Andreas Reif

Meta-analysis of genome-wide association studies of attention-deficit/hyperactivity disorder

OBJECTIVEnAlthough twin and family studies have shown attention-deficit/hyperactivity disorder (ADHD) to be highly heritable, genetic variants influencing the trait at a genome-wide significant level have yet to be identified. As prior genome-wide association studies (GWAS) have not yielded significant results, we conducted a meta-analysis of existing studies to boost statistical power.nnnMETHODnWe used data from four projects: a) the Childrens Hospital of Philadelphia (CHOP); b) phase I of the International Multicenter ADHD Genetics project (IMAGE); c) phase II of IMAGE (IMAGE II); and d) the Pfizer-funded study from the University of California, Los Angeles, Washington University, and Massachusetts General Hospital (PUWMa). The final sample size consisted of 2,064 trios, 896 cases, and 2,455 controls. For each study, we imputed HapMap single nucleotide polymorphisms, computed association test statistics and transformed them to z-scores, and then combined weighted z-scores in a meta-analysis.nnnRESULTSnNo genome-wide significant associations were found, although an analysis of candidate genes suggests that they may be involved in the disorder.nnnCONCLUSIONSnGiven that ADHD is a highly heritable disorder, our negative results suggest that the effects of common ADHD risk variants must, individually, be very small or that other types of variants, e.g., rare ones, account for much of the disorders heritability.

Toward a molecular architecture of personality

Epidemiological studies provided a large body of evidence that personality dimensions are influenced by genetic factors and that the genetic component is highly complex, polygenic, and epistatic. However, consistent findings on the genetic basis of personality have yet remained sparse. In recent years, molecular genetics has begun to identify specific genes coding in particular for components of the serotonergic and dopaminergic neurotransmitter systems representing quantitative trait loci (QTLs) for behavioral traits. The QTL concept suggests that complex traits are not attributable to single genes. According to this polygenic model, the genetic basis of personality and behavior and its pathological variations thus results from additive or nonadditive interactions of various genes. As the number of suitable candidate genes constantly increases, the QTL model provides a reasonable explanation for the genetic basis of personality and its disorders. In this review, the current knowledge on the impact of a large number of candidate gene polymorphisms (e.g. variations in serotonin and dopamine receptor and serotonin transporter genes) on personality and temperament is summarized. Additionally, investigations of gene-gene and gene-environment interactions in humans and animals, which currently intensify the identification of genes that underlie behavioral variations, are examined. The findings converge on the notion that a probabilistic rather than deterministic impact of genes on the expression of behavior will contribute to the demystification of behavioral disorders.

Differences in inflammatory pain in nNOS-, iNOS- and eNOS-deficient mice

To assess the relative importance of the isoforms of nitric oxide synthase (NOS) in inflammatory pain, we directly compared pain behaviour and paw thickness after intraplantar injection of complete Freunds adjuvant (CFA) in wild‐type (WT) mice and in mice lacking either inducible (iNOS), endothelial (eNOS) or neuronal NOS (nNOS). In mice deficient for nNOS, thermal hyperalgesia was reduced by approximately 50% compared to wild type mice at 4 and 8h after CFA injection, and mechanical hypersensitivity was absent. The only change in pain behaviour in iNOS and eNOS deficient mice compared to WT mice was a more rapid recovery from thermal hyperalgesia. A compensatory up‐regulation of nNOS in dorsal root ganglia (DRG) and spinal cords of iNOS and eNOS knockout mice was excluded using RT‐PCR. However, an increase of iNOS gene expression was found in spinal cords of eNOS and nNOS deficient mice. To study the downstream effects of nNOS deficiency on DRG neurones, we assessed their immunoreactivity for calcitonin gene‐related peptide (CGRP) and cytokines. We found a significant reduction in the CFA induced increase in CGRP immunoreactive neurones as well as in CGRP gene expression in nNOS deficient mice, whereas the percentage of cells immunopositive for tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) was unchanged. These results support the proposed role of nNOS in sensitization of DRG neurones, and might indicate that CGRP is involved in this process.

Major Depression: A Role for Hippocampal Neurogenesis?

Since its discovery in mammals, adult neurogenesis, the process of generating functional neurons from neural progenitor cells in the adult brain, has inspired numerous animal studies. These have revealed that adult neurogenesis is a highly regulated phenomenon. Enriched environment, exercise and learning for instance, are positive regulators while stress and age are major negative regulators. Stressful life events are not only shown to reduce adult neurogenesis levels but are also discussed to be a key element in the development of various neuropsychiatric disorders such as depression. Interestingly, altered monoaminergic brain levels resulting from antidepressant treatment are shown to have a strong reinforcing effect on adult neurogenesis. Additionally, disturbed adult neurogenesis, possibly resulting in a malfunctioning hippocampus, may contribute to the cognitive deficits and reduced hippocampal volumes observed in depressed patients. Hence, the question arises as to whether disturbed adult neurogenesis and the etiopathogenesis of depression are causally linked. In this chapter, we discuss the possible causal interrelation of disturbed adult neurogenesis and the etiopathogenesis of depression as well as the possibility that adult neurogenesis is not exclusively linked to depression but is also linked to other psychiatric disorders including schizophrenia and neurodegenerative diseases like Alzheimers disease. Additionally, we look at the functional relevance of adult neurogenesis in different species, upon which we base our discussion as to whether adult neurogenesis could be causally linked to the development of certain brain disorders in humans, or whether it is only an epiphenomenon.

Replication and meta-analysis of TMEM132D gene variants in panic disorder.

A recent genome-wide association study in patients with panic disorder (PD) identified a risk haplotype consisting of two single-nucleotide polymorphisms (SNPs) (rs7309727 and rs11060369) located in intron 3 of TMEM132D to be associated with PD in three independent samples. Now we report a subsequent confirmation study using five additional PD case–control samples (n=1670 cases and n=2266 controls) assembled as part of the Panic Disorder International Consortium (PanIC) study for a total of 2678 cases and 3262 controls in the analysis. In the new independent samples of European ancestry (EA), the association of rs7309727 and the risk haplotype rs7309727–rs11060369 was, indeed, replicated, with the strongest signal coming from patients with primary PD, that is, patients without major psychiatric comorbidities (n=1038 cases and n=2411 controls). This finding was paralleled by the results of the meta-analysis across all samples, in which the risk haplotype and rs7309727 reached P-levels of P=1.4e−8 and P=1.1e−8, respectively, when restricting the samples to individuals of EA with primary PD. In the Japanese sample no associations with PD could be found. The present results support the initial finding that TMEM132D gene contributes to genetic susceptibility for PD in individuals of EA. Our results also indicate that patient ascertainment and genetic background could be important sources of heterogeneity modifying this association signal in different populations.

The novel brain-specific tryptophan hydroxylase-2 gene in panic disorder

Panic disorder is a common psychiatric disorder characterized by recurrent anxiety attacks and anticipatory anxiety. Due to the severity of the symptoms of the panic attacks and the frequent additional occurrence of agoraphobia, panic disorder is an often debilitating disease. Elevation of central serotonin levels by drugs such as clomipramine represents one of the most effective treatment options for panic disorder. This points to an important role of dysregulation of the serotonergic system in the genetic etiology of panic disorder. The novel brain-specific 5-HT synthesizing enzyme, tryptophan hydroxylase-2 (TPH2), which represents the rate-limiting enzyme of 5-HT production in the brain, may therefore be of particular importance in panic disorder. We focused on the putative transcriptional control region of TPH2 and identified two novel common single nucleotide polymorphisms (SNPs) of TPH2 in and close to this region. Moreover, a recently described loss-of-function mutation of TPH2 which results in an 80% reduction of serotonin production, was assessed. In an analysis of the putative transcriptional control region SNPs in a sample of panic disorder patients and controls no association of the disorder with the TPH2 SNPs or haplotypes was found. Moreover, the loss-of-function R441H mutation of TPH2 was not present in the panic disorder patients. The results of this first study of TPH2 in panic disorder argue against an importance of allelic variation of TPH2 in the pathogenesis of panic disorder with or without agoraphobia.

Adult neurogenesis in serotonin transporter deficient mice

SummarySerotonin (5-HT) is a regulator of morphogenetic activities during early brain development and neurogenesis, including cell proliferation, migration, differentiation, and synaptogenesis. The 5-HT transporter (5-HTT, SLC6A4) mediates high-affinity reuptake of 5-HT into presynaptic terminals and thereby fine-tunes serotonergic neurotransmission. Inactivation of the 5-HTT gene in mice reduces 5-HT clearance resulting in persistently increased concentrations of synaptic 5-HT. In the present study, we investigated the effects of elevated 5-HT levels on adult neurogenesis in the hippocampus of 5-HTT deficient mice, including stem cell proliferation, survival, and differentiation. Using an in vivo approach, we showed an increase in proliferative capacity of hippocampal adult neural stem cells in aged 5-HTT knockout mice (∼14.5 months) compared to wildtype controls. In contrast, in vivo and additional in vitro analyses of younger adult 5-HTT knockout mice (∼7 weeks and ∼3.0 months) did not reveal significant changes in proliferation of neural stem cells or survival of newborn cells. We showed that the cellular fate of newly generated cells in 5-HTT knockout mice is not different with respect to the total number and percentage of neurons or glial cells from wildtype controls. Our findings indicate that elevated synaptic 5-HT concentration throughout early development and later life of 5-HTT deficient mice does not induce adult neurogenesis in adult mice, but that elevated 5-HT levels in aged mice influence stem cell proliferation.

Bipolar disorder risk alleles in children with ADHD

Bipolar disorder (BD) and attention deficit/hyperactivity disorder (ADHD) may share common genetic risk factors as indicated by the high co-morbidity of BD and ADHD, their phenotypic overlap especially in pediatric populations, the high heritability of both disorders, and the co-occurrence in families. We therefore examined whether known polygenic BD risk alleles are associated with ADHD. We chose the eight best SNPs of the recent genome-wide association study (GWAS) of BD patients of German ancestry and the nine SNPs from international GWAS meeting a ‘genome-wide significance’ level of αxa0=xa05xa0×xa010−8. A GWAS was performed in 495 ADHD children and 1,300 population-based controls using HumanHap550v3 and Human660xa0W-Quadv1 BeadArrays. We found no significant association of childhood ADHD with single BD risk alleles surviving adjustment for multiple testing. Yet, risk alleles for BD and ADHD were directionally consistent at eight of nine loci with the strongest support for three SNPs in or near NCAN,BRE, and LMAN2L. The polygene analysis for the BP risk alleles at all 14 loci indicated a higher probability of being a BD risk allele carrier in the ADHD cases as compared to the controls. At a moderate power to detect association with ADHD, if true effects were close to estimates from GWAS for BD, our results suggest that the possible contribution of BD risk variants to childhood ADHD risk is considerably lower than for BD. Yet, our findings should encourage researchers to search for common genetic risk factors in BD and childhood ADHD in future studies.

Pterin interactions with distinct reductase activities of NO synthase.

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

IMpACTing on adult ADHD research

Franke, Barbara Reif, Andreas Editorial Netherlands Eur Neuropsychopharmacol. 2013 Jun;23(6):413-5. doi: 10.1016/j.euroneuro.2013.04.003.