Effect of suicide on expression of GR mRNA transcripts in the DLPFC.

<p>Suicide-positive and suicide-negative control cases, bipolar disorder cases and schizophrenia cases were compared. A) Pan GR mRNA expression; B) GR-1B mRNA expression; C) GR-1C mRNA expression; D) GR-1H mRNA expression. For all transcripts, GR transcript expression was decreased in suicide-negative schizophrenia cases relative to suicide-positive schizophrenia cases and relative to controls (all suicide-negative). For the GR-1C mRNA transcript, expression was decreased in bipolar disorder as well as schizophrenia in suicide-negative cases relative to both suicide-positive cases and controls. Green, orange and blue diamonds represent control, bipolar disorder and schizophrenia cases respectively. Red bars represent group means. Abbreviations: BP- bipolar disorder, SCZ- schizophrenia. * <i>p</i>≤0.05, ** <i>p</i><0.005, *** <i>p</i><0.0005.</p

Endpoint PCR primers used for detection of GR exon 1 mRNA transcript variants.

<p>Endpoint PCR primers used for detection of GR exon 1 mRNA transcript variants.</p

Demographic details of cases from the Stanley Array cohort used to quantify GR exon 1 mRNA transcript variant expression.

<p>Abbreviations: M- male, F- female, L- left, R- right, PMI- post-mortem interval, RIN- RNA integrity number. Data quoted are mean (range) +/− standard deviation.</p

Effects of rs10052957 (Tth111l) and rs6190 (R23K) genotypes on GR mRNA expression.

<p>A) A significant main effect of rs10052957 genotype on GR-1B mRNA expression was identified (ANCOVA F(2, 81) = 5.32, <i>p</i><0.001), with significant decreases in GR-1B expression in individuals with CC genotype (n = 46) relative to those with TC genotype (n = 40; 18.4% decrease, <i>p</i><0.05) and TT genotype (n = 9; 31.8% decrease, <i>p</i><0.005). B) An effect of rs6190 genotype on GR-1C mRNA expression was also observed (Mann-Whitney U-test, z = 2.58, <i>p</i><0.01), with a 24.8% decrease in GG homozygotes (n = 91) relative to GA heterozygotes (n = 4). Error bars represent SEM. * <i>p</i><0.05, ** <i>p</i><0.005.</p

Taqman primers/probes used for qPCR analysis of pan GR mRNA and GR exon 1 mRNA transcript variants. HK- housekeeping gene.

<p>Taqman primers/probes used for qPCR analysis of pan GR mRNA and GR exon 1 mRNA transcript variants. HK- housekeeping gene.</p

Effect of rs41423247 (Bcl1) genotype on GRα protein expression in the DLPFC.

<p>There were significant main effects of genotype on IR band 2 intensity (ANCOVA, F(2, 87) = 4.85, <i>p</i><0.05) and total IR bands 1–5 (ANOVA F(2, 86) = 3.28, <i>p</i><0.05). The abundance of IR band 2 (67 kDa GRα) was significantly decreased in rs41423247 GG homozygotes (n = 11; 26.5% decrease, <i>p</i><0.05) and GC heterozygotes (n = 43; 23.4% decrease, <i>p</i><0.01) relative to CC homozygotes (n = 41). The total GRα abundance (IR bands 1–5) was decreased (15.1%) in GC heterozygotes relative to CC homozygotes (<i>p</i><0.05). * p<0.05, ** p = 0.006.</p

Details of NR3C1 SNPs genotyped in this study.

<p>Abbreviations: UCSC- University of California Santa Cruz, MAF- minor allele frequency, HWE- Hardy-Weinberg Equilibrium.</p

Endpoint PCR detection of GR exon 1 mRNA transcript variants in the DLPFC.

<p>A) strong amplification of GR-1B, GR-1C and GR-1H variants was evident in DLPFC cDNA, B) weaker amplification of GR-1E and GR-1F variants was observed in the DLPFC, but strong amplification was observed in universal human cDNA, C) GR-1A and GR-1D variants were detected neither in the DLPFC nor in universal cDNA. UNI- universal human cDNA.</p

Characterisation of Genetic Variation in <i>ST8SIA2</i> and Its Interaction Region in NCAM1 in Patients with Bipolar Disorder

<div><p>Alpha-2,8-sialyltransferase 2 (ST8SIA2) is an enzyme responsible for the transfer of polysialic acid (PSA) to glycoproteins, principally the neuronal cell adhesion molecule (NCAM1), and is involved in neuronal plasticity. Variants within <i>ST8SIA2</i> have previously shown association with bipolar disorder, schizophrenia and autism. In addition, altered PSA-NCAM expression in brains of patients with schizophrenia or bipolar disorder indicates a functional dysregulation of glycosylation in mental illness. To explore the role of sequence variation affecting PSA-NCAM formation, we conducted a targeted re-sequencing study of a ∼100 kb region – including the entire ST8SIA2 gene and its region of interaction with NCAM1 – in 48 Caucasian cases with bipolar disorder using the Roche 454 platform. We identified over 400 DNA variants, including 47 putative novel variants not described in dbSNP. Validation of a subset of variants via Sequenom showed high reliability of Roche 454 genotype calls (97% genotype concordance, with 80% of novel variants independently verified). We did not observe major loss-of-function mutations that would affect PSA-NCAM formation, either by ablating ST8SIA2 function or by affecting the ability of NCAM1 to be glycosylated. However, we identified 13 SNPs in the UTRs of <i>ST8SIA2</i>, a synonymous coding SNP in exon 5 (rs2305561, P207P) and many additional non-coding variants that may influence splicing or regulation of <i>ST8SIA2</i> expression. We calculated nucleotide diversity within <i>ST8SIA2</i> on specific haplotypes, finding that the diversity on the specific “risk” and “protective” haplotypes was lower than other non-disease-associated haplotypes, suggesting that putative functional variation may have arisen on a spectrum of haplotypes. We have identified common and novel variants (rs11074064, rs722645, 15∶92961050) that exist on a spectrum of haplotypes, yet are plausible candidates for conferring the effect of risk and protective haplotypes via multiple enhancer elements. A Galaxy workflow/pipeline for sequence analysis used herein is available at: <a href="https://main.g2.bx.psu.edu/u/a-shaw-neura/p/next-generation-resources" target="_blank">https://main.g2.bx.psu.edu/u/a-shaw-neura/p/next-generation-resources</a>.</p></div

Genotype concordance between 454-generated genotype calls and those directly genotyped via Illumina GoldenGate or Illumina 660 W.

<p>Genotype calls are divided into two categories: all calls; or confident genotype calls based on Genotype Quality (GQ) ≥ 10. Call accuracy gives the concordance between the SNP genotype derived from 454 data and Illumina-generated data across all samples excluding missing data, and the call rate incorporates missing data on an individual basis. These are calculated for all genotypes, and heterozygous (het) calls only.</p