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The human brain is the most complicated and complex organ in the human body, responsible for not only regulating basic physiological processes such as metabolism and sensation, but also higher cognitive functions such as learning and emotion. As with all other organs of the human body, this complexity in function is built upon the framework of interconnected cells, whose individual activity/function is driven by gene transcription and protein expression. Contemporary models on brain function have been based on the static assumption that cells of the central nervous system operate under constant and non-varying genomes (i.e. that all brain cells contain two copies of each autosome and two sex chromosomes). The body of work contained in this dissertation challenges this notion, and describes the inherently mosaic composition individual cells in the human brain at the genomic level. This dissertation describes the experimental tools used to uncover DNA content variation (DCV) in the mouse and human brain, identifies a novel brain region which exhibits one type of DCV (aneuploid mosaicism), clarifies a cell-cycle based neurodegenerative model of Alzheimer's disease, and characterizes DCV in the normal human brain. The importance of DCV for basic neuroscience is underscored by the observation that DCV changes are associated in a region specific manner in neurodegenerative disease, notably Alzheimer's disease.
The human cerebral cortex makes up approximately 82% of the total brain mass, has 52 distinct Brodmann areas, and contains approximately 16 billion neurons. In recent years, neuroscientists, geneticists, bioengineers, and bioinformaticians, by working in collaboration have only begun to scratch the surface towards understanding the enormous cellular complexity and heterogeneity that exists in our brains. My thesis work in has focused on the investigation of the immense diversity that comprises both the genomic and the transcriptomic landscapes of the human brain through the use of traditional, and newly engineered, single-cell technologies. Neuronal genomic mosaicism--the phenomenon wherein neurons possess unique somatically altered genomes--was first identified as mosaic aneuploidies, a gain or loss of an entire chromosome. In recent years, multiple labs have now demonstrated that the somatic genomic changes also include LINE-1 retrotransposons, both large (>10 megabases (Mb)) and small (
Cells of the soma, especially of the brain, generate genomic variations with region-specific differences in frequency, which leads to somatic mosaicism. This postzygotic phenomenon is, among others, a consequence of DNA damage or defective repair and may contribute to neurogenetic disorders. The present work provides two innovative approaches to investigate the role of retrotransposons and DNA double-strand breaks (DSBs) in the formation of somatic mosaicism in the human brain. Retrotransposons, including SVA and LINE-1, are mobile genetic elements that replicate in the genome by the "copy-and-paste" mechanism. Recent NGS-based studies demonstrated that the retrotransposon machinery is active in the human brain. This raises the question of whether SVA and LINE-1, respectively their presence at orthologous loci, can be used to track somatic differences in brain regions. For this purpose, a subtractive kinetic enrichment technique called Representational Difference Analysis (RDA) coupled with NGS is established. In addition, chromosomal DSB hotspots and their regional differences in the brain will be investigated. For one type of DSB repair, SINE/LINE information is known to be used in the context of non-homologous end-joining, i.e. typical signatures of SINE/LINE integrations at DSB sites are generated. To describe the 'breakome', a DSB labeling system based on Breaks Labeling In Situ and Sequencing (BLISS) is implemented. The RDA provides evidence for somatic mosaicism caused by differential retrotransposition of LINE-1 and SVAs in the human brain. In this context, SVAs as 'presence/absence' markers can reflect the development of telencephalon and metencephalon. De novo SVA and LINE-1 insertions have chromosome-wide rates and preferential integration in GC- and TE-rich regions and genes that tend to be involved in neural functions. The 'breakome' results show DSB hotspots occurring across the brain or in a brain region-specific manner. As a result, several known and novel recurrent DSB cluster (RDC) associated genes are detectable and can be linked to neurological diseases. Moreover, (epi-) genetic predictors of DSB formation can be identified, including DNA-binding proteins that play a role in DSB repair. Interestingly, retrotransposons and DSBs frequently occur in close proximity to each other, suggesting a possible involvement of mobile DNA in the induction or repair of DSBs. In summary, the methods presented in this work can be applied in various research areas, such as cell lineage tracing experiments or the analysis of potentially pathogenic DNA damage in the context of neurological or tumor diseases.
DNA copy number variations (CNVs) have previously been reported in human cortical neurons from non-diseased patients, but these alterations do not appear to be consistent from cell to cell and appear to be rare among neurons overall. Interestingly, Alzheimer's disease patients appear to have a higher prevalence of CNVs than non-diseased, although the biological significance of this observation is still largely unknown. Single-cell whole-genome next-generation sequencing holds promise to investigate these variations and the regions in which they occur in an unbiased manner. Unlike recent advances in single-cell RNA-seq, however, library preparation for single-cell DNA-seq suffers from extremely limited throughput. Furthermore, it is difficult to assess the significance of individual variations from whole-genome sequencing alone, particularly when control samples from non-diseased patients also show some variation at lower frequency. A potential solution is a multi-omics approach, in which information is collected about multiple species of biomolecules simultaneously from each sample, which taken together aid the interpretation of individual observations with respect to biological significance. This dissertation describes the design and development of a technology to physically separate DNA and RNA and to prepare sequencing libraries from each in parallel from limited starting samples without splitting, which we called Gel-seq. Thirty-two paired DNA and RNA sequencing libraries were successfully prepared from a variety of human and mouse cells lines and from mouse liver tissue using Gel-seq. Sample types could be clearly distinguished from each other based on either genomic copy number or transcriptomic profiles. This dissertation also describes the design and development of a technology to prepare a thousand single-cell whole-genome sequencing libraries in a single run. A proof-of-concept was performed with 87 cells from human and mouse lines. Copy number profiles agreed with bulk, and 96% and 92% of human and mouse cells, respectively, clustered correctly within their cell line based on copy number profile alone. These technologies will help to enable the unbiased characterization of genomic alterations not only in neurodegenerative disorders, but potentially also in other conditions associated with mosaic genomic backgrounds, such as cancer, microbiome disorders, or infectious diseases.
Heritable human genome editing - making changes to the genetic material of eggs, sperm, or any cells that lead to their development, including the cells of early embryos, and establishing a pregnancy - raises not only scientific and medical considerations but also a host of ethical, moral, and societal issues. Human embryos whose genomes have been edited should not be used to create a pregnancy until it is established that precise genomic changes can be made reliably and without introducing undesired changes - criteria that have not yet been met, says Heritable Human Genome Editing. From an international commission of the U.S. National Academy of Medicine, U.S. National Academy of Sciences, and the U.K.'s Royal Society, the report considers potential benefits, harms, and uncertainties associated with genome editing technologies and defines a translational pathway from rigorous preclinical research to initial clinical uses, should a country decide to permit such uses. The report specifies stringent preclinical and clinical requirements for establishing safety and efficacy, and for undertaking long-term monitoring of outcomes. Extensive national and international dialogue is needed before any country decides whether to permit clinical use of this technology, according to the report, which identifies essential elements of national and international scientific governance and oversight.
In The Birth of the Mind , award-winning cognitive scientist Gary Marcus irrevocably alters the nature vs. nurture debate by linking the findings of the Human Genome project to the development of the brain. Startling findings have recently revealed that the genome is much smaller than we once thought, containing no more than 30,000-40,000 genes. Since this discovery, scientists have struggled to understand how such a tiny number of genes could contain the instructions for building the human brain, arguably the most complex device in the known universe. Synthesizing up-to-the-minute biology with his own original findings on child development, Marcus is the first to resolve this apparent contradiction by chronicling exactly how genes create the infinite complexities of the human mind. Along the way, he dispels the common misconceptions people harbor about genes, and explores the stunning implications of this research for the future of genetic engineering. Vibrantly written and completely accessible to the lay reader, The Birth of the Mind will forever change the way we think about our origins and ourselves.
In this landmark work, the author team led by Dr. Sean Carroll presents the general principles of the genetic basis of morphological change through a synthesis of evolutionary biology with genetics and embryology. In this extensively revised second edition, the authors delve into the latest discoveries, incorporating new coverage of comparative genomics, molecular evolution of regulatory proteins and elements, and microevolution of animal development. An accessible text, focusing on the most well-known genes, developmental processes and taxa. Builds logically from developmental genetics and regulatory mechanisms to evolution at different genetic morphological levels. Adds major insights from recent genome studies, new evo-devo biology research findings, and a new chapter on models of variation and divergence among closely related species. Provides in-depth focus on key concepts through well-developed case studies. Features clear, 4-color illustrations and photographs, chapter summaries, references and a glossary. Presents the research of Dr. Carroll, a pioneer in the field and the past president of the Society for Developmental Biology.
Genomically identical cells have long been assumed to comprise the human brain, with post-genomic mechanisms giving rise to its enormous diversity, complexity, and disease susceptibility. However, the identification of neural cells containing somatically generated mosaic aneuploidy - loss and/or gain of chromosomes from a euploid complement - and other genomic variations including LINE1 retrotransposons and regional patterns of DNA content variation (DCV), demonstrate that the brain is genomically heterogeneous. The effects of constitutive aberrations, as observed in Down syndrome, implicate roles for defined mosaic genomes relevant to cellular survival, differentiation potential, stem cell biology, brain organization, and neuropathological processes. Analyses of genomic mosaicism in sporadic Alzheimer's disease (AD) provide evidence for potential functional mosaic changes, as dramatic genomic alterations in the AD frontal cortex manifested via a significant increase in DCV. The resulting somatic locus-specific amplification of amyloid precursor protein supports mosaicism as a factor in AD pathogenesis, while microfluidic quantitative (q)PCR analyses of single cortical AD neurons reveal the variability of somatic changes that occur within the brain of a single individual. Given the range of genomic variation that has been observed, understanding of the precise phenotypes and functions produced by genomic mosaicism in either diseased or normal brains is limited. However, the ablation of programmed cell death leading to increased observance of extreme karyotypes in cortical neural progenitor cells supports the functional non-equivalence of varied mosaic forms, as extremely aneuploid cells are targeted for elimination while cells with mild aneuploidies survive. Induction of increased neural mosaic aneuploidy through fetal exposure to substances of abuse demonstrates the fragility of the individual cellular genome and the vulnerability of the brain to induced mosaicism with pathogenic potential, highlighting the consequences of compromised somatic genomic integrity.
A psychologist offers a detailed study of the genetic underpinnings of human thought, looking at the small number of genes that contain the instructions for building the vastly complex human brain to determine how these genes work, common misconceptions about genes, and their implications for the future of genetic engineering. 30,000 first printing.