Matthew Hill

The 5-HT2C receptor and antipsychoticinduced weight gain – mechanisms and genetics:

The mechanisms underlying weight gain resulting from antipsychotic drugs are not fully understood, although antagonism of the 5-HT2C receptor is likely to contribute. Animal studies indicate that the drugs most likely to cause weight gain, clozapine and olanzapine, have direct effects on the NPY-containing neurons of the hypothalamus; these neurons mediate the effects of the circulating anorexigenic hormone leptin on the control of food intake. The substantial differences between individuals in the extent of antipsychotic-induced weight gain suggest that genetic factors may be important. We have been studying pharmacogenetic correlates and find that a common 5-HT2C receptor promoter region polymorphisms demonstrates strong associations with weight gain in two first episode psychotic samples. In both series, we have found further association of antipsychotic drug-induced weight gain with a common and functional polymorphism of the gene for leptin. Along with initial BMI, these two pharmacogenetic factors account for almost 30% of the variance in drug-induced weight gain. Interestingly, the 5-HT2C polymorphism appears to determine levels of circulating leptin, providing a potential mechanism underlying the genetic association of the 5-HT2C receptor with weight gain. We have undertaken functional studies of haplotypes of the 5-HT2C promoter region and find the allele associated with protection from weight gain results in reduced promoter activity. These findings demonstrate the value of pharmacogenetics in determining liability to a major side effect of antipsychotic treatment, and indicate both the molecular and physiological mechanisms underlying this side effect.

Knockdown of the psychosis susceptibility gene ZNF804A alters expression of genes involved in cell adhesion

Genome-wide association studies have convincingly implicated several novel genes in susceptibility to schizophrenia and bipolar disorder. The first genome-wide significant association with the broad phenotype of psychosis was with a polymorphism in the ZNF804A gene. However, the biological function(s) of ZNF804A have, to date, been entirely unknown. In this study, we manipulated the expression of ZNF804A in neural progenitor cells derived from human cortical neuroepithelium and assessed its effects on the cellular transcriptome. Gene ontology analysis of differentially expressed genes indicated a significant effect of ZNF804A knockdown on the expression of genes involved in cell adhesion, suggesting a role for ZNF804A in processes such as neural migration, neurite outgrowth and synapse formation. Several highly significant gene expression changes were confirmed in repeat cell culture experiments. Most consistent gene expression changes were seen for C2ORF80, a gene of as-yet-unknown function, and STMN3, a gene involved in neurite outgrowth and axonal and dendritic branching. These data, generated in a hypothesis-free manner, provide a basis for more targeted investigations of ZNF804A function.

Evidence That Schizophrenia Risk Variation in the ZNF804A Gene Exerts Its Effects During Fetal Brain Development

OBJECTIVEnThe single-nucleotide polymorphism rs1344706, located within an intron of the ZNF804A gene, exhibits genome-wide significant association with schizophrenia. Although genotype at rs1344706 is associated with altered functional brain connectivity, the molecular mechanisms mediating its association with schizophrenia have not been clearly defined. Given its location in noncoding sequence, the authors tested association between rs1344706 and ZNF804A expression in adult and fetal human brain.nnnMETHODnHighly quantitative measures of relative allelic expression were used to assess the effect of rs1344706 genotype on the mRNA expression of ZNF804A in the dorsolateral prefrontal cortex, hippocampus, and substantia nigra of the adult human brain and in human brain tissue from the first and second trimester of gestation.nnnRESULTSnGenotype at rs1344706 had no significant effect on the regulation of ZNF804A in any of the three adult brain regions examined. In contrast, rs1344706 genotype had a significant effect on ZNF804A allelic expression in second-trimester fetal brain, with the schizophrenia risk (T) allele associated with reduced ZNF804A expression.nnnCONCLUSIONSnThe T allele of rs1344706 is associated with a relative decrease in ZNF804A expression during the second trimester of fetal brain development. These data provide evidence for a schizophrenia risk mechanism that is operational in (and possibly specific to) the fetal brain.

The emerging roles of TCF4 in disease and development

Genome-wide association studies have identified common variants in transcription factor 4 (TCF4) as susceptibility loci for schizophrenia, Fuchs endothelial corneal dystrophy, and primary sclerosing cholangitis. By contrast, rare TCF4 mutations cause Pitt-Hopkins syndrome, a disorder characterized by intellectual disability and developmental delay, and have also been described in patients with other neurodevelopmental disorders. TCF4 therefore sits at the nexus between common and rare disorders. TCF4 interacts with other basic helix-loop-helix proteins, forming transcriptional networks that regulate the differentiation of several distinct cell types. Here, we review the role of TCF4 in these seemingly diverse disorders and discuss recent data implicating TCF4 as an important regulator of neurodevelopment and epithelial-mesenchymal transition.

5-HT2C receptor gene polymorphisms associated with antipsychotic drug action alter promoter activity

Polymorphisms within the 5-HT(2C) receptor gene promoter have been associated with several physiological and psychiatric phenotypes. Notably, the -759T allele has been associated with resistance to antipsychotic induced weight gain. This study assessed the activity of four promoter haplotypes expressed as luciferase constructs in the SH-SY5Y human neuroblastoma cell line in the presence or absence of a constitutively active 5-HT(2C) receptor. The presence of either -759T or -697C alleles reduced promoter activity. In addition a haplotype associated with resistance to antipsychotic induced weight showed under activity compared to the most common haplotype. In the presence of the 5-HT(2C) receptor a similar pattern of promoter activity was observed. Both -759C/T and -697G/C polymorphic sites are likely to play a role in basal promoter activity. Resistance to weight gain may, in part, be mediated by the consequences of reduced 5-HT(2C) receptor expression.

Transcriptional consequences of schizophrenia candidate miR-137 manipulation in human neural progenitor cells

MIR137, transcribed as the microRNA miR-137, is one of the leading candidate schizophrenia susceptibility genes to arise from large genome-wide association studies (GWAS) of the disorder. Recent data suggest that miR-137 modulates the expression of other schizophrenia susceptibility genes. Although bioinformatic resources are available with which to predict genes regulated by individual microRNA, there has been a lack of empirical data on genome-wide gene expression changes following miR-137 manipulation. We have therefore performed a genome-wide assessment of transcriptional changes in a human neural progenitor cell line after miR-137 over-expression and inhibition in order to elucidate molecular pathways by which genetic perturbation of miR-137 could promote susceptibility to schizophrenia. Bioinformatically-predicted miR-137 targets showed a small but highly significant down-regulation following miR-137 over-expression. Genes that were significantly down-regulated in association with miR-137 over-expression were enriched for involvement in neuronal differentiation. Differentially expressed genes that were confirmed by qPCR included others at genome-wide significant risk loci for schizophrenia (MAD1L1 and DPYD) and BDNF. These data point to molecular pathways through which genetic variation at the MIR137 locus could confer risk for schizophrenia.

Effects of cis-regulatory variation differ across regions of the adult human brain

Cis-regulatory variation is considered to be an important determinant of human phenotypic variability, including susceptibility to complex disease. Recent studies have shown that the effects of cis-regulatory polymorphism on gene expression can differ widely between tissues. In the present study, we tested whether the effects of cis-regulatory variation can also differ between regions of the adult human brain. We used relative allelic expression to measure cis-effects on the RNA expression of five candidate genes for neuropsychiatric illness (ZNF804A, NOS1, RGS4, AKT1 and TCF4) across multiple discrete brain regions within individual subjects. For all five genes, we observed significant differences in allelic expression between brain regions in several individual subjects, suggesting regional differences in the effects of cis-regulatory polymorphism to be a common phenomenon. As well as highlighting an important caveat for studies of regulatory polymorphism in the brain, our findings indicate that it is possible to delineate brain areas in which cis-regulatory variants are active. This may provide important insights into the fundamental biology of neuropsychiatric phenotypes with which such variants are associated.

Allelic differences in nuclear protein binding at a genome-wide significant risk variant for schizophrenia in ZNF804A.

Allelic differences in nuclear protein binding at a genome-wide significant risk variant for schizophrenia in ZNF804A

Functional consequences of two HTR2C polymorphisms associated with antipsychotic-induced weight gain

BACKGROUNDnGenetic variation in the promoter region of HTR2C encoding for the 5-HT(2C) receptor is associated with antipsychotic-induced weight gain. Several studies have investigated the regulatory potential of associated variants using gene-reporter systems. Establishing associated polymorphisms as causal variants may aid in the identification of the molecular mechanisms of phenotypic variation.nnnAIMS & METHODSnTo this end we examined the binding of nuclear factors from rat hypothalamus to two polymorphisms in HTR2C, rs3813929 (-759C/T) and rs518147 (-697C/G) using electromobility shift assays. For rs518147, allele-specific RNA folding was also investigated.nnnRESULTSnBoth polymorphisms bound nuclear factors, identifying the sequence fragments as regulatory elements. Importantly, rs3813929 (-759C/T) altered DNA-protein interactions with the weight gain-resistant allele abolishing the formation of two complexes. The formation of allele-specific RNA loops was also observed for rs518147.nnnCONCLUSIONnThese data establish rs3813929 (-759C/T) as a functional polymorphism and suggest disruption of DNA-protein interactions as a mechanism by which HTR2C expression is perturbed leading to an influence on antipsychotic-induced weight gain.

Functional analysis of intron 8 and 3 ' UTR variable number of tandem repeats of SLC6A3: differential activity of intron 8 variants

Association studies have found that variation in the dopamine transporter gene (SLC6A3) is important in the susceptibility to attention-deficit hyperactivity disorder (ADHD) and response to methylphenidate treatment. An understanding of the biological mechanisms underlying these associations is still inconclusive. We assessed the relative activity of variable number tandem repeat (VNTR) alleles of SLC6A3 under basal and stimulated cellular conditions, as well as in the presence of pharmacological blockade of the dopamine transporter using gene-reporter constructs. The intron 8 VNTR 5-repeat allele is more active than the 6-repeat allele. In the presence of forskolin, both alleles were significantly induced. Blockade of the dopamine transporter did not influence activity of either allelic construct. No difference in activity between 9- and 10-repeat alleles of the 3′-untranslated region VNTR was observed under any experimental condition. These data suggest that the intron 8 VNTR is a functional variant with an ADHD susceptibility allele having reduced activity. The lack of enhanced allele-specific activity in response to treatment regimes suggests that differential activity under basal conditions is the primary mode of action.