Biology subjects

Vaccarino, F. M.

Publications and source records attributed to Vaccarino, F. M..

17 recordsLinked to original sources

bioRxiv2026

Somatic mutation landscape revealed by non-invasive iPSC derivation from urine cells

Somatic mutations that arise post-zygotically create genetic diversity among normal human cells and provide key insights into human development and aging. Fibroblast-derived induced pluripotent stem cells (iPSCs) have proved to be a useful system for disease modelling; however, due to their clonal nature, iPSC lines carry somatic mutations inherited from the founder cells, raising concerns about their genomic integrity. At the same time, this clonality enables single-cell-level discovery of somatic mutations and the reconstruction of developmental lineages. In living individuals, though, this approach requires invasive biopsies and is limited to skin-derived lineages. Here, we generated 33 urine-derived iPSC lines from four males representing two father- son relationships, performed shallow whole-genome sequencing of the lines and analyzed somatic mutations. Derived iPSCs representing single cells from urine carried a few hundred of somatic single-nucleotide variants per genome, dominated by endogenous, clock-like mutational signatures and lacking environmental imprints such as UV-associated mutations. Copy-number analysis identified somatic CNVs in most of the lines and revealed higher CNV burdens in fathers than in sons, consistent with age-related structural mosaicism. Shared mutations across lines enabled reconstruction of cell lineage phylogenetic trees. In summary, urine-derived iPSCs showed genomic alterations comparable to those in fibroblast-derived iPSC lines and represent a valuable non-invasive alternative for disease modeling. Overall, this study provides the first genome-wide characterization of somatic mutations in urine-derived iPSCs and establishes them as a practical and non-invasive platform for charting somatic mutation landscapes and tracing developmental lineages in living humans.

genomics↗

bioRxiv2025

A comprehensive view of somatic mosaicism by single-cell DNA analysis

Single-cell DNA sequencing offers a powerful means of studying somatic mosaicism but requires careful analysis to mitigate DNA amplification-related artifacts. We performed primary template-directed amplification (PTA) and sequencing of 102 nuclei from postmortem lung and colon tissues of a 74-year-old male. Single-cell mutation burdens and spectra were validated by duplex sequencing and revealed heterogeneity across organs and cells, including signatures of APOBEC activity and tobacco exposure. Cells from both tissues exhibited chromosomal aneuploidies, loss of chromosome Y, and chromosomal rearrangements including rearrangements of the T-cell receptor loci indicative of T-cells. Shared embryonic mutations between cells enabled reconstruction of cellular ancestries from the zygote, which were validated by bulk sequencing. Collectively, we demonstrate a comprehensive approach for single-cell genomics that yields an expansive view of diverse somatic mutation types from development through aging across diverse tissues--insights that are obscured in bulk sequencing and only partially captured by other single-cell methods.

genomics↗

bioRxiv2025

Single cell whole genome and transcriptome sequencing links somatic mutations to cell identity and ancestry

The role of somatic mutations in human development and disease is obscured by difficulties in characterizing mutations at the single cell level and identifying cell types carrying them. Here we analysed somatic genomes of clonal iPSC lines and of single-cells after whole-genome amplification (scWGA) by PTA and ResolveOme from skin fibroblasts, blood and urine of a live donor. Mutation burden and spectra converged across approaches, revealing heterogeneous mutational footprints across cells driven by environmental exposures (UV damage and chemotherapy) and lymphocyte differentiation. Aneuploidies in single cells were detected by all the approaches and were orthogonally validated by Strand-seq. Uniquely, ResolveOme enabled cell-type identification using single-cell transcriptomes. Using a newly developed method accounting for noise and allele drop-out in scWGA, we de novo reconstructed the cell phylogenetic tree for this donor. Together, scWGA establishes a powerful foundation for comprehensive, cell type-aware, lineage-aware profiling of somatic mutations at single cell level.

genomics↗

bioRxiv2024

Transgenerational transmission of post-zygotic mutations suggests symmetric contribution of first two blastomeres to human germline

Little is known about the origin of germ cells in humans. We previously leveraged post-zygotic mutations to reconstruct zygote-rooted cell lineage ancestry trees in a phenotypically normal woman, termed NC0. Here, by sequencing the genome of her children and their father, we analyzed the transmission of early pre-gastrulation lineages and corresponding mutations across human generations. We found that the germline in NC0 is polyclonal and is founded by at least two cells likely descending from the two blastomeres arising from the first zygotic cleavage. Analyses of public data from several multi-children families and from 1,934 familial quads confirmed this finding in larger cohorts, revealing that known imbalances of up to 90:10 in early lineages allocation in somatic tissues are not reflected in transmission to offspring, establishing a fundamental difference in lineage allocation between the soma and the germline. Analyses of all the data consistently suggest that germline has a balanced 50:50 lineage allocation from the first two blastomeres.

genetics↗

bioRxiv2024

Early Developmental Origins of Cortical Disorders Modeled in Human Neural Stem Cells

The implications of the early phases of human telencephalic development, involving neural stem cells (NSCs), in the etiology of cortical disorders remain elusive. Here, we explored the expression dynamics of cortical and neuropsychiatric disorder-associated genes in datasets generated from human NSCs across telencephalic fate transitions in vitro and in vivo. We identified risk genes expressed in brain organizers and sequential gene regulatory networks across corticogenesis revealing disease-specific critical phases, when NSCs are more vulnerable to gene dysfunctions, and converging signaling across multiple diseases. Moreover, we simulated the impact of risk transcription factor (TF) depletions on different neural cell types spanning the developing human neocortex and observed a spatiotemporal-dependent effect for each perturbation. Finally, single-cell transcriptomics of newly generated autism-affected patient-derived NSCs in vitro revealed recurrent alterations of TFs orchestrating brain patterning and NSC lineage commitment. This work opens new perspectives to explore human brain dysfunctions at the early phases of development. One-sentence summaryThe temporal analysis of gene regulatory networks in human neural stem cells reveals multiple early critical phases associated with cortical disorders and neuropsychiatric traits.

neuroscience↗

bioRxiv2024

Specification of human regional brain lineages using orthogonal gradients of WNT and SHH in organoids

The repertory of neurons generated by progenitor cells depends on their location along antero-posterior and dorso-ventral axes of the neural tube. To understand if recreating those axes was sufficient to specify human brain neuronal diversity, we designed a mesofluidic device termed Duo-MAPS to expose induced pluripotent stem cells (iPSC) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison of single cell transcriptomes with fetal human brain revealed that Duo-MAPS-patterned organoids generated the major neuronal lineages of the forebrain, midbrain, and hindbrain. Morphogens crosstalk translated into early patterns of gene expression programs predicting the generation of specific brain lineages. Human iPSC lines from six different genetic backgrounds showed substantial differences in response to morphogens, suggesting that interindividual genomic and epigenomic variations could impact brain lineages formation. Morphogen gradients promise to be a key approach to model the brain in its entirety.

neuroscience↗

bioRxiv2024

Interneuron loss and microglia activation by transcriptome analyses in the basal ganglia of Tourette syndrome

Tourette syndrome (TS) is a disorder of high-order integration of sensory, motor, and cognitive functions afflicting as many as 1 in 150 children and characterized by motor hyperactivity and tics. Despite high familial recurrence rates, a few risk genes and no biomarkers have emerged as causative or predisposing factors. The syndrome is believed to originate in basal ganglia, where patterns of motor programs are encoded. Postmortem immunocytochemical analyses of brains with severe TS revealed decreases in cholinergic, fast-spiking parvalbumin, and somatostatin interneurons within the striatum (caudate and putamen nuclei). Here, we performed single cell transcriptomic and chromatin accessibility analyses of the caudate nucleus from 6 adult TS and 6 control post-mortem brains. The data reproduced the known cellular composition of the adult human striatum, including a majority of medium spiny neurons (MSN) and small populations of GABAergic and cholinergic interneurons. Comparative analysis revealed that interneurons were decreased by roughly 50% in TS brains, while no difference was observed for other cell types. Differential gene expression analysis suggested that mitochondrial function, and specifically oxidative metabolism, in MSN and synaptic function in interneurons are both impaired in TS subjects. Furthermore, such an impairment was coupled with activation of immune response pathways in microglia. Also, our data explicitly link gene expression changes to changes in cis-regulatory activity in the corresponding cell types, suggesting de-regulation as a factor for the etiology of TS. These findings expand on previous research and suggest that impaired modulation of striatal function by interneurons may be the origin of TS symptoms.

neuroscience↗

bioRxiv2023

Enhancer-driven regulatory network of forebrain human development provides insights into autism

Cell differentiation is orchestrated by transcription factors (TFs) binding to enhancers, shaping gene regulatory networks that drive neuronal lineage specification. Deciphering these enhancer-driven networks in human forebrain development is essential for understanding the genetic basis of neurodevelopmental disorders. Through integrative epigenomic and transcriptomic analyses of human forebrain organoids derived from 10 individuals with autism spectrum disorder (ASD) and their neurotypical fathers, we constructed a comprehensive enhancer-driven gene regulatory network (GRN) of early neurodevelopment. This GRN revealed hierarchical regulatory transitions guiding neuronal differentiation and was experimentally validated via CRISPR interference (CRISPRi) and loss-of-function analyses. A subnetwork linked ASD-associated transcriptomic alterations to dysregulated TF activity, implicating FOXG1, BHLHE22, EOMES, and NEUROD2 as key regulators of excitatory neuron specification in macrocephalic ASD. These findings suggest that ASD disrupts enhancer-driven regulatory frameworks, altering neuronal cell fate decisions in the developing fetal brain.

genomics↗

bioRxiv2023

Characterization of enhancer activity in early human neurodevelopment using Massively parallel reporter assay (MPRA) and forebrain organoids

AO_SCPLOWBSTRACTC_SCPLOWRegulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of [~]7,000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of [~]35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community. AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWEnhancers are non-coding elements that play a crucial role in the regulation of gene expression during brain development. Despite the availability of various techniques available to identify enhancers, their functional activity is relatively less understood, leaving a gap in our understanding of how enhancer behavior might regulate complex transitions of neurodevelopment. To address this, we utilized forebrain organoids, a 3D model system which closely mimics the complex cellular environment of the developing human brain, and employed Massively Parallel Reporter Assay (MPRA) to validate enhancer activity at various stages of forebrain differentiation, from induced pluripotent stem cells (iPSCs) to neuronal progenitors and cortical neurons. Our study provides a comprehensive catalog of over 2,300 enhancers, showcasing their temporal activity profiles during early neuronal development and offering valuable insights into their likely biological functions. This research advances our understanding of enhancer dynamics in brain development and offers new avenues for further investigations in this field.

genomics↗

bioRxiv2023

Evaluating performance and applications of sample-wise cell deconvolution methods on human brain transcriptomic data

Sample-wise deconvolution methods have been developed to estimate cell-type proportions and gene expressions in bulk-tissue samples. However, the performance of these methods and their biological applications has not been evaluated, particularly on human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk-tissue RNAseq, single-cell/nuclei (sc/sn) RNAseq, and immunohistochemistry. A total of 1,130,767 nuclei/cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expression. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk-tissue or single-cell eQTLs alone. Differential gene expression associated with multiple phenotypes were also examined using the deconvoluted data. Our findings, which were replicated in bulk-tissue RNAseq and sc/snRNAseq data, provided new insights into the biological applications of deconvoluted data.

genomics↗

bioRxiv2023

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development. One Sentence SummaryWe identify 46,802 enhancers and 164 psychiatric disorder variants with regulatory effects in the developing cortex and organoids.

genomics↗

bioRxiv2022

Clonally selected lines after CRISPR/Cas editing are not isogenic

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (i) editing cultured cells; (ii) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, while cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, while detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and copy number alterations acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

genomics↗

bioRxiv2022

ASD modelling in organoids reveals imbalance of excitatory cortical neuron subtypes during early neurogenesis

There is no clear genetic etiology or convergent pathophysiology for autism spectrum disorders (ASD). Using cortical organoids and single-cell transcriptomics, we modeled alterations in the formation of the forebrain between sons with idiopathic ASD and their unaffected fathers in thirteen families. Alterations in the transcriptome suggest that ASD pathogenesis in macrocephalic and normocephalic probands involves an opposite disruption of the balance between the excitatory neurons of the dorsal cortical plate and other lineages such as the early-generated neurons from the putative preplate. The imbalance stemmed from a divergent expression of transcription factors driving cell fate during early cortical development. While we did not find probands genomic variants explaining the observed transcriptomic alterations, a significant overlap between altered transcripts and reported ASD risk genes affected by rare variants suggests a degree of gene convergence between rare forms of ASD and developmental transcriptome in idiopathic ASD.

neuroscience↗

bioRxiv2021

All2: A tool for selecting mosaic mutations from comprehensive multi-cell comparisons

Accurate discovery of somatic mutations in a cell is a challenge that partially lays in immaturity of dedicated analytical approaches. Approaches comparing cells genome to a control bulk sample miss common mutations, while approaches to find such mutations from bulk suffer from low sensitivity. We developed a tool, All2, which enables accurate filtering of mutations in a cell from exhaustive comparison of cells genomes to each other without data for bulk(s). Based on all pair-wise comparisons, every variant call (point mutation, indel, and structural variant) is classified as either a germline variant, mosaic mutation, or false positive. As All2 allows for considering dropped-out regions, it is applicable to whole genome and exome analysis of cloned and amplified cells. By applying the approach to a variety of available data, we showed that its application reduces false positives, enables sensitive discovery of high frequency mutations, and is indispensable for conducting high resolution cell lineage tracing. All2 is freely available at https://github.com/abyzovlab/All2.

bioinformatics↗

bioRxiv2020

Comprehensive identification of somatic nucleotide variants in human brain tissue

Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. Here, the Brain Somatic Mosaicism Network conducted a coordinated, multi-institutional study to: (i) examine the ability of existing methods to detect simulated somatic single nucleotide variants (SNVs) in DNA mixing experiments; (ii) generate multiple replicates of whole genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual; (iii) devise strategies to discover somatic SNVs; and (iv) apply various approaches to validate somatic SNVs. These efforts led to the identification of 43 bona fide somatic SNVs that ranged in variant allele fractions from ~0.005 to ~0.28. Guided by these results, we devised best practices for calling mosaic SNVs from 250X whole genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrated that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. Thus, this study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

genomics↗

bioRxiv2020

Early developmental asymmetries in cell lineage trees in living individuals

Post-zygotic mosaic mutations can be used to track cell lineages in humans. By using cell cloning and induced pluripotent cell lines, we analyzed early cell lineages in two living individuals (a patient and a control), and a postmortem human specimen. Of ten reconstructed post-zygotic divisions, none resulted in balanced contributions of daughter lineages to tissues. In both living individuals one of two lineages from the first cleavage was dominant across tissues, with 90% frequency in blood. We propose that the efficiency of DNA repair contributes to lineage imbalance. Allocation of lineages in postmortem brain correlated with anterior-posterior axis, associating lineage history with cell fate choices in embryos. Recurrence of germline variants as mosaic suggested that certain loci may be particularly susceptible to mutagenesis. We establish a minimally invasive framework for defining cell lineages in any living individual, which paves the way for studying their relevance in health and disease.

genomics↗

bioRxiv2019

Machine learning reveals bilateral distribution of somatic L1 insertions in human neurons and glia

Active retrotransposons in the human genome (L1, Alu and SVA elements) can create genomic mobile element insertions (MEIs) in both germline and somatic tissue1. Specific somatic MEIs have been detected at high levels in human cancers2, and at lower to medium levels in human brains3. Dysregulation of somatic retrotransposition in the human brain has been hypothesized to contribute to neuropsychiatric diseases4, 5. However, individual somatic MEIs are present in small proportions of cells at a given anatomical location, and thus standard whole-genome sequencing (WGS) presents a difficult signal-to-noise problem, while single-cell approaches suffer from limited scalability and experimental artifacts introduced by enzymatic whole-genome amplification6. Previous studies produced widely differing estimates for the somatic retrotransposition rates in human brain3, 6-8. Here, we present a highly precise machine learning method (RetroSom) to directly identify somatic L1 and Alu insertions in <1% cells from 200x deep WGS, which allows circumventing the restrictions of whole-genome amplification. Using RetroSom we confirmed a lower rate of retrotransposition for individual somatic L1 insertions in human neurons. We discovered that anatomical distribution of somatic L1 insertion is as widespread in glia as in neurons, and across both hemispheres of the brain, indicating retrotransposition occurs during early embryogenesis. We characterized two of the detected brain-specific L1 insertions in great detail in neurons and glia from a donor with schizophrenia. Both insertions are within introns of genes active in brain (CNNM2, FRMD4A) in regions with multiple genetic associations with neuropsychiatric disorders9-11. Gene expression was significantly reduced by both somatic insertions in a reporter assay. Our results provide novel insights into the potential for pathological effects of somatic retrotransposition in the human brain, now including the large glial fraction. RetroSom has broad applicability in all disease states where somatic retrotransposition is expected to play a role, such as autoimmune disorders and cancer.

bioinformatics↗