Eric Vilain - US grants

DESCRIPTION (provided by applicant): In mammals, sex determination is the embryonic process that determines the developmental fate of the bipotential gonad into either testis or ovary. It is triggered by the presence, in males, or the absence in females, of Sry, a Y-linked gene encoding a transcription factor. Disorders of human sex determination cause defects in gonadal function and can result in a spectrum of abnormalities in the internal and external genitalia, ranging from mild sexual ambiguities to complete sex reversal. Although several sex-determining genes have been identified in humans and mouse models, the vast majority of XY patients with disorders of sex determination are not explained genetically, suggesting the existence of other genetic factors involved in this process. In addition, the molecular mechanisms of known sex-determining genes are poorly understood. Our overarching goal is to decipher the molecular events underlying the differentiation of the embryonic gonad, and therefore the process of sex determination. To achieve this objective, we will investigate a mouse model of disorders of sex development. We will identify novel genetic factors protecting against XY sex reversal in the C57BL/6J-YPOS mouse model in which the combination of a Y chromosome originating from a domesticus strain (YPOS) and a C57BL/6J background results in disrupted testicular development. Since our preliminary results show that a congenic region from mouse chromosome 11 protects against sex reversal in the C57BL/6J-YPOS model, we will test the hypothesis that this congenic region carries one or several genes that differ between C57BL/6J and the donor, congenic, fragment and that the difference is responsible for the protection. We will narrow down the congenic region by creating sub-congenic areas and identifying a minimal congenic fragment associated with the protection phenotype (Aim 1). We will also screen for and select candidate genes, investigate their expression profile and their functional relationship with known sex-determining genes and test if the alteration of their expression causes modifications in embryonic gonadal development (Aim 2). Finally, we will investigate the molecular mechanisms of XY sex reversal in the C57BL/6J-YPOS model and analyze the molecular and cellular nature of protective effect from the congenic region on gonadal development (Aim 3). Dissecting the molecular pathway of mammalian sex determination will be crucial in understanding the basic sex differences in gonadal development and the pathophysiology of human disorders of sex development. PUBLIC HEALTH RELEVANCE: One of the most defining moment of our lives is when, in the womb, we embark on a male or female path, and what triggers this moment is when the gene Sry is turned on in males, or stays off in females;yet, many molecular events that happen after Sry action remain poorly understood, and in humans, disruption of sexual development occurs at a frequency of 0.5% to 1%. As only 25% of human pathologies of sex determination are explained genetically, we propose to identify new genes involved in this process by using a mouse model of abnormal sex development. This proposal will elucidate basic questions about how males and females become different, and will improve genetic classification and diagnostic methods of patients born with disorders of sex development.

DESCRIPTION (provided by applicant): Male and female brains are different. The overarching goal of the proposed studies is to understand the molecular mechanisms of sex differences in the brain, focusing on the substantia nigra (SN) as a model. It is well established that testosterone, secreted by the testes, before or after birth, acts on the male brain to masculinize specific neural networks, which results in specific masculine behaviors. The classical view is that gonadal androgens are the only factors involved in the masculinization of the brain. Discovery of differential gene expression in the mammalian brain before the gonads start producing androgens led us to propose an alternative hypothesis. We propose to explore that sex differences in brain function and behavior may be caused in part by genetic factors not produced by the gonads. We have demonstrated specific expression of the Y-linked, testis-determining factor Sry, in two regions of the adult brain: the substantia nigra (SN) and the mammillary bodies. Reduction of Sry expression in the SN resulted in a strong decrease of tyrosine hydroxylase as well as in sensorimotor impairment. In order to understand the role of Sry in sex differences in SN function, we will investigate the expression profile of Sry in the adult brain, identify the cell types in which Sry is expressed, measure the level of expression of Sry and test whether it correlates with TH expression (Specific Aim 1). We will explore the consequences of down-regulating and upregulating Sry in the SN on TH-positive neurons and the sensorimotor behaviors they control, and whether Sry action in males compensates for the lack (or lower levels) of a female-specific factor. Specifically, we will test if this female factor could be estradiol, or dosage of the X chromosome (Specific Aim 2). Finally, we will elucidate the molecular mechanisms of action of Sry on dopaminergic neurons using NT2N cells as a model of dopaminergic neurons (Specific Aim 3). This proposal intends to improve our general understanding of sex differences in brain function. The characterization of the molecular mechanisms by which male and female brains differ impacts our understanding of sex differences in psychiatric and neurological disorders such as Parkinson's disease, attention deficit disorder, or depression.

DESCRIPTION (provided by applicant): Disorders of sex development (DSD) are phenotypically heterogeneous, ranging from minor genital malformations (hypospadias, cryptorchidism, hypertrophy of the clitoris) to genital ambiguity. In the aggregate, DSD have an estimated incidence of about 1%. DSD can result in severe consequences for behavioral health, fertility, cancer risk and quality of life. For families, the birth of a child with a DSD, and the accompanying uncertainty about future psychological and sexual development, is believed to be extraordinarily stressful. Recently, the debate over clinical management of DSD, in particular gender assignment and genital surgery, has intensified; yet the scientific data on patient outcomes have remained very incomplete. Major obstacles to optimal clinical management of DSD include gaps in understanding of pathophysiology, impeding precise diagnostic categorization, along with the absence of prospective longitudinal studies of health and quality of life outcomes. This proposal delivers a platform for hypothesis-based research on the mechanisms of sex development and evidence-based care for patients and families affected by DSD. Aim 1 identifies novel genetic mechanisms of sex development and improves understanding of the pathophysiology and molecular diagnosis of DSD by a step-wise approach of specific DNA capture and sequencing, analysis of copy number variants, and identification of novel candidate genes for DSD. Aim 2 delivers standardized tools for reliable phenotypic descriptions across multiple study sites and investigators, including radiological, biochemical, histological evaluations and descriptions of genital phenotype and post- surgical appearance and function, facilitating interpretation of genetic, gender, and quality of life outcomes Aim 3 identifies short and medium-term outcomes by delivering a comprehensive psychosocial and health-related quality of life assessment battery using psychometrically robust measures suitable for use in routine clinical care, a necessary step leading to evidence-based psychosocial treatment protocols. Aim 4 builds a sustainable research infrastructure and ensures rapid translation of new evidence into ongoing clinical practice by integrating standardized DSD diagnostic and treatment protocols and fostering the transfer of best practices in healthcare delivery across network sites. This registry provides the analytic platform by which data are collected, analyzed, and shared among researchers and sites, while a collaborative network will supply the foundation for multidisciplinary basic, clinical, and translational research. This unique combination of genetic, phenotypic and psychosocial approaches will transform participating sites into self-sustaining DSD centers of excellence for clinical care along with the added value of a registry serving as a critical resource for hypothesis-driven research.

DESCRIPTION (provided by applicant): Klinefelter Syndrome (KS) is a common chromosomal abnormality of males who have two X chromosomes XXY, rather than one, XY. XXY males experience a variety of congenital developmental problems, including infertility, lower levels of androgens, increased risk for obesity and metabolic disease, increased risk for autoimmune diseases, and cognitive features including alterations in executive function and delayed language development. The long-term objectives of this project are to identify X chromosome genes that cause behavioral features of Klinefelter Syndrome, using novel mouse models. An overarching question is to separate the direct effects of X chromosome genes that cause Klinefelter Syndrome traits, from those caused by lower testosterone levels in XXY individuals. A novel mouse model produces XXY, XY, and XX mice that have either testes or ovaries, so that sex chromosomal effects can be identified that do not require testicular secretions, or occur when testicular secretions do not explain differences. Mice will be compared in a series of Klinefelter Syndrome-relevant behavioral measurements that assess executive functions, development of vocalizations, and partner preference. The expression levels of six specific X chromosome genes, which are the major candidates for causing the features of Klinefelter Syndrome, will be directly manipulated to assess which is/are likely the causal genes in the mouse model.

DESCRIPTION (provided by applicant): Undiagnosed diseases take a disproportionate toll on the health care system and on affected patients and families. Our proposal builds a collaborative network of researchers and healthcare providers, all with a stake in improving healthcare and outcomes for persons affected by various rare genetic disorders. Our approach will synergize basic and clinical research with the use of cutting-edge phenotyping technologies, an array of world class experts, and the translation of whole exome sequencing to the bedside. This will result in the development of a diagnostic process, rapidly translating clinical evidence into improved healthcare delivery. The Undiagnosed Disease Network (UDN) will provide the foundation to stimulate additional multi-and interdisciplinary basic, translational, and clinical research. Investigating rare diseases involving multiple systems and incorporating comprehensive genomic data into clinical care creates considerable challenges, from the interpretation of vast amounts of genetic variants to their relevance to the symptoms, to the communication issues linked to their disclosure, and to their impact on clinical management. Our proposal delivers a platform for a UDN Clinical Site that functions locally and as part of a network to tackle the incorporation of genomic information into the clinical workflow, analyze patients' symptoms in a standardized and reproducible fashion, and perform research investigations to elucidate further the mechanisms of undiagnosed diseases. We will reach these overarching goals by implementing the following specific aims: Aim 1: Create a UDN clinic model that functions locally and network-wide Aim 2: Investigate the clinical phenotypes of new and rare disorders Aim 3: Investigate the underlying mechanisms of new and rare disorders Aim 4: Build a network-wide sustainable infrastructure for translational research on new and rare disorders Our project integrates the resources of (1) the infrastructure of a Clinical and Translational Science Institute, allowing for a state-of-the-art clinical investigation of complex, multisystemic disorders, within a maximum of a week stay; (2) an experienced, team of clinicians from all specialty fields, that delivers clinical use of whole- exome sequencing, and it integration in the diagnostic process of rare, undiagnosed disorders; (3) the UCLA Clinical Genomic Center that offers bioinformatics data handling, and clinical laboratory exome sequencing, that will interpret and report clinically relevant DNA variants; (4) expertise in the investigation of environmental effects on clinical symptoms; (5) a registry infrastructure with longstanding experience in standardization of phenotypic and genotypic information; (6) access to a large, ethnically varied population and (7) a sustainable approach, with provisions to care for un/underinsured patients. Overall, our approach is designed to work cooperatively with the other Clinical Sites, the Coordinating Center, and the IRP-UDP, by capitalizing on our experiences in medical genetics, genetic counseling, clinical exome sequencing, and statistical genomics.

PROJECT SUMMARY Disorders/Differences of Sex Development (DSD) is an umbrella term covering congenital conditions in which chromosomal, gonadal, or anatomic sex development is atypical. DSD are phenotypically heterogeneous, ranging from genital malformations (hypospadias, cryptorchidism, clitoral hypertrophy) to genital ambiguity. DSD have a collective incidence of about 1% and can result in serious consequences for fertility, cancer risk and quality of life across the lifespan. Debate over clinical management, in particular gender assignment and genital or gonadal surgery, has intensified; yet scientific data informing best practices remain limited. Clinical care in DSD is hampered by a fragmented research agenda and lack of standardization, leaving fundamental gaps in knowledge of DSD pathology and links between treatment options and desired outcomes. Major obstacles include gaps in understanding of pathophysiology (impeding precise diagnostic categorization), the absence of prospective longitudinal studies of psychosocial outcomes, and the potential moderating influence of biomedical, psychosocial and legal factors on medical decision making. This project is the first of its kind, globally, to prospectively study the variable pathways from DSD diagnosis and clinical management to psychosocial adaptation. This goal will be accomplished by exploiting the infrastructure and robust collaboration of the DSD?Translational Research Network (DSD-TRN). The Network comprises a consortium of 12 interdisciplinary healthcare teams across the nation in conjunction with patient stakeholder and bioethics representation. Our guiding principle is that evidence-based standardization of diagnostic and treatment protocols will be associated with higher rates of definitively diagnosed DSD, reduced variation in clinical practice, enhanced patient/family healthcare-related experiences, and improved psychosocial outcomes for patients and their families. The proposed project will deliver evidence needed to raise the quality of healthcare in DSD to levels observed for other rare diseases. Specific aims include: 1. Genetics. Improving and expanding the molecular diagnosis of DSD; 2. Psychosocial. Identifying diagnostic, clinical care, and family risk and resilience factors associated with variability in psychological outcomes of patients with DSD and their families; 3. Determinants of clinical management. Identifying biomedical, legal, and psychosocial determinants of clinical management decisions.