Daniel Geschwind - US grants

The left and right hemispheres of the human cerebral cortex are functionally and anatomically distinct. Although recent work has identified genes whose expression underlies the rostral-caudal segmentation of the nervous system, no gene (s) that contributes to the lateralization of the cerebral hemispheres has been identified. Since the development of cerebral asymmetry (laterality) underlies language and other uniquely human cognitive abilities, and its disruption has been implicated in a number of pathologic conditions, the identification of such genes is of great importance to the clinical and basic neurosciences. The experiments outlined in this proposal seek to identify and characterize genes that are asymmetrically expressed in the developing human left and right cerebral hemispheres. Since no such gene has been previously identified, this work has the potential to open an entirely new avenue of neurobiological research. The preliminary results are encouraging, 2 candidate asymmetrically expressed genes have been identified thusfar. Representational Difference Analysis (RDA) has been used to subtract the right and left posterior perisylvian regions from each other during fetal development, proximal to the emergence of gross cerebral asymmetry. Analysis of approximately 1600 RDA subtraction products using cDNA microarray technology and a 2-color fluorescent detection system has resulted in the identification of 198 candidate clones for asymmetrical expression. Partial sequencing identified a subset of genes for further characterization, some of which share intriguing homologies with genes involved in visceral asymmetry or nervous system pattern formation. Further secondary screening using RT-PCR or Northern blotting has confirmed the differential expression of 2of these genes: one that is enriched on the right and the other that is enriched on the left. Many of clones identified in this initial screen appear to represent novel genes, about 25 percent of which have no EST (expressed sequence tag) matches in the nucleic acid and protein databases. This and other data suggest that the utility of further microarray screening of this RDA is far from saturated. Therefore, cDNA microarrays will be used to evaluate an additional 7680 RDA subtraction clones for asymmetric expression, followed by a second stage screening using the step-wise approach that we have used to identify the 2 differentially expressed clones mentioned above. Once these clones are identified, full-length cDNAs will be obtained, and the developmental expression pattern of several of these genes will be studied in the nervous system and in non- nervous system tissues, using a combination of techniques including Northern blotting, in situ hybridization and immunohistochemistry. In addition, the evolutionary conservation of asymmetrically expressed genes in non-human species will be investigated. The availability of molecular markers correlated with the development of cerebral asymmetry will greatly serve the study of the development and evolution of cognitive functions such as language, and disorders that disrupt them.

The left and right hemispheres of the human cerebral cortex are functionally and anatomically distinct. Although recent work has identified genes whose expression underlies the rostral-caudal segmentation of the nervous system, no gene (s) that contributes to the lateralization of the cerebral hemispheres has been identified. Since the development of cerebral asymmetry (laterality) underlies language and other uniquely human cognitive abilities, and its disruption has been implicated in a number of pathologic conditions, the identification of such genes is of great importance to the clinical and basic neurosciences. The experiments outlined in this proposal seek to identify and characterize genes that are asymmetrically expressed in the developing human left and right cerebral hemispheres. Since no such gene has been previously identified, this work has the potential to open an entirely new avenue of neurobiological research. The preliminary results are encouraging, 2 candidate asymmetrically expressed genes have been identified thusfar. Representational Difference Analysis (RDA) has been used to subtract the right and left posterior perisylvian regions from each other during fetal development, proximal to the emergence of gross cerebral asymmetry. Analysis of approximately 1600 RDA subtraction products using cDNA microarray technology and a 2-color fluorescent detection system has resulted in the identification of 198 candidate clones for asymmetrical expression. Partial sequencing identified a subset of genes for further characterization, some of which share intriguing homologies with genes involved in visceral asymmetry or nervous system pattern formation. Further secondary screening using RT-PCR or Northern blotting has confirmed the differential expression of 2of these genes: one that is enriched on the right and the other that is enriched on the left. Many of clones identified in this initial screen appear to represent novel genes, about 25 percent of which have no EST (expressed sequence tag) matches in the nucleic acid and protein databases. This and other data suggest that the utility of further microarray screening of this RDA is far from saturated. Therefore, cDNA microarrays will be used to evaluate an additional 7680 RDA subtraction clones for asymmetric expression, followed by a second stage screening using the step-wise approach that we have used to identify the 2 differentially expressed clones mentioned above. Once these clones are identified, full-length cDNAs will be obtained, and the developmental expression pattern of several of these genes will be studied in the nervous system and in non- nervous system tissues, using a combination of techniques including Northern blotting, in situ hybridization and immunohistochemistry. In addition, the evolutionary conservation of asymmetrically expressed genes in non-human species will be investigated. The availability of molecular markers correlated with the development of cerebral asymmetry will greatly serve the study of the development and evolution of cognitive functions such as language, and disorders that disrupt them.

DESCRIPTION (adapted from applicant's abstract):The core features of idiopathic basal ganglia calcifications (IBGC) or Fahr's disease are dystonia, parkinsonism and neurobehavioral abnormalities that are associated with calcifications visible on CT scan of the brain. Familial IBGC shows mostly an autosomal dominant mode of inheritance. The investigators have mapped a locus on chr.14q in one large multiplex family. In two other families linkage to chr.14 has been excluded, demonstrating genetic heterogeneity. The minimal critical region (MCR) on chr.14 is 15 or probably 10cM. The investigators propose to narrow down the MCR by collecting additional family members of the original pedigree as well as other families. Physical mapping and candidate screening for mutations will be pursued as the region is narrowed to identify the IBGC gene. A genome scan will be performed in families who are not linked to the chr.14 locus.

DESCRIPTION (provided by applicant): Autism is a devastating neuropsychiatric condition with unknown pathophysiology and for which there are no current effective treatments. Autism spectrum disorders are not uncommon and have an estimated incidence of approximately 1/1000. Although autism has a multifactorial etiology, it has a large genetic component. Recent genome scans have identified several potential chromosomal loci, but clear evidence for genetic heterogeneity has emerged, and the regions identified remain wide and in most cases only suggestive linkage to these regions has been found. Therefore, while a genetic approach to understanding autism etiology is likely to be fruitful, collaborative efforts involving large pooled samples will be necessary to achieve maximal power to identify disease critical regions narrow enough to permit positional cloning of autism susceptibility genes. A collaborative effort to produce an open data and biomaterials resource for the research community, the autism genetic resource exchange (AGRE), has been formed to facilitate the identification of autism susceptibility genes. This collaborative multi-site proposal seeks to expand and study the AGRE sample so as to identify and narrow autism susceptibility loci. Three hundred new multiplex families with autism spectrum disorders will be ascertained, for a total of 550 families in AGRE. A tiered, whole genome scan initially at 10 cM resolution, followed by fine mapping will be conducted to identify novel autism loci and more definitively confirm those previously identified. Phenotypic information will be used to help stratify the population so as to limit heterogeneity, as well as permit quantitative trait analysis focused on several heritable neurobehavioral components of autism. In parallel, karyotyping, and FISH using subtelomeric probes, and probes for several specific regions where chromosomal anomalies are highly associated with autism will be done. Finally, we have developed a novel, inexpensive, microarray-based method for SNP genotyping that will be used for association analyses, to permit narrowing of susceptibility regions identified. All phenotypic and genotype data will be made accessible via the Internet on a rolling basis, further enhancing the value of this resource to the community.

[unreadable] DESCRIPTION (provided by applicant): Autism is a severe and chronic neuropsychiatric disorder that typically reveals itself in the first 36 months of life. While individual behavioral profiles can vary widely, there are core domains revealing deficits in social interaction and relations, language and communication skills, and behavioral control. Twin studies and high sibling relative risk ratios indicate a strong genetic component to this disorder, which was once thought to be rare, but autism spectrum disorders are now thought to have a 1/250 prevalence or more. Genetic studies have been challenged by sample heterogeneity and limited by small sample sizes. This supplement to parent grant 1 R01 MH64547-01, "A Genome Wide Search for Autism Susceptibility Loci," supports the efforts of the Autism Genetic Resource Exchange (AGRE), a large collaborative effort to create a bank of genotypic and phenotypic data from multiplex autism families and to share this data with the scientific community. Specifically, this supplement would fund the recruitment, collection of biomaterials, and state-of-the-science phenotypic profiling (ADI-R, ADOS, Peabody Picture Vocabulary Test, Raven Progressive Matrices, Vineland Adaptive Behavior Scales, and medical history and neurological exams) of 400 North American multiplex families affected by autism. The work will be carried out by the AGRE team of diagnosticians, clinicians, administrators and phlebotomists under subcontract to the University of California, Los Angeles. In order to facilitate recruitment and accommodate the strained circumstances of multiplex families, the majority of diagnostic work is done in the family home. Biological samples (DNA, cell lines, and serum) are placed in the NIMH repository and all phenotypic data is placed in an internet accessible database maintained by the Autism Genetic Resource Exchange. Data are marked only with a confidential sample ID so that families remain anonymous and data become immediately available, with no holdback period, to the entire scientific community through approved access to the password-protected databases for biomaterials and/or phenotypic data. This effort accelerates recruitment, exceeds previously stated goals for sample size and, by adding to the already substantial number of previously collected families pledged by AGRE to the NIMH repository, creates the world's largest and most powerful genetic resource for the study of autism. This work is a direct response to Title 1 of the Pediatric Health Act of 2000 which authorizes and mandates an increased NIH commitment to autism gene banking. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The identification of the genetic bases of dementias remains an important goal of research whose aims are to improve our understanding and develop new therapeutics for these diseases. The discovery of tau mutations in inherited cases of frontotemporal dementia (FTD) demonstrated that tau dysregulation can cause neurodegenerative disease. But, tau mutations have not been described in AD, and tau mutations account for about 15% of familial FTD cases. An evolving body of work from many groups suggests that genes involved in modifying tau, especially those that modulate tau phosphorylation are enticing candidates for contributing to the genetic risk for these AD and FTD. In cases where tau abnormalities have not been identified on a pathological or genetic basis, dysregulation in key machinery involved in protein processing and folding apparatus may provide additional genetic risk elements. The goal of this proposal is to identify and confirm these novel genetic risk factors in Frontotemporal Dementia (FTD) and Alzheimer's Disease (AD) using a novel re-sequencing approach that allows massively parallel, cost-effective gene re-sequencing in patients recruited by a multi-center collaborative group of NIA funded ADCs, fully consistent with the stated RFA goals. This approach will allow a tour de force assessment of the hypothesis that these pathways that have been implicated in neurodegenerative disease, and for whom there is a strong underlying biological and pathophysiological rationale are involved, rather than simply testing one gene at a time. Some of these studies will be completed in understudied ethnic minorities, providing important data on sequence variants in populations that have not been studied in this detail and for whom such data is typically not yet available in public or commercial databases. The biomaterials and clinical data from these patients, along with the genetic data obtained will be deposited with the NACC and NCRAD, thus providing an invaluable genetic resource. This data will be integrated with other potential SNPs in a larger set of cases and controls, so as to make maximum use of haplotype information. Association studies will be performed to identify disease-associated variants in FTD and in each ethnic cohort of AD subjects, so as to identify any population specific risk variants that can provide important data that will serve as the foundation for future studies in these groups.

DESCRIPTION (provided by applicant): Furthering knowledge of the molecular basis of human cognitive specializations is of critical importance for developing an improved understanding and treatments for a wide variety of neurodevelopmental and neurodegenerative diseases in humans. Among the most important functional specializations of the human cerebral cortex are the perisylvian language regions, which are asymetric in humans. Surprisingly little is known about the biological processes that underlie this functional and structural compartmentilization of language regions in humans, their lateralization and presence in other potential model organisms. This proposal is a renewal of our previously funded work, in which we began to screen a large number of potentially asymmetric expreesed genes using microarray technology, followed by confirmation using in situ hybridization. This process of confirmation is ongoing and we will continue to identlfy and characterize genes that are differentially expressed by the developing left and right cerebral hemispheres in the developing human brain. Based on data obtained in our completed studies and Preliminary Results, we have modified our initial studies in this proposal to include the identification of genes enriched in anterior and posterior peri-sylvian language regions, as well as those expressed asymmetrically in adult posterior peri-sylvian regions. Representational Difference Analysis-coupled microarray screening will be used to identify genes differentially expressed within these language related regions, which will be confirmed using Northern Blotting and In Situ hybridization. A subset of differentially expressed genes will be further studied in detail at different developmental stages and in different brain regions at the RNA and protein level to probe their functional relationship with other brain structures and circuits. Cross species comparisons, in mice and non-human primate species will be performed to investigate the evolutionary conservation of genes that are enriched in language-related cortex, or asymmetrically expressed in the developing human cerebral cortex. This will provide insight into the potential role of these genes in the development and evolution of language and related human cognitive specializations and the relationship of these regions in lower species to homologus human structures. This work will inform the study of human neurodevelopmental disorders that are related to speech and language, such as autism and development dyslexia, as well as probe the utility and limitations of animal models for these and related neurodevelopmental disorders.

DESCRIPTION (provided by applicant): Many disorders of language are due to genetic aberrations; spoken and written language ability have significant genetic components in the general population. However, normal variation in language and language disorders tend to be complex, in that they are caused by multiple genes interacting with the environment. Recently a mutation in the FOXP2 gene was identified in a family with a speech and language development disorder including orofacial dyspraxia and grammar deficiencies. FoxP2 is a transcriptional represser in the forkhead binding domain containing family. Following the divergence of chimpanzees to humans, 2 coding variations resulting in amino acid changes occurred in FOXP2. These changes have undergone positive selection in the human lineage, therefore, it is hypothesized that these amino acid changes have in part led to the ability of humans to communicate with a spoken language. The functions of these changes at a systems or molecular level are not known. In addition, although Fox genes are known transcription factors, there are no known targets of FoxP2 in the brain. We expect that targets of the Foxp2 transcription factor will elucidate important players in the development of language and cognitive skills, as well as other features of brain development. We propose to perform chromatin immunoprecipitation (ChIP) with an antibody to FoxP2 followed by hybridization to a microarray with promoter sequences. We will specifically examine cerebral cortex and basal ganglia, regions where FoxP2 is known to be expressed in human during fetal brain development and that are abnormal in individuals with FoxP2 mutations. Gene expression analysis at the mRNA level will be used to confirm the Fox binding targets in vitro, and in situ hybridization and immunocytochemistry will be used to study co-expression of targets with FoxP2 in vivo. The effect of the 2 human specific changes will also be studied in vitro using site directed mutagenesis in a human neural cell line. Completion of the experiments described in this proposal will elucidate potential pathways through which FoxP2 acts, as well as the potential function of the human-specific variants with broad implications for our understanding of normal human brain development and disorders of language. This proposal has a strong screening component in an area where no molecular mechanisms have been identified and fits well within the R21 framework.

One of the most important functional specializations of the human cerebral cortex is that of the perisylvian cortex and the other subcortical regions with which it is connected. These regions are involved in human higher cognition and behavior, including language. Surprisingly little is known about the biological processes that underlie the development of perisylvian cortical regions in humans, their asymmetry, and presence in other potential model organisms. This proposal is an extension of the Pi's Merit Award, in which we have worked successfully to identify key genes involved in human higher cognition by virtue of their asymmetric expression or enrichment in perisylvian cortex, including CNTNAP2 and other extracellular adhesion molecules that are also related to neuropsychlatric disease. In parallel, we have developed an entirely novel approach to elucidate the complex structure of the transcriptome, and successfully applied this to adult human brain. We propose to apply these methods in conjunction with NextGen sequencing to perform digital gene expression in anatomically defined interconnected human language cortex and its homologues in nonhuman primates. This work will put gene products in a clear functional context, enabling characterization of the set of genes most central to this aspect of human brain organization, rather than relying on less structured means of prioritizing genes for follow-up. Putative differentially expressed genes and key hub genes within the networks will be confirmed using qRT-PCR and In Situ hybridization. Cross species comparisons, in mice and non-human primate species will continue to be performed to investigate the evolutionary conservation of genes that are central hubs of the modules that are enriched in languagerelated cortex in adults, or asymmetrically expressed in the developing human cerebral cortex. This will provide insight into the potential role of these genes in the development and evolution of language and related human cognitive specializations and the relationship of these regions in lower species to homologous human structures. All of this will clearly inform the study of human neurodevelopmental disorders that are related to speech and language, such as autism or schizoprenia, as we and others have already demonstrated, and provide proper context for the use of animal models for these disorders.

[unreadable] DESCRIPTION (provided by applicant): This proposal aims to provide funding for a pre-doctoral training program in Neurobehavioral Genetics (NBG). The completion of human genome sequencing provides an extraordinary opportunity to identify the genetic basis of disorders of brain function. Progress in this endeavor will be speeded by bridging several longstanding dichotomies; between nervous system mechanisms and behavior, between neurology and psychiatry/psychology, between diseases and non-disease traits, and between humans and model organisms. The goal of this proposal is to achieve such bridging by providing a unified and multidisciplinary training to PhD candidates from a wide range of backgrounds, including neuroscience, psychology, human genetics, neuroimaging, and pharmacology. If funded, this program would be the only training program focusing on mammalian or complex genetics at UCLA, thus serving a critical training niche for a large number of students from a variety of primary disciplines. Integrated training in genomics, neuroscience and phenomics is a critical step in the development of efficient, higher throughput genetic investigation of brainrelated phenotypes. The program will emphasize the importance of systematic delineation and assessment of nervous system phenotypes, including the integration of traditional clinical and cognitive evaluations with recently available phenotyping tools such as neuroimaging and gene expression profiling. This predoctoral training program will be closely integrated with a post-doctoral NBG training program which we anticipate will be funded through a T32 grant from NINDS beginning in 2004. The interactions of predoctoral students with postdoctoral fellows and faculty from disciplines that they would ordinarily not interact with in their primary department or program through the shared coursework and seminar series will provide a unique and special training environment for these predoctoral candidates. The ambitious goals of the program are achievable because the program faculty is very strong in virtually all of the areas that are relevant to neurobehavioral genetics, and because the faculty members have long embraced, in their research, the integrative and crossdisciplinary approach that is at the heart of the program. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The identification of the genetic bases of dementias remains an important goal of research whose aims are to improve our understanding and develop new therapeutics for these diseases. The discovery of tau mutations in inherited cases of frontotemporal dementia (FTD) demonstrated that tau dysregulation can cause neurodegenerative disease. But, tau mutations have not been described in AD, and tau mutations account for about 15% of familial FTD cases. An evolving body of work from many groups suggests that genes involved in modifying tau, especially those that modulate tau phosphorylation are enticing candidates for contributing to the genetic risk for these AD and FTD. In cases where tau abnormalities have not been identified on a pathological or genetic basis, dysregulation in key machinery involved in protein processing and folding apparatus may provide additional genetic risk elements. The goal of this proposal is to identify and confirm these novel genetic risk factors in Frontotemporal Dementia (FTD) and Alzheimer's Disease (AD) using a novel re-sequencing approach that allows massively parallel, cost-effective gene re-sequencing in patients recruited by a multi-center collaborative group of NIA funded ADCs, fully consistent with the stated RFA goals. This approach will allow a tour de force assessment of the hypothesis that these pathways that have been implicated in neurodegenerative disease, and for whom there is a strong underlying biological and pathophysiological rationale are involved, rather than simply testing one gene at a time. Some of these studies will be completed in understudied ethnic minorities, providing important data on sequence variants in populations that have not been studied in this detail and for whom such data is typically not yet available in public or commercial databases. The biomaterials and clinical data from these patients, along with the genetic data obtained will be deposited with the NACC and NCRAD, thus providing an invaluable genetic resource. This data will be integrated with other potential SNPs in a larger set of cases and controls, so as to make maximum use of haplotype information. Association studies will be performed to identify disease-associated variants in FTD and in each ethnic cohort of AD subjects, so as to identify any population specific risk variants that can provide important data that will serve as the foundation for future studies in these groups.

Affect; Alleles; Area; autism spectrum disorder; Autistic Disorder; Basal Ganglia; base; Behavioral; Biocompatible Materials; Blood; Brain; Candidate Disease Gene; Cell Line; Cells; Child; Chromosome abnormality; Chromosomes; Clinical; Cognition; Cognitive; cohort; Cohort Studies; Copy Number Polymorphism; Cytogenetic Analysis; Data; density; Development; disorder risk; endophenotype; Family; Family member; Fluorescent in Situ Hybridization; Functional Imaging; Functional Magnetic Resonance Imaging; Genes; Genetic; Genetic Risk; Genetic Status; Genotype; Haplotypes; Heterogeneity; Infant; infant outcome; Karyotype determination procedure; Language; Language Development; Learning; Link; Maps; Measurement; Measures; Methods; Microscopic; mirror neuron system; Molecular; Molecular Cytogenetics; Multivariate Analysis; neuropsychiatry; novel; Parents; Phenotype; proband; programs; prospective; Prospective Studies; Quantitative Trait Loci; Questionnaires; relating to nervous system; Research; Research Personnel; Resolution; Risk; Sampling; Siblings; SNP genotyping; social; social communication; Speech; Study Subject; Subgroup; System; Testing; Time; Variant

PROJECT SUMMARY (See instructions): Recent major discoveries in the genetics of frontotemporal lobar degeneration (FTLD) have accelerated the understanding of the pathogenetic mechanisms underlying FTLD and related disorders. These include the discovery of mutations in progranulin (GRN) as an important cause of FTLD and the finding of TAR DNA binding protein 43 (TDP43/TARDBP) as a major component of the intraneuronal inclusions in FTLD. Over the past years, the Genetics Core has 1) screened a significant number of PPG patients for mutations in known genes causing dementia (including MAPT), identifying known and novel mutations and a novel risk factor for neurodegeneration; 2) characterized the genotype for most of the known risk factors for dementia, including APOE and MAPT haplotypes; and 3) started a productive collaboration with the laboratory of Rosa Rademakers at Mayo Clinic Jacksonville, a leading group in the study of FTD genetics. We will continue to collect DNA and RNA from peripheral blood from patients and controls with FTD-spectrum disorders evaluated through the PPG, and to screen select cases for mutation in all the dementia-causing genes. Indepth analysis of peripheral progranulin level will be performed in collaboration with the Rademakers lab.Cell lines will be created and stored in the NIH-funded AD National Cell Repository. We will also assess a panel of common polymorphisms in several genes that have been reported to modulate the dementia risk, memory performance, or social behavior, RNA from peripheral blood will be collected and used for gene expression studies. Finally, we propose to identify new loci associated with FTD and AD using novel mapping methods. These genetic data will be integrated with clinical, pathological and imaging data to achieve the core aims of the PPG projects, which are advancing our understanding of the diagnosis, characterization, and genetic architecture of neurodegenerative dementia and, in conjunction with the other sections of the current Program Project, building an extremely well characterized series of patients with neurodegenerative dementia and controls, a potentially invaluable resource for the field.

DESCRIPTION (provided by applicant): The identification of the genetic bases of dementias remains an important goal of research whose aims are to improve our understanding and develop new therapeutics for these diseases. The discovery of tau mutations in inherited cases of frontotemporal dementia (FTD) demonstrated that tau dysregulation can cause neurodegenerative disease. But, tau mutations have not been described in AD, and tau mutations account for about 15% of familial FTD cases. An evolving body of work from many groups suggests that genes involved in modifying tau, especially those that modulate tau phosphorylation are enticing candidates for contributing to the genetic risk for these AD and FTD. In cases where tau abnormalities have not been identified on a pathological or genetic basis, dysregulation in key machinery involved in protein processing and folding apparatus may provide additional genetic risk elements. The goal of this proposal is to identify and confirm these novel genetic risk factors in Frontotemporal Dementia (FTD) and Alzheimer's Disease (AD) using a novel re-sequencing approach that allows massively parallel, cost-effective gene re-sequencing in patients recruited by a multi-center collaborative group of NIA funded ADCs, fully consistent with the stated RFA goals. This approach will allow a tour de force assessment of the hypothesis that these pathways that have been implicated in neurodegenerative disease, and for whom there is a strong underlying biological and pathophysiological rationale are involved, rather than simply testing one gene at a time. Some of these studies will be completed in understudied ethnic minorities, providing important data on sequence variants in populations that have not been studied in this detail and for whom such data is typically not yet available in public or commercial databases. The biomaterials and clinical data from these patients, along with the genetic data obtained will be deposited with the NACC and NCRAD, thus providing an invaluable genetic resource. This data will be integrated with other potential SNPs in a larger set of cases and controls, so as to make maximum use of haplotype information. Association studies will be performed to identify disease-associated variants in FTD and in each ethnic cohort of AD subjects, so as to identify any population specific risk variants that can provide important data that will serve as the foundation for future studies in these groups.

DESCRIPTION (provided by applicant): Autism is a devastating neuropsychiatric condition with unknown pathophysiology. Autism spectrum disorders (ASD) have an estimated incidence of 1/200 and thus are more common than many other childhood disorders. Although ASD have a multifactorial etiology, it has a large genetic component. It is also becoming clear that comprehensive efforts involving large sample sizes and methods to reduce heterogeneity are necessary to achieve maximal power to identify disease critical regions narrow enough to permit positional cloning of autism susceptibility genes. The investigators in this application aim to continue their collaborative effort that has produced and enhanced a highly successful open data and biomaterials resource for the research community, the Autism Genetic Resource Exchange (AGRE). This collaborative network application involving six research sites and the AGRE DCC, will systematically and comprehensively investigate the genetics of ASD to identify rare mutations, chromosomal abnormalities, and common variation contributing to ASD susceptibility. Specifically, they will enrich existing resources by adding 400 simplex families, including 200 families of African American descent, in addition to phenotype enrichment. The large overall sample size permits stratification of families based on analysis of heritable quantitative and qualitative endophenotypes. It further allows independent confirmation of loci as demonstrated for chromosome 17 and provides adequate sample for whole genome association studies to find loci with adequate power. The investigators will perform follow up linkage studies to confirm several new loci identified based on autism-related endophenotypes or co- variants, such as language delay, sex, and head circumference. In parallel, comparative genomic hybridization (CGH) using 500k SNP arrays will be performed, yielding the highest resolution molecular karyotypes and providing a resource on genome wide copy number variation (CNV) in ASD. CNV identified will be followed in family members and controls using QPCR and FISH. The use of SNP genotyping for CNV detection in ASD probands further provides for significant economies, since additional genotyping need only be conducted in the parents for efficient whole genome association studies. Regions or genes identified by linkage will be followed up by efficient, staged dense SNP genotyping. CNV assessment and WGA will also yield candidates that will be integrated with the linkage results for focused confirmatory studies, including re-sequencing to identify and confirm potentially causal genetic variants. Genetic risk factors identified in the mostly white European sample will be tested for association in the African American sample to determine whether these cohorts share the same genetic risk factors. All phenotypic and genotype data will be made accessible via the Internet on a rolling basis, including minority families, further enhancing the value of this resource to the community.

DESCRIPTION (provided by applicant): In response to the RFA-OD-09-005, Recovery Act Limited Competition: Biomedical Research Core Centers to Enhance Research Resources (P30), the aims of this new application are to support our hiring and providing an appropriate start-up package for an outstanding clinician scientist as a tenure-track faculty at UCLA and to develop resources to support research projects within the context of a specialized new biomedical research core center. The proposed core activities will be conducted primarily within the context of: 1) the UCLA Center for Autism Research and Treatment (CART), which is one of six NIH Autism Centers of Excellence (ACE) and also one of five ACE Network grants (PI, Geschwind);and will involve a joint appointment for the successful candidate within 2) the Center for Neurobehavioral Genetics (CNG), housing an internationally recognized program in neurogenetics. Both of these centers have been developed within the Semel Institute for Neuroscience and Human Behavior (Semel Institute;formerly the Neuropsychiatric Institute), which provides support and cohesion for a wide range of clinical and basic research activities related to mental health. Lastly, there is broad support for this center across the large, highly collaborative UCLA neuroscience community overseen by the Brain Research Institute (BRI) and the UCLA medical center. As a leading center for autism research, we have taken a multidisciplinary approach to the study of autism spectrum disorders (ASD), integrating our work at many levels, from early diagnosis, genetics and brain imaging to cognition, behavior, psychopharmacology and other treatment modalities. Of particular relevance to this proposed core center application, CART investigators have engaged in strategic planning during the course of our annual retreats. Several years ago, a major outcome of this process was the clearly recognized need to expand our infant research capabilities and to develop a program in electrophysiology. Thus, our highest current priority, and the programmatic goal of this center application is to expand our early infant research and to add expertise in electrophysiologic methods for the study of infants and young, minimally verbal children. This is especially important, given that quantifiable, electrophysiologic brain phenotypes may help us understand some of the neurobiology and the heterogeneity in autism, especially in conjunction with careful deep phenotyping, genetics and neuroimaging. PUBLIC HEALTH RELEVANCE: There is a clearly recognized need to expand infant research capabilities and to develop a program in electrophysiology, which is the programmatic goal of this center application. We want to expand our early infant research and add expertise in electrophysiologic methods for the study of infants and young, minimally verbal children. This is especially important, given that quantifiable, electrophysiologic brain phenotypes may help us understand some of the neurobiology and the heterogeneity in autism, especially in conjunction with careful deep phenotyping, genetics and neuroimaging.

The availability of genome sequence for a wide variety of experimental organisms and man has challenged us to use this information in the most effective manner. This Core focuses on the application of genome-level analyses in neuroscientific investigation, most based on microarray related technologies, but also supporting a burgeoning use of next-generation sequencing-based approaches, such as RNA sequencing (RNA-seq) and ChlP-seq. For the foreseeable future, expertise in both microarray and sequencing applications need to be maintained within the Core, as both of these technologies are the Core platforms used in functional genomics today. The nervous system poses specific challenges, such as unprecedented tissue heterogeneity and the need to study the genome in relation to circuits and behavior. Further, the analysis of large-scale data sets requires specific expertise in bioinformatics that most neuroscience laboratories lack. It is not practical for most laboratories to become fully proficient in all of the aspects of experimental design and statistics to amplification and hybridization technology that is necessary to perform adequately powered microarray experiments. Furthermore, although Core facilities exist to perform microarray hybridizations, the downstream bioinformatic follow up and statistical support needed to bring a study to completion over a series of months or even years is sorely absent for members of the IDDRC. This is why in the past, many performed such experiments, but many experiments did not lead to publications because the analytic know how was lacking. Further, how to move from a list of genes to biological knowledge poses additional challenges that most laboratories cannot meet alone. The recent advent of high-throughput next-generation sequencing (NGS) is having a major impact in the sciences, especially in biomedical research. Since its introduction to the market in 2005, massively parallel sequencing has dramatically altered genomic research. The degree of throughput and the decreasing cost per base, along with a relatively low error rate, have made it possible to obtain genomic sequence information on a previously unimaginable scale and at a cost that is dramatically lower than that achievable with traditional Sanger sequencing. In addition, these new systems are extending the field to new applications, previously out of reach for conventional sequencing, such as gene expression profiling, small non-coding RNA profiling, structural variant analysis, and analysis of epigenetic modifications of histones and DNA. Soon, whole-genome sequencing will replace existing targeted array technologies and reveal new insights into transcriptomes, genetic and genomic variation, and allow for systematic epigenetic profiling. In particular, RNA-seq is expected to completely change the landscape in the field of gene expression analysis, due to the powerful combination of increased throughput, unprecedented detail, and significantly lower cost. Given the enormous amount of data generated in any NGS experiment, issues related to informatics have been magnified by 1-2 orders of magnitude. Specifically, this is due to massively increased storage and processing capacity for NGS data, as well as the need for mapping reads to the genome for every run. Thus, now more than ever, cores with both data storage, processing and bioinformatics capability are necessary to help support modern neuroscience research.

DESCRIPTION (provided by applicant): Our current understanding of autism spectrum disorders (ASD) delineates a highly heritable, yet etiologically heterogeneous disease. Forward genetic approaches to find disease associated mutations or common variation have been successful and continue to offer considerable power. Yet, given the accumulating evidence for very significant heterogeneity and environmental influences, complementary approaches to classic forward genetics become necessary. Genetic polymorphism and mutation data to date have identified dozens of causal or contributory variants, yet our preliminary data from autism brain suggest that common molecular pathways are involved in a significant subset of cases. This convergence at the tissue level suggests that other mechanisms, specifically epigenetic changes, combined with genetic background, are contributing to such final common pathways. We propose to further test this hypothesis by taking a comprehensive and integrative genome-wide approach to assessing brain gene-expression, miRNA levels and the related, causal epigenetic mechanisms in ASD etiology. The work proposed here, which brings together three principal investigators with a publication track record and clear expertise in all of the methods and approaches necessary, comprises a unique international collaborative group capable of performing this work using state of the art techniques. We will perform RNA-seq analyses of four cerebral cortical regions and cerebellum from ASD cases and controls, to assess mRNA, miRNA, and splicing isoform regulation. In parallel, we will identify key differences in chromatin state and DNA methylation across multiple brain regions in the same ASD and control individuals used in the expression analyses using ChIP-Seq and MeDIP . We will thus assess the mechanisms by which changes in DNA methylation, histone modification, and DNA sequence contribute to the observed differences in gene expression, and we will explore the hypothesis that epigenetic processes mediate susceptibility for ASD via long-term changes in transcriptional regulation. This work, which represents an unprecedented effort to unify these often disparate data (usually produced without integration in mind), will delineate potential shared molecular pathways in ASD and the underlying mechanism of these differences at the level of miRNA, the chromatin regulatory apparatus, and DNA methylation. In addition, these data will be made available to the community in a web-based format to be of maximum utility.

Genetic and phenotypic heterogeneity in autism and autism spectrum disorders (ASD) pose significant challenges for research focused on defining biomarkers, developmental trajectory and treatment and outcomes. This provides a key rationale for requiring Fragile X testing and high resolution karyotyping using SNP arrays on all subjects enrolled in ACE Centers or Networks, as we will continue to in this ACE. In our previous ACE Center Project II, we hypothesized that studying ASD endophenotypes, would aid in identification of more homogeneous patient subgroups and hasten identification of genetic loci. We showed how a common ASD susceptibility variant in CNTNAP2 modulates brain function, connecting gene to brain to endophenotype for the first time in ASD. We also identified several cases of rare, large copy number variation (CNV) and smaller variants of less certain pathogenecity. Here we propose to continue to genetically characterize all ACE probands, hypothesizing that identifying certain etiological subclasses may provide more homogeneous populations that will be more predictive of trajectory and outcome. We will integrate identification of CNV with gene expression data to identify dysregulated genes within and near CNVs, thus improving classification of pathogenecity, and identify those with mutations currently undetectable by structural variant analysis alone. We will take a systems approach to functionally group these genes into biological pathways, and thus to group patients by shared molecular defects. We will then relate shared molecular pathway defects in the patient subsets to the phenotypic biomarker measurements collected in projects l-IV. We will test the relationship between known and newly discovered genetic variants and measures of behavior, eye-tracking/pupillometry, EEG, and brain imaging at both single time points and examining longitudinal trajectories, as well as their influence on response to treatment. In this way we seek to connect genetic variation to measures of brain function as a means of unraveling the genetic and phenotypic heterogeneity observed in ASD, and to develop improved predictors of diagnosis and treatment response.

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ ABSTRACT DESCRIPTION: See instructions. State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. Autism Spectrum Disorder (ASD) is a common, often devastating neuropsychiatric condition with largely unknown pathophysiology. Although ASD has a multifactorial etiology, it encompasses a large genetic component. The investigators in this proposal aim to continue and enhance our collaborative effort that has produced significant advances in our understanding of ASD over the last four years and generated highly successful, open data and biomaterials resources for the research community, the NIMH Genetics Initiative and the Autism Genetic Resource Exchange (AGRE). Our Network has met or exceeded our original aims. We have built patient resources for research, identified rare and common ASD susceptibility alleles, defined models of ASD genetic susceptibility, provided evidence for convergent pathophysiology, and led development of animal and cell culture models. Here we propose to take a major new direction, filling a significant gap in ASD research, by recruiting underserved subjects of self-reported African ancestry (African-American; AA), an important population that has not previously been well-represented in ASD genetics research. Our Network involves six research sites and the AGRE DCC, collaborating in a systematic, comprehensive investigation of ASD genetics in order to identify rare mutations, chromosomal abnormalities, and common variation contributing to ASD susceptibility in the AA population. Specifically, we will enrich existing resources by recruiting at least 600 AA probands and additional family members. Our recruitment plan includes an embedded health disparities project that will evaluate access to care for AAs with ASD and clarify factors influencing participation of AA individuals in genetic research. We will employ novel methods to define the ancestral origin of specific chromosomal segments and ascertain the background on which susceptibility alleles occur. We will perform follow up GWA on ASD-related endophenotypes or co-variates, such as language delay, sex and head circumference. In parallel, we will conduct whole exome sequencing (WES) and analysis of copy number variation (CNV) using 2.5M SNP arrays yielding high resolution molecular karyotypes and providing a resource on genome-wide CNV and coding sequence variation (SNV) in ASD. Gene expression profiling and network analysis will be used to prioritize variants. Genetic risk factors identified in the mostly European samples will be tested for association in the AA sample to determine whether these cohorts share the same genetic risk factors, using a sample size providing power to replicate previous associations and to identify rare, recurrent CNV and SNV. The observation of new forms or different population frequencies of ASD-related variation in this sample as well as the sharing of most CNV and SNV with other cohorts are both outcomes that will have great significance for future studies and clinical care. As has been our practice, our Network will make all phenotypic and genotype data accessible via the internet on a rolling basis, further enhancing the value of this resource to the community.

Abstract Schizophrenia (SCZ) and bipolar disorder (BP) are the major adult psychotic disorders; uncertainty about their relationship is a central issue in psychiatry. By discovering variants with a high impact on either or both disorders (or on their endophenotypes) we can transform our understanding of their biology. This multisite project, from investigators with a track record of successful collaboration, will leverage exceptional, extensively phenotyped pedigree and population samples and integrate a combination of bioinformatics and experimental genomics approaches to identify such variants and demonstrate their relationship to these diseases. We will proceed through three steps: (1) Detect novel variants by whole genome sequencing (WGS) of discovery samples from ethnically homogenous populations; (2) Prioritize genome regions for further studies of these variants, using bioinformatics and functional genomics analyses; (3) Identify disease related variants through (a) imputation-based association analyses in very large case/control samples from the same populations and (b) validation of associated and predicted-deleterious variants using novel high-throughput functional assays. The WGS discovery sample includes SCZ, BP, and control individuals from Finland (FIN) and the Netherlands (NL). We will analyze their variants using a pipeline already implemented by our group to analyze the completed WGS of BP pedigrees from recently bottlenecked Latin American founder populations. The majority of our WGS samples derive from such founder populations; as we have shown previously this creates a high probability of detecting deleterious variants in these samples. WGS will detect a huge number of variants, most of unknown functional significance. We will thus narrow our focus to regions (coding and non-coding) most likely relevant to SCZ and BP, using two approaches: (1) Bioinformatics to prioritize regions previously implicated in these disorders (e.g. GWAS or CNV loci, or variants highlighted in prior sequencing studies including that of our BP pedigrees) and candidate regulatory regions (through analyses of ENCODE or other reference data); (2) Sequencing-based assessments of transcriptomic and epigenomic variation that we will conduct in two unique samples relevant to our phenotypes: fibroblasts from affected and unaffected members of our BP pedigrees; and neuronal and neuronal progenitor cells from reference individuals. We will then leverage very large, genotyped case/control samples available from the same populations as our discovery samples (or closely related ones) to accurately impute the novel variants from our prioritized regions; this will enable us to identify associations with SCZ, BP, and/or endophenotypes. Finally, in an initial validation study, we will use a novel high-throughput sequence-based reporter assay to evaluate the function of putative regulatory variants highlighted by our bioinformatics pipeline or by our association analyses.

DESCRIPTION (provided by applicant): This proposal uses proteomics to better understand Alzheimer's disease pathogenesis with a large-scale, unbiased, and direct approach to discover and validate novel disease processes in postmortem AD brain, and to prioritize new targets for early stage therapeutic intervention. The AD proteome mediates the effects of aging, genetics and other risk factors and contains unidentified protein targets for therapies. The approach leverages the strengths of a national team of collaborating AD Centers and associated studies of aging, an innovative proteomics platform, advanced systems biology, and model systems to produce new treatment targets. The first aim will identify novel proteomic targets selectively altered in asymptomatic AD brain. Brains will be analyzed by mass spectrometry (MS), yielding discovery proteomes to compare 1) controls free of AD and other pathologies; 2) asymptomatic controls with AD pathology; 3) non-demented mildly impaired cases with AD pathology, 4) definite AD, and 5) other neurodegenerative diseases. Protein changes in synapses, insoluble aggregates, glial and neuron-specific nuclei, and select posttranslational modifications will be determined. Bioinformatics will be used with available large-scale data to identify potentially druggable targets in key networks and cellular processes. The second aim will validate candidate proteomic targets in postmortem brains from independent community and clinic-based cohorts and determine relationships with clinicopathological features, including cognition. Absolute levels of candidate proteins will be quantified using selected reaction monitoring MS. The third aim will establish links between the validated proteome and AD pathogenesis and druggability. The most promising candidates will be studied for effects on neuronal viability and interactions with Ass and tau using cell culture and drosophila models. These results and other data will drive selection of the most promising candidates to advance to mouse models to assess therapeutic potential.

? DESCRIPTION (provided by applicant): Little is currently known about the number, proportion, or lineage of distinct cell types in the developing human fetal brain. Knowledge of such a component list and its functional genomic foundations is crucial for understanding the function of this complex system, its evolution, and how it is disrupted in disease. We hypothesize that comprehensive single-cell mRNA expression profiles provide an accurate and efficient rubric for a first generation classification schema that can be integrated with lineage, morphology and connectivity. We will use unsupervised learning algorithms to cluster 10,000 single cell transcriptomes derived from RNAseq of the human fetal cortical anlage, providing an unbiased model to identify and understand the resultant cell classes. We will validate these cell class determinations using in situ hybridization. We will use marker genes identified in this analysis to perform lineage tracing using cell-type specific reporters engineered via genome editing, and obtain single cell transcriptional profiles at different stages of development to determine lineage relationships. Finally, we will analyze intact fetal cerebral hemispheres and mouse-human chimeras processed with the CLARITY technique to provide 3D localization and morphology of a subset of cell types. To markedly improve scalability of this approach, we will apply enhanced Bessel Beam Tomography for spectral image acquisition, increasing imaging speed by 2-3 orders of magnitude while maintaining high resolution. In parallel, we will develop automated image analysis algorithms to enable cell detection and comprehensive morphological assessment in an automated and correctable fashion. Overall, completion of these studies will permit, among many future advances: (1) The generation of an unbiased rubric of cell class based on integration of morphological, transcriptomic, and connectivity data used to understand cell type diversity and function; (2) Connection of cellular function, morphology, and connectivity to specific genes and proteins; (3) Refinement of the cellular basis of transcriptomic disease signatures and understanding of the individual cellular mechanisms of disease (4) The construction of connectivity diagrams based on discovered circuit components allowing development of realistic computational models of brain function and dysfunction; (5) Optimization of cell types generated by in vitro stem cell models based on gold standard in vivo cell class definition; and (6) Advancement of light microscopy techniques for rapid, spectral high resolution imaging of postmortem human and mouse brain including automated processing, which in addition to permitting high throughput brain mapping, will significantly advance mechanistic studies in mouse models and pathological studies in human. Completion of these aims will provide a proof of principle, multiple tools, and a core framework on which to build a more comprehensive knowledge of cell classes in the human brain.

SUMMARY: PROJECT 2 There is a lack of understanding in the field of mental health of the mechanisms by which environmental or maternal factors influence disease susceptibility, specifically with neurodevelopmental disorders such as schizophrenia (SZ). The long-term objective of Project 2 is to connect the molecular pathways disrupted in the offspring after maternal immune activation (MIA) in the mother to cellular, circuit and neurobehavioral alterations that lead to SZ and perhaps other allied neurodevelopmental disorders. To accomplish this objective, the project will undertake four specific aims. First, the Geschwind lab, in collaboration with the Nonhuman Primate (NHP) Core, will identify the transcriptional signature of MIA in the PFC, ACC, HC and VC in an extensively characterized NHP model. The lab will use advanced, next-generation sequencing methods to measure genome-wide transcriptome changes in four relevant brain regions in a well-characterized 4-year-old cohort of NHPs exposed in utero to MIA and compare this with matched controls. Second, the research team will characterize and integrate the transcriptional signature caused by MIA in the PFC, ACC, HC, and VC in a validated mouse model at four developmental ages to compare to changes observed during psychosis and to determine the developmental progression of changes in gene expression. The work will focus on the same 4 regions as in the NHP using RNAseq methods, but have the additional advantage of spanning the full time course over the development of molecular and behavioral alterations so as to develop causal models and identify changes shared across species. Third, the project will identify changes in the transcriptome in the PFC, ACC, HC, and VC in well-characterized individuals with SZ and matched controls. The same RNAseq methods used in Aims 1 and 2 will be applied so as to ensure direct comparability between the species, SZ patients and controls for the first time. In the fourth aim, a systems biology framework (WGCNA) will be used to integrate NHP, mouse and human transcriptional data. Using a powerful network analysis, WGCNA, the project will identify key co-expression modules associated with MIA across species and time, as well as the regulatory network that drives these changes, and determine whether these changes in gene expression correlate with the magnitude of cortical inflammation (Project 1), dysregulation of subcortical DA (Project 1), changes in anatomical and functional connectivity (Project 2), changes in synaptic connectivity (Project 4), and changes in expression of immune molecules in the brain (Project 4). This will enable development of true causal models that connect molecular pathway alterations at the level of gene expression to the ontogeny of cellular changes, anatomical changes and behavioral changes that follow in utero exposure to MIA. The single-platform, cross-species design will enable a more-definitive determination of the relationship of these changes to SZ in humans and provide a framework for assessment of other neuropsychiatric disorders, such as Austism Spectrum Disorder.

ABSTRACT Genetic and genomic investigations have yielded important findings as to the genetic contributions to major psychiatric illnesses, illustrating significant etiological heterogeneity, as well as cross-disorder overlap. It has also become clear that understanding how this genetic variation leads to alterations in brain development and function that underlies psychiatric disease pathophysiology will be greatly advanced by a roadmap of the transcriptomic and epigenetic landscape of the human cerebral cortex across key developmental windows. Here, we propose, via a highly collaborative group of investigators, each with distinct areas of expertise and research focus, to create a scaffold of genomic data for understanding ASD pathophysiology, and psychiatric disorders more broadly. The work proposed here represents an ambitious multi-PI project (Yale, UCLA, and UCSF) that brings together three principal investigators and collaborators with strong publication records and expertise in all approaches necessary to perform this work using state-of-the-art and novel methodologies. We will perform time-, region-, and cell type-specific molecular profiling of control and ASD brains (Aim 1), including RNA-seq based transcriptomics, identifying cis-regulatory elements via ChIP-seq, and use Hi-C to determine the 3D chromatin architecture and physical relationships that underlie transcriptional regulation in three major regions implicated in neuropsychiatric disease (frontal and temporal cortex and striatum) across five major epochs representing disease-relevant stages in human brain development. This will include complementary genomic analyses in controls and matched post mortem ASD brain to identify genetic mechanisms underlying processes altered in ASD brain. We will address cellular heterogeneity via fluorescence-activated nuclear sorting (FANS) so as to profile neurons and non-neural cells separately, which will complement the whole tissue analyses. We will analyze and integrate these datasets to identify regional, developmental, and ASD-related processes to gain insight into underlying mechanisms, harmonizing these multi-omic data with other psychENCODE studies, as well as other large scale data sets, such as BrainSpan, ENCODE, GTEx and Roadmap Epigenomics Project (Aim 2). We will perform integrated analysis of germ-line ASD variations identified in more than 1000 families from the Simons Simplex Collection to characterize causal enrichments in developmental periods, brain regions, and cell types to better characterize the mechanisms by which genetic variation in humans alters brain development and function in health and disease (Aim 3). Completion of these aims will lead to a well-integrated resource across major periods in human cortical and striatal development that will permit generation of concrete testable hypotheses of ASD mechanisms, and inform our pathophysiological understanding of other related neuropsychiatric disorders.

? DESCRIPTION (provided by applicant):Large-scale genomic investigations have begun to illuminate the genetic contributions to major psychiatric illnesses. In autism spectrum disorder (ASD), rare large effect variants provide novel causal anchors to understand its neurobiological basis, and to understand convergence and divergence in disease mechanisms. These findings, not unlike other psychiatric disorders, also emphasize extreme genetic heterogeneity. We and others have shown that gene and protein networks provide an organizing framework for understanding heterogeneous psychiatric disease genetic risk in a unified biological context. The emergence of large-scale genomic and epigenetic data from human brain, and growing knowledge of genetic variation involved in ASD and other psychiatric diseases, coupled with advances in methodology, provides an unprecedented opportunity for comprehensive integrative analyses. We bring together a collaborative group of investigators, each with distinct areas of expertise critical for understanding psychiatric diseases, that have not been combined before, to develop a framework for integrative genomic network analysis. The work proposed here represents an ambitious multi-PI project (UCLA/UW, MGH/Harvard, and Johns Hopkins) that brings together 3 principal investigators and collaborators with strong publication records and expertise in all approaches necessary to perform this work using state of the art and novel methodologies. Through close collaboration we aim to develop a comprehensive framework and test optimal methods for integration of gene expression and protein-protein interaction (PPI) networks in the brain with genetic and epigenetic data - networks that will be iteratively refined using experimental data. We will construct networks representing multiple brain regions in adulthood and development through rigorous combination of multiple transcriptomic data sets from ASD and control brain, developing and validating methods for integration of splicing and expression levels within gene networks (Aim 1). These networks will be refined to inform tissue specific PPI inference, validated via experimental tissue-specific PPI (Aim 2). We will further identify causal drivers by integration of genetic and epigenetic data, identifying QTL effects on RNA, splicing and protein levels (Aim 3). We will experimentally validate network regulatory predictions for a subset of putative causal drivers prioritizing network hubs and ASD associated genes (Aim 4). In addition, we will use our networks to predict high likelihood risk genes, whose relationship to ASD will be assessed using data from large-scale sequencing in ASD and related psychiatric disease cohorts, as well as our own focused experimental validation via multiplex inversion probe (MIPs) sequencing (Aim 4). Completion of these aims will lead to more valid and comprehensive CNS networks thereby significantly advancing our understanding of ASD associated variants and causal neurobiological pathways. As is our usual practice, our data, networks, and all code will be made freely available in a web-based format to be of maximum utility to the community.

Project Summary Core C establishes an infrastructure for rigorous methodologies to study the neurobiological heterogeneity in ASD by integrating genetic testing with neuroimaging (structural and functional MRI) and neurophysiology (EEG) across projects. Core C will be led by Drs. Geschwind and Jeste, who will jointly design the experiments for correlating genetic variation with core measures of neural function. Dr. Geschwind will take major responsibility for genetic data-generation while Dr. Jeste will oversee electrophysiology studies. Drs. Bookheimer and Dapretto will oversee MRI methods. Drs. Geschwind and Jeste will coordinate efforts within Core C, between Core C and Core B, and between Core C and Projects I-IV through regular monthly meetings. Informed by our experience and success in ACE-I and II, we will strengthen an infrastructure that will support acquisition and analysis of excellent quality genetics, EEG and MRI, and we will ensure optimal participant retention using techniques and approaches optimized for infants and children with ASD. The core will provide a shared and thus more efficient infrastructure for projects using genetic analyses (all Projects), EEG (Projects I, II, III, IV) and resting state, structural and functional MRI (Projects I, III, IV). Core C personnel will contribute their expertise in acquisition and analysis of these datasets, utilizing new techniques in connectivity analysis using graph theory and machine-learning approaches for understanding the heterogeneity in key domains in ASD, including baseline connectivity, sensorimotor function and social communication/language, consistent with the Center's central themes. By consolidating all services within the core we maximize efficiency and consistency in implementing these techniques across projects. Through this integrative process, we will begin to move the field towards a more biologically based classification, which in turn we expect to improve our clinical care by improving prognostication, treatment choice and outcomes.

Genetic approaches have been successful in identifying causal genetic factors, both common and rare, that contribute to risk for autism spectrum disorder (ASD), providing a crucial starting point for mechanistic neurobiological investigations. However, moving towards an integrated mechanistic understanding of ASD at a molecular, cellular, and circuit level faces substantial challenges, such as extreme genetic heterogeneity and the lack of causal frameworks with which to connect different levels of analysis of nervous system function in model systems or patients. Nearly a decade ago, we reasoned that gene and protein networks would provide an organizing framework for understanding heterogeneous psychiatric disease genetic risk in a unified context and inform disease modeling; indeed there is now substantial evidence supporting convergence of major effect risk genes during mid-fetal cortical development. Furthermore, related functional genomic studies, including in those with a major gene form of ASD (dup)15q11-13, show shared patterns of transcriptional and chromatin dysregulation in post-mortem ASD brain, further supporting biological convergence. Where and how this occurs, and what biological mechanism(s) it reflects is not known. To address this, we propose an ambitious project that addresses several major challenges in establishing causal linkages between genetic risk and CNS structure and function in ASD. The work proposed in this multi-PI U01 involves a team of four principal investigators and co-investigators from UCLA and Stanford with the expertise necessary to perform this work using state of the art methodologies, ranging from developing and characterizing in vitro models of human brain development, stem cells, physiology, genomics, physics, and behavior. Through close collaboration, we will develop and analyze in vitro human stem cell based models that are differentiated from induced pluripotent stem cells and assembled into organized 3D brain cultures called human forebrain spheroids (hFS). These hFS contain the major cell classes of the developing forebrain, including progenitors, radial glia, cortical interneurons, glutamatergic neurons, and non-reactive astrocytes, and form functional synapses. We will model the effects of six major effect ASD risk loci in hFS with molecular, genomic, and physiological analyses to assess convergence at each level of analysis. We will also conduct comparisons of physiology using three rodent models based on the same genes modeled in vitro with the aim of integrating phenotypes to develop predictive models and compare with in vivo rodent models. We will analyze the relationship of molecular alterations and basic cellular and synaptic features with potential emergent or dynamic network features in control-derived hFS and compare these features with hFS harboring ASD risk mutations and test a subset of causal relationships based on network model predictions. Completion of these aims will lead to a more clear understanding of the power and limitations of model systems and computational models, while uncovering potential areas of convergence in different genetic forms of ASD.

DESCRIPTION (provided by applicant): This proposal uses proteomics to better understand Alzheimer's disease pathogenesis with a large-scale, unbiased, and direct approach to discover and validate novel disease processes in postmortem AD brain, and to prioritize new targets for early stage therapeutic intervention. The AD proteome mediates the effects of aging, genetics and other risk factors and contains unidentified protein targets for therapies. The approach leverages the strengths of a national team of collaborating AD Centers and associated studies of aging, an innovative proteomics platform, advanced systems biology, and model systems to produce new treatment targets. The first aim will identify novel proteomic targets selectively altered in asymptomatic AD brain. Brains will be analyzed by mass spectrometry (MS), yielding discovery proteomes to compare 1) controls free of AD and other pathologies; 2) asymptomatic controls with AD pathology; 3) non-demented mildly impaired cases with AD pathology, 4) definite AD, and 5) other neurodegenerative diseases. Protein changes in synapses, insoluble aggregates, glial and neuron-specific nuclei, and select posttranslational modifications will be determined. Bioinformatics will be used with available large-scale data to identify potentially druggable targets in key networks and cellular processes. The second aim will validate candidate proteomic targets in postmortem brains from independent community and clinic-based cohorts and determine relationships with clinicopathological features, including cognition. Absolute levels of candidate proteins will be quantified using selected reaction monitoring MS. The third aim will establish links between the validated proteome and AD pathogenesis and druggability. The most promising candidates will be studied for effects on neuronal viability and interactions with Ass and tau using cell culture and drosophila models. These results and other data will drive selection of the most promising candidates to advance to mouse models to assess therapeutic potential.

ABSTRACT Autism Spectrum Disorder (ASD) is a common neuropsychiatric condition with largely unknown pathophysiology, but with a substantial genetic contribution. Over the last 8.5 years, the investigators in this highly successful Network have contributed significant advances in our understanding of ASD and enhanced open data and biomaterials resources for the research community via the NIMH Genetics Initiative and the AGRE resource. In our last renewal, we took an important new direction to fill a critical gap in ASD research by recruiting subjects of self-reported African ancestry (African-American; AA), a group not previously represented in ASD genetics research. We have made substantial progress on our aims and the project is in a critical phase: by obtaining a larger cohort via continued recruiting, we will be well powered to conduct the first comprehensive assessment of the coding and non-coding genome via whole genome sequencing (WGS). This proposal involving six research sites and the AGRE DCC, will systematically investigate the genetics of ASD to identify rare single nucleotide variation (SNV), structural variation (SV), and common variation contributing to ASD susceptibility in this population. Specifically, we will enrich existing resources by recruiting at least 700 AA probands and additional family members to bring our cohort to 1300 AA probands. We have successfully conducted a health disparities project that that confirms significant diagnostic delays despite well-articulated parental concerns. In the next phase, we propose to improve early diagnosis, facilitated by the application of a novel heritable, quantitative biomarker, which we hypothesize will improve access to care. We will leverage acquisition of WGS of all family members via other funding sources, at no cost to this proposal, to characterize the contributions of genetic risk for ASD in AA individuals, including novel rare risk variants for ASD, which will also benefit genetic studies outside of this population by improving classification of rare variation in ASD in European (EU) and other ethnicities. We will use innovative methods to define local ancestry to ascertain the background on which susceptibility alleles occur. We will perform analysis of rare de novo and transmitted variants, and perform gene-based burden tests, which are well powered in this cohort. Gene expression profiling, eQTL, and network analysis will be used to prioritize variants. Genetic risk factors identified in cohorts studied to date (EU) will be tested for association in the AA sample to determine whether these cohorts share the same genetic risk factors, using a sample size that provides power to replicate previous associations and identify rare, recurrent CNV and SNV. We will perform follow up GWA on ASD-related endophenotypes or covariates, such as sex and social responsiveness. The observation of new forms or different population frequencies of ASD-related variation in this sample, or alternatively, the sharing of most variation with other cohorts are both outcomes that will have great significance for future studies and clinical care. As has been our practice, all data will be shared on a rolling basis, further enhancing this genetic resource for the community. .

In this project, we will develop novel methods for finding brain-specific enhancers, build regulatory networks, deconvolve brain-region-specific regulation, and relate differential enhancer signals to variations in the human population. We will then apply these analytical methods to the psychENCODE data corpus, integrating these data with GTEx, ENCODE, and CommonMind data, annotating GWAS SNPs associated with psych disease, prioritizing the discovered regulatory elements for validation, and visualizing all psychENCODE data in an integrated fashion. We will then validate these predicted regulatory elements using large-scale genomic assays in neuroblastoma cells, iPSC cells, and neuronal precursor cells differentiated into neuronal lineages, and using a microfluidics platform capable of culturing neuronal cells and neuronal organoids. The results from these studies will further our understanding of the genetic regulatory basis for neuronal function in both normal and neuropsychiatric disease states.

Abstract We request administrative supplemental funding to support the enrollment, inclusion, and molecular analyses of a total of 295 African-American families at two new point-of-care facilities affiliated with the Emory site of this network --the Marcus Autism Center Diagnostics Program (located in the same building as the Marcus Autism Center/Emory research program) and Hughes Spalding Children?s Hospital (located six miles away) -- over three years (Years 2-4) of our current UCLA-led ACE Network. We anticipate that this additional recruitment (an increase of 42% overall) will (a) increase power of hypothesis-testing in all Aims of the project, (b) increase socio- economic diversity and representability of the African-American population, particularly of its low-income subsections (a critical goal given hypotheses tested in Aim 2 and in light of recent health disparity findings), and (c) set the foundation for additional community engagement and subsequent minority African-American subject recruitment for this study and for other network studies in the future. !

In this project, we will develop novel methods for finding brain-specific enhancers, build regulatory networks, deconvolve brain-region-specific regulation, and relate differential enhancer signals to variations in the human population. We will then apply these analytical methods to the psychENCODE data corpus, integrating these data with GTEx, ENCODE, and CommonMind data, annotating GWAS SNPs associated with psych disease, prioritizing the discovered regulatory elements for validation, and visualizing all psychENCODE data in an integrated fashion. We will then validate these predicted regulatory elements using large-scale genomic assays in neuroblastoma cells, iPSC cells, and neuronal precursor cells differentiated into neuronal lineages, and using a microfluidics platform capable of culturing neuronal cells and neuronal organoids. The results from these studies will further our understanding of the genetic regulatory basis for neuronal function in both normal and neuropsychiatric disease states.

Progressive supranuclear palsy (PSP) is the most common frontotemporal lobar degeneration associated with tau pathology. While rare pathogenic variants, common risk factors, and ? more recently ? rare risk-associated variants have been identified in PSP, a significant proportion of the heritability for neurodegenerative tauopathies and other frontotemporal lobar degenerations remains unexplained, strongly suggesting that additional genetic risk factors await discovery. In this application, we propose to identify novel genetic variation associated with PSP using a multi-stage strategy. First, we will detect variants through whole-genome sequencing of neuropathologically characterized PSP. Second, we will prioritize pathological brain tissue samples for a multidimensional screen that includes transcriptional, proteomics, and epigenetic assays. Through recursive application of a prioritization algorithm, regions and variants most likely to have a high impact on disease risk will be identified. Finally, we will follow up on these variants using a high-throughput functional screen. This project taps unprecedented pathologic resources of PSP, leverages a pathologic and genetic infrastructure created with support from private foundations, and offers to transform our understanding of the genetic architecture of PSP and to advance towards the biology and downstream effects of this prototypical tauopathy downstream effects of this prototypical tauopathy.