Matthew W. State - US grants

The goal of this proposal is to use molecular cytogenetic and molecular genetic tools to identify genes involved in Tourette syndrome (TS) and related phenotypes. TS is a neuropsychiatric disorder defined by the combination of persistent vocal and motor tics. Despite strong evidence for a genetic etiology, linkage studies undertaken over several decades have not yet identified a single gene clearly implicated in TS- spectrum disorders, which include chronic tics (CT) and tic- related Obsessive Compulsive Disorder (OCD). We have undertaken an alternative strategy for disease-gene identification based on the hypothesis that a small subset of patients presenting with this genetically heterogeneous disorder have their symptoms as the result of a structural or functional disruption of a gene of major effect due to a cytogenetic abnormality. We further propose that the identification of such a gene may illuminate biological pathways involved in more common forms of TS. Our lab has identified two patients with TS-related phenotypes: (1) a patient with CT and OCD and an inversion inv(18)(q21q22); and, (2) a patient with OCD and a balanced translocation t(18;2)(q22;p25). These abnormalities map approximately 500 kilobases (kb) and 4 megabases (Mb) respectively from a chromosome 18q22 breakpoint that segregates with TS, CT and OCD in a previously-described family (Bhogosian-Sell et al. 1996). We have obtained cell lines from this pedigree as well and have initiated an exhaustive molecular characterization of the 4.5 Mb interval defined by the three 18q22 breakpoints. Our preliminary data also demonstrates that the 18q22 inversion is altering the replication timing at this locus. This change in the epigenetic properties of the patient's inverted chromosome 18 suggests that the rearrangement is influencing the regulation of gene expression across this region. The data has prompted two associated avenues of investigation: (1) an assessment of the extent of epigenetic changes in all three 18q22 rearrangements; and, (2) an evaluation of the epigenetic properties of the 18q22 region in cytogenetically-normal subjects with TS-spectrum phenotypes. We now propose: (1) to complete the fine-mapping of the three chromosome 18q22 breakpoints at the nucleotide level; (2) to identify known and novel transcripts within the interval defined by these abnormalities; (3) to prioritize genes based on functional and structural data and to conduct mutation screening on selected candidates; (4) to investigate epigenetic phenomena in the region in cytogenetically-affected cases as well as in cytogenetically-normal TS, OCD and CT patients; and, (5) to perform screening karyotypes on well-characterized TS spectrum patients from the Yale Child Study Center and Genetics Clinic as the basis for future mapping experiments.

[unreadable] DESCRIPTION (provided by applicant): Tourette Disorder (TD) is a developmental neuropsychiatric syndrome defined by the presence of chronic vocal and motor tics that affects as many as 1 in 100 school aged children. Despite considerable evidence for a genetic contribution, no disease-related genes have been identified. SLIT and Trk-like family 1 (SLITRK1) has recently been found to be a strong candidate for involvement in TD, initially through the mapping of a de novo chromosomal abnormality in the only affected member of a three-generation pedigree. Mutation screening of 204 probands subsequently identified: 1) a truncating frameshift mutation that was present in two affected, and absent in three unaffected family members, and could not be found in 3600 control chromosomes; 2) A non-synonymous substitution at a highly conserved amino acid in the transmembrane domain in 2 affected siblings, not present in 4000 control chromosomes; and 3) in two unrelated individuals, the identical single base substitution at a highly conserved nucleotide in the binding domain for the brain expressed microRNA-189 (miR-189), not present in 4296 control chromosomes. Expression analysis demonstrates that SLITRK1 mRNA and miR-189 overlap in brain regions thought relevant to the pathogenesis of TD. Overexpression of wild-type Slitrkl, but not the frameshift mutant, in developing cortical neurons promotes dendritic elongation. We now propose to investigate further the role of SLITRK1 in TD through a continued cross-disciplinary collaboration involving pediatric psychiatry, genetics and neurobiology. Specifically we aim to: 1) search for additional mutations in SLITRK1 in an expanded group of patients with TD and related disorders; 2) further characterize the expression of SLITRK1 RNA and protein in developing mouse and human brain; 3) elaborate the function of wildtype and mutant SLITRK1 in developing cortical neurons; and 4) identify proteins that interact with SLITRK1 as a prelude to mutation screening of these genes. SLITRK1 is a gene implicated in some cases of Tourette Disorder (TD) by the finding of rare DNA sequence changes in a small number of patients that have not been found in unaffected persons. The purpose of this study is to better understand .what causes TD by identifying additional abnormalities in the SLITRK1 gene and by investigating the impact of these unusual genetic changes on the developing brain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal is in response to a request for applications to address "Genomic Profiling of Mental Disorders," and proposes intensive genomic profiling and functional analysis of Contactin Associated Protein 2 (CNTNAP2) as well as the presynaptic cytomatrix protein Piccolo (PCLO), in an effort to clarify their roles in Autism Spectrum Disorders (ASD). In addition, the application proposes to deeply sequence genes in the Contactin and Contactin Associated pathways, as well as genes coding for proteins known to interact with PCLO. We propose to leverage ongoing collaborations among the State, De Camilli and Giraldez labs at Yale to allow for rapid functional assays (both in vitro and in vivo) of identified rare sequence mutations, which will be used to clarify genomic findings and to confirm the relevance of rare mutations for disease risk. The organizing principles supporting this application are as follows: 1) Rare genetic variation is likely to play a significant role in the etiology of ASD;2) Methods of detecting rare (MAF<5%) and very rare (MAF<1%) variation in large numbers of patients and controls are now economically and technically feasible;3) Rare and very rare variations, including those in highly conserved coding regions, are widespread throughout the genomes of both affected and unaffected individuals;4) Rare and very rare alleles of intermediate effect will likely require association as opposed to linkage strategies to evaluate disease risk;and 5) That among the most pressing issues at this stage in the success of genomic profiling of mental disorders are differentiating disease-related mutations from low frequency neutral mutations and addressing the methodolgical challenges of assessing disease association in the case of rare and very rare alleles. This application proposes to address these issues as follows: 1) Deep-resequencing using next generation technologies to identify rare and very rare mutations in a large, extremely well-characterized and carefully ethnically matched case-control sample;2) To use quantitative ASD phenotyping in addition to categorical diagnoses in an effort to enhance the power of case-control association of rare variants;3) To leverage well established association methodologies, including genomic control, mutation burden analyses for very rare mutations, correction for multiple comparisons and replication samples, to decrease type 1 error;4) To use both in vitro and in vivo methods to evaluate the functional consequences of rare mutations identified in cases and controls, specifically in the genes CNTNAP2 and PCLO;and importantly, 5) To test the hypothesis that functional assays stratifying rare functional from neutral variation in CNTNAP2 will clarify the results of association analyses, as has been successfully employed in other rare variant studies (1). PUBLIC HEALTH RELEVANCE: This study seeks to identify the role of specific genes in Autism Spectrum Disorders. The research plan utilizes high throughput sequencing technologies and combines these with molecular in vitro and in vivo biological studies in an effort to clarify the precise contribution of two brain expressed molecules Contactin Associated Protein 2 and Picollo as well as related genes to Autism and related conditions.

DESCRIPTION (provided by applicant): While there has been great progress in understanding the genomic architecture of autism, only a moderate number of the hundreds of genes and genomic regions thought to be involved in ASD have been identified. Next-generation sequencing (NGS) has proven its utility to rapidly identify variants underlying ASD, and this approach is being carried out in ca. 6,000 independent ASD samples through multiple studies. There is an urgent need to develop a framework to integrate and expand these current studies, and to jointly analyze emerging data to maximize the identification of valid ASD loci, because validated risk variants present opportunities for genetic counseling, understanding pathogenesis, and drug development. The Autism Sequencing Consortium (ASC) represents a coordinated effort by more than 20 independent groups to rapidly identify and validate ASD risk genes, which represent lead targets for neurobiological analyses and drug discovery. The long-term goal of the ASC is to make use of genetics to identify therapeutic targets in ASD, while contributing to translating such research findings to clinical practice. The overall objective of tis proposal is to rapidly identify ASD genes representing lead targets for high impact neurobiological studies and drug discovery. Our central hypothesis - formulated based on data with SNV, indels, and CNV, as well as review of medical genetic conditions in ASD and targeted sequencing in ASD - is that multiple independent rare variants account for a very significant proportion of risk to ASD. Our rationale for this proposal is that the identification of genetic variants conferring high-risk risk to ASD and associated neurodevelopmental disorders can form the bases of studies to understand pathogenesis as well as the bases for novel therapies. Moreover, such variants have direct implications for patients and their families in terms of etiological diagnosis, genetic counseling and patient care. These objectives will be accomplished with the following Specific Aims: 1) Maintain the infrastructure to support the ASC objectives; 2) Deploy pipelines for data cleaning and harmonization and variant calling; 3) Implement novel statistical methods for identifying ASD-associated genes; and, 4) Carry out whole-exome sequencing of 3,000 ASD subjects and parents. This contribution is significant because it represents the first step in research to understand pathogenesis of ASD and to the development of pharmacological strategies for treatment of core symptoms of ASD and etiologically related neurodevelopmental disorders. The research proposed in this application is innovative, in our opinion, because it involves an entirely new model of sharing data before publication, uses state-of-the-art methods for calling diverse types of variants in NGS data, incorporates novel methods for updating variant calling and sharing data, and includes highly innovative statistical methods to identify risk loci. This is a new and substantively different approach to gene discovery in ASD that departs significantly from the status quo and provides the means to achieve these important goals.

DESCRIPTION (provided by applicant): The development of human brain is an immensely complex process, which is likely reflected in the complexity of the underlying transcriptional processes. Gene expression and its precise spatio-temporal regulation, particularly by histone modifications and non-coding RNAs, are crucial for normal human brain development and are thought to be altered in major developmental psychiatric disorders, such as autism spectrum disorders (ASD). Moreover, changes in the developmental brain transcriptome are likely the major contributors to the evolution of the most distinctly human aspects of cognition, some of which are also affected in ASD and other psychiatric disorders However, our understanding of transcriptional and epigenetic processes involved in the development, evolution and dysfunction of the human brain is still elusive. Furthermore, most of our knowledge of transcriptional processes in the human brain is limited to the expression of protein coding genes. Given that the genomes of humans and other mammals have approximately the same protein-coding complexity, there is likely an additional reservoir of transcriptional complexity, especially in organs such as the brain, which has many structurally and functionally distinct regions in humans. This view is corroborated by recent findings of the ENCODE consortium, which found many cis-acting regulatory regions and that 60% of the human genome is transcribed, with a majority of the transcripts belonging to non-coding RNAs. Moreover, these and other studies have also uncovered pervasive involvement of regulatory DNA variations in common human diseases and evolution. However, how these findings on non-coding elements in cell lines relate to the complexity of human brain development and dysfunction is still largely unknown. The objective of this proposal is to employ unbiased and genome-wide approaches to (1) discover and characterize developmentally regulated and human-specific non-coding functional genomic elements in multiple regions of the developing human and non-human primate brains, (2) and elucidate their role(s) in the molecular pathophysiology of ASD, by using genomic analyses of post-mortem ASD brains, by screening for de novo mutations in ASD quartets, and by modeling functional consequences of ASD-associated elements in the developing mouse brain.

DESCRIPTION (provided by applicant): While there has been great progress in understanding the genomic architecture of autism, only a moderate number of the hundreds of genes and genomic regions thought to be involved in ASD have been identified. Next-generation sequencing (NGS) has proven its utility to rapidly identify variants underlying ASD, and this approach is being carried out in ca. 6,000 independent ASD samples through multiple studies. There is an urgent need to develop a framework to integrate and expand these current studies, and to jointly analyze emerging data to maximize the identification of valid ASD loci, because validated risk variants present opportunities for genetic counseling, understanding pathogenesis, and drug development. The Autism Sequencing Consortium (ASC) represents a coordinated effort by more than 20 independent groups to rapidly identify and validate ASD risk genes, which represent lead targets for neurobiological analyses and drug discovery. The long-term goal of the ASC is to make use of genetics to identify therapeutic targets in ASD, while contributing to translating such research findings to clinical practice. The overall objective of tis proposal is to rapidly identify ASD genes representing lead targets for high impact neurobiological studies and drug discovery. Our central hypothesis - formulated based on data with SNV, indels, and CNV, as well as review of medical genetic conditions in ASD and targeted sequencing in ASD - is that multiple independent rare variants account for a very significant proportion of risk to ASD. Our rationale for this proposal is that the identification of genetic variants conferring high-risk risk to ASD and associated neurodevelopmental disorders can form the bases of studies to understand pathogenesis as well as the bases for novel therapies. Moreover, such variants have direct implications for patients and their families in terms of etiological diagnosis, genetic counseling and patient care. These objectives will be accomplished with the following Specific Aims: 1) Maintain the infrastructure to support the ASC objectives; 2) Deploy pipelines for data cleaning and harmonization and variant calling; 3) Implement novel statistical methods for identifying ASD-associated genes; and, 4) Carry out whole-exome sequencing of 3,000 ASD subjects and parents. This contribution is significant because it represents the first step in research to understand pathogenesis of ASD and to the development of pharmacological strategies for treatment of core symptoms of ASD and etiologically related neurodevelopmental disorders. The research proposed in this application is innovative, in our opinion, because it involves an entirely new model of sharing data before publication, uses state-of-the-art methods for calling diverse types of variants in NGS data, incorporates novel methods for updating variant calling and sharing data, and includes highly innovative statistical methods to identify risk loci. This is a new and substantively different approach to gene discovery in ASD that departs significantly from the status quo and provides the means to achieve these important goals.

? DESCRIPTION (provided by applicant): Autism Spectrum Disorder (ASD) is characterized by impairments in social communication and restricted or repetitive behavior or interests. The application of genomic technologies has led to the identification of many of the genes underlying ASD, presenting the opportunity to assess the insight these risk genes can give into the etiology of ASD. In this proposal we aim to: 1) Generate a list of ASD-associated genes; 2) Identify points of convergence between these genes in biological data (e.g. gene regulation and expression); and 3) Validate these points of convergence in model systems. Since ASD is a human neurodevelopmental disorder we will prioritize biological data that is collected longitudinally across development from human brain tissue. In our prior work we have demonstrated that de novo mutations, specifically copy number variants (CNVs) and loss of function (LoF) point mutations, are strongly associated with ASD. Furthermore, these mutations cluster at ASD risk genes and loci in cases but not in controls. By comparing the distribution of these mutations between cases and controls we can identify the points of mutational clustering that represent ASD risk loci (e.g. CNVs at the 500kbp 16p11.2 locus and LoFs at the gene CHD8). We have developed a statistical framework to assess this clustering as well as incorporating evidence from inherited variants and case-control data. This framework is called the Transmitted and De novo Associated Test (TADA). In Aim 1 we will develop this test further to incorporate all the available CNV, exome, genome, and targeted sequencing data into a single ASD gene list, ranked by the degree of ASD association. Previously we used the top nine ASD risk genes as seeds for gene co-expression networks and assessed the validity of these networks by their ability to incorporate 120 independent ASD risk genes. By limiting the co- expression input data to narrow windows of development and specific brain regions we could identify the spatiotemporal networks with the greatest enrichment, for example pre-frontal cortex in mid-fetal development. In Aim 2, we propose a similar approach, but using the DAWN (Detecting Association With Networks) method developed by our group. DAWN uses the narrow windows of co-expression data as before, but is able to incorporate evidence from other datasets such as gene regulation, and protein-protein interaction (PPI). By seeding the DAWN networks with the highest confidence genes we will assess the spatiotemporal networks that best predict other ASD genes. ASD shows a significant sex bias implicating an interaction between ASD etiology and sexually dimorphic factors. Building on our work of identifying sexually dimorphic transcripts in the developing human brain we will test their enrichment within specific networks identified by DAWN. To validate the ASD-associated networks, in Aim 3 we will identify the gene that best represents each network and assess if disrupting it also disrupts the other genes within the network. We will disrupt each gene using CRISPR/Cas9 in both mice and human-derived iPSCs and assess the genes disrupted using RNA-Seq.

Project Summary/Abstract The past decade has seen outstanding advances in the genetics of autism spectrum disorder (ASD), however only a moderate number of the hundreds of genes and genomic regions thought to be involved in ASD have been identified. Advances have come largely from the study of rare genetic variants, especially de novo variation, including single nucleotide variation (SNV), insertion/deletions (indels), copy number variation (CNV), and larger chromosomal imbalances. A portion of the progress for ASD has come through the efforts of the Autism Sequencing Consortium (ASC), which represents a coordinated effort by more than 40 independent groups to rapidly identify ASD risk genes. Here we propose to continue the work of the ASC, largely by continued production and analysis of sequence data from ASD subjects and their families. The ASC benefits from substantial leveraging of resources, including the Exome Aggregation Consortium (ExAC) centered at the Broad Institute (BI) and whole-exome sequencing (WES) of ASC samples, supported by an NHGRI Center Grant to BI, to make this renewal as low cost as possible. We also plan new avenues of research, such as integrating whole genome sequence (WGS) data and building on ideas that have emerged from the study of common variants to understand the interplay of common and rare variants to impact risk. Through this new research we will accelerate our overall objective, which is the identification of ASD genes, thereby facilitating our long-term goal of building the foundation from which therapeutic targets for ASD emerge. Our rationale is that the identification of genes conferring significant risk to ASD and associated neurodevelopmental disorders can form the basis of studies to understand pathogenesis, as well as the basis for novel therapies. Moreover, such variants have direct implications for patients and their families in terms of etiological diagnosis, genetic counseling and patient care. Our central hypothesis ? formulated based on results over the past decade ? is that rare and common variation contributes additively to risk for ASD, but only certain rare variants confer substantial risk. The objectives will be accomplished with the following Specific Aims: 1) Produce and/or analyze WES of 30,000 new ASD subjects, parents and other controls, for a total of more than 50,000 samples; 2) Develop and apply approaches to find ?hidden? risk variants, and, 3) Use results from common and rare variant studies to describe the interplay of such variation in ASD risk. This contribution is significant because it represents the first step in research to understand pathogenesis of ASD and to the development of pharmacological strategies for treatment of core symptoms of ASD and etiologically related neurodevelopmental disorders. The research proposed is innovative, in our opinion, because it uses groundbreaking and novel statistical methods for identifying risk variants and for integrating rare and common variation. This is a new and substantively different approach to gene discovery in ASD that departs significantly from the status quo and provides the means to achieve these important goals.

ABSTRACT Recent advances in genetics and genomics have identified hundreds of coding variants that increase risk for major neuropsychiatric disorders, such autism spectrum disorder. Work to clarify the contribution of non- coding variants is also underway and is expected to accelerate rapidly in the next few years. While these advances have considerably improved our understanding of the genetic landscape neuropsychiatric disorders, a deeper understanding of molecular pathophysiology is still missing. This knowledge gap is due to, in part, the heterogeneity of risk loci involved, their potential roles in regulating expression of a large number of genes, the pleiotropic nature of risk genes, and the high likelihood that neuropsychiatric disorders result from dysfunctional circuitry involving multiple cell types and brain regions, altogether making the identification of molecular and cellular mechanisms underlying a disease problematic, especially in the context of the protractive and complex nature of brain development. Therefore, the discovery and characterization of the full spectrum of functional genomic elements active in the human brain, as well as their activity/expression patterns across the spatiotemporal dimensions, is essential for clarifying when, where, and what cell types are relevant to the etiology and treatment of neuropsychiatric disorders. This is particularly so in the context of non-coding variants, which are difficult to annotate, yet potentially hold the promise of providing highly specific spatial, temporal, and cell type specific information. To address this knowledge gap and to continue our contributions to the PsychENCODE Consortium, we propose four specific aims that identify gene regulatory and cell type-specific mechanisms of human neurodevelopment. In Aim 1, we identify functional genomic elements across single cells (nuclei), cell types, regions and developmental time points of neurotypical human and macaque postmortem brains. In Aim 2, we map the spatio-temporal proteome of neurotypical human and macaque postmortem brains. In Aim 3, we perform integrative identification of functional genomic elements and proteomics in diseased brains and iPSC-derived neural cells. In Aim 4, we integrate results from Aims 1-3, as well as with independent genetic datasets of neuropsychiatric populations, to identify non-coding elements, genes, or molecular pathways that will lead to a better understanding of the underlying pathophysiological mechanisms of neuropsychiatric disorders. Finally, these mechanisms will be functionally characterized in model systems. Data from this proposal will also serve as a critical new resource for members of the community, with which they can intersect their results and draw deeper and more meaningful conclusions, especially as the wealth of genomic data from neuropsychiatric disorders continues to accumulate.