Drug sensitivity testing on patient-derived sarcoma cells predicts patient response to treatment and identifies c-Sarc inhibitors as active drugs for translocation sarcomas.

BACKGROUND: Heterogeneity and low incidence comprise the biggest challenge in sarcoma diagnosis and treatment. Chemotherapy, although efficient for some sarcoma subtypes, generally results in poor clinical responses and is mostly recommended for advanced disease. Specific genomic aberrations have been identified in some sarcoma subtypes but few of them can be targeted with approved drugs. METHODS: We cultured and characterised patient-derived sarcoma cells and evaluated their sensitivity to 525 anti-cancer agents including both approved and non-approved drugs. In total, 14 sarcomas and 5 healthy mesenchymal primary cell cultures were studied. The sarcoma biopsies and derived cells were characterised by gene panel sequencing, cancer driver gene expression and by detecting specific fusion oncoproteins in situ in sarcomas with translocations. RESULTS: Soft tissue sarcoma cultures were established from patient biopsies with a success rate of 58%. The genomic profile and drug sensitivity testing on these samples helped to identify targeted inhibitors active on sarcomas. The cSrc inhibitor Dasatinib was identified as an active drug in sarcomas carrying chromosomal translocations. The drug sensitivity of the patient sarcoma cells ex vivo correlated with the response to the former treatment of the patient. CONCLUSIONS: Our results show that patient-derived sarcoma cells cultured in vitro are relevant and practical models for genotypic and phenotypic screens aiming to identify efficient drugs to treat sarcoma patients with poor treatment options.

Susceptibility to infections, without concomitant hyper-IgE, reported in 1976, is caused by hypomorphic mutation in the phosphoglucomutase 3 (PGM3) gene.

Phosphoglucomutase 3 (PGM3) is an enzyme converting N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate, a precursor important for glycosylation. Mutations in the PGM3 gene have recently been identified as the cause of novel primary immunodeficiency with a hyper-IgE like syndrome. Here we report the occurrence of a homozygous mutation in the PGM3 gene in a family with immunodeficient children, described already in 1976. DNA from two of the immunodeficient siblings was sequenced and shown to encode the same homozygous missense mutation, causing a destabilized protein with reduced enzymatic capacity. Affected individuals were highly prone to infections, but lack the developmental defects in the nervous and skeletal systems, reported in other families. Moreover, normal IgE levels were found. Thus, belonging to the expanding group of congenital glycosylation defects, PGM3 deficiency is characterized by immunodeficiency, with or without increased IgE levels, and with variable forms of developmental defects affecting other organ systems.

Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels.

BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas.

Background: Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts. Methods: Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1-rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21). Results: Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between the assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNA-sequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay. Conclusions: The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.

Evaluation of NTRK immunohistochemistry as a screening method for NTRK gene fusion detection in non-small cell lung cancer.

PURPOSE: The small molecule inhibitors larotrectinib and entrectinib have recently been approved as cancer agnostic drugs in patients with tumours harbouring a rearrangement of the neurotrophic tropomyosin receptor kinase (NTRK). These oncogenic fusions are estimated to occur in 0.1-3 % of non-small cell lung cancers (NSCLC). Although molecular techniques are most reliable for fusion detection, immunohistochemical analysis is considered valuable for screening. Therefore, we evaluated the newly introduced diagnostic immunohistochemical assay (clone EPR17341) on a representative NSCLC cohort. METHODS: Cancer tissue from 688 clinically and molecularly extensively annotated NSCLC patients were comprised on tissue microarrays and stained with the pan-TRK antibody clone EPR17341. Positive cases were further analysed with the TruSight Tumor 170 RNA assay (Illumina). Selected cases were also tested with a NanoString NTRK fusion assay. For 199 cases, NTRK RNA expression data were available from previous RNA sequencing analysis. RESULTS: Altogether, staining patterns for 617 NSCLC cases were evaluable. Of these, four cases (0.6 %) demonstrated a strong diffuse cytoplasmic and membranous staining, and seven cases a moderate staining (1.1 %). NanoString or TST170-analysis could not confirm an NTRK fusion in any of the IHC positive cases, or any of the cases with high mRNA levels. In the four cases with strong staining intensity in the tissue microarray, whole section staining revealed marked heterogeneity of NTRK protein expression. CONCLUSION: The presence of NTRK fusion genes in non-small cell lung cancer is exceedingly rare. The use of the immunohistochemical NTRK assay will result in a small number of false positive cases. This should be considered when the assay is applied as a screening tool in clinical diagnostics.

PCM1 and schizophrenia: a replication study in the Northern Swedish population.

Previous studies implicated centrosomal dysfunction as a source of various neuropsychiatric disorders, including schizophrenia (SZ). Two recent reports [Gurling et al., 2006; Datta et al., 2008. Mol Psychiatry] described an association between polymorphisms in the PCM1 gene and SZ in a UK/Scottish population. In this study, we aimed to replicate these findings in a Northern Swedish association sample of 486 research subjects with SZ and 512 unrelated control individuals. We genotyped 12 previously described SNP markers and carried out haplotype analyses using the same multi-marker haplotypes previously reported. Though we could not replicate the association with SNPs rs445422 and rs208747, we did observe a significant protective association with intronic SNP rs13276297. Furthermore, we performed a meta-analysis comprising 1,794 SZ patients and 1,553 controls, which confirmed the previously reported association with rs445422 and rs208747. These data provide further evidence that PCM1-though certainly not a major risk factor in the Northern Swedish population-cannot be ruled out as a contributor to SZ risk and/or protection, and deserves further replication in larger populations to elucidate its role in disease etiology.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.

Detailed analysis of the serotonin transporter gene (SLC6A4) shows no association with bipolar disorder in the Northern Swedish population.

Through active reuptake of serotonin into presynaptic neurons, the serotonin transporter (5-HTT) plays an important role in regulating serotonin concentrations in the brain, and it is the site of binding for tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). Therefore it has been hypothesized that this transporter is involved in the etiology of bipolar (BP) disorder. Inconsistent association study results for the SLC6A4 gene encoding 5-HTT reported in literature emphasize the need for more systematic and detailed analyses of this candidate gene. We performed an extensive analysis of SLC6A4 on DNA of 254 BPI patients and 364 control individuals from a Northern Swedish isolated population. This analysis consisted of a HapMap LD-based association study including three widely investigated polymorphisms (5-HTTVNTR, 5-HTTLPR, and rs3813034), a copy-number variation (CNV) analysis and a mutation analysis of the complete coding sequence and the 3'-UTR of SLC6A4. No single marker showed statistically significant association with BPI, nor did any of the haplotypes. In the mutation analysis 13 novel variants were detected, including 2 amino acid substitutions M389V and I587L, but these are probably not implicated in risk for BP. No deletions or duplications were detected in the CNV analysis. We conclude that variation in the SLC6A4 gene or its regulatory regions does not contribute to the susceptibility for BP disorder in the Northern Swedish population.

Daphnia magna and ecotoxicogenomics: gene expression profiles of the anti-ecdysteroidal fungicide fenarimol using energy-, molting- and life stage-related cDNA libraries.

In the present study, the existing life stage-specific cDNA library was extended with energy- and molting-related genes using Suppression Subtractive Hybridization PCR and a microarray for the aquatic test organism Daphnia magna was created. A gene set of 2455 fragments was produced belonging to different pathways such as carbohydrate and lipid metabolism, O2 transport and heme metabolism, immune response, embryo development, cuticula metabolism and visual perception pathways. Using this custom microarray, gene expression profiles were generated from neonates exposed to three concentrations of the anti-ecdysteroidal fungicide fenarimol (0.5, 0.75, 1 microg/ml) during 48 h and 96 h. In total, 59 non-redundant genes were differentially expressed, of which more genes were down- than up-regulated. The gene expression data indicated a main effect on molting specific pathways. At the highest concentration, a set of proteolytic enzymes - including different serine proteases and carboxypeptidases - were induced whereas different cuticula proteins were down-regulated (48 h). Moreover, effects on embryo development were demonstrated at the gene expression as well as at the organismal level. The embryo development related gene vitellogenin was differentially expressed after 96 h of exposure together with a significant increase in embryo abnormalities in the offspring. This study suggests that this Daphnia magna microarray is of great further value for the elucidation of molecular mechanisms of toxicity and for the future development of specific biomarkers for hazard characterization.

Effluent impact assessment using microarray-based analysis in common carp: a systems toxicology approach.

Effluents are a main source of direct and continuous input of pollutants to the aquatic environment, and can cause ecotoxicological effects at different levels of biological organization. Since gene expression responses represent the primary interaction site between environmental contaminants and biota, they provide essential clues to understand how chemical exposure can affect organismal health. The aim of the present study was to investigate the applicability of a microarray approach for unraveling modes of action of whole effluent toxicity and impact assessment. A chronic toxicity test with common carp (Cyprinus carpio) was conducted where fish were exposed to a control and 100% effluent for 21 days under flow-through conditions. Microarray analysis revealed that effluent treatment mainly affected molecular pathways associated with the energy balance of the fish, including changes in carbohydrate and lipid metabolism, as well as digestive enzyme activity. These gene expression responses were in clear agreement with, and provided additional mechanistic information on various cellular and higher level effects observed for the same effluent. Our results demonstrate the benefit of toxicogenomic tools in a "systems toxicology" approach, involving the integration of adverse effects of chemicals and stressors across multiple levels of biological complexity.

Expression profiling of endocrine-disrupting compounds using a customized Cyprinus carpio cDNA microarray.

Exposure to a variety of anthropogenic compounds has been shown to interfere with normal development, physiology, and reproduction in a wide range of organisms, both in laboratory studies and wildlife. We have developed a Cyprinus carpio cDNA microarray consisting of endocrine-related genes. In the current study, we investigated the applicability of this microarray (1) to study the molecular effects induced by exposure to a variety of endocrine-disrupting compounds (EDCs) in fish and (2) to discriminate the specific transcriptional profiles associated with these compounds. To that purpose, gene expression profiles were generated in livers of juvenile carp exposed to 14 Organization of Economical Cooperation Development (OECD)-recommended reference EDCs (17beta-estradiol, 17alpha-ethinylestradiol, 4-nonylphenol, bisphenol A, tamoxifen, 17alpha-methyltestosterone, 11-ketotestosterone, dibutyl phthalate, flutamide, vinclozolin, hydrocortisone, CuCl(2), propylthiouracil, and a mixture of L-triiodothyronine and L-thyroxine). Our results show that, in addition to some expression similarities between analogous acting substances, each individual compound produced its own unique expression pattern on the array, distinct from the profiles generated by the other compounds. In addition, we were able to identify a minimal subset of genes, which also allowed to discriminate between the different compounds. Overall, our findings suggest that the developed cDNA array has great promise to screen new and existing chemicals on their endocrine-disruptive potential and to identify distinct classes of EDCs.

Patterns of gene expression in carp liver after exposure to a mixture of waterborne and dietary cadmium using a custom-made microarray.

Gene expression changes in carp liver tissue were studied after acute (3 and 24h) and subchronic (7 and 28 days) exposure to a mixture of waterborne (9, 105 and 480 microg/l) and dietary (9.5, 122 and 144 microg/g) cadmium, using a custom-made microarray. Suppression subtractive hybridization-PCR (SSH-PCR) was applied to isolate a set of 643 liver genes, involved in multiple biological pathways, such as energy metabolism (e.g. glucokinase), immune response (e.g. complement C3) and stress and detoxification (e.g. cytochrome P450 2F2, glutathione-S-transferase pi). These genes were subsequently spotted on glass-slides for the construction of a custom-made microarray. Resulting microarray hybridizations indicated a highly dynamic response to cadmium exposure. At low exposure concentrations (9 microg/l through water and 9.5 microg/g dry weight through food) mostly energy-related genes (e.g. glucokinase, elastase) were influenced, while a general stress response was obvious through induction of several stress-related genes, including hemopexin and cytochrome P450 2F2, at high cadmium concentrations. In addition, fish exposed to the highest cadmium concentrations showed liver damage after 7 days of exposure, as measured by elevated alanine transaminase activity in plasma and increased liver water content (wet-to-dry weight ratio). Moreover, decreased hematocrit and growth were found at the end of the experiment. Altogether this study clearly demonstrated the importance of varying exposure conditions for the characterization of the molecular impact of cadmium and showed that microarray results can provide important information, required to unravel the molecular events and responses related to cadmium exposure.

Effects of the antidepressant mianserin in zebrafish: molecular markers of endocrine disruption.

Due to their environmental occurrence and intrinsic biological activity, human pharmaceuticals have received increasing attention from environmental and health agencies. Of particular, ecotoxicological concern are drugs that affect nervous- and endocrine-systems. Zebrafish genome-wide oligo arrays are used to collect mechanistic information on mianserin-induced changes in gene expression in zebrafish. Gene expression analysis in brain and gonad tissue clearly demonstrated the estrogenic activity of mianserin and its potency to disrupt normal endocrine (estrogenic) signaling, based on induction of molecular biomarkers of estrogenicity (e.g., vitellogenin1 and zona pellucida proteins). The possible mechanism underlying this estrogenic activity of mianserin is disturbance of the Hypothalamo-Pituitary-Gonadal (HPG) axis by direct interference of mianserin with the serotonergic and adrenergic systems in the brain of zebrafish. Taking into account the importance of the HPG-axis, and considering the concept of 'critical window of exposure', our results reveal the importance for more elaborate testing of endocrine disruptive effects of aquatic antidepressants at different lifestages and during longer exposure periods (e.g., life cycle studies). Although there is a low concordance between the gene expression results in this study and previous cDNA microarray hybridizations, the global mechanistic expression patterns are similar in both platforms. This argues in favor of pathway-driven analysis of gene expression results compared to gene-per-gene analysis.

Neuropharmaceuticals in the environment: mianserin-induced neuroendocrine disruption in zebrafish (Danio rerio) using cDNA microarrays.

Because of their environmental occurrence and high biological activity, human pharmaceuticals have received increasing attention from environmental and health agencies. A major bottleneck in their risk assessment is the lack of relevant and specific effect data. We developed an approach using gene expression analysis in quantifying adverse effects of neuroendocrine pharmaceuticals in the environment. We studied effects of mianserin on zebrafish (Danio rerio) gene expression using a brain-specific, custom microarray, with real-time polymerase chain reaction as confirmation. After exposure (0, 25, and 250 microg/L) for 2, 4, and 14 d, RNA was extracted from brain tissue and used for microarray hybridization. In parallel, we investigated the impact of exposure on egg production, fertilization, and hatching. After 2 d of exposure, microarray analysis showed a clear effect of mianserin on important neuroendocrine-related genes (e.g., aromatase and estrogen receptor), indicating that antidepressants can modulate neuroendocrine processes. This initial neuroendocrine effect was followed by a "late gene expression effect" on neuronal plasticity, supporting the current concept regarding the mode of action for antidepressants in mammals. Clear adverse effects on egg viability were seen after 14 d of exposure at the highest concentration tested. Based on the specific molecular impact and the effects on reproduction, we conclude that further investigation of the adverse effects on the brain-liver-gonad axis is needed for a correct ecological risk assessment of antidepressants.

HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings.

Diagnostics of primary immunodeficiency diseases: a sequencing capture approach.

Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of "next generation" sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.

Sequencing of DISC1 pathway genes reveals increased burden of rare missense variants in schizophrenia patients from a northern Swedish population.

In recent years, DISC1 has emerged as one of the most credible and best supported candidate genes for schizophrenia and related neuropsychiatric disorders. Furthermore, increasing evidence--both genetic and functional--indicates that many of its protein interaction partners are also involved in the development of these diseases. In this study, we applied a pooled sample 454 sequencing strategy, to explore the contribution of genetic variation in DISC1 and 10 of its interaction partners (ATF5, Grb2, FEZ1, LIS-1, PDE4B, NDE1, NDEL1, TRAF3IP1, YWHAE, and ZNF365) to schizophrenia susceptibility in an isolated northern Swedish population. Mutation burden analysis of the identified variants in a population of 486 SZ patients and 514 control individuals, revealed that non-synonymous rare variants with a MAF<0.01 were significantly more present in patients compared to controls (8.64% versus 4.7%, P = 0.018), providing further evidence for the involvement of DISC1 and some of its interaction partners in psychiatric disorders. This increased burden of rare missense variants was even more striking in a subgroup of early onset patients (12.9% versus 4.7%, P = 0.0004), highlighting the importance of studying subgroups of patients and identifying endophenotypes. Upon investigation of the potential functional effects associated with the identified missense variants, we found that ∼90% of these variants reside in intrinsically disordered protein regions. The observed increase in mutation burden in patients provides further support for the role of the DISC1 pathway in schizophrenia. Furthermore, this study presents the first evidence supporting the involvement of mutations within intrinsically disordered protein regions in the pathogenesis of psychiatric disorders. As many important biological functions depend directly on the disordered state, alteration of this disorder in key pathways may represent an intriguing new disease mechanism for schizophrenia and related neuropsychiatric diseases. Further research into this unexplored domain will be required to elucidate the role of the identified variants in schizophrenia etiology.

Support for NRG1 as a susceptibility factor for schizophrenia in a northern Swedish isolated population.

CONTEXT: Neuregulin 1 (NRG1), a growth factor involved in neurodevelopment, myelination, neurotransmitter receptor expression, and synaptic plasticity, first joined the list of candidate genes for schizophrenia when a 7-marker haplotype at the 5' end of the gene (Hap(ICE)) was shown to be associated with the disorder in the Icelandic population. Since then, more genetic and functional evidence has emerged, which supports a role for NRG1 in the development of schizophrenia. OBJECTIVE: To determine the contribution of NRG1 to susceptibility for schizophrenia in a northern Swedish isolated population. DESIGN: Detailed linkage disequilibrium (LD)-based patient-control association study. This is the first study to type and analyze the 7 Hap(ICE) markers and a set of 32 HapMap tagging single-nucleotide polymorphisms (SNPs) that represents variants with a minor allele frequency of at least 1% and fully characterizes the LD structure of the 5' part of NRG1. SETTING: Outpatient and inpatient hospitals. PARTICIPANTS: A total of 486 unrelated patients with schizophrenia and 514 unrelated control individuals recruited from a northern Swedish isolated population. MAIN OUTCOME MEASURES: Association between markers and disease. RESULTS: Analysis of the Hap(ICE) markers showed the association of a 7-marker and 2-microsatellite haplotype, different from the haplotypes associated in the Icelandic population and overrepresented in northern Swedish control individuals. Subsequently, a more detailed analysis that included all 37 genotyped SNPs was performed by investigating haplotypic association, dependent and independent of LD block structure. We found significant association with 5 SNPs located in the second intron of NRG1 (.007

Gene expression analysis of estrogenic compounds in the liver of common carp (Cyprinus carpio) using a custom cDNA microarray.

Exposure to a variety of compounds with estrogenic activity has been shown to interfere with normal developmental and reproductive processes in various vertebrate species. The aim of this study was to determine the transcriptional profile of the natural estrogen, 17 beta-estradiol, and three synthetic estrogenic compounds (4-nonylphenol, bisphenol A, ethinylestradiol) in the liver of common carp, using a custom cDNA microarray. For that purpose, fish were aqueously exposed to three concentrations of each chemical for 24 or 96 h. Microarray analysis revealed that a total of 185 different gene transcripts were differentially expressed following exposure to at least one of the estrogen(-like) concentrations. We were able to identify a common set of 28 gene fragments, whose expression was significantly modified in the same way by the three xenoestrogens and 17 beta-estradiol. Although several of these gene expression effects corroborated past literature data, we also discovered some novel target genes of (xeno)estrogen exposure, providing interesting insights into the molecular basis of estrogenic effects. In addition, each of the four compounds induced gene expression changes that were not, or only partially, shared by the other chemicals, suggesting that not all chemicals with estrogenic activity act alike. These results demonstrate the potential of our custom Cyprinus carpio microarray to detect common estrogen-like activity as well as to identify unique compound-associated effects of (estrogenic) endocrine disruptors in fish.

Use of suppression subtractive hybridization PCR for the development of cDNA arrays for the detection of endocrine disruption in carp (Cyprinus carpio).

The potential of a variety of xenobiotic compounds to modulate or disrupt the endocrine system of humans and wildlife is now widely recognized. In the present study, we developed a molecular tool for the evaluation of endocrine disruption in common carp (Cyprinus carpio). Suppression Subtractive Hybridization PCR was applied for the isolation of a relevant gene set, consisting of gender- and hormone-responsive gene fragments. This resulted in 398 different gene fragments that were most related to endocrine functioning. To investigate the applicability of this gene collection for studying endocrine disruption in fish, the gender-related genes were spotted on a cDNA macroarray, and expression profiles were generated for 17beta-estradiol (E2) and cortisol. Therefore, fish were injected with these hormones, and after 24 h and 96 h RNA was extracted and used for macroarray hybridizations. E2 exposure resulted in a total of 35 differentially expressed genes, whereas cortisol only affected 3 genes spotted on the macroarray. These results indicate the discriminating power of the developed array, and its usefulness to describe the toxicological mode of action of endocrine disruptive chemicals.