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Background/Objectives: Genetic newborn screening (NBS) has already entered the phase of common practice in many countries. In Germany, spinal muscular atrophy (SMA), severe combined immunodeficiency (SCID) and sickle cell disease (SCD) are currently a mandatory part of NBS. Here, we describe the experience of six years of genetic NBS including the prevalence of those three diseases in Germany. Methods: Samples and nucleic acids were extracted from dried blood spot cards, commonly used for NBS. A qPCR assay was used to detect disease-causing variants for SMA and SCD, and the detection of T-cell receptor excision circles (TRECs) was performed for SCID screening. Results: The results of the NBS of over 1 million newborns for SMA, approximately 770,000 for SCID and over 410,000 for SCD are discussed in detail. In these newborns, we have identified 121 cases of SMA, 15 cases of SCID and syndrome-based immunodeficiencies and 77 cases of SCD or β-thalassemia. Conclusions: The flexibility of multiplex qPCR is assessed as an effective tool for incorporating different molecular genetic markers for screening. The processing of dried blood spot (DBS) filter cards for molecular genetic assays and the assays are described in detail; turn-around times and cost estimations are included to give an insight into the processes and discuss further options for optimization. The identified cases are in the range expected for the total number of screened newborns, but present a more exact view on the actual prevalences for Germany. [ABSTRACT FROM AUTHOR]

Background: Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD). Methods: This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined. Results: Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS. Conclusions: Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per μl may not be reliable and should therefore be avoided. [ABSTRACT FROM AUTHOR]

Abstract Background The hypothalamic-pituitary-adrenal (HPA) axis is essential to control physiological stress responses in mammals. Its dysfunction is related to several mental disorders, including anxiety and depression. The aim of this study was to identify genetic loci underlying the endocrine regulation of the HPA axis. Method High (HAB) and low (LAB) anxiety-related behaviour mice were established by selective inbreeding of outbred CD-1 mice to model extremes in trait anxiety. Additionally, HAB vs. LAB mice exhibit comorbid characteristics including a differential corticosterone response upon stress exposure. We crossbred HAB and LAB lines to create F1 and F2 offspring. To identify the contribution of the endocrine phenotypes to the total phenotypic variance, we examined multiple behavioural paradigms together with corticosterone secretion-based phenotypes in F2 mice by principal component analysis. Further, to pinpoint the genomic loci of the quantitative trait of the HPA axis stress response, we conducted genome-wide multipoint oligogenic linkage analyses based on Bayesian Markov chain Monte Carlo approach as well as parametric linkage in three-generation pedigrees, followed by a two-dimensional scan for epistasis and association analysis in freely segregating F2 mice using 267 single-nucleotide polymorphisms (SNPs), which were identified to consistently differ between HAB and LAB mice as genetic markers. Results HPA axis reactivity measurements and behavioural phenotypes were represented by independent principal components and demonstrated no correlation. Based on this finding, we identified one single quantitative trait locus (QTL) on chromosome 3 showing a very strong evidence for linkage (2ln (L-score) > 10, LOD > 23) and significant association (lowest Bonferroni adjusted p < 10-28) to the neuroendocrine stress response. The location of the linkage peak was estimated at 42.3 cM (95% confidence interval: 41.3 - 43.3 cM) and was shown to be in epistasis (p-adjusted < 0.004) with the locus at 35.3 cM on the same chromosome. The QTL harbours genes involved in steroid synthesis and cardiovascular effects. Conclusion The very prominent effect on stress-induced corticosterone secretion of the genomic locus on chromosome 3 and its involvement in epistasis highlights the critical role of this specific locus in the regulation of the HPA axis.

Genomic copy number variants (CNVs) have been implicated in multiple psychiatric disorders, but not much is known about their influence on anxiety disorders specifically. Using next-generation sequencing (NGS) and two additional array-based genotyping approaches, we detected CNVs in a mouse model consisting of two inbred mouse lines showing high (HAB) and low (LAB) anxiety-related behavior, respectively. An influence of CNVs on gene expression in the central (CeA) and basolateral (BLA) amygdala, paraventricular nucleus (PVN), and cingulate cortex (Cg) was shown by a two-proportion Z-test (p = 1.6 x 10-31), with a positive correlation in the CeA (p = 0.0062), PVN (p = 0.0046) and Cg (p = 0.0114), indicating a contribution of CNVs to the genetic predisposition to trait anxiety in the specific context of HAB/LAB mice. In order to confirm anxiety-relevant CNVs and corresponding genes in a second mouse model, we further examined CD-1 outbred mice. We revealed the distribution of CNVs by genotyping 64 CD 1 individuals using a high-density genotyping array (Jackson Laboratory). 78 genes within those CNVs were identified to show nominally significant association (48 genes), or a statistical trend in their association (30 genes) with the time animals spent on the open arms of the elevated plus-maze (EPM). Fifteen of them were considered promising candidate genes of anxiety-related behavior as we could show a significant overlap (permutation test, p = 0.0051) with genes within HAB/LAB CNVs. Thus, here we provide what is to our knowledge the first extensive catalogue of CNVs in CD-1 mice and potential corresponding candidate genes linked to anxiety-related behavior in mice. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behavior (HAB) versus low anxiety-related behavior, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behavior. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity--an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioral consequence of these differences revealed that intracerebroventricular NPS reversed the hyperanxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date. [ABSTRACT FROM AUTHOR]

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait ''anxiety''. We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4). Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy. [ABSTRACT FROM AUTHOR]

Background: To investigate neurobiological correlates of trait anxiety, CD1 mice were selectively bred for extremes in anxiety-related behavior, with high (HAB) and low (LAB) anxiety-related behavior mice additionally differing in behavioral tests reflecting depression-like behavior. Methodology/ Principal Findings: In this study, microarray analysis, in situ hybridization, quantitative real-time PCR and immunohistochemistry revealed decreased expression of the vasopressin gene (Avp) in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of adult LAB mice compared to HAB, NAB (normal anxiety-related behavior) and HABxLAB F1 intercross controls, without detecting differences in receptor expression or density. By sequencing the regions 2.5 kbp up- and downstream of the Avp gene locus, we could identify several polymorphic loci, differing between the HAB and LAB lines. In the gene promoter, a deletion of twelve bp Δ(22180-2191) is particularly likely to contribute to the reduced Avp expression detected in LAB animals under basal conditions. Indeed, allele-specific transcription analysis of F1 animals revealed a hypomorphic LAB-specific Avp allele with a reduced transcription rate by 75% compared to the HAB-specific allele, thus explaining line-specific Avp expression profiles and phenotypic features. Accordingly, intra-PVN Avp mRNA levels were found to correlate with anxiety-related and depression-like behaviors. In addition to this correlative evidence, a significant, though moderate, genotype/phenotype association was demonstrated in 258 male mice of a freely-segregating F2 panel, suggesting a causal contribution of the Avp promoter deletion to anxiety-related behavior. Discussion: Thus, the identification of polymorphisms in the Avp gene promoter explains gene expression differences in association with the observed phenotype, thus further strengthening the concept of the critical involvement of centrally released AVP in trait anxiety. [ABSTRACT FROM AUTHOR]

Following secretion from the posterior pituitary, the neuropeptide vasopressin (AVP) stimulates the kidney to retain water, and when released centrally it can contribute to anxiety- and depression-like behaviours. We hypothesized that CD1 mice bred for low trait anxiety (LAB) suffer from a deficit in AVP. Both osmotically stimulated peripheral secretion and intra-paraventricular nucleus (PVN) release of AVP were found decreased in LAB animals compared with normal anxiety (NAB) or high anxiety (HAB) controls. Consequently, in addition to their extreme non-anxiety, LAB mice showed signs of central diabetes insipidus (cDI), including increased fluid intake and reduced urine osmolality, as well as a pathological increase in plasma osmolality upon water deprivation. These cDI symptoms were attenuated by administration of a selective AVP V2 receptor agonist. A single nucleotide polymorphism (SNP) in exon 1 (C(+ 40)T) of the Avp gene of LAB animals causes an amino acid substitution in the signal peptide of the AVP precursor, and is likely to impair processing and trafficking of the precursor, as suggested by reduced axonal transport of AVP from the hypothalamic PVN, finally contributing to cDI symptoms and low trait anxiety. In an F2 panel, this SNP co-segregated with fluid intake and showed a partial contribution to low anxiety-related behaviour, indicated by its co-segregation with time spent on the open arms of the elevated plus-maze in a subset of F2 mice. Thus, the SNP-associated deficit in plasma and central AVP contributes to signs of cDI and, at least partially, to low trait anxiety, both features being typical of LAB animals. [ABSTRACT FROM AUTHOR]

For >15 generations, CD1 mice have been selectively and bidirectionally bred for either high-anxiety-related behavior (HAB-M) or low-anxiety-related behavior (LAB-M) on the elevated plus-maze. Independent of gender, HAB-M were more anxious than LAB-M animals in a variety of additional tests, including those reflecting risk assessment behaviors and ultrasound vocalization, with unselected CD1 "normal" control (NAB-M) and cross-mated (CM-M) mice displaying intermediate behavioral scores in most cases. Furthermore, in both the forced-swim and tail-suspension tests, LAB-M animals showed lower scores of immobility than did HAB-M and NAB-M animals, indicative of a reduced depression-like behavior. Using proteomic and microarray analyses, glyoxalase-I was identified as a protein marker, which is consistently expressed to a higher extent in LAB-M than in HAB-M mice in several brain areas. The same phenotype-dependent difference was found in red blood cells with NAB-M and CM-M animals showing intermediate expression profiles of glyoxalase-I. Additional studies will examine whether glyoxalase-I has an impact beyond that of a biomarker to predict the genetic predisposition to anxiety- and depression-like behavior. [ABSTRACT FROM AUTHOR]

Background Evidence has been provided linking neurogenesis to mood disorders. Notably, it has been shown that chronic experimental stress resulting in enhanced depression-like behavior decreases neurogenesis in the dentate gyrus (DG), while antidepressants reverse these stress-induced effects. However, in most studies ''normal'' animals reflecting physiology rather than pathophysiology are used. Therefore, we aimed to investigate neurogenesis in the DG of a mouse model of high-trait anxiety and comorbid depression (HAB), which mimics important features of human psychopathology, and their normal anxiety/ depression (NAB) controls. Methods BrdU (5-bromo-2'-deoxyuridine) was administered to female HABs and NABs. Mice were sacrificed at 15 and 42 days post BrdU to study cell proliferation and survival respectively. At 42 d the mice were subjected to a forced swim test. Double labelling of BrdU and c-Fos (a marker for neuronal activation) was performed to observe if newborn cells functionally integrated into the DG network. Furthermore, effects of the selective serotonin reuptake inhibitor (SSRI) fluoxetine on depression-like behavior and cell survival were assessed. Finally, gene array studies were conducted in the DG. Results Compared to NABs, HAB mice displayed enhanced depression-like behavior in the forced swim test and reduced newborn cell proliferation and survival in the DG. Double-labelling of BrdU and c-Fos revealed that some of the newborn cells in the survival paradigm functionally integrated in NABs, while no such evidence was found in HABs. Gene array studies revealed lower abundance of cyclin-dependent kinase 5 (Cdk5) and brain-derived neurotrophic factor (BDNF) in HABs which might contribute to reduced neurogenesis. Finally, although chronic treatment with fluoxetine reduced the depression-like behavior exclusively in female HABs, it did not alter cell survival or functional integration of newborn neurons in the DG. Conclusion Taken together, the enhanced depression-like behavior in a psychopathological animal model is accompanied by decreased hippocampal neurogenesis as well as BDNF and CdK5 expression possibly contributing to the phenotype. Since the antidepressant effect of fluoxetine is discerned from neurogenesis, it is suggested that mechanisms other than adult neurogenesis underlie the therapeutic action of SSRIs in the HAB model, which, however, has to be confirmed by using additional SSRIs. Thus, the present data do not support the idea that neurogenesis is a prerequisite for therapeutic actions common to all antidepressants. Moreover, these findings highlight the importance of psychopathological animal models in order to investigate molecular mechanisms of effective antidepressants. [ABSTRACT FROM AUTHOR]

Although gene-environment interactions are known to significantly influence psychopathology-related disease states, only few animal models cover both the genetic background and environmental manipulations. Therefore, we have taken advantage of the bidirectionally inbred high (HAB) and low (LAB) anxiety-related behavior mouse lines to generate HAB × LAB F1 hybrids that intrinsically carry both lines' genetic characteristics, and subsequently raised them in three different environments-standard, enriched (EE) and chronic mild stress (CMS). Assessing genetic correlates of trait anxiety, we focused on two genes already known to play a role in HAB vs. LAB mice, corticotropin releasing hormone receptor type 1 (Crhr1) and high mobility group nucleosomal binding domain 3 (Hmgn3). While EE F1 mice showed decreased anxiety-related and increased explorative behaviors compared to controls, CMS sparked effects in the opposite direction. However, environmental treatments affected the expression of the two genes in distinct ways. Thus, while expression ratios of Hmgn3 between the HAB- and LAB-specific alleles remained equal, total expression resembled the one observed in HAB vs. LAB mice, i.e., decreased after EE and increased after CMS treatment. On the other hand, while total expression of Crhr1 remained unchanged between the groups, the relative expression of HAB- and LAB-specific alleles showed a clear effect following the environmental modifications. Thus, the environmentally driven bidirectional shift of trait anxiety in this F1 model strongly correlated with Hmgn3 expression, irrespective of allele-specific expression patterns that retained the proportions of basic differential HAB vs. LAB expression, making this gene a match for environment-induced modifications. An involvement of Crhr1 in the bidirectional behavioral shift could, however, rather be due to different effects of the HAB- and LAB-specific alleles described here. Both candidate genes therefore deserve attention in the complex regulation of anxiety-related phenotypes including environment-mediated effects.