Analysis of genetic diversity in patients with major psychiatric disorders versus healthy controls: A molecular-genetic study of 1698 subjects genotyped for 100 candidate genes (549 SNPs).

BACKGROUND: This study analyzed the extent to which irregularities in genetic diversity separate psychiatric patients from healthy controls. METHODS: Genetic diversity was quantified through multidimensional "gene vectors" assembled from 4 to 8 polymorphic SNPs located within each of 100 candidate genes. The number of different genotypic patterns observed per gene was called the gene's "diversity index". RESULTS: The diversity indices were found to be only weakly correlated with their constituent number of SNPs (20.5 % explained variance), thus suggesting that genetic diversity is an intrinsic gene property that has evolved over the course of evolution. Significant deviations from "normal" diversity values were found for (1) major depression; (2) Alzheimer's disease; and (3) schizoaffective disorders. Almost one third of the genes were correlated with each other, with correlations ranging from 0.0303 to 0.7245. The central finding of this study was the discovery of "singular genes" characterized by distinctive genotypic patterns that appeared exclusively in patients but not in healthy controls. Neural Nets yielded nonlinear classifiers that correctly identified up to 90 % of patients. Overlaps between diagnostic subgroups on the genotype level suggested that (1) diagnoses-crossing vulnerabilities are likely involved in the pathogenesis of major psychiatric disorders; (2) clinically defined diagnoses may not constitute etiological entities. CONCLUSION: Detailed analyses of the variation of genotypic patterns in genes along with the correlation between genes lead to nonlinear classifiers that enable very robust separation between psychiatric patients and healthy controls on the genotype level.

Association of norepinephrine transporter methylation with in vivo NET expression and hyperactivity-impulsivity symptoms in ADHD measured with PET.

Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a robust genetic influence. The norepinephrine transporter (NET) is of particular interest as it is one of the main targets in treatment of the disorder. As ADHD is a complex and polygenetic condition, the possible regulation by epigenetic processes has received increased attention. We sought to determine possible differences in NET promoter DNA methylation between patients with ADHD and healthy controls. DNA methylation levels in the promoter region of the NET were determined in 23 adult patients with ADHD and 23 healthy controls. A subgroup of 18 patients with ADHD and 18 healthy controls underwent positron emission tomography (PET) with the radioligand (S,S)-[18F]FMeNER-D2 to quantify the NET in several brain areas in vivo. Analyses revealed significant differences in NET methylation levels at several cytosine-phosphate-guanine (CpG) sites between groups. A defined segment of the NET promoter ("region 1") was hypermethylated in patients in comparison with controls. In ADHD patients, a negative correlation between methylation of a CpG site in this region and NET distribution in the thalamus, locus coeruleus, and the raphe nuclei was detected. Furthermore, methylation of several sites in region 1 was negatively associated with the severity of hyperactivity-impulsivity symptoms. Our results point to an epigenetic dysregulation in ADHD, possibly due to a compensatory mechanisms or additional factors involved in transcriptional processing.

Genome-wide autozygosity is associated with lower general cognitive ability.

Inbreeding depression refers to lower fitness among offspring of genetic relatives. This reduced fitness is caused by the inheritance of two identical chromosomal segments (autozygosity) across the genome, which may expose the effects of (partially) recessive deleterious mutations. Even among outbred populations, autozygosity can occur to varying degrees due to cryptic relatedness between parents. Using dense genome-wide single-nucleotide polymorphism (SNP) data, we examined the degree to which autozygosity associated with measured cognitive ability in an unselected sample of 4854 participants of European ancestry. We used runs of homozygosity-multiple homozygous SNPs in a row-to estimate autozygous tracts across the genome. We found that increased levels of autozygosity predicted lower general cognitive ability, and estimate a drop of 0.6 s.d. among the offspring of first cousins (P=0.003-0.02 depending on the model). This effect came predominantly from long and rare autozygous tracts, which theory predicts as more likely to be deleterious than short and common tracts. Association mapping of autozygous tracts did not reveal any specific regions that were predictive beyond chance after correcting for multiple testing genome wide. The observed effect size is consistent with studies of cognitive decline among offspring of known consanguineous relationships. These findings suggest a role for multiple recessive or partially recessive alleles in general cognitive ability, and that alleles decreasing general cognitive ability have been selected against over evolutionary time.

Expression analysis in a rat psychosis model identifies novel candidate genes validated in a large case-control sample of schizophrenia.

Antagonists of the N-methyl-D-aspartate (NMDA)-type glutamate receptor induce psychosis in healthy individuals and exacerbate schizophrenia symptoms in patients. In this study we have produced an animal model of NMDA receptor hypofunction by chronically treating rats with low doses of the NMDA receptor antagonist MK-801. Subsequently, we performed an expression study and identified 20 genes showing altered expression in the brain of these rats compared with untreated animals. We then explored whether the human orthologs of these genes are associated with schizophrenia in the largest schizophrenia genome-wide association study published to date, and found evidence for association for 4 out of the 20 genes: SF3B1, FOXP1, DLG2 and VGLL4. Interestingly, three of these genes, FOXP1, SF3B1 and DLG2, have previously been implicated in neurodevelopmental disorders.

Neuropsychological effects of the CSMD1 genome-wide associated schizophrenia risk variant rs10503253.

The single-nucleotide polymorphism (SNP) rs10503253, located within the CUB and Sushi multiple domains-1 (CSMD1) gene on 8p23.2, was recently identified as genome-wide significant for schizophrenia (SZ), but is of unknown function. We investigated the neurocognitive effects of this CSMD1 variant in vivo in patients and healthy participants using behavioral and imaging measures of brain structure and function. We compared carriers and non-carriers of the risk 'A' allele on measures of neuropsychological performance typically impaired in SZ (general cognitive ability, episodic and working memory and attentional control) in independent samples of Irish patients (n = 387) and controls (n = 171) and German patients (205) and controls (n = 533). Across these groups, the risk 'A' allele at CSMD1 was associated with deleterious effects across a number of neurocognitive phenotypes. Specifically, the risk allele was associated with poorer performance on neuropsychological measures of general cognitive ability and memory function but not attentional control. These effects, while significant, were subtle, and varied between samples. Consistent with previous evidence suggesting that CSMD1 may be involved in brain mechanisms related to memory and learning, these data appear to reflect the deleterious effects of the identified 'A' risk allele on neurocognitive function, possibly as part of the mechanism by which CSMD1 is associated with SZ risk.

Schizophrenia shows a unique metabolomics signature in plasma.

Schizophrenia is a severe complex mental disorder affecting 0.5-1% of the world population. To date, diagnosis of the disease is mainly based on personal and thus subjective interviews. The underlying molecular mechanism of schizophrenia is poorly understood. Using targeted metabolomics we quantified and compared 103 metabolites in plasma samples from 216 healthy controls and 265 schizophrenic patients, including 52 cases that do not take antipsychotic medication. Compared with healthy controls, levels of five metabolites were found significantly altered in schizophrenic patients (P-values ranged from 2.9 × 10(-8) to 2.5 × 10(-4)) and in neuroleptics-free probands (P-values ranging between 0.006 and 0.03), respectively. These metabolites include four amino acids (arginine, glutamine, histidine and ornithine) and one lipid (PC ae C38:6) and are suggested as candidate biomarkers for schizophrenia. To explore the genetic susceptibility on the associated metabolic pathways, we constructed a molecular network connecting these five aberrant metabolites with 13 schizophrenia risk genes. Our result implicated aberrations in biosynthetic pathways linked to glutamine and arginine metabolism and associated signaling pathways as genetic risk factors, which may contribute to patho-mechanisms and memory deficits associated with schizophrenia. This study illustrated that the metabolic deviations detected in plasma may serve as potential biomarkers to aid diagnosis of schizophrenia.

Association of a variant in the muscarinic acetylcholine receptor 2 gene (CHRM2) with nicotine addiction.

Genetic factors contribute to the overall risk of developing nicotine addiction, which is the major cause of preventable deaths in western countries. However, knowledge regarding specific polymorphisms influencing smoking phenotypes remains scarce. In the present study we provide evidence that a common single nucleotide polymorphism (SNP) in the 5' untranslated region of CHRM2, the gene coding for the muscarinic acetylcholine receptor 2 is associated with nicotine addiction. CHRM2 was defined as a candidate gene for nicotine addiction based on previous evidence that linked variations in CHRM2 to alcohol and drug dependence. A total of more than 5,500 subjects representative of the German population were genotyped and assessed regarding their smoking habits. The impact of three SNPs in CHRM2 on smoking behavior/nicotine addiction was investigated using logistic regression models or a quasi-Poisson regression model, respectively. We found the T allele of SNP rs324650 to be associated with an increased risk of smoking/nicotine dependence according to three different models, the recessive models of regular or heavy smokers vs. never-smokers (odds ratio 1.17 in both analyses) and according to the Fagerström index of nicotine addiction. In the analysis stratified by gender this association was only found in females. Our data provide further evidence that variations in CHRM2 may be associated with the genetic risk of addiction in general or with certain personality traits that predispose to the development of addiction. Alternatively, variations in CHRM2 could modulate presynaptic auto-regulation in cholinergic systems and may thereby affect an individual's response to nicotine more specifically.

Analysis of 10 independent samples provides evidence for association between schizophrenia and a SNP flanking fibroblast growth factor receptor 2.

We and others have previously reported linkage to schizophrenia on chromosome 10q25-q26 but, to date, a susceptibility gene in the region has not been identified. We examined data from 3606 single-nucleotide polymorphisms (SNPs) mapping to 10q25-q26 that had been typed in a genome-wide association study (GWAS) of schizophrenia (479 UK cases/2937 controls). SNPs with P<0.01 (n=40) were genotyped in an additional 163 UK cases and those markers that remained nominally significant at P<0.01 (n=22) were genotyped in replication samples from Ireland, Germany and Bulgaria consisting of a total of 1664 cases with schizophrenia and 3541 controls. Only one SNP, rs17101921, was nominally significant after meta-analyses across the replication samples and this was genotyped in an additional six samples from the United States/Australia, Germany, China, Japan, Israel and Sweden (n=5142 cases/6561 controls). Across all replication samples, the allele at rs17101921 that was associated in the GWAS showed evidence for association independent of the original data (OR 1.17 (95% CI 1.06-1.29), P=0.0009). The SNP maps 85 kb from the nearest gene encoding fibroblast growth factor receptor 2 (FGFR2) making this a potential susceptibility gene for schizophrenia.

Systems biology and complex neurobehavioral traits.

There is evidence for a strong genetic component in the etiology of schizophrenia, as demonstrated by family, twin and adoption studies suggesting a heritability of about 80%. There are several approaches in the search for genetic risk factors such as linkage or association studies. Additionally, much effort was done in refining the phenotype including neuropsychology, neurophysiology, imaging or the generation of animal models. Genes becoming associated with schizophrenia have to be tested for functionality including e.g. metabolomics, transcriptomics, proteomics, generation of transgenic mice, analysis of protein-protein interactions, allele-specific RNA expression analysis, analysis of neuronal and stem cell cultures, as well as post mortem studies and behavioral studies in rodents. This amount of data requires complex data analysis. A system's perspective can help in the analysis of the structural and functional complexity of the brain. New tools will be needed for a more complex and systemic view. The systems biology approach could be a pivotal tool in understanding of complex behavior and diseases in future.

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3.

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. OBJECTIVES: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal.

N-Methyl-D-aspartate (NMDA) receptors, members of the glutamate receptor channel superfamily, are generally inhibited by alcohol. The expression and alternative splicing of the obligatory NR1 subunit is altered by alcohol exposure, emphasizing the involvement of the NR1 subunit, which is coded by the GRIN1 gene, in alcohol-mediated effects. We performed an association study in patients with alcohol dependence with the GRIN1 locus. Two independent case control samples consisting of a total of 442 alcohol-dependent patients and 442 unrelated controls were included. There was no overall difference in allele or genotype frequency between patients and controls. However, the 2108A allele and A-containing genotypes were over-represented in the patients with a history of withdrawal-induced seizures when compared to healthy volunteers (allele: chi(2) = 5.412, df = 1, P = 0.020) or an independent sample of patients without a history of seizures (allele: chi(2) = 4.185, df = 1, P = 0.041). Age at onset, years of alcohol dependence, and a history of delirium tremens did not differ between genotype or allele groups. These findings support the hypothesis that the GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal. This novel finding warrants replication.

M129V variation in the prion protein may influence cognitive performance.

Correlations between general intelligence (g) and brain volume are about 0.40, and the correlation between g and white matter volume has been reported to be largely due to genetic factors. Establishing that the correlation between brain volumes and cognitive abilities is mediated by shared genetic factors is only the first step in unveiling the relation between them. We have recently shown that methionine at codon 129 in the prion protein is associated with white matter reduction in a group of healthy volunteers and schizophrenic patients. The present study examines the influence of the same genetic variation on psychometric cognitive performance measurements in 335 community-based healthy volunteers. The polymorphism was associated with Full Scale IQ (genotype: F=4.38, df=2/317, P=0.013; allele: F=8.04, df=1/658, P=0.005), as measured by HAWIE-R (German version of the Wechsler Adult Intelligence Scale, Revised). Genotype accounted for 2.7% of the total variability in Full Scale IQ (partial eta2=0.027). An exploratory analysis revealed association with several HAWIE-R subscales; the association with the Digit Symbol subtest remained significant after correction for multiple testing. In summary, we deliver evidence for an association of a common genetic variation in the prion protein gene with cognitive performance. However, independent replications are needed before firm conclusions can be drawn.

Cloning and characterization of an alternatively spliced form of SR protein kinase 1 that interacts specifically with scaffold attachment factor-B.

Serine/arginine protein kinases have been conserved throughout evolution and are thought to play important roles in the regulation of mRNA processing, nuclear import, germline development, polyamine transport, and ion homeostasis. Human SRPK1, which was first identified as a kinase specific for the SR family of splicing factors, is located on chromosome 6p21.2-p21.3. We report here the cloning and characterization of SRPK1a, which is encoded by an alternatively processed transcript derived from the SRPK1 gene. SRPK1a contains an insertion of 171 amino acids at its NH(2)-terminal domain and is similar to SRPK1 in substrate specificity and subcellular localization. Moreover, both isoforms can induce alternative splicing of human tau exon 10 in transfected cells. Using the yeast two-hybrid assay, we found that the extended NH(2)-terminal domain of SRPK1a interacts with Scaffold Attachment Factor-B, a nuclear scaffold-associated protein. Confirmation of this interaction was provided by in vitro binding assays, as well as by co-immunoprecipitation from 293T cells doubly transfected with SRPK1a and SAF-B. Our studies suggest that different SRPK family members are uniquely regulated and targeted and thus the multiple SRPK kinases present in higher eukaryotes may perform specialized and differentiable functions.

Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors.

Tau is a microtubule-associated protein whose transcript undergoes regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult-specific and encodes a microtubule-binding domain. Mutations increasing the inclusion of exon 10 result in the production of tau protein which predominantly contains four microtubule-binding repeats and were shown to cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we show that exon 10 usage is regulated by CDC2-like kinases CLK1, 2, 3, and 4 that phosphorylate serine-arginine-rich proteins, which in turn regulate pre-mRNA splicing. Cotransfection experiments suggest that CLKs achieve this effect by releasing specific proteins from nuclear storage sites. Our results show that changing pre-mRNA-processing pathways through phosphorylation could be a new therapeutic concept for tauopathies.

The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites.

We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein. Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B. rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2beta and tau minigenes in cotransfection experiments. This effect can be reversed by rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purine-rich sequence. Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing.

The ER repeat protein YT521-B localizes to a novel subnuclear compartment.

The characterization of distinct subnuclear domains suggests a dynamic nuclear framework supporting gene expression and DNA replication. Here, we show that the glutamic acid/arginine-rich domain protein YT521-B localizes to a novel subnuclear structure, the YT bodies. YT bodies are dynamic compartments, which first appear at the beginning of S-phase in the cell cycle and disperse during mitosis. Furthermore, in untreated cells of the human cell line MCF7 they were undetectable and appeared only after drug- induced differentiation. YT bodies contain transcriptionally active sites and are in close contact to other subnuclear structures such as speckles and coiled bodies. YT bodies disperse upon actinomycin D treatment, whereas other transcriptional inhibitors such as alpha-amanitin or DRB have little effect. On the basis of our experiments, we propose that YT521-B may participate in the assembly of genes into transcription centers, thereby allowing efficient regulation of gene expression.

Effects of chronic kombucha ingestion on open-field behaviors, longevity, appetitive behaviors, and organs in c57-bl/6 mice: a pilot study.

Kombucha is a lightly fermented tea beverage popularly consumed as a self-prescribed folk-remedy for numerous ailments. Kombucha is claimed to enhance cognition, aid weight loss, and prolong life. This pilot study reports longevity, general health, and open-field exploratory behavioral outcomes from a 3-y longitudinal study of 64 C57-BL/6 mice (males and females), half of which chronically drank kombucha, and all of which experienced natural mortality. Compared by MANOVA to controls, mice that drank kombucha showed greater vertical exploration (P = 0.001) and a sex-interactive effect in novel object manipulation (P = 0.049). MANOVA of kombucha-drinking mice compared to controls detected differences in appetitive behaviors (food consumption, P < 0.001; beverage consumption, P = 0. 008), and gross body weight (P < 0.001). Appetitive behaviors changed with the addition of voluntary exercise on a running wheel, with differing patterns of change noted for males and females. Both male and female mice who drank kombucha lived longer than controls (P < 0.001), with the greatest variability among the male mice (sex interactive effect, P < 0.001). Comparable effects and mechanisms in humans remain uncertain, as do health safety issues, because serious health problems and fatalities have been reported and attributed to drinking kombucha.

Effect of exposure to photographs of thin models on self-consciousness in female college students.

The purpose of this study was to assess the immediate influence of brief exposure to images taken from print media on the general self-consciousness and body self-consciousness of 67 college women. After viewing photographs of either thin female models or control photographs, the women completed the Self-consciousness Scale and the Body Self-consciousness Questionnaire. Although alpha was .45, the college women who looked at images of thin female models gave immediate ratings significantly (p < .001) higher on both general Self-consciousness and Body Self-consciousness than those who looked at control images.

The in vivo minigene approach to analyze tissue-specific splicing.

The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements (cis-factors), their interacting protein components (trans-factors) or the cell type to alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections. Here, we give a detailed description of this technique that allows analysis of large gene fragments (up to 10-12 kb) under in vivo condition. Furthermore, we provide a summary of 44 genes currently investigated to demonstrate the general feasibility of this technique.

The interaction and colocalization of Sam68 with the splicing-associated factor YT521-B in nuclear dots is regulated by the Src family kinase p59(fyn).

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5-20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein-protein interaction domain. The Src family kinase p59(fyn)-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59(fyn) dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.