The vast majority of somatic mutations in plants are layer-specific.

BACKGROUND: Plant meristems are structured organs consisting of distinct layers of stem cells, which differentiate into new plant tissue. Mutations in meristematic layers can propagate into large sectors of the plant. However, the characteristics of meristematic mutations remain unclear, limiting our understanding of the genetic basis of somaclonal phenotypic variation. RESULTS: Here, we analyse the frequency and distribution of somatic mutations in an apricot tree. We separately sequence the epidermis (developing from meristem layer 1) and the flesh (developing from meristem layer 2) of several fruits sampled across the entire tree. We find that most somatic mutations (> 90%) are specific to individual layers. Interestingly, layer 1 shows a higher mutation load than layer 2, implying different mutational dynamics between the layers. The distribution of somatic mutations follows the branching of the tree. This suggests that somatic mutations are propagated to developing branches through axillary meristems. In turn, this leads us to the unexpected observation that the genomes of layer 1 of distant branches are more similar to each other than to the genomes of layer 2 of the same branches. Finally, using single-cell RNA sequencing, we demonstrate that layer-specific mutations were only transcribed in the cells of the respective layers and can form the genetic basis of somaclonal phenotypic variation. CONCLUSIONS: Here, we analyse the frequency and distribution of somatic mutations with meristematic origin. Our observations on the layer specificity of somatic mutations outline how they are distributed, how they propagate, and how they can impact clonally propagated crops.

Genomic Mosaicism of the Brain: Origin, Impact, and Utility.

Genomic mosaicism describes the phenomenon where some but not all cells within a tissue harbor unique genetic mutations. Traditionally, research focused on the impact of genomic mosaicism on clinical phenotype-motivated by its involvement in cancers and overgrowth syndromes. More recently, we increasingly shifted towards the plethora of neutral mosaic variants that can act as recorders of cellular lineage and environmental exposures. Here, we summarize the current state of the field of genomic mosaicism research with a special emphasis on our current understanding of this phenomenon in brain development and homeostasis. Although the field of genomic mosaicism has a rich history, technological advances in the last decade have changed our approaches and greatly improved our knowledge. We will provide current definitions and an overview of contemporary detection approaches for genomic mosaicism. Finally, we will discuss the impact and utility of genomic mosaicism.

Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration.

Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human postmortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.

Extraction and Purification of Single Nuclei from Frozen Human Brain Tissue.

Resolving the complexity of the human brain at the level of single cells is essential to gaining an understanding of the immense diversity of cell types and functional states in both healthy and diseased brains. To exploit fully the technologies available for such studies, one must extract and isolate pure nuclei from unfixed postmortem tissue while preserving the molecules to be interrogated. Currently, nuclei are necessary substitutes for individual brain cells, since myriad cell types/sub-types constituting the human brain are embedded within the neuropil-a complex milieu of interconnected cells, processes, and synapses-which precludes intact and selective isolation of single brain cells. Here, we describe a protocol for the extraction and purification of intact single nuclei from frozen human brain tissue along with modifications to accommodate numerous downstream analyses, particularly for transcriptomic applications.

Colistin Resistance Onset Strategies and Genomic Mosaicism in Clinical Acinetobacter baumannii Lineages.

The treatment of multidrug-resistant Gram-negative infections is based on colistin. As result, COL-resistance (COL-R) can develop and spread. In Acinetobacter baumannii, a crucial step is to understand COL-R onset and stability, still far to be elucidated. COL-R phenotypic stability, onset modalities, and phylogenomics were investigated in a clinical A. baumannii sample showing a COL resistant (COLR) phenotype at first isolation. COL-R was confirmed by Minimum-Inhibitory-Concentrations as well as investigated by Resistance-Induction assays and Population-Analysis-Profiles (PAPs) to determine: (i) stability; (ii) inducibility; (iii) heteroresistance. Genomics was performed by Mi-Seq Whole-Genome-Sequencing, Phylogenesis, and Genomic Epidemiology by bioinformatics. COLRA. baumannii were subdivided as follows: (i) 3 A. baumannii with stable and high COL MICs defining the "homogeneous-resistant" onset phenotype; (ii) 6 A. baumannii with variable and lower COL MICs displaying a "COL-inducible" onset phenotype responsible for adaptive-resistance or a "subpopulation" onset phenotype responsible for COL-heteroresistance. COL-R stability and onset strategies were not uniquely linked to the amount of LPS and cell envelope charge. Phylogenomics categorized 3 lineages clustering stable and/or unstable COL-R phenotypes with increasing genomic complexity. Likewise, different nsSNP profiling in genes already associated with COL-R marked the stable and/or unstable COL-R phenotypes. Our investigation finds out that A. baumannii can range through unstable or stable COLR phenotypes emerging via different "onset strategies" within phylogenetic lineages displaying increasing genomic mosaicism.

Altered cleavage plane orientation with increased genomic aneuploidy produced by receptor-mediated lysophosphatidic acid (LPA) signaling in mouse cerebral cortical neural progenitor cells.

The brain is composed of cells having distinct genomic DNA sequences that arise post-zygotically, known as somatic genomic mosaicism (SGM). One form of SGM is aneuploidy-the gain and/or loss of chromosomes-which is associated with mitotic spindle defects. The mitotic spindle orientation determines cleavage plane positioning and, therefore, neural progenitor cell (NPC) fate during cerebral cortical development. Here we report receptor-mediated signaling by lysophosphatidic acid (LPA) as a novel extracellular signal that influences cleavage plane orientation and produces alterations in SGM by inducing aneuploidy during murine cortical neurogenesis. LPA is a bioactive lipid whose actions are mediated by six G protein-coupled receptors, LPA1-LPA6. RNAscope and qPCR assessment of all six LPA receptor genes, and exogenous LPA exposure in LPA receptor (Lpar)-null mice, revealed involvement of Lpar1 and Lpar2 in the orientation of the mitotic spindle. Lpar1 signaling increased non-vertical cleavage in vivo by disrupting cell-cell adhesion, leading to breakdown of the ependymal cell layer. In addition, genomic alterations were significantly increased after LPA exposure, through production of chromosomal aneuploidy in NPCs. These results identify LPA as a receptor-mediated signal that alters both NPC fate and genomes during cortical neurogenesis, thus representing an extracellular signaling mechanism that can produce stable genomic changes in NPCs and their progeny. Normal LPA signaling in early life could therefore influence both the developing and adult brain, whereas its pathological disruption could contribute to a range of neurological and psychiatric diseases, via long-lasting somatic genomic alterations.

Brain cell somatic gene recombination and its phylogenetic foundations.

A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration-approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements.

Genomic landscape of the global oak phylogeny.

The tree of life is highly reticulate, with the history of population divergence emerging from populations of gene phylogenies that reflect histories of introgression, lineage sorting and divergence. In this study, we investigate global patterns of oak diversity and test the hypothesis that there are regions of the oak genome that are broadly informative about phylogeny. We utilize fossil data and restriction-site associated DNA sequencing (RAD-seq) for 632 individuals representing nearly 250 Quercus species to infer a time-calibrated phylogeny of the world's oaks. We use a reversible-jump Markov chain Monte Carlo method to reconstruct shifts in lineage diversification rates, accounting for among-clade sampling biases. We then map the > 20 000 RAD-seq loci back to an annotated oak genome and investigate genomic distribution of introgression and phylogenetic support across the phylogeny. Oak lineages have diversified among geographic regions, followed by ecological divergence within regions, in the Americas and Eurasia. Roughly 60% of oak diversity traces back to four clades that experienced increases in net diversification, probably in response to climatic transitions or ecological opportunity. The strong support for the phylogeny contrasts with high genomic heterogeneity in phylogenetic signal and introgression. Oaks are phylogenomic mosaics, and their diversity may in fact depend on the gene flow that shapes the oak genome.

Modeling Brain Somatic Mosaicism With Cerebral Organoids, Including a Note on Mutant Microglia.

The brain is a genomic mosaic. Cell-to-cell genomic differences, which are the result of somatic mutations during development and aging, contribute to cellular diversity in the nervous system. This genomic diversity has important implications for nervous system development, function, and disease. Brain somatic mosaicism might contribute to individualized behavioral phenotypes and has been associated with several neuropsychiatric and neurodegenerative disorders. Therefore, understanding the causes and consequences of somatic mosaicism in neural circuits is of great interest. Recent advances in 3D cell culture technology have provided new means to study human organ development and various human pathologies in vitro. Cerebral organoids ("mini-brains") are pluripotent stem cell-derived 3D culture systems that recapitulate, to some extent, the developmental processes and organization of the developing human brain. Here, I discuss the application of these neural organoids for modeling brain somatic mosaicism in a lab dish. Special emphasis is given to the potential role of microglial mutations in the pathogenesis of neurodegenerative diseases.

Mosaic APP Gene Recombination in Alzheimer's Disease-What's Next?

A first example of somatic gene recombination (SGR) within the human brain was recently reported, involving the well-known Alzheimer's disease (AD)-related gene amyloid precursor protein (APP). SGR was characterized by the creation of APP genomic complementary DNA (gencDNA) sequences that were identified in prefrontal cortical neurons from both normal and sporadic Alzheimer's disease (SAD) brains. Notably, SGR in SAD appeared to become dysregulated, producing many more numbers and forms of APP gencDNAs, including 11 single-nucleotide variations (SNVs) that are considered pathogenic APP mutations when they occur in families, yet are present mosaically among SAD neurons. APP gene transcription, reverse transcriptase (RT) activity, and DNA strand-breaks were shown to be three key factors required for APP gencDNA production. Many mechanistic details remain to be determined, particularly how APP gencDNAs are involved in AD initiation and progression. The possibility of reducing disease-related SGR through the use of RT inhibitors that are already FDA-approved for HIV and Hepatitis B treatment represents both a testable hypothesis for AD clinical trials and a genuine therapeutic option, where none currently exists, for AD patients.

Synthesis and Characterization of Specific Reverse Transcriptase Inhibitors for Mammalian LINE-1 Retrotransposons.

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.

Genomic mosaicism: A neglected factor that promotes variability in asthma diagnosis.

To elucidate the genetic architecture of asthma continues to be a challenge for molecular biologists and medical researchers. However, powerful genomic technologies are at disposal to help decipher complete human genomes; the genetic variability in asthma hinders the discovery of common molecular markers for this disease. In this context, we purpose to explore genomic mosaicism on asthma cells' biology as a strategy to discover key mechanisms, which can complement or re-define asthma diagnosis. Recent evidences showed that genomic mosaicism could be a normal event. In brains, each neuron may harbor hundreds of genetic alterations, which may contribute to neuronal diversity. Thus, can mosaicism be a natural motor of diversity in asthma? Why this genetic event is little described in scientific literature? To discuss these questions, we perform a critical review about the normality of genomic mosaicism; moreover, we examine the difficulty of current experimental approaches to detect different genotypes in cell populations of one individual.

Submegabase copy number variations arise during cerebral cortical neurogenesis as revealed by single-cell whole-genome sequencing.

Somatic copy number variations (CNVs) exist in the brain, but their genesis, prevalence, forms, and biological impact remain unclear, even within experimentally tractable animal models. We combined a transposase-based amplification (TbA) methodology for single-cell whole-genome sequencing with a bioinformatic approach for filtering unreliable CNVs (FUnC), developed from machine learning trained on lymphocyte V(D)J recombination. TbA-FUnC offered superior genomic coverage and removed >90% of false-positive CNV calls, allowing extensive examination of submegabase CNVs from over 500 cells throughout the neurogenic period of cerebral cortical development in Mus musculus Thousands of previously undocumented CNVs were identified. Half were less than 1 Mb in size, with deletions 4× more common than amplification events, and were randomly distributed throughout the genome. However, CNV prevalence during embryonic cortical development was nonrandom, peaking at midneurogenesis with levels triple those found at younger ages before falling to intermediate quantities. These data identify pervasive small and large CNVs as early contributors to neural genomic mosaicism, producing genomically diverse cellular building blocks that form the highly organized, mature brain.

Genomic mosaicism in the developing and adult brain.

Since the discovery of DNA, the normal developing and functioning brain has been assumed to be composed of cells with identical genomes, which remains the dominant view even today. However, this pervasive assumption is incorrect, as proven by increasing numbers of reports within the last 20 years that have identified multiple forms of somatically produced genomic mosaicism (GM), wherein brain cells-especially neurons-from a single individual show diverse alterations in DNA, distinct from the germline. Critically, these changes alter the actual DNA nucleotide sequences-in contrast to epigenetic mechanisms-and almost certainly contribute to the remarkably diverse phenotypes of single brain cells, including single-cell transcriptomic profiles. Here, we review the history of GM within the normal brain, including its major forms, initiating mechanisms, and possible functions. GM forms include aneuploidies and aneusomies, smaller copy number variations (CNVs), long interspersed nuclear element type 1 (LINE1) repeat elements, and single nucleotide variations (SNVs), as well as DNA content variation (DCV) that reflects all forms of GM with greatest coverage of large, brain cell populations. In addition, technical considerations are examined, along with relationships among GM forms and multiple brain diseases. GM affecting genes and loci within the brain contrast with current neural discovery approaches that rely on sequencing nonbrain DNA (e.g., genome-wide association studies (GWAS)). Increasing knowledge of neural GM has implications for mechanisms of development, diversity, and function, as well as understanding diseases, particularly considering the overwhelming prevalence of sporadic brain diseases that are unlinked to germline mutations. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol, 2018.

Regional mosaic genomic heterogeneity in the elderly and in Alzheimer's disease as a correlate of neuronal vulnerability.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by fibrillary aggregates of Aß peptide and tau protein. The distribution of these pathological hallmarks throughout the brain is not random; it follows a predictive pattern that is used for pathological staging. However, most etiopathogenetic concepts, irrespective of whether they focus on Aß or tau pathology, leave a key question unanswered: what is the explanation for the different vulnerabilities of brain regions in AD? The pattern of regional progression of neurofibrillary degeneration in AD to some extent inversely recapitulates ontogenetic and phylogenetic brain development. Accordingly, degeneration preferentially affects brain areas that have recently been acquired or restructured during anthropoid evolution, which means that the involvement of a neurodevelopmental mechanism is highly likely. Since evolutionary expansion of the neocortex is based on a substantial extension of the mitotic activity of progenitor cells, we propose a conceptual link between neurogenesis in anthropoid primates and a higher risk of accumulating mitotic errors that give rise to genomic aberrations commonly referred to as DNA content variation (DCV). If increased rates of DCV make neurons more vulnerable to AD-related pathology, one might expect there to be a higher rate of DCV in areas that are affected very early during the course of AD, as compared to areas which are hardly affected or are affected only during the most advanced stages. Therefore, in the present study, we comparatively analyzed the DCV in five different cortical areas that are affected during the early stage (entorhinal cortex), the intermediate stage (temporal, frontal, and parietal association cortex), and the late stage (primary sensory occipital cortex) of AD in both normal elderly subjects and AD patients. On average, we observed about 10 % neuronal mosaic DCV in the normal elderly and a two- to threefold increase in DCV in AD patients. We were able to demonstrate, moreover, that the neuronal DCV in the cerebral cortex of the normal elderly as well as the increased neuronal DCV in AD patients are not randomly distributed but instead show systematic regional differences which correspond to differences in vulnerability. These findings provide additional evidence that mosaic genomic heterogeneity may play a key role in AD pathology.

Cohesion and the aneuploid phenotype in Alzheimer's disease: A tale of genome instability.

Neurons are postmitotic cells that are in permanent cell cycle arrest. However, components of the cell cycle machinery that are expressed in Alzheimer's disease (AD) neurons are showing features of a cycling cell and those attributed to a postmitotic cell as well. Furthermore, the unique physiological operations taking place in neurons, ascribed to "core cell cycle regulators" are also key regulators in cell division. Functions of these cell cycle regulators include neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. In this review, we focus on cohesion and cohesion related proteins in reference to their neuronal functions and how impaired centromere/cohesion dynamics may connect cell cycle dysfunction to aneuploidy in AD.