Promoter haplotypes of the corticotropin-releasing hormone encoding gene modulate the physiological stress response in vitro and in vivo.

The corticotropin-releasing hormone (CRH) is a neuropeptide mediating stress responses. CRH exerts effects via the hypothalamus pituitary adrenal axis as well as immediate effects on the sympathetic-adrenal-medullary system. Genetic variants of the CRH promoter were previously found to be associated with altered CRH promoter activity and physiological reactions. Functional characterization of three CRH promoter haplotypes have been performed in vitro using a reporter gene assay under different stimulation conditions. Furthermore, 232 healthy subjects were genotyped and the influence of CRH haplotypes on basal parameters such as post-awakening cortisol and blood pressure as well as on stress reactivity measured after socially evaluated cold pressor test (SeCPT) was investigated. In vitro, CRH haplotype 2 showed the highest promoter activity under baseline conditions and after forskolin stimulation compared with other haplotypes. Forskolin treatment resulted in a two fold increase of haplotype 2 promoter activity compared with the baseline condition. Cell line-dependent promoter activation was found after hydrocortisone treatment. In vivo, CRH haplotype 2 carriers showed significant higher baseline blood pressure (p = .002) and blood pressure after SeCPT (p < .001), but did not differ in cortisol levels. This study provides converging evidence for the importance of CRH promoter variants on physiological stress response parameters.

The chromosome 15q14 locus for bipolar disorder and schizophrenia: is C15orf53 a major candidate gene?

Bipolar disorder (BD) and schizophrenia are complexly inherited and highly heritable disorders with currently unknown etiologies. Recently, two independent genome-wide association studies for BD identified a small region on chromosome 15q14-15.1, pointing to a locus close to the gene C15orf53. Previously, this genomic region was also found to co-segregate with periodic catatonia (SCZD10, OMIM %605419), an unsystematic schizophrenia according to Leonhard's classification, in several multiplex families, thus pointing to overlapping etiologies of both conditions. A susceptibility locus on chromosome 15q14-15.1 was narrowed down to a 4.38 Mb region in these affected families followed by mutation and segregation analyses of C15orf53. Association analysis of individuals affected by BD and/or SCZD10 (n = 274) and controls (n = 230) and expression analyses in distinct post-mortem human limbic brain tissues were conducted. C15orf53 revealed no mutations in our SCZD10 family members, but segregation of two common haplotypes was found. No association of identified haplotypes was found in our case-control samples. Gene expression could be demonstrated for immune-system-derived cells but not for the post-mortem human limbic brain tissue. Our results indicate that C15orf53 is probably neither causative for the etiology of BD nor for SCZD10 in our samples.

Biological and psychosocial environmental risk factors influence symptom severity and psychiatric comorbidity in children with ADHD.

Attention-deficit/hyperactivity disorder (ADHD) is a genetically as well as environmentally determined disorder with a high rate of psychiatric comorbidity. In this study, non-genetic biological and psychosocial risk factors for ADHD symptom severity and comorbid disorders were assessed in 275 children with ADHD, aged 5-13 years, mean age 9.7 (SD 1.9). Pre-/perinatal biological and lifetime psychosocial risk factors as well as data on parental ADHD were obtained. A different pattern of risk factors emerged for inattentive and hyperactive-impulsive ADHD symptoms. Inattentive symptoms were strongly influenced by psychosocial risk factors, whereas for hyperactive-impulsive symptoms, predominantly biological risk factors emerged. Hyperactive-impulsive symptoms also were a strong risk factor for comorbid oppositional defiant (ODD) and conduct disorder (CD). Smoking during pregnancy was a risk factor for comorbid CD but not ODD and further differential risk factors were observed for ODD and CD. Comorbid anxiety disorder (AnxD) was not related to ADHD symptoms and additional biological and psychosocial risk factors were observed. This study adds to the body of evidence that non-genetic biological and psychosocial risk factors have an impact on ADHD symptom severity and differentially influence comorbid disorders in ADHD. The findings are relevant to the prevention and treatment of ADHD with or without comorbid disorders.

DIRAS2 is associated with adult ADHD, related traits, and co-morbid disorders.

Several linkage analyses implicated the chromosome 9q22 region in attention deficit/hyperactivity disorder (ADHD), a neurodevelopmental disease with remarkable persistence into adulthood. This locus contains the brain-expressed GTP-binding RAS-like 2 gene (DIRAS2) thought to regulate neurogenesis. As DIRAS2 is a positional and functional ADHD candidate gene, we conducted an association study in 600 patients suffering from adult ADHD (aADHD) and 420 controls. Replication samples consisted of 1035 aADHD patients and 1381 controls, as well as 166 families with a child affected from childhood ADHD. Given the high degree of co-morbidity with ADHD, we also investigated patients suffering from bipolar disorder (BD) (n=336) or personality disorders (PDs) (n=622). Twelve single-nucleotide polymorphisms (SNPs) covering the structural gene and the transcriptional control region of DIRAS2 were analyzed. Four SNPs and two haplotype blocks showed evidence of association with ADHD, with nominal p-values ranging from p=0.006 to p=0.05. In the adult replication samples, we obtained a consistent effect of rs1412005 and of a risk haplotype containing the promoter region (p=0.026). Meta-analysis resulted in a significant common OR of 1.12 (p=0.04) for rs1412005 and confirmed association with the promoter risk haplotype (OR=1.45, p=0.0003). Subsequent analysis in nuclear families with childhood ADHD again showed an association of the promoter haplotype block (p=0.02). rs1412005 also increased risk toward BD (p=0.026) and cluster B PD (p=0.031). Additional SNPs showed association with personality scores (p=0.008-0.048). Converging lines of evidence implicate genetic variance in the promoter region of DIRAS2 in the etiology of ADHD and co-morbid impulsive disorders.

Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder.

Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A(2A) receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A(2A) receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

Characterization of a glucocorticoid receptor gene (GR, NR3C1) promoter polymorphism reveals functionality and extends a haplotype with putative clinical relevance.

Hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis has been associated with the etiology of major depression. One of the factors underlying altered glucocorticoid signaling might be variability of the glucocorticoid receptor gene (GR, NR3C1). GR polymorphisms have been associated with variability in glucocorticoid sensitivity and endocrine responses to psychosocial stress. Furthermore, a common GR SNP (rs10482605), located in the promoter region, has been associated with major depression. We performed functional characterization of this SNP in vitro using a reporter gene assay under different stimulation conditions. Furthermore, we genotyped 219 subjects previously genotyped for four common GR SNPs to further characterize GR haplotype structure. The minor C allele of the rs10482605 SNP showed reduced transcriptional activity under unstimulated conditions and under different stimulation conditions in two brain derived cell lines. Linkage analyses revealed that the rs10482605 SNP is in high linkage disequilibrium with a A/G SNP in exon 9beta (rs6198), associated with relative glucocorticoid resistance and increased GRbeta mRNA stability. We provide evidence that two functional GR SNPs in linkage disequilibrium are responsible for both regulation of GR expression and mRNA stability. This newly characterized haplotype could increase the risk for the development of stress related disorders, including major depression.

Glucocorticoid sensitivity in fibromyalgia patients: decreased expression of corticosteroid receptors and glucocorticoid-induced leucine zipper.

In fibromyalgia (FM) patients, differences in glucocorticoid receptor (GR) affinity and disturbances associated with loss of hypothalamic-pituitary-adrenal (HPA) axis resiliency have been observed. Based on these studies, we investigated whether FM would be associated with abnormalities in glucocorticoid (GC) sensitivity. Salivary and blood samples were collected from 27 FM patients and 29 healthy controls. Total plasma cortisol and salivary free cortisol were quantified by ELISA and time-resolved fluorescence immunoassay, respectively. GR sensitivity to dexamethasone was evaluated through IL-6 inhibition in stimulated whole blood. The corticosteroid receptors, GR alpha and mineralocorticoid receptor, as well as the glucocorticoid-induced leucine zipper (GILZ) and the FK506 binding protein 5 mRNA expression were assessed in peripheral blood mononuclear cells (PBMCs) by real-time RT-PCR. Furthermore, the corticosteroid receptors were analysed for polymorphism. We observed lower basal plasma cortisol levels (borderline statistical significance) and a lower expression of corticosteroid receptors and GILZ in FM patients when compared to healthy controls. The MR rs5522 (I180V) minor allele was found more often in FM patients than in controls and this variant was recently associated with a mild loss of receptor function. The lower GR and MR expression and possibly the reduced MR function may be associated with an impaired function of the HPA axis in these patients which, compounded by lower anti-inflammatory mediators, may sustain some of symptoms that contribute to the clinical picture of the syndrome.

The brain-specific protein MLC1 implicated in megalencephalic leukoencephalopathy with subcortical cysts is expressed in glial cells in the murine brain.

The human MLC1 gene (also known as KIAA0027 and WKL1) and its murine orthologue (Mlc1) encode a putative transmembrane protein expressed primarily in brain. Recessive mutations within human MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), whereas a missense mutation resulting in a methionine substitution within a transmembrane leucine string of MLC has been implicated in catatonic schizophrenia in a large pedigree. To gain insight into the function of the MLC protein and to elucidate the pathophysiology of these severe neurodegenerative disorders, information on the cellular and regional distribution of the murine Mlc1, as well as the developmental pattern of Mlc1 expression in brain, is required. Using in situ hybridization (ISH), Mlc1 mRNA was exclusively detected in glial cells of the adult murine brain, such as astrocytes, Bergmann glia, and ependymal cells. ISH, Northern blot analysis, and quantitative real-time polymerase chain reaction (PCR) demonstrated that Mlc1 mRNA is broadly distributed in the adult mouse brain, with highest concentrations of expression in the cerebellum and olfactory bulb. Furthermore, differential expression patterns during brain development were revealed. Overall brain Mlc1 mRNA concentrations exhibited a substantial increase in the perinatal period reaching adult concentrations at postnatal day 5. At the cellular level, highest Mlc1 expression was found during the pre- and perinatal period in multipotential neural precursor cells, especially in the subventricular zone of the lateral ventricle, whereas in adulthood highest Mlc1 mRNA concentrations were revealed in Bergmann glia cells. Because the temporal expression profile of Mlc1 indicates that, in contrast to developing and mature astrocytes, oligodendrocytes are devoid of Mlc1 expression, white matter tract abnormalities observed in these disorders may result from a primary astrocytic defect. Detailed information on Mlc1 expression in brain is likely to lead to a better understanding of Mlc1 involvement in the pathogenesis of both MLC and catatonic schizophrenia.

Structural and functional characterization of the human PAX7 5'-flanking regulatory region.

The human PAX7 gene is a member of the paired box containing gene family of transcription factors implicated in development of the skeletal muscle of the trunk and limbs as well as elements of the central nervous system. To understand the molecular mechanisms involved in its expression, we have localized the transcription start sites in adult skeletal muscle and functionally characterized the 5'-flanking regulatory region responsible for PAX7 expression in this tissue. The major transcription start was identified 664 bp upstream from the ATG codon using primer extension and 5'-rapid amplification of cDNA ends (5'-RACE). Analysis of the 5'-flanking sequence revealed the absence of a TATA-box and the presence of an inverted CCAAT-box. Several consensus sites for common transcriptional regulators including Oct-1, NF1, AP2, AP4, CREB, Sp1, Nkx2.5, and MyoD are present in the promoter region. To determine the sites critical for the function of the PAX7 promoter, a series of deletion fragments of the 5'-flanking region were cloned adjacent to luciferase reporter gene and expressed in RD, Cos-7 and JAR cell lines. The maximal promoter activity was achieved by a fragment extending from the position -403 to +373. No strong positive or negative regulatory elements were discovered by adding of further sequences (up to 2.97 kb). A polymorphic (CCT)(n) repeat sequence was found 107 bp upstream of the transcription initiation site. PCR-based systematic screening for length variations in 227 unrelated individuals of a Caucasian population showed a bimodal distribution of three alleles containing 8, 10 or 11 repeat units. When different variants of this PAX7 gene-linked polymorphic region (PAX7-LPR) were fused to a luciferase reporter gene and transfected into RD cells, the variant with 11 repeat units revealed higher transcriptional efficiency compared to the 8 or 10 repeat alleles.