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S.1.1 Genetic approaches to psychiatricdiseases
Molecular Neuropsychopharmacology
Lectures
IS.1.11 Genetic approaches to psychiatric diseases D. Goldman. Laboratory of Neurogeneties, NIAAA, NIH,
Roekville, AID 20852, USA Heritabilities of most psychiatric diseases are moderate to high, indicating that gene searches are likely to detect origins of vulnerability. Frequency and effect sizes of several validated functional genetic variants that contribute to complex behaviors vary widely, indicating that different gene search strategies are required. Some genes, such as the serotonin transporter and MAOA, have both variants that are common and probabilistic in their action as w e l l as variants that are uncommon and more highly deterministic in their action (associated w i t h a h i g h odds ratio). Several disorders, including autism and schizophrenia are probably polygenic, based on concordance patterns in relatives at different degrees of genetic relationship (e.g. high monozygotic/dizygotic twin concordance ratios). Other psychiatric diseases, such as alcoholism, are not demonstrably polygenic in their inheritance patterns, but genetic heterogeneity is already evident, based on heterogeneity in clinical features, intermediate phenotypes, and in the variety of genes in which functional genetic variants are already being implicated in risk (ADH1B, ALDH2, COMT, HTTLPR, G A B A A alpha 2. receptor). Genetic analysis of psychiatric diseases is increasingly directed towards neurobiologic domains accessed through intermediate phenotypes, including measures of personality and cognitive function, brain imaging, and chemistry. Frequently, the effects of individual genes on intermediate phenotypes are more clearcut, as compared to gene effects in complex behaviors. This emphasizes the challenge to understand gene/gene and gene/environment interactions in psychiatric disorders, and the challenge to identify disease subtypes. Three apparently robust examples of closer correlation of gene to intermediate phenotype than gene to disease include 1) COMT, which has been linked to vulnerability to schizophrenia, substance dependencies and other diseases but is more clearly linked to frontallymediated executive cognitive fur~tion and to variation in anxiety and pain/stress resiliency; and 2) HTTLPR which is linked, in environmentally modified fashion
to anxiety/dysphoria but more strongly to amygdale metabolic response to cognitive fear challenge, and 3) the linkage of alcohol metabolic enzyme variants to alcoholinduced flushing, and thereby to risk of alcoholism itsel£ COMT Val158Met is a common, functional enzyme polymorphism that is common across populations. Its two alleles themselves frequently reside on ancientlyderived chromosomes whose haplotype patterns are further predictive of COMT expression and function. The COMT Val158 allele and a haplotype carrying this allele, was linked to schizophrenia in some, but not all, studies. The mechanism would appear to be diminished frontal cognitive function, as shown by a combination of studies measuring cognitive performance or brain metabolic response during cognitive tasks in diverse clinical populations: schizophrenics, well siblings of schizophrenics, controls, and head injured patients. Different clinical groups have different baseline performances in frontal cognitive tasks but in general C O M T Val158 homozygotes and heterozygotes perform less well, coherent w i t h lower frontal dopamine levels. Val158 has also been linked to substance abuse but generally the Met158 allele, or a haplotype carrying this allele, is more abundant in substance abusers. This may be because the Met 158 allele is associated with higher trait anxiety, especially in women, and with diminished resiliency to pain and stress, as shown both by measures of pain threshold in a large cohort of w o m e n followed prospectively for temporomandibular joint pain and by a neuroimaging study of the brain's opioid system activation after a pain/stress challenge. The serotonin transporter is a pharmacotherapeutic target in disorders of anxiety/dysphoria, and a functional promoter polymorphism HTTLPR plays a role in armiety/dysphoria and appears to also have some predictive value for SSRI response in Major Depression. However, each copy of the "S" allele only contributes approximately an 0.1 SD increase in trait anxiety. The effect of the S allele to increase anxiety responses was more evident in an imaging study in w h i c h amygdala activation was measured w h e n research participants assessed fearful faces. HTTLPR genotype has now also been correlated to amygdale volume, and shown to be predictive &responses to other emotionally evocative pictures. Recently another variation has been found at the serotonin transporter gene, and linked to behavior. A n uncommon amino acid
Molecular Neuropsychopharmacology
$2
substitution Ile425Val was studied in two families and in these families Va1425, the gain-of-function variant, led to various severe psychopathologies including Asperger's syndrome, anorexia, and-especially- treatment-resistant OCD. Within the HTTLPR promoter region itself, a n e w functional A > G SNP was discovered, and using this new information H T T was linked to OCD both in a large case/control sample and in a transmission equilibrium test (TDT) sample consisting of parent child trios. Again, it was the gain of function LAJLA genotype that showed linkage to OCD.
concepts: histone modifications S•-.1.-2]toMolecular investigate the impact of stress on the brain J.M.H.M. Reul, S.K. Droste, Y. Chandramohan. Henry
Wellcome Laboratories for Integratioe Neuroscience and Endocrinology, Unioersity of Bristol, Bristol, United Kingdom Evidence has been accumulating that aberrant stress coping is an etiological factor in the development of stress-related psychiatric diseases such as major depression and anxiety disorders. It is also becoming increasingly clear that aberrant stress coping involves defective molecular and cellular mechanisms in the brain. Therefore, to understand the pathophysiology of stressrelated psychiatric diseases, we need to gain insight into how stressful environmental stimuli affect brain function. It is thought that c h a n t s in gene expression are involved in the neuroplasticity processes underlying stress coping. Gene expression is controlled by transcription factors whose activity is governed by a variety of signal transduction cascades. Control of gene expression is tight and normally most of the genome is silent with the chromatin being structurally organized in nucleosomes. The nucleosomes consist of highly organized complexes of D N A and histone molecules and represent a barrier to transcription by blocking access of transcription factors. Nuclear receptors such as the glucocorticoid receptor (GR) are an exception to this rule as they are able to access their hormone responsive elements and "unlock" the nucleosome rendering it accessible for molecules involved in chromatin remodeling and gene transcription [1]. Recently, substantial progress has been made in obtaining insight into the role of chromatin remodeling in the control of gene expression. The concept has arisen that distinct post-translational modifications in the N - t e r m i n a l tails of histone molecules play a decisive role in chromatin remodeling and making the underlying genome accessible for transcription factors and other molecules involved in gene transcription [2].
Phosphorylation of histone H3 at S e r l 0 has been receiving most attention. This modification has a dual "personality" [2] as it has been shown to be involved in both mitosis and transcriptional activation (of f.i. immediate-early genes (IEGs)) [3]. In transcriptional activation, the histone H3 molecules are additionally acetylated at L y s l 4 (i.e. P(Serl0)-Ac(Lysl4)-H3) and, actually, phosphorylation at Serl 0 is thought to facilitate acetylation at Lys14 (3), although this synergism is still controversial [4]. In addition, it appears that the S e r l 0 residue in histone H3 comprises a converging point of multiple signal transduction pathways and kinases such as the mitogen-activated protein kinase (MAPK) cascade and the subsequent activation of the extracellular-regulated kinases (-ERKs) and the mitogen- and stress-induced kinases 1 and 2 (MSK1/2). Recently, we could demonstrate for the first time that stressful stimuli induce histone H3 modifications necessary for transcriptional activation [5]. We discriminated between phosphorylated histone H3 (P(Serl0)-H3)immunoreactive cells in the dentate g y m s (DG) of rats and mice being either engaged in mitosis or in transcriptional activation [5] on the basis of morphology (condensed chromosomes versus speckled pattern) and localization (subgranular zone vs granular cell layer). The speckled staining pattern has been shown to be associated with transcriptional activation (e.g., ref. [6]). Accordingly, use of an anti-P(Serl 0)-Ac(Lys 14)-H3 antibody also presented a speckled pattern in nuclei of granular neurons, but no staining of mitotic cells were seen [5] (Y. Chandramohan, S.K. Droste, J.M,H.M, Reul, in preparation, and in this volume). Thus, with the P(Serl0)-H3 and P(Ser10)Ac(Lys14)-H3 antibodies we can visualize granular neurons w i t h activated transcription of formerly silent genes. Stained neurons were also observed in the neocortex and amygdala. To study whether psychologically stressful challenges known to be processed by the hippocampus would affect the number of "transcriptionally activated" neurons, rats and mice were submitted to a forced swim test (15 m i n in 25°C-water). Under baseline conditions the number of stained neurons in the rat and mouse D G is low but increases dramatically after exposing animals to forced swimming peaking between 8 and 2 4 h [5]. The increase in P(Serl0)-H3 + D G neurons after forced swimming was only seen in mature (NeuN+) neurons but not in immature (NeuN-) neurons [5] suggesting that only mature neurons are recruited in the adaptive response to stress. Exposure to physical stressors such as ether or cold was ineffective, but exposure to a novel environment, a m i l d psychological stressor, resulted in a marked increase in phosphorylation and phospho-acetylation of histone H3 in dentate gyrus granule neurons (Y. Chandramohan, S.K. Droste, J.M.H.M. Reul, in preparation, and in this volume).
Lectures
IS.1.11 Genetic approaches to psychiatric diseases D. Goldman. Laboratory of Neurogeneties, NIAAA, NIH,
Roekville, AID 20852, USA Heritabilities of most psychiatric diseases are moderate to high, indicating that gene searches are likely to detect origins of vulnerability. Frequency and effect sizes of several validated functional genetic variants that contribute to complex behaviors vary widely, indicating that different gene search strategies are required. Some genes, such as the serotonin transporter and MAOA, have both variants that are common and probabilistic in their action as w e l l as variants that are uncommon and more highly deterministic in their action (associated w i t h a h i g h odds ratio). Several disorders, including autism and schizophrenia are probably polygenic, based on concordance patterns in relatives at different degrees of genetic relationship (e.g. high monozygotic/dizygotic twin concordance ratios). Other psychiatric diseases, such as alcoholism, are not demonstrably polygenic in their inheritance patterns, but genetic heterogeneity is already evident, based on heterogeneity in clinical features, intermediate phenotypes, and in the variety of genes in which functional genetic variants are already being implicated in risk (ADH1B, ALDH2, COMT, HTTLPR, G A B A A alpha 2. receptor). Genetic analysis of psychiatric diseases is increasingly directed towards neurobiologic domains accessed through intermediate phenotypes, including measures of personality and cognitive function, brain imaging, and chemistry. Frequently, the effects of individual genes on intermediate phenotypes are more clearcut, as compared to gene effects in complex behaviors. This emphasizes the challenge to understand gene/gene and gene/environment interactions in psychiatric disorders, and the challenge to identify disease subtypes. Three apparently robust examples of closer correlation of gene to intermediate phenotype than gene to disease include 1) COMT, which has been linked to vulnerability to schizophrenia, substance dependencies and other diseases but is more clearly linked to frontallymediated executive cognitive fur~tion and to variation in anxiety and pain/stress resiliency; and 2) HTTLPR which is linked, in environmentally modified fashion
to anxiety/dysphoria but more strongly to amygdale metabolic response to cognitive fear challenge, and 3) the linkage of alcohol metabolic enzyme variants to alcoholinduced flushing, and thereby to risk of alcoholism itsel£ COMT Val158Met is a common, functional enzyme polymorphism that is common across populations. Its two alleles themselves frequently reside on ancientlyderived chromosomes whose haplotype patterns are further predictive of COMT expression and function. The COMT Val158 allele and a haplotype carrying this allele, was linked to schizophrenia in some, but not all, studies. The mechanism would appear to be diminished frontal cognitive function, as shown by a combination of studies measuring cognitive performance or brain metabolic response during cognitive tasks in diverse clinical populations: schizophrenics, well siblings of schizophrenics, controls, and head injured patients. Different clinical groups have different baseline performances in frontal cognitive tasks but in general C O M T Val158 homozygotes and heterozygotes perform less well, coherent w i t h lower frontal dopamine levels. Val158 has also been linked to substance abuse but generally the Met158 allele, or a haplotype carrying this allele, is more abundant in substance abusers. This may be because the Met 158 allele is associated with higher trait anxiety, especially in women, and with diminished resiliency to pain and stress, as shown both by measures of pain threshold in a large cohort of w o m e n followed prospectively for temporomandibular joint pain and by a neuroimaging study of the brain's opioid system activation after a pain/stress challenge. The serotonin transporter is a pharmacotherapeutic target in disorders of anxiety/dysphoria, and a functional promoter polymorphism HTTLPR plays a role in armiety/dysphoria and appears to also have some predictive value for SSRI response in Major Depression. However, each copy of the "S" allele only contributes approximately an 0.1 SD increase in trait anxiety. The effect of the S allele to increase anxiety responses was more evident in an imaging study in w h i c h amygdala activation was measured w h e n research participants assessed fearful faces. HTTLPR genotype has now also been correlated to amygdale volume, and shown to be predictive &responses to other emotionally evocative pictures. Recently another variation has been found at the serotonin transporter gene, and linked to behavior. A n uncommon amino acid
Molecular Neuropsychopharmacology
$2
substitution Ile425Val was studied in two families and in these families Va1425, the gain-of-function variant, led to various severe psychopathologies including Asperger's syndrome, anorexia, and-especially- treatment-resistant OCD. Within the HTTLPR promoter region itself, a n e w functional A > G SNP was discovered, and using this new information H T T was linked to OCD both in a large case/control sample and in a transmission equilibrium test (TDT) sample consisting of parent child trios. Again, it was the gain of function LAJLA genotype that showed linkage to OCD.
concepts: histone modifications S•-.1.-2]toMolecular investigate the impact of stress on the brain J.M.H.M. Reul, S.K. Droste, Y. Chandramohan. Henry
Wellcome Laboratories for Integratioe Neuroscience and Endocrinology, Unioersity of Bristol, Bristol, United Kingdom Evidence has been accumulating that aberrant stress coping is an etiological factor in the development of stress-related psychiatric diseases such as major depression and anxiety disorders. It is also becoming increasingly clear that aberrant stress coping involves defective molecular and cellular mechanisms in the brain. Therefore, to understand the pathophysiology of stressrelated psychiatric diseases, we need to gain insight into how stressful environmental stimuli affect brain function. It is thought that c h a n t s in gene expression are involved in the neuroplasticity processes underlying stress coping. Gene expression is controlled by transcription factors whose activity is governed by a variety of signal transduction cascades. Control of gene expression is tight and normally most of the genome is silent with the chromatin being structurally organized in nucleosomes. The nucleosomes consist of highly organized complexes of D N A and histone molecules and represent a barrier to transcription by blocking access of transcription factors. Nuclear receptors such as the glucocorticoid receptor (GR) are an exception to this rule as they are able to access their hormone responsive elements and "unlock" the nucleosome rendering it accessible for molecules involved in chromatin remodeling and gene transcription [1]. Recently, substantial progress has been made in obtaining insight into the role of chromatin remodeling in the control of gene expression. The concept has arisen that distinct post-translational modifications in the N - t e r m i n a l tails of histone molecules play a decisive role in chromatin remodeling and making the underlying genome accessible for transcription factors and other molecules involved in gene transcription [2].
Phosphorylation of histone H3 at S e r l 0 has been receiving most attention. This modification has a dual "personality" [2] as it has been shown to be involved in both mitosis and transcriptional activation (of f.i. immediate-early genes (IEGs)) [3]. In transcriptional activation, the histone H3 molecules are additionally acetylated at L y s l 4 (i.e. P(Serl0)-Ac(Lysl4)-H3) and, actually, phosphorylation at Serl 0 is thought to facilitate acetylation at Lys14 (3), although this synergism is still controversial [4]. In addition, it appears that the S e r l 0 residue in histone H3 comprises a converging point of multiple signal transduction pathways and kinases such as the mitogen-activated protein kinase (MAPK) cascade and the subsequent activation of the extracellular-regulated kinases (-ERKs) and the mitogen- and stress-induced kinases 1 and 2 (MSK1/2). Recently, we could demonstrate for the first time that stressful stimuli induce histone H3 modifications necessary for transcriptional activation [5]. We discriminated between phosphorylated histone H3 (P(Serl0)-H3)immunoreactive cells in the dentate g y m s (DG) of rats and mice being either engaged in mitosis or in transcriptional activation [5] on the basis of morphology (condensed chromosomes versus speckled pattern) and localization (subgranular zone vs granular cell layer). The speckled staining pattern has been shown to be associated with transcriptional activation (e.g., ref. [6]). Accordingly, use of an anti-P(Serl 0)-Ac(Lys 14)-H3 antibody also presented a speckled pattern in nuclei of granular neurons, but no staining of mitotic cells were seen [5] (Y. Chandramohan, S.K. Droste, J.M,H.M, Reul, in preparation, and in this volume). Thus, with the P(Serl0)-H3 and P(Ser10)Ac(Lys14)-H3 antibodies we can visualize granular neurons w i t h activated transcription of formerly silent genes. Stained neurons were also observed in the neocortex and amygdala. To study whether psychologically stressful challenges known to be processed by the hippocampus would affect the number of "transcriptionally activated" neurons, rats and mice were submitted to a forced swim test (15 m i n in 25°C-water). Under baseline conditions the number of stained neurons in the rat and mouse D G is low but increases dramatically after exposing animals to forced swimming peaking between 8 and 2 4 h [5]. The increase in P(Serl0)-H3 + D G neurons after forced swimming was only seen in mature (NeuN+) neurons but not in immature (NeuN-) neurons [5] suggesting that only mature neurons are recruited in the adaptive response to stress. Exposure to physical stressors such as ether or cold was ineffective, but exposure to a novel environment, a m i l d psychological stressor, resulted in a marked increase in phosphorylation and phospho-acetylation of histone H3 in dentate gyrus granule neurons (Y. Chandramohan, S.K. Droste, J.M.H.M. Reul, in preparation, and in this volume).