Investigation of putative regulatory loci relevant to the pathogenesis of psychiatric illness

The genetic contribution to the aetiology of psychiatric illness is well-established; however, few variants that alter the encoded protein have been irrefutably identified as causative, leading to the hypothesis that variants affecting gene regulation may play a pathogenic role. This thesis focuses on two genes, Neuregulin 1 (NRG1) and Disrupted in Schizophrenia 1 (DISC1), for which there is strong genetic evidence for involvement in psychiatric illness, as well as evidence for altered expression in patients. Association analysis was carried out to assess the involvement of six intronic NRG1 single nucleotide polymorphisms (SNPs) in schizophrenia and bipolar disorder in two independent samples from the Scottish (Scottish 2; n = 307 control subjects, 303 schizophrenic patients, and 239 bipolar disorder patients and German populations (n = 397 control subjects, 396 schizophrenic patients, and 400 bipolar disorder patients). These SNPs form two haplotypes, one encompassing the 5’ and promoter region of the gene and the other located at the 3’ end of the gene, that were previously associated with schizophrenia and bipolar disorder in a Scottish sample (Scottish 1). The location of these haplotypes, together with the prior evidence for altered NRG1 expression in schizophrenia, suggested the potential involvement of regulatory variants. On combining the Scottish 1 and Scottish 2 samples (combined n = 765 control subjects, 682 schizophrenic patients and 601 bipolar disorder patients), a two- SNP haplotype spanning both coding and non-coding regions in the 3’ region was associated with schizophrenia (p = 0.0037, OR=1.3, 95% CI: 1.1-1.6) and the combined schizophrenia and bipolar disorder case group (p = 0.0080, OR=1.2, 95% CI: 1.1-1.5), with both these associations remaining significant after permutation analysis (p = 0.022 and p = 0.044, respectively). To further understanding of how DISC1, a leading candidate gene for schizophrenia that has also been implicated in other psychiatric disorders, is regulated the previously uncharacterised promoter region was assessed both bioinformatically and in vitro using the dual luciferase reporter assay. The region was found to lack canonical promoter motifs but to contain a CpG island, consistent with DISC1’s ubiquitous pattern of expression. A region located 300bp to -177bp relative to the transcription start site (TSS) was identified as contributing positively to DISC1 promoter activity, whilst a region -982bp to -301bp relative to the TSS was found to confer a repressive effect. FOXP2, a transcription factor which is mutated in a rare speech and language disorder and implicated in autism pathogenesis, was found to repress transcription from the DISC1 promoter. Two pathogenic FOXP2 point mutations reduced this transcriptional repression. Preliminary evidence for a bi-directional regulatory relationship between DISC1 and FOXP2 was observed: a mouse model of schizophrenia that carries a Disc1 L100P amino acid substitution and shows altered developmental Disc1 expression was also found to show altered developmental expression of Foxp2. These results further understanding of two genes whose altered expression might contribute to the pathogenesis of psychiatric illness

Preliminary investigation of miRNA expression in individuals at high familial risk of bipolar disorder

AbstractBipolar disorder (BD) is a highly heritable psychiatric disorder characterised by recurrent episodes of mania and depression. Many studies have reported altered gene expression in BD, some of which may be attributable to the dysregulated expression of miRNAs. Studies carried out to date have largely studied medicated patients, so it is possible that observed changes in miRNA expression might be a consequence of clinical illness or of its treatment. We sought to establish whether altered miRNA expression might play a causative role in the development of BD by studying young, unmedicated relatives of individuals with BD, who are at a higher genetic risk of developing BD themselves (high-risk individuals). The expression of 20 miRNAs previously implicated in either BD or schizophrenia was measured by qRT-PCR in whole-blood samples from 34 high-risk and 46 control individuals. Three miRNAs, miR-15b, miR-132 and miR-652 were up-regulated in the high-risk individuals, consistent with previous reports of increased expression of these miRNAs in patients with schizophrenia. Our findings suggest that the altered expression of these miRNAs might represent a mechanism of genetic susceptibility for BD. Moreover, our observation of altered miRNA expression in the blood prior to the onset of illness provides hope that one day blood-based tests may aid in the risk-stratification and treatment of BD

Expression of DISC1-Interactome Members Correlates with Cognitive Phenotypes Related to Schizophrenia

Cognitive dysfunction is central to the schizophrenia phenotype. Genetic and functional studies have implicated Disrupted-in-Schizophrenia 1 (DISC1), a leading candidate gene for schizophrenia and related psychiatric conditions, in cognitive function. Altered expression of DISC1 and DISC1-interactors has been identified in schizophrenia. Dysregulated expression of DISC1-interactome genes might, therefore, contribute to schizophrenia susceptibility via disruption of molecular systems required for normal cognitive function. Here, the blood RNA expression levels of DISC1 and DISC1-interacting proteins were measured in 63 control subjects. Cognitive function was assessed using neuropsychiatric tests and functional magnetic resonance imaging was used to assess the activity of prefrontal cortical regions during the N-back working memory task, which is abnormal in schizophrenia. Pairwise correlations between gene expression levels and the relationship between gene expression levels and cognitive function and N-back-elicited brain activity were assessed. Finally, the expression levels of DISC1, AKAP9, FEZ1, NDEL1 and PCM1 were compared between 63 controls and 69 schizophrenic subjects. We found that DISC1-interactome genes showed correlated expression in the blood of healthy individuals. The expression levels of several interactome members were correlated with cognitive performance and N-back-elicited activity in the prefrontal cortex. In addition, DISC1 and NDEL1 showed decreased expression in schizophrenic subjects compared to healthy controls. Our findings highlight the importance of the coordinated expression of DISC1-interactome genes for normal cognitive function and suggest that dysregulated DISC1 and NDEL1 expression might, in part, contribute to susceptibility for schizophrenia via disruption of prefrontal cortex-dependent cognitive functions

DNA methylation in a Scottish family multiply affected by bipolar disorder and major depressive disorder

Background: Bipolar disorder (BD) is a severe, familial psychiatric condition. Progress in understanding the aetiology of BD has been hampered by substantial phenotypic and genetic heterogeneity. We sought to mitigate these confounders by studying a multi-generational family multiply affected by BD and major depressive disorder (MDD), who carry an illness-linked haplotype on chromosome 4p. Within a family, aetiological heterogeneity is likely to be reduced, thus conferring greater power to detect illness-related changes. As accumulating evidence suggests that altered DNA methylation confers risk for BD and MDD, we compared genome-wide methylation between (i) affected carriers of the linked haplotype (ALH) and married-in controls (MIs), (ii) well unaffected haplotype carriers (ULH) and MI, (iii) ALH and ULH and (iv) all haplotype carriers (LH) and MI.Results: Nominally significant differences in DNA methylation were observed in all comparisons, with differences withstanding correction for multiple testing when the ALH or LH group was compared to the MIs. In both comparisons, we observed increased methylation at a locus in FANCI, which was accompanied by increased FANCI expression in the ALH group. FANCI is part of the Fanconi anaemia complementation (FANC) gene family, which are mutated in Fanconi anaemia and participate in DNA repair. Interestingly, several FANC genes have been implicated in psychiatric disorders. Regional analyses of methylation differences identified loci implicated in psychiatric illness by genome-wide association studies, including CACNB2 and the major histocompatibility complex. Gene ontology analysis revealed enrichment for methylation differences in neurologically relevant genes.Conclusions: Our results highlight altered DNA methylation as a potential mechanism by which the linked haplotype might confer risk for mood disorders. Differences in the phenotypic outcome of haplotype carriers might, in part, arise from additional changes in DNA methylation that converge on neurologically important pathways. Further work is required to investigate the underlying mechanisms and functional consequences of the observed differences in methylation

Gene co-expression within the DISC1-interactome.

a<p>Corrected by permutation analysis (10⁵ permutations).</p

Relationship between the expression levels of DISC1-interactome genes and their pairwise interactions and cognitive performance.

a<p>PC = principal component.</p>b<p>The model explaining the most variation within each PC was determined by backward removal multiple regression analysis including main effects and gene-by-gene interaction terms.</p

Relationship between the expression levels of DISC1-interactome genes and cognitive performance.

a<p>PC = principal component.</p>b<p>The model explaining the most variation within each PC was determined by backward removal multiple regression analysis including main effects only.</p

Graphical depiction of the principal components derived from performance on the neurocognitive tests.

<p>Four principal components were defined by principal components analysis. Each principal component was named according to the specific cognitive functions assessed by the tests that loaded onto it.</p

Relationship of gene expression levels to activity in the prefrontal cortex elicited by the N-back task.

a<p>PC = principal component.</p>b<p>Corrected by permutation analysis (10⁵ permutations).</p

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally