD1 intron 3 trinucleotide repeat that is annotated as rs71534387 and the intron 5 SNP rs10503223 displayed the most robust associations (Fig 1; p = 0.03/0.02, respectively; t tests). The Bonferroni-corrected thresholds for two sided t tests are p < 0.012 and p < 0.024 with r = 0.6 and r = 0.8, respectively; these nominally-significant associations thus did not definitively surpass Bonferroni correction for the numbers of markers tested. These two markers displayed D' values of 0.53 in individuals who donated the brain samples studied here. rs71534387 data comes from 104 individuals who were either hetero or homozygous for the 200 bp vs 206 bp alleles. These nominally-significant associations corresponded to 18/25 differences in levels of expression. Expression is lower in individuals with longer rs71534387 repeat lengths and in rs10503223 = G homozygotes. These differences in postmortem brain expression help to support heterozygous and/or homozygous csmd1 knockout mice as models for common CSMD1 allelic variation of relevance for addiction and, possibly, schizophrenia-related phenotypes. The schizophrenia-associated SNP rs10503253 itself did not provide the nominally-significant associations noted for the nearby rs71534387 trinucleotide repeat, however.PLOS ONE | DOI:10.1371/journal.pone.0120908 July 14,5 /CSMD1 Variants and AddictionFig 1. Differential expression of CSMD1 mRNA in cerebral cortical samples from individuals with different rs71534387 SSLP genotypes. Mean +/standard error of the mean (SEM) of relative expression of CSMD1 mRNA in cerebral cortic.
