Joseph Cubells - US grants

DESCRIPTION: (Adapted from applicant's abstract) Cocaine-induced paranoia and psychotic symptoms (CIPPS) are associated with violence and greater morbidity in cocaine users. Since the phenotype appears only after chronic cocaine use, CIPPS provides a direct human example of cocaine-induced sensitization, an understanding of which is critical to addiction science. Genetic factors are important in CIPPS. CIPPS symptoms comprise a relatively homogeneous phenotypic spectrum. Thus, users who develop CIPPS, and those who do not, each represent more homogeneous groups of cocaine users, in whom genetic investigations are more likely to be fruitful than in those merely meeting dependence criteria. To examine the genetics of CIPPS, this work will: (1) Collect DNA samples from unrelated cocaine-using subjects in whom the CIPPS phenotype has been thoroughly evaluated, and from family trios containing probands carefully assessed for CIPPS. (2) Replicate and extend previous findings from this laboratory on the relationship of allelic variation at genes expressed in catecholamine neurons and CIPPS. (3) Determine relationships between genotypes, biochemical phenotypes, and behavioral phenotypes in African- and European-Americans. (4) Use methods previously developed for idiopathic psychoses to evaluate psychotic realm experiences and related psychological phenomena in abstinent cocaine users. Important strengths of the proposed work are (1) Development of unique DNA resources Suitable for extensive future study, including a first-of-its-kind family collection. (2) A focus on candidate genes supported by previous positive results and neurobiological considerations. (3) Use of haplotypes as well as single genotypes in genetic analysis. (4) Application of well-validated measures of psychosis: the Bell Object Relations and Reality Testing Inventory, the Scale for Assessment of Positive Symptoms, as well as the Cocaine Experience Questionnaire. (5) The use of a repeated measures design in phenotypic assessment. (5) Possible identification of phenotypes that do not depend on cocaine intoxication for expression. This program of research will improve our understanding of the genetics underlying human brain responses to chronic cocaine exposure, thus leading to better diagnosis and treatments for cocaine dependence and other addictive disorders.

DESCRIPTION (provided by applicant): K02 Independent Scientist Career Development Award for Joseph F. Cubells,MD, PhD This proposal seeks support for the candidate's continued development as a research psychiatrist specializing in human genetics of drug-abuse-related phenotypes. The overarching hypothesis organizing both the Research Plan and the Career Plan is that sequence variation at specific loci will exert measurable effects on specific drug-abuse-related phenotypes. This hypothesis is predicated on the importance of identifying and characterizing appropriate phenotypes, which will reflect the effects of one or a few genes with high penetrance. Three projects are proposed in the Research Plan. Project 1, which is well under way, is the candidate's R01-funded research on the human genetics of cocaine-induced psychosis (DA 12422). The project seeks to collect DNA samples from unrelated individuals with cocaine dependence, and from family trios containing cocaine-dependent probands; these probands are carefully characterized for cocaine-induced psychotic symptoms, and for axis-1 substance-abuse and psychiatric diagnoses, as well as for plasma levels of the catecholamine-synthetic enzyme dopamine beta-hydroxylase. Project 2 will build on work accomplished in Project 1, to examine how linkage disequilbrium among nearby polymorphisms influences phenotypic variation in quantitative traits. Project 3 will elucidate genetic influences on nicotine-dependence-related phenotypes by examining the influence of functional variation at the COMT locus on human neurocognitive function and acoustic startle responses in smoking and nonsmoking subjects, with and without schizophrenia. The Career Development Plan will entail collaboration with colleagues at Yale University and the University of Connecticut, whose expertises range from evaluation of complex phenotypes, to molecular genetics, to statistical genetics. In addition, the candidate plans specific coursework, both at Yale, and at off-campus sites, to strengthen his expertise in a both molecular, statistical and clinical genetics. The latter goal will be accomplished by the candidate directing the newly-established Yale Human Behavioral Genetics Clinic, which offers psychiatric and substance-abuse services to patients with genetic illnesses.

The hypothalamic-pituitary-adrenal (HPA) axis mediates many adaptations to stress, and HPA dysfunction occurs in depression and other stress-related disorders. During pregnancy, HPA-related mechanisms mediate stress-related communication between the maternal and fetal compartments through the placenta, which secretes corticotrophin-releasing hormone (CRH). The overarching hypothesis guiding Project 2 is that fetal and encoding key components of the hypothalamic-pituitary-adrenal (HPA) axis substantially influence fetal vulnerability to intrauterine exposures and maternal emotional distress by modulating (i) the sensitivity of the fetus to the milieu, and (ii) fetal-maternal communication mediated by the placenta. We will use family-based association methods (FBAT and PBAT) to address that hypothesis, by pursuing' the following Specific Aims: (1) examine the effects of maternal stress and fetal genotypes on placental expression of HPA-related genes, (2) examine the relationship among fetal polymorphisms in HPA-related genes and maternal and fetal serum concentrations of CRH, CRHBP and cortisol, (3) test the association of fetal genotypes and haplotypes of HPA-axis related genes with fetal and neonatal outcome (uterine blood flow, birth weight and infant cortisol response after inoculation and strange situation), and (4) test for gene x environment interactions, the environment being low vs. high maternal stress during pregnancy on the outcomes examined in Specific Aims 2 and 3. Public-health relevance: Increasing evidence suggests that maternal depression and other stress-related disorders occurring during pregnancy negatively impact fetal and infant outcomes. HPA-related mechanisms appear to be critical mediators and modulators of the relationship between maternal stress and depression, and such outcomes. Understanding whether and how variation at specific HPA-axis-related genes alters the vulnerability of offspring to maternal stress and depression may elucidate the role of the gene products they encode in negative (and positive) fetal and infant outcomes. Such knowledge will facilitate early-intervention strategies for at-risk children, and their mothers

DESCRIPTION (provided by applicant): Dopamine beta-hydroxylase (DbH) converts dopamine (DA) to norepinephrine (NE), and can be assayed in the plasma, where its activity is under genetic control by an oligogenic mechanism. While linkage studies based on phenotypic markers, and molecular association studies, suggest the structural gene DBH is a major quantitative trait locus regulating plasma DbH activity, this hypothesis has never been tested by molecular linkage analysis. There is evidence for at least one additional locus contributing to the regulation of plasma DbH activity, but the identity of this locus is unknown. Identifying the loci responsible for regulating plasma DbH is important for genetic research on schizophrenia (SCZ), because several lines of evidence suggest that differences in plasma DbH activity associate with differences in the phenotypic presentation of SCZ. In addition, abundant evidence suggests that alteration in DA- and NE-medicated neuro-transmission influence core phenotypes relevant to SCZ psychopathology, such as executive and working memory. We therefore hypothesize that DBH is a modifying gene in SCZ, that interacts with susceptibility loci to alter the symptoms and course of the illness. This application proposes 4 aims: (1) to conduct linkage analysis of plasma DbH activity in a set of SCZ pedigrees from which plasma samples are available;(2) to conduct follow-up association analysis of that linkage study to identify loci and specific variants responsible for regulating plasma DbH;(3) to conduct a linkage analysis of SCZ in a larger collection of SCZ families, conditional on genotypes at 2 SNPs at DBH already established to associate strongly with plasma DbH activity;and (4) to conduct association analyses in the probands of those families to test the hypothesis that DbH-regulating SNPs associate with altered symptomatic profiles and course of illness. The proposed work is significant for the public health because SCZ is a common disorder that usually disables people just as they enter adulthood, leading to huge costs in productivity, large burdens on healthcare and criminal-justice systems, and enormous human suffering by patients and their families. Identifying predictors of varying outcomes in SCZ would open new avenues for development of targeted interventions that could significantly ameliorate the impact of SCZ on individuals and society.

The overarching goal of this project is to identify hypothalamic-pituitary-adrenal (HPA)-axis-related[unreadable] parameters as potential predictors and/or biomarkers of disease progression and response to treatment for[unreadable] major depression in treatment-naTve patients. We will develop candidate parameters related to the HPA-axis,[unreadable] and more specifically, to glucocorticoid receptor (GR) function. Those parameters may include genotypes or[unreadable] haplotypes at GR-related loci, differences in expression of GR chaperone genes, measurements of GR[unreadable] function in vivo and in vitro, or some combination of these. Once our genetic investigations have identified[unreadable] putative predictors of treatment response, they will be integrated with data from neuro-imaging, transporteroccupancy[unreadable] studies, clinical assessments and other data for inclusion in an overall model of response[unreadable] predictors to be developed in the Special Scientific Procedures Core.[unreadable] The specific aims of this project include examination of relationships among HPA-axis-associated[unreadable] markers, measured at the genotypic, mRNA-expression, biochemical and systemic levels. More specifically[unreadable] we will investigate how sequence variation at the genetic level in GR receptor- regulating genes associate[unreadable] with mRNA expression in monocytes isolated from patients at multiple timepoints, and to glucocorticoid[unreadable] receptor function measured in vitro (i.e., in monocytes) as well as in vivo (using the combined[unreadable] dexamethasone suppression/CRH stimulation (DEX-CRH) test).[unreadable] Integrating multiple levels of analysis may help to identify genetic and molecular mechanisms for HPAaxis[unreadable] dysregulation and its normalization in response to anti-depressant treatment, thereby suggesting[unreadable] specific predictors for response to antidepressant treatments or disease progression. The elucidation of[unreadable] molecular mechanisms for the normalization of HPA-axis hyperactivity that accompanies successful[unreadable] antidepressant treatment may also be an important step in the development of novel antidepressants.[unreadable] This project will interact closely with the Operations and Clinical Assessment Core by coordinating all[unreadable] necessary blood draws and endocrine challenge tests and through a shared database integrating genetic[unreadable] and phenotypic data, the Research Methods Core by genotyping polymorphisms in all candidate genes[unreadable] relevant for this project and providing detailed information on their population-specific haplotypic structure,[unreadable] and the Special Scientific Procedures Core by generating multi-level HPA-axis related data for inclusion in[unreadable] the overall predictive model for treatment outcome.

DESCRIPTION (provided by applicant): Project Summary: Dopamine 2-hydroxylase (DBH) catalyzes synthesis of the neurotransmitter norepinephrine (NE) from dopamine (DA), and thereby regulates levels of both molecules. DBH is detectable in human serum, where its activity largely reflects sequence variation at the structural locus DBH. We identified a single nucleotide polymorphism (SNP) 970 bp upstream of the transcriptional start site (-970C>T) that accounts for 30-50% of the total variance in serum DBH activity, with the "T" allele being associated with lower activity than the "C" allele, in a co-dominant pattern. Our work on a population-genetic level, and molecular experiments from another laboratory provide strong evidence that -970C>T is a true regulatory variant, affecting transcription of DBH. However, the molecular experiments suggested that the low-serum-DBH T allele associates with greater transcription. Thus, how -970C>T regulates DBH expression in vivo is not clear. The present project proposes to use BAC transgenesis, followed by crossing with Dbh -/- mice lacking endogenous DBH expression, to investigate DBH expression in brain, adrenal medulla and serum, associated with either the T or C allele in an otherwise identical human-sequence context of ~170 kb. This project will inform multiple areas of public health research, including studies of psychosis, mood disorders, drug addiction, neuro-degenerative disorders, and disorders of human blood pressure, as variation in serum DBH and/or -970C>T associates with important aspects of human phenotypes related to all of those disorders. The proposed work will produce a valuable and innovative animal model for investigation of many aspects of genetic variation in NE-mediated neurotransmission. PUBLIC HEALTH RELEVANCE: Project Narrative: Dopamine 2-hydroxylase (DBH) is an enzyme that synthesizes the neural signaling molecule norepinephrine (NE), which is in turn involved in human psychiatric, neurological and blood-pressure related disorders. A sequence variant (polymorphism) in the human DBH gene strongly regulates levels of DBH, and influences all of the foregoing human disorders. This project will put the two versions (alleles) of the human DBH gene into mice that lack their own Dbh. This will allow functional study of this important human polymorphism in a mouse model.

Project Summary In this revised proposal, we plan to examine the physiological and synaptic properties of pluripotent stem cell (iPSC)-derived neurons, as well as schizophrenia (SCZ)-related physiological phenotypes, gathered from patients with 22q11 Deletion Syndrome (22q11DS). The syndrome associates with a 20-30 fold increase in the risk for schizophrenia. 20-30% of patients with 22q11DS develop SCZ by early adulthood. The acoustic startle response (ASR) is an evolutionarily conserved reflex, aspects of which differ in SCZ compared to healthy controls. Prior work on non-22q11DS individuals at high risk for SCZ based on their phenotypic characteristics (i.e., those with prodromal symptoms) suggest that the latency of ASR predicts conversion to SCZ. Mismatch negativity (MMN) is an evoked potential in response to unusual or ?oddball? acoustic stimuli imbedded within a train of repetitive acoustic stimuli. Impaired generation of an enhanced response to the oddball stimuli is the well-replicated MMN abnormality seen in SCZ. Our proposed work will examine ASR measures and MMN in older adolescents and young adults with 22q11DS (and healthy controls) to test the hypothesis that latency of the ASR and/or MMN will predict severity of prodromal symptoms, and ultimately conversion to SCZ, in this genetically defined high-risk group. Simultaneously we will study potential cellular mechanisms related to ASR and MMN in iPSC-derived neurons from 22q11DS patients exhibiting extreme values of latency to startle in the ASR. We hypothesize that doing so will identify potential cellular mechanisms underlying the phenotypic impact of the 22q11 deletion (including elevated risk for SCZ). This research will thus shed light on how genetic mechanisms alter cellular properties relevant to clinical and physiological differences observed in SCZ and the SCZ prodrome.