BackgroundWe have previously reported linkage of markers on chromosome 1q22 to schizophrenia, a finding supported by several independent studies. Within this linkage region, we have identified significant linkage disequilibrium between schizophrenia and markers within the gene for carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON). Prior sequencing of the ten exons of CAPON failed to reveal a coding mutation associated with illness.Methods and FindingsWe screened a human fetal brain cDNA library and identified a new isoform of CAPON that consists of the terminal two exons of the gene, and verified the expression of the predicted corresponding protein in human dorsolateral prefrontal cortex (DLPFC). We examined the expression levels of both the ten-exon CAPON transcript and this new isoform in postmortem brain samples from the Stanley Array Collection. Quantitative real-time PCR analysis of RNA from the DLPFC in 105 individuals (35 with schizophrenia, 35 with bipolar disorder, and 35 psychiatrically normal controls) revealed significantly (p < 0.005) increased expression of the new isoform in both schizophrenia and bipolar disorder. Furthermore, this increased expression was significantly associated (p < 0.05) with genotype at three single-nucleotide polymorphisms previously identified as being in linkage disequilibrium with schizophrenia.ConclusionBased on the known interactions between CAPON, neuronal nitric oxide synthase (nNOS), and proteins associated with the N-methyl-D-aspartate receptor (NMDAR) complex, overexpression of either CAPON isoform would be expected to disrupt the association between nNOS and the NMDAR, leading to changes consistent with the NMDAR hypofunctioning hypothesis of schizophrenia. This study adds support to a role of CAPON in schizophrenia, produces new evidence implicating this gene in the etiology of bipolar disorder, and suggests a possible mechanism of action of CAPON in psychiatric illness.

To what extent and in what way do gene promoters and their transacting regulatory proteins coevolve? In this and in earlier publications we show that the Bicoid-dependent promoters of the segmentation genes hunchback and tailless in species of higher Diptera (Drosophila, Musca, Calliphora, and Lucilia) are different with respect to the copy number, spacing, sequence, and orientation of Bicoid binding sites. At the same time there are significant amino acid differences in the Bicoid homeodomain. To test these interspecific differences, we used a series of functional assays, starting with the analysis of Bicoid binding affinities of individual sites, through to transgene rescue experiments, to compare within-species with between-species mixtures of Bicoid homeodomains and hunchback or tailless promoters. We observed that components taken from different species interact with less efficiency compared with those taken from within the same species. Our interpretation is that such interspecific incompatibilities are a consequence of interactive genetic elements coevolving one with another, hence maintaining functional compatibility within each species. At the same time such a process allows differences to accumulate between species regarding the precise molecular basis whereby the common function is effected.

Interacting genetic elements need to coevolve if their joint function is to be maintained; for example, the correct binding of transcriptional regulators to defined binding sites in gene promoters needs to be maintained during evolution to ensure proper function. As part of a wider investigation into the molecular coevolution of the Dipteran homeodomain-bearing regulator bicoid (bcd) and Bcd-dependent promoters, we present data on the functional, structural, and sequence differences between the promoters of the segmentation gene hunchback (hb), in several species of Cyclorrhaphan (higher) Diptera. The result of phenocopying hb mutations using RNA interference (RNAi) in Musca domestica shows broadly similar functions to the hb gene in Drosophila melanogaster. However, the Bcd-binding sites in the hb promoters of Drosophila, Musca, and the two blowfly species Lucilia sericata and Calliphora vicina differ in copy number, sequence, orientation, and spacing. Furthermore, all promoters are subject to rapid turnover by slippage-like processes leading to high densities of short repetitive motifs. A study of polymorphism among six strains of M. domestica reveals that turnover by slippage also occurs in the promoter, untranslated leader, and exonic coding sequences of hb, but to different extents. We discuss these results in terms of the known interspecific differences in bcdand the potential coevolution of selected compensatory mutations in trans and cis in response to continuous promoter restructuring.

Objective The authors previously demonstrated significant association between markers within NOS1AP and schizophrenia in a set of Canadian families of European descent, as well as significantly increased expression in schizophrenia of NOS1AP in unrelated postmortem samples from the dorsolateral prefrontal cortex. In this study the authors sought to apply novel statistical methods and conduct additional biological experiments to isolate at least one risk allele within NOS1AP. Method Using the posterior probability of linkage disequilibrium (PPLD) to measure the probability that a single nucleotide polymorphism (SNP) is in linkage disequilibrium with schizophrenia, the authors evaluated 60 SNPs from NOS1AP in 24 Canadian families demonstrating linkage and association to this region. SNPs exhibiting strong evidence of linkage disequilibrium were tested for regulatory function by luciferase reporter assay. Two human neural cell lines (SK-N-MC and PFSK-1) were transfected with a vector containing each allelic variant of the SNP, the NOS1AP promoter, and a luciferase gene. Alleles altering expression were further assessed for binding of nuclear proteins by electrophoretic mobility shift assay. Results Three SNPs produced PPLDs >40%. One of them, rs12742393, demonstrated significant allelic expression differences in both cell lines tested. The allelic variation at this SNP altered the affinity of nuclear protein binding to this region of DNA. Conclusions The A allele of rs12742393 appears to be a risk allele associated with schizophrenia that acts by enhancing transcription factor binding and increasing gene expression.

To further understand the evolutionary dynamics of the regulatory interactions underlying development, we expand on our previous analysis of hunchback and compare the structure and function of the tailless enhancer between Musca domestica and Drosophila melanogaster. Our analysis shows that although the expression patterns and functional protein domains of tll are conserved between Musca and Drosophila, the enhancer sequences are unalignable. Upon closer investigation, we find that these highly diverged enhancer sequences encode the same regulatory information necessary for Bicoid, Dorsal, and the terminal system to drive tll expression. The binding sites for these transcription factors differ in the sequence, number, spacing, and position between the Drosophila and Musca tll enhancers, and we were unable to establish homology between binding sites from each species. This implies that the Musca and Drosophila Bcd-binding sites have evolved de novo in the 100 million years since these species diverged. However, in transgenic Drosophila embryos the Musca tll enhancer is able to drive the same expression pattern as endogenous Drosophila tll. Therefore, during the rapid evolution of enhancer sequences individual binding sites are continually lost and gained, but the transcriptional output is maintained by compensatory mutations in cis and in trans.