Transcriptional regulation of neurodevelopmental and metabolic pathways by NPAS3.

The basic helix-loop-helix PAS (Per, Arnt, Sim) domain transcription factor gene NPAS3 is a replicated genetic risk factor for psychiatric disorders. A knockout (KO) mouse model exhibits behavioral and adult neurogenesis deficits consistent with human illness. To define the location and mechanism of NPAS3 etiopathology, we combined immunofluorescent, transcriptomic and metabonomic approaches. Intense Npas3 immunoreactivity was observed in the hippocampal subgranular zone-the site of adult neurogenesis--but was restricted to maturing, rather than proliferating, neuronal precursor cells. Microarray analysis of a HEK293 cell line over-expressing NPAS3 showed that transcriptional targets varied according to circadian rhythm context and C-terminal deletion. The most highly up-regulated NPAS3 target gene, VGF, encodes secretory peptides with established roles in neurogenesis, depression and schizophrenia. VGF was just one of many NPAS3 target genes also regulated by the SOX family of transcription factors, suggesting an overlap in neurodevelopmental function. The parallel repression of multiple glycolysis genes by NPAS3 reveals a second role in the regulation of glucose metabolism. Comparison of wild-type and Npas3 KO metabolite composition using high-resolution mass spectrometry confirmed these transcriptional findings. KO brain tissue contained significantly altered levels of NAD(+), glycolysis metabolites (such as dihydroxyacetone phosphate and fructose-1,6-bisphosphate), pentose phosphate pathway components and Kreb's cycle intermediates (succinate and α-ketoglutarate). The dual neurodevelopmental and metabolic aspects of NPAS3 activity described here increase our understanding of mental illness etiology, and may provide a mechanism for innate and medication-induced susceptibility to diabetes commonly reported in psychiatric patients.

Interacting haplotypes at the NPAS3 locus alter risk of schizophrenia and bipolar disorder.

The neuronal PAS domain 3 (NPAS3) gene encodes a neuronal transcription factor that is implicated in psychiatric disorders by the identification of a human chromosomal translocation associated with schizophrenia and a mouse knockout model with behavioural and hippocampal neurogenesis defects. To determine its contribution to the risk of psychiatric illness in the general population, we genotyped 70 single-nucleotide polymorphisms across the NPAS3 gene in 368 individuals with bipolar disorder, 386 individuals with schizophrenia and 455 controls. Modestly significant single-marker and global and individual haplotypes were identified in four discrete regions of the gene. The presence of both risk and protective haplotypes at each of these four regions indicated locus and allelic heterogeneity within NPAS3 and suggested a model whereby interactions between variants across the gene might contribute to susceptibility to illness. This was supported by predicting the most likely haplotype for each individual at each associated region and then calculating an NPAS3-mediated 'net genetic load' value. This value differed significantly from controls for both bipolar disorder (P=0.0000010) and schizophrenia (P=0.0000012). Logistic regression analysis also confirmed the combinatorial action of the four associated regions on disease risk. In addition, sensitivity/specificity plots showed that the extremes of the genetic loading distribution possess the greatest predictive power-a feature suggesting multiplicative allele interaction. These data add to recent evidence that the combinatorial analysis of a number of relatively small effect size haplotypes may have significant power to predict an individual's risk of a complex genetic disorder such as psychiatric illness.

Chromosome abnormalities, mental retardation and the search for genes in bipolar disorder and schizophrenia.

Genetic factors contribute to schizophrenia and bipolar disorder, and linkage and association studies have been successful in identifying several candidate genes. However these genes explain only a very small part of the total population risk and the psychoses appear to be very heterogeneous with several models of genetic inheritance relevant to different groups of patients, including some cases caused by multiple common genetic variants, while others are single gene disorders. Studying chromosomal abnormalities is a useful strategy for identifying genes in illness, and patients with both mental retardation and psychosis form a special group where large chromosomal abnormalities detected by routine cytogenetic analysis are more prevalent than in patients with schizophrenia or bipolar disorder alone, or in the general population. Studying these patients provides valuable opportunities to identify genes contributing to psychoses. This review of the literature on large chromosomal rearrangements in patients with mental retardation and psychotic illness illustrates how schizophrenia and bipolar phenotypes are associated with a large number of different chromosomal disruptions. Recent genome wide association studies have identified an excess of small chromosomal deletions and duplications in schizophrenia, adding further support to the importance of chromosomal structural variation in psychotic illness. The genes GRIK4 and NPAS3, each associated with psychosis in patients with mental retardation are discussed to illustrate the value of rare cytogenetic events as a means to signpost neurobiological pathways of general importance for illness in the wider population.

A common variant in the 3'UTR of the GRIK4 glutamate receptor gene affects transcript abundance and protects against bipolar disorder.

Underactivity of the glutamatergic system is an attractive model for the pathophysiology of several major mental illnesses. We previously described a chromosome abnormality disrupting the kainate class ionotropic glutamate receptor gene, GRIK4/KA1, in an individual with schizophrenia and learning disability (mental retardation). We also demonstrated in a case-control study that two physically separated haplotypes within this gene were significantly associated with increased risk of schizophrenia and decreased risk of bipolar disorder, respectively. The latter protective haplotype was located at the 3' end of the gene. We now report the identification from carriers of the protective haplotype of a deletion variant within the 3' untranslated region of the gene. The deletion allele also was found to be negatively associated with bipolar disorder in both initial (P = 0.00000019) and replication (P = 0.0107) case-control studies. Expression studies indicated that deletion-carrying mRNA transcripts were relatively more abundant. We postulate that this may be a direct consequence of the differences in the RNA secondary structures predicted for the insertion and deletion alleles. These data suggest a mechanism whereby the genetic protective effect is mediated through increased kainate receptor expression.

Association analysis of the chromosome 4p15-p16 candidate region for bipolar disorder and schizophrenia.

Several independent linkage studies have identified chromosome 4p15-p16 as a putative region of susceptibility for bipolar disorder (BP), schizophrenia (SCZ) and related phenotypes. Previously, we identified two subregions (B and D) of the 4p15-p16 region that are shared by three of four 4p-linked families examined. Here, we describe a large-scale association analysis of regions B and D (3.8 and 4.5 Mb, respectively). We selected 408 haplotype-tagging single nucleotide polymorphisms (SNPs) on a block-by-block basis from the International HapMap project and tested them in 368 BP, 386 SCZ and 458 control individuals. Nominal significance thresholds were determined using principal component analysis as implemented in the program SNPSpD. In region B, overlapping SNPs and haplotypes met the region-wide threshold (P

Association of Neuregulin 1 with schizophrenia and bipolar disorder in a second cohort from the Scottish population.

Neuregulin 1 (NRG1) is a strong candidate for involvement in the aetiology of schizophrenia. A haplotype, initially identified as showing association in the Icelandic and Scottish populations, has shown a consistent effect size in multiple European populations. Additionally, NRG1 has been implicated in susceptibility to bipolar disorder. In this first study to select markers systematically on the basis of linkage disequilibrium across the entire NRG1 gene, we used haplotype-tagging single-nucleotide polymorphisms to identify single markers and haplotypes associated with schizophrenia and bipolar disorder in an independently ascertained Scottish population. Haplotypes in two regions met an experiment-wide significance threshold of P=0.0016 (Nyholt's SpD) and were permuted to correct for multiple testing. Region A overlaps with the Icelandic haplotype and shows nominal association with schizophrenia (P=0.00032), bipolar disorder (P=0.0011), and the combined case group (P=0.0017). This region includes the 5' exon of the NRG1 GGF2 isoform and overlaps the expressed sequence tag (EST) cluster Hs.97362. However, no haplotype in Region A remains significant after permutation analysis (P>0.05). Region B contains a haplotype associated with both schizophrenia (P=0.00014), and the combined case group (P=0.000062), although it does not meet Nyholt's threshold in bipolar disorder alone (P=0.0022). This haplotype remained significant after permutation analysis in both the schizophrenia and combined case groups (P=0.024 and P=0.016, respectively). It spans a approximately 136 kb region that includes the coding sequence of the sensory and motor neuron derived factor (SMDF) isoform and 3' exons of all other known NRG1 isoforms. Our study identifies a new of NRG1 region involved in schizophrenia and bipolar disorder in the Scottish population.

Cytogenetic and genetic evidence supports a role for the kainate-type glutamate receptor gene, GRIK4, in schizophrenia and bipolar disorder.

In the search for the biological causes of schizophrenia and bipolar disorder, glutamate neurotransmission has emerged as one of a number of candidate processes and pathways where underlying gene deficits may be present. The analysis of chromosomal rearrangements in individuals diagnosed with neuropsychiatric disorders is an established route to candidate gene identification in both Mendelian and complex disorders. Here we describe a set of genes disrupted by, or proximal to, chromosomal breakpoints (2p12, 2q31.3, 2q21.2, 11q23.3 and 11q24.2) in a patient where chronic schizophrenia coexists with mild learning disability (US: mental retardation). Of these disrupted genes, the most promising candidate is a member of the kainate-type ionotropic glutamate receptor family, GRIK4 (KA1). A subsequent systematic case-control association study on GRIK4 assessed its contribution to psychiatric illness in the karyotypically normal population. This identified two discrete regions of disease risk within the GRIK4 locus: three single single nucleotide polymorphism (SNP) markers with a corresponding underlying haplotype associated with susceptibility to schizophrenia (P=0.0005, odds ratio (OR) of 1.453, 95% CI 1.182-1.787) and two single SNP markers and a haplotype associated with a protective effect against bipolar disorder (P=0.0002, OR of 0.624, 95% CI 0.485-0.802). After permutation analysis to correct for multiple testing, schizophrenia and bipolar disorder haplotypes remained significant (P=0.0430, s.e. 0.0064 and P=0.0190, s.e. 0.0043, respectively). We propose that these convergent cytogenetic and genetic findings provide molecular evidence for common aetiologies for different psychiatric conditions and further support the 'glutamate hypothesis' of psychotic illness.

SUSPECTS: enabling fast and effective prioritization of positional candidates.

UNLABELLED: SUSPECTS is a web-based server which combines annotation and sequence-based approaches to prioritize disease candidate genes in large regions of interest. It uses multiple lines of evidence to rank genes quickly and effectively while limiting the effect of annotation bias to significantly improve performance. AVAILABILITY: SUSPECTS is freely available at http://www.genetics.med.ed.ac.uk/suspects/ SUPPLEMENTARY INFORMATION: A quick-start guide in Macromedia Flash format is available at http://www.genetics.med.ed.ac.uk/suspects/help.shtml and Excel spreadsheets detailing the comparative performance of the software are included as Supplementary material.

Cytogenetics and gene discovery in psychiatric disorders.

The disruption of genes by balanced translocations and other rare germline chromosomal abnormalities has played an important part in the discovery of many common Mendelian disorder genes, somatic oncogenes and tumour supressors. A search of published literature has identified 15 genes whose genomic sequences are directly disrupted by translocation breakpoints in individuals with neuropsychiatric illness. In these cases, it is reasonable to hypothesise that haploinsufficiency is a major factor contributing to illness. These findings suggest that the predicted polygenic nature of psychiatric illness may not represent the complete picture; genes of large individual effect appear to exist. Cytogenetic events may provide important insights into neurochemical pathways and cellular processes critical for the development of complex psychiatric phenotypes in the population at large.

Chromosomal abnormalities and mental illness.

Linkage studies of mental illness have provided suggestive evidence of susceptibility loci over many broad chromosomal regions. Pinpointing causative gene mutations by conventional linkage strategies alone is problematic. The breakpoints of chromosomal abnormalities occurring in patients with mental illness may be more direct pointers to the relevant gene locus. Publications that describe patients where chromosomal abnormalities co-exist with mental illness are reviewed along with supporting evidence that this may amount to an association. Chromosomal abnormalities are considered to be of possible significance if (a) the abnormality is rare and there are independent reports of its coexistence with psychiatric illness, or (b) there is colocalisation of the abnormality with a region of suggestive linkage findings, or (c) there is an apparent cosegregation of the abnormality with psychiatric illness within the individual's family. Breakpoints have been described within many of the loci suggested by linkage studies and these findings support the hypothesis that shared susceptibility factors for schizophrenia and bipolar disorder may exist. If these abnormalities directly disrupt coding regions, then combining molecular genetic breakpoint cloning with bioinformatic sequence analysis may be a method of rapidly identifying candidate genes. Full karyotyping of individuals with psychotic illness especially where this coexists with mild learning disability, dysmorphism or a strong family history of mental disorder is encouraged.

Epigenetic targeting in the mouse zygote marks DNA for later methylation: a mechanism for maternal effects in development.

The transgenic sequences in the mouse line TKZ751 are demethylated on a DBA/2 inbred strain background but become highly methylated at postimplantation stages in offspring of a cross with a BALB/c female. In the reciprocal cross the transgene remains demethylated suggesting that imprinted BALB/c methylation modifiers or egg cytoplasmic factors are responsible for this striking maternal effect on de novo methylation. Reciprocal pronuclear transplantation experiments were carried out to distinguish between these mechanisms. The results indicate that a maternally-derived oocyte cytoplasmic factor from BALB/c marks the TKZ751 sequences at fertilization; this mark and postzygotic BALB/c modifiers are both required for de novo methylation of the target sequences at postimplantation stages. Using genetic linkage analyses we mapped the maternal effect to a locus on chromosome 17. Moreover, seven postzygotic modifier loci were identified that increase the postimplantation level of methylation. Analysis of interactions between the maternal and the postzygotic loci shows that both are needed for de novo methylation in the offspring. The combined experiments thus reveal a novel epigenetic marking process at fertilization which targets DNA for later methylation in the foetus. The most significant consequence is that the genotype of the mother can influence the epigenotype of the offspring by this marking process. A number of parental and imprinting effects may be explained by this epigenetic marking.

A dominant modifier of transgene methylation is mapped by QTL analysis to mouse chromosome 13.

The single-copy hepatitis B virus transgene in the E36 transgenic mouse strain undergoes methylation changes in a parent-of-origin, tissue, and strain-specific fashion. In a C57BL/6 background, the paternally transmitted transgene is methylated in 30% of cells, whereas it is methylated in more than 80% of cells in (BALB/c x C57BL/6) F1 mice. We established previously that several genetic factors were likely to contribute to the transgene methylation profile, some with demethylating and some with de novo methylating activities. Using quantitative trait loci (QTL) mapping, we have now localized one major modifier locus on chromosome 13 (Mod13), which explains a 30% increase in the methylation level of this transgene with no effect on the flanking endogenous sequences. No other QTL could be identified, except for a demethylating activity of low significance located on chromosome 12. Recombinant inbred mice containing a BALB/c allele of Mod13 were then used to show that the presence of Mod13 is sufficient to induce de novo methylation. A segregation between de novo methylation and repression of transgene expression was uncovered, suggesting that this genetic system is also useful for the identification of factors that interpret methylation patterns in the genome.

Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices.

Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum.

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

Imprinting mechanisms.

A number of recent studies have provided new insights into mechanisms that regulate genomic imprinting in the mammalian genome. Regions of allele-specific differential methylation (DMRs) are present in all imprinted genes examined. Differential methylation is erased in germ cells at an early stage of their development, and germ-line-specific methylation imprints in DMRs are reestablished around the time of birth. After fertilization, differential methylation is retained in core DMRs despite genome-wide demethylation and de novo methylation during preimplantation and early postimplantation stages. Direct repeats near CG-rich DMRs may be involved in the establishment and maintenance of allele-specific methylation patterns. Imprinted genes tend to be clustered; one important component of clustering is enhancer competition, whereby promoters of linked imprinted genes compete for access to enhancers. Regional organization and spreading of the epigenotype during development is also important and depends on DMRs and imprinting centers. The mechanism of cis spreading of DNA methylation is not known, but precedent is provided by the Xist RNA, which results in X chromosome inactivation in cis. Reading of the somatic imprints could be carried out by transcription factors that are sensitive to methylation, or by methyl-cytosine-binding proteins that are involved in transcriptional repression through chromatin remodeling.