Annapurna Poduri - US grants

DESCRIPTION (provided by applicant): Epilepsy affects approximately one percent of the population and one in 200 children. Epilepsy syndromes with proven or likely genetic cause contribute substantially to the causes of epilepsy, especially in children. Over the last half century, there has been increasing recognition that genetic factors play an important role in predisposing individuals to epilepsy. The traditional approach of linkage analysis in families with well-defined epilepsy syndromes has been successful in identifying the genes responsible for some of these syndromes. To date, most of these genes encode ion channel subunits and provide an explanation for only a small proportion of epilepsy. We hypothesize that by studying familial forms of epilepsy with both dominant and recessive inheritance, we will discover novel genes and novel processes that set the stage for epilepsy in the developing brain. Such discovery will deepen our understanding of the developmental processes and pathways important in epilepsy and may also identify novel approaches to rational pharmacological treatment for patients with epilepsy. We will first perform rigorous phenotyping methods to classify individuals in families with individual diagnoses and familial epilepsy syndromes. We will use genome-wide markers of genetic variability (short tandem repeat polymorphisms and single nucleotide polymorphisms) to perform analyses of linkage between disease status and genomic loci, and we will further analyze the regions with evidence of linkage with positional cloning and high-throughput sequencing to identify specific genetic mutations in these families. Once this is achieved, we will screen other families and sporadic individuals with the same epilepsy phenotypes for mutations in these genes. The candidate is a board-certified child neurologist with additional clinical neurophysiology/pediatric EEG training. She will perform this research at Children's Hospital Boston under the supervision of Dr. Christopher Walsh, a renowned expert in the field of neurogenetics with extensive clinical and research experience in the genetics of brain malformations, with co-mentorship from Dr. Ruth Ottman, a pioneer in the field of epilepsy genetics who has developed the phenotyping methodologies now accepted as standard for epilepsy genetics research. In addition to the combined training under these mentors, the candidate will also participate in the Epilepsy Phenome Genome Project and receive additional training from a renowned group of national experts in epilepsy genetics. The experience and skills she will garner during this training period will set the stage for an independent career in clinical neuroscience research. PUBLIC HEALTH RELEVANCE: Epilepsy is a common condition, affecting approximately one in one hundred people. While the causes of epilepsy are varied, genetics play an important role in the development of epilepsy in many individuals and may affect several individuals in a family. The goal of this project is to gain deeper insight into the fundamental causes of epilepsy by studying the genetics of familial forms of epilepsy.

PROJECT SUMMARY Brain malformations comprise a group of genetic developmental brain disorders that present in childhood with intellectual disability and epilepsy and other neurologic features, causing substantial morbidity, mortality, and health care costs. To date, over 1000 genes have been associated with brain malformations. The long term goal of this project is to create a resource of well-categorized and expertly curated genes and variants responsible for causing brain malformations, to help clinicians navigate diagnosis and inform management. To this end we have assembled a group of clinician-investigators with broad, complementary expertise in brain malformations in the domains of gene discovery, neurobiology, clinical phenotype, radiographic presentation, and treatment. We have also included experts in bioinformatics and colleagues deeply involved in ClinGen and ClinVar to facilitate integration of our findings into these existing organizational frameworks. Important for this proposal, our group is comprised of individuals with a long-standing history of collaboration across institutions, nations, and generations. In Aim 1 of this proposal we will survey the literature to curate genes associated with brain malformations, assess the strength of the evidence for these associations using ClinGen criteria, and organize them into biologically and clinically useful groups. In Aim 2 of this proposal, we will extend our efforts to curate variants encountered in clinical and research exomes recruited via the Brain Development and Genetics Clinic, a specialty multidisciplinary clinic at Boston Children?s Hospital devoted to the diagnosis, treatment and counseling of patients with brain malformations. Variant interpretations will be contributed to ClinVar. In Aim 3 of this proposal, we will perform deep curation of genes and variants from the literature and publicly available databases for clinically important subgroups of brain malformations, including those associated with focal cortical dysplasia (FCD), hemimegalencephaly (HME), and polymicrogyria (PMG) with megalencephaly, all recently associated with de novo germline or post-zygotic variants that result in activation of the mTOR-PI3K- AKT pathway. Successfully completed, this project will provide a blueprint for best practices in the clinical application of genomics to the care of patients with brain malformations: selecting appropriate diagnostic testing, interpreting variants, and choosing appropriate management.

Project Summary Sudden infant death syndrome (SIDS) and sudden unexplained death in childhood (SUDC), which we study together under the rubric of sudden unexpected death in pediatrics (SUDP), is a major cause of infant and child mortality. While Safe Sleep efforts aim to minimize risks in the sleep environment in SIDS, it is recognized that affected children also possess intrinsic vulnerabilities that increase their susceptibility to sudden death. As external factors have been addressed, the persistence of SUDP attests to the significance of these intrinsic vulnerabilities. SUDP has long been considered ?idiopathic,? like other conditions with elusive and likely multifactorial etiologies. Our group approaches SUDP as a constellation of undiagnosed diseases. We hypothesize that the intrinsic biological factors leading to SUDP include neurodevelopmental, epilepsy-related, cardiac, metabolic, respiratory, and infectious mechanisms, and that these mechanisms have a discoverable genetic basis. We take a multidisciplinary approach that mirrors undiagnosed disease programs, with extensive phenotyping and comprehensive genomic analysis to identify unrecognized disease mechanisms responsible for SUDP. Our group has previously found serotonin deficits in the brainstem of SIDS infants, malformations of the hippocampus in SIDS and SUDC cases, and shown that our diagnostic approach increases the likelihood of implicating natural causes in the assessment of these deceased children. The research in this application seeks preliminary data on novel genes and genomic mechanisms underlying sudden death through an analysis informed by our program's approach to phenotyping. The proposed research will investigate whether a complex genetic architecture plays a major role in SUDP. This hypothesis will be pursued by combining rich phenotypic data from SUDP cases with exome sequencing analysis. We will ascertain and comprehensively phenotype SUDP cases and their families (Aim 1), and then analyze exome data from these well-phenotyped proband-parent trios, to determine genetic mechanisms associated with SUDP (Aim 2). A highly novel aspect of this research is the opportunity to gain population- based insights due to the unprecedented forensic-academic partnership we have established with the Massachusetts Office of the Chief Medical Examiner (OCME) to assess all children dying suddenly and unexpectedly under the age of 3 years in Massachusetts. The potential impact of this research is the elucidation of genetic mechanisms involved in sudden unexplained deaths in children under the age of three years. This research carries the further promise of contributing to advancements in specific predictive algorithms and genetic markers for infants at risk for SUDP, and advancing the forensic molecular autopsy in establishing a major cause of mortality. The preliminary data gained in this research will lead to the refinement of hypotheses to be explored in future research.

Epilepsy is a common condition, affecting 1 in 26 individuals, for which the role of genetics is well recognized. Since its discovery in 2008, PCDH19 has been amongst the most prominent single gene causes of epilepsy. Mutations in PCDH19 cause X-linked ?female limited epilepsy,? with refractory childhood-onset seizures, intellectual disability, and autism. PCDH19 encodes a central nervous system protocadherin predicted to mediate cell adhesion, but its role in neurodevelopment is not established. PCDH19 mutations can be de novo or inherited from mildly affected/unaffected mothers or, curiously, unaffected fathers. The female predominance is hypothesized to be due to ?obligate mosaicism.? Reports of rare symptomatic mosaic males are consistent with this hypothesis. However, recently noted subtle behavioral features in ?carrier? fathers suggest that a mosaic state is not required for all phenotypic manifestations. We will address, using these unusual genetics as a clue, the functional role of PCDH19 and its role in epilepsy. We will conduct genotype- phenotype studies to understand the importance of mutation location, type, and inheritance on PCDH19- related dysfunction. We will then harness a carefully curated set of human PCDH19 mutations, from the PCDH19 Registry that we founded, into zebrafish mechanistic models. There are currently no established animal models of PCDH19-induced epilepsy. Zebrafish represent an established vertebrate model system with genetic tractability and identifiable seizures. Our preliminary data from CRISPR/Cas9 genome-edited zebrafish suggest that loss of function of pcdh19 results in seizures. With these models, we will study the cell types involved and the specific mechanisms by which loss or alteration of pcdh19 results in disease. We hypothesize that the location and type of PCDH19 mutation correlates with phenotypic severity in humans and that zebrafish models based on human mutations will display seizures. We thus pursue a novel approach to study the mechanisms involved in PCDH19 dysfunction, with models based on mutations that we have accrued through our Registry. We will aim to correlate the severity of PCDH19-related phenotypes with location and type of patient mutations; establish pcdh19 zebrafish models based on human mutations and characterize their epilepsy phenotypes; and identify the effects of mutations on cell migration, adhesion, and excitability in mutant pcdh19 zebrafish. Our research will develop the first in vivo animal models for PCDH19-related epilepsy. Through our zebrafish models, we will investigate the neurodevelopmental role of PCDH19 and develop drug screens for PCDH19- related epilepsy in the emerging era of precision medicine. The broader impacts of this study are that it will (1) provide insight into protocadherin-related epilepsies, (2) establish a human-to-zebrafish paradigm for translational research in genetic epilepsy, and (3) inform the modeling of other mosaic neurological disorders.