Stephanie L. Bielas, Ph.D. - US grants

DESCRIPTION (provided by applicant): The combination of human genetics, animal models and the recent addition of induced pluripotent stem cells (iPSCs) to the human neurodevelopmental disease modeling toolbox has the potential to greatly expand our understanding of human disease mechanisms. To date, a major challenge to understanding human neurodevelopmental disorders has been the lack of affected tissue. The capacity of iPSCs to differentiate the full complement of neural tissue, from neural progenitors (NP) to mature cortical neurons, from patient iPSC opens an exciting new avenue to understanding unique human features of disease. In this application, I propose to tailor this new technology to model defects in neurogenesis which underlie autosomal recessive primary microcephaly (MCPH). MCPH is a neurodevelopmental disorder characterized by a great reduction of head growth in utero and is accompanied by nonprogressive mental retardation. MCPH is the result of cerebral cortex hypoplasia and generalized diminution of an otherwise architecturally normal brain, a phenotype that is thought to result from defective NP proliferation early in development. MCPH is well suited for this new modeling approach as NPs differentiate early in iPSC differentiation protocols and proliferation can be evaluated within the context of neural rosettes. To correlate iPSCs in vitro modeling data to in vivo brain development, I propose to utilize a combination of patient iPSCs, transgenic mouse iPSCs and animal models. To test the sensitivity of iPSC modeling to convey unique mechanistic information, I propose to model two genetic causes for MCPH that should perturb the same set of cells in different ways or with varying severity. To gain a more comprehensive understanding of the role of in neurogenesis in the molecular pathology of MCPH according to the techniques described above I am proposing to model both Nucleoporin 107 (NUP107) and abnormal spindle-like microcephaly associated (ASPM). I recently identified Nucleoporin 107 (NUP107), a gene not previously linked to human disease, as a causative gene for MCPH. To pursue the proposed research, I have generated iPSCs from Nup107 patient and control fibroblasts and chimeric mice for a conditional NUP107 gene trap allele (NUP107GT). I have also identified a novel ASPM mutation, the gene most commonly mutated in MCPH, for which iPSC modeling will be pursued as an independent investigator. The level of mechanistic understanding that can be gained from this modeling approach for MCPH will lay the foundation that can lead to new therapies and insights into how the normal human brain develops. PUBLIC HEALTH RELEVANCE: Primary microcephaly is a neurodevelopmental disorder presumably due to altered neurogenesis and causing a great reduction in brain growth. I have identified a new causative gene, NUP107, and propose to study induced-pluripotent stem cells and knockout mice to model this human disease.

? DESCRIPTION (provided by applicant): The incidence of children with inherited neurodevelopmental disorders (NDD) is high in low- and middle- income countries (LMIC) and becoming an enormous burden on heath care resources. While individual inherited NDD are rare, in aggregate they affect millions of people. Whole exome sequencing (WES) has risen to the forefront of genetic testing in High and Middle Income Countries (HMIC) based on its potential to uncover genetic causes for inherited NDD conditions, while circumventing bottlenecks caused by candidate gene screening approaches. Dr. Girisha at Kasturba Medical College at Manipal University, India and Dr. Bielas in the Department of Human Genetics at University of Michigan Medical School, US will build an ongoing collaboration to implement WES technology in the Department of Medical Genetics at Manipal University. This collaboration will upgrade the research capacity to support the use of WES for diagnosis of known causes of NDD. We will supplement education of medical genetics professionals and facilitate the incorporation of genetic counseling into genetic diagnoses. The World Health Organization recommends incorporating genetic counseling into the continuum of medical genetics care as it is predicted to lead to a reduction in the burden of NDD. The proposed infrastructure development will lay the foundation for future applications aimed at identifying novel genetic causes of NDD, developing research capacity to functionally test novel deleterious alleles and developing best practices for clinical application of WES in India.

ABSTRACT The incidence of children with inherited neurodevelopmental disorders (NDDs) is high in LMICs, and an enormous burden on heathcare resources. While individual inherited NDDs are rare, in aggregate they affect millions of people. Identifying the genetic etiology of NDDs is beneficial to families, communities and science. Genetic diagnosis allows families to recognize risk of recurrence, and act on anticipatory prognoses. Genetic discoveries drive public health policy aimed at reducing disease burden through community genetics. Genes that cause NDDs provide molecular insights into normal brain development and pathogenesis of disorders. Whole exome sequencing (WES) has risen to the forefront of genetic testing in HMIC, based on its potential to uncover the genetic basis of inherited NDDs, but is infrequently used in LMICs. An ongoing collaboration between Dr. Shukla at Kasturba Medical College at Manipal University, India and Dr. Bielas in the Department of Human Genetics at University of Michigan, US, has developed a sustainable strategy to use WES-based testing for genetic diagnosis of NDDs in India. With a diagnostic yield on par with HMIC, WES-based genetic testing will be an important tool in address the elevated burden of inherited NDD in India. We propose experiments to delineate genetic diversity of South-East Asian ancestry. For two genes we identified as novel genetic etiologies of NDDs, the same pathogenic variant was detected in unrelated affected Indian families, indicative of a founder effect with higher carrier frequency in the Indian population. This finding highlights the uneven representation of diverse populations in genomic studies. The lack of parity in sequence representation and functional studies originating from India, is a scientific and health challenge that negatively impact interpretation of genetic variants. We hypothesize that disparities in representation of diverse populations in genomic sequencing studies impact interpretation of deleterious alleles and genetic diagnosis of NDDs in India, which contribute to inequity in genomic medicine globally. We will address this challenge by defining genetic diversity in a larger cohort of South-East Asian ancestry (Aim1), functionally characterizing variants to support their classification as pathogenic (Aim 2) and reduce uneven representation of diverse populations in genomic medicine by developing a searchable web-based platform to make de-identified genetic diversity identified in Indian ancestry publically available (Aim 3). Our experimental strategy prioritizes educational exchange and research infrastructure, that fosters sustainable strategies to tackle these important problems.

During cortical development, neural progenitor cells (NPCs) produce mature neuronal subtypes in a defined temporal order. Restriction of NPC multipotency determines the cortical neuron composition of the six-layer cortex and is governed by changes to NPC chromatin. Abnormal production of cortical progeny underlies the pathology of neurodevelopmental disorders with features of autism. Chromatin remodeling genes are often identified in autism spectrum disorder (ASD), therefore, neuronal epigenetic mechanisms are likely to be essential for corticogenesis. Developmental remodeling of histone modifications across the chromatin landscape permits spatial and temporal regulation of transcription circuitry that restricts NPC multipotency. One chromatin modification is histone H2A lysine 119 mono-ubiquitination (H2AUb1), an evolutionarily conserved repressive histone modification of the Polycomb group (PcG) proteins. We recently identified human de novo dominant pathogenic variants in the PcG protein ASXL3 (Additional sex comb-like 3) as the genetic basis of neurodevelopmental disorders with syndromic features of autism and intellectual disability. ASXL3 clearly plays a central role in mammalian brain function. We propose experiments to delineate mechanisms of ASXL3 regulation in corticogenesis. We have shown that ASXL3 is a component of the Polycomb repressive deubiquitinase complex (PR-DUB), which deubiqutinates H2AUb1. Pathogenic human ASXL3 variants alter the genome-wide H2AUb1 levels and affect transcriptional regulation in patient-derived cells. We have confirmed and extended this finding in mouse and human neural progenitor cells (NPCs). Although H2AUb1 was described more than three decades ago, its functions in transcriptional regulation and epigenetic repression are less well understood than other histone modifications. We hypothesize that ASXL3-dependent deubiquitination activity plays a critical role in specifying NPC transcriptional programs that contribute to the neuronal diversity of the cortex, and, ultimately, higher brain function. We will define the cortical developmental mechanisms regulated by ASXL3 and H2AUb1 by: (Aim 1) using an existing Asxl3 mutant mice, (Aim 2) determining the genome-wide distribution of excess H2AUb1 in NPCs and the epigenomic mechanisms of corticogenesis using genetically- engineered mice, and (Aim 3) testing the conservation of ASXL3 pathology and PR-DUB activity in human cerebral organoid models of neural development. Our experimental strategy will establish the epigenetic foundation of cortical development, identify paradigms for cortical neurogenesis in NPCs, and, ultimately, unveil the mechanisms of dysregulation that leads to ASD pathology.