Biology subjects

Lucente, D.

Publications and source records attributed to Lucente, D..

11 recordsLinked to original sources

bioRxiv2025

CRISPR-engineered deletion of POGZ alters transcription factor binding at promoters of genes involved in synaptic signaling

One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDD) has been that loss-of-function (LoF) mutations in many genes that impact chromatin and transcriptional regulation confer substantial liability to NDD. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the direct or indirect regulatory targets of POGZ, or the mechanisms by which loss of this chromatin modifier alters early neuronal development and synaptic functions. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and biallelic POGZ deletions. In hiPSC-derived neural stem cells (NSC) and Neurogenin 2-induced neurons (iN), POGZ LoF altered the expression of genes associated with critical cellular processes and neuronal functions, including synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes in neuronal development associated with NDD and POGZ regulation, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iN. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with biallelic deletions, but not heterozygous lines, suggesting subtle effects in neuronal development associated with haploinsufficiency. Overall, these shared molecular consequences suggest key points of convergence that connect epigenetic regulation to neuronal function in the etiology of neurodevelopmental pathologies.

genomics↗

bioRxiv2025

Huntington's disease LIG1 modifier variant increases ligase fidelity and suppresses somatic CAG repeat expansion

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by inheriting an expanded CAG repeat tract in the huntingtin gene (HTT) that further expands in somatic cells over an individuals lifetime. Genome-wide association studies have provided critical insight into factors that modify the course of disease. These include DNA repair genes that alter the rate of somatic expansion and other genes that do not appear to directly influence this process. One modifier gene is DNA ligase 1 (LIG1), in which a variant specifying a lysine to asparagine substitution (K845N) is associated with a profound (7-8 year) delay in the onset of motor signs. Here, we have taken a multifaceted approach to gain insight into the protective nature of this variant in HD. We demonstrate using in vitro ligase assays and enzyme kinetics that K845N enhances discrimination towards mismatched substrates and increases repair fidelity. Consistent with increased ligation fidelity, K845N confers protection against oxidative stress in cell-based assays. Finally, we demonstrate that the mouse LIG1 K843N orthologue suppresses somatic CAG expansion in HD knock-in mice. Overall, our data provide evidence that altered LIG1 function due to the K845N substitution may contribute to HD clinical delay by slowing somatic expansion in the brain and protecting the genome globally against damage. Significantly, our results provide a mechanistic foundation for considering DNA ligase fidelity as a therapeutic target in HD and potentially in other trinucleotide repeat disorders. Significance StatementWe analyzed a missense variant in DNA Ligase 1 (K845N) that is associated with a profound delay in the onset of Huntingtons disease (HD). We find that K845N enhances substrate discrimination towards mismatched substrates, thus increasing repair fidelity, conferring protection against oxidative stress and slows somatic expansion of the HD CAG repeat. Our observations provide insight into underlying mechanisms of disease modification and suggest avenues that can be harnessed for disease-modifying therapeutic intervention. Classification: Biological Sciences, Genetics

genetics↗

bioRxiv2024

Genetic modifiers of somatic expansion and clinical phenotypes in Huntington's disease reveal shared and tissue-specific effects

Huntingtons disease (HD), due to expansion of a CAG repeat in HTT, is representative of a growing number of disorders involving somatically unstable short tandem repeats. We find that overlapping and distinct genetic modifiers of clinical landmarks and somatic expansion in blood DNA reveal an underlying complexity and cell-type specificity to the mismatch repair-related processes that influence disease timing. Differential capture of non-DNA-repair gene modifiers by multiple measures of cognitive and motor dysfunction argues additionally for cell-type specificity of pathogenic processes. Beyond trans modifiers, differential effects are also illustrated at HTT by a 5-UTR variant that promotes somatic expansion in blood without influencing clinical HD, while, even after correcting for uninterrupted CAG length, a synonymous sequence change at the end of the CAG repeat dramatically hastens onset of motor signs without increasing somatic expansion. Our findings are directly relevant to therapeutic suppression of somatic expansion in HD and related disorders and provide a route to define the individual neuronal cell types that contribute to different HD clinical phenotypes.

genetics↗

bioRxiv2023

Resolution of ring chromosomes, Robertsonian translocations, and complex structural variants from long-read sequencing and telomere-to-telomere assembly

The capacity to resolve structural variants (SVs) at sequence resolution in highly repetitive genomic regions has long been intractable. Consequently, the properties, origins, and functional effects of multiple classes of complex rearrangement are unknown. To resolve these challenges, we leveraged recent technical milestones: 1) Oxford-Nanopore (ONT) sequencing; 2) the gapless Telomere-to-Telomere (T2T) genome assembly; and 3) a novel tool to discover large-scale rearrangements from long-reads. We applied these technologies across 13 patients with ring chromosomes, Robertsonian translocations, and complex balanced SVs that were unresolved by short-read sequencing. We resolved 10 of 13 events, including ring chromosomes, the complex SVs, and a Robertsonian translocation. Multiple breakpoints were localized to highly repetitive regions inaccessible to short-read alignment, such as acrocentric p-arms, ribosomal DNA arrays, and telomeric repeats, and involved complex structures such as a deletion-inversion and interchromosomal dispersed duplications. We also leveraged ONT native methylation detection to discover phased differential methylation in a gene promoter proximal to a ring fusion site, suggesting a long-range positional effect with heterochromatin spreading. Breakpoint sequences were consistent with common mechanisms of SV formation, including microhomology-mediated mechanisms, non-homologous end-joining, and non-allelic homologous recombination. These methods provide some of the first glimpses into the sequence resolution of ring chromosomes and Robertsonian translocations and illuminate the structural diversity of chromosomal rearrangements with implications for molecular diagnosis and genome biology. HighlightsO_LICryptic rearrangements revealed in repetitive and previously inaccessible regions C_LIO_LIAligning long-reads to telomere-to-telomere assembly enabled breakpoint discovery C_LIO_LIFirst sequence resolution of a Robertsonian translocation breakpoint C_LIO_LIHaplotype-specific methylation changes associated with ring formation C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/555775v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@188cef8org.highwire.dtl.DTLVardef@cc2ed5org.highwire.dtl.DTLVardef@116b7e6org.highwire.dtl.DTLVardef@837f73_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1, a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1, making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.

genetics↗

bioRxiv2022

Tissue and cell-type specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models

Recurrent deletion and duplication of [~]743 kilobases of unique genomic sequence and segmental duplications at chromosome 16p11.2 underlie a reciprocal genomic disorder (RGD; OMIM 611913 and 614671) associated with neurodevelopmental and psychiatric phenotypes, including intellectual disability, autism spectrum disorder (ASD), and schizophrenia (SCZ). To define molecular alterations associated with the 16p11.2 RGD, we performed transcriptome analyses of mice with reciprocal copy number variants (CNVs) of the syntenic chromosome 7qF3 region and human neuronal models derived from isogenic human induced pluripotent stem cells (hiPSCs) carrying CRISPR-engineered CNVs at 16p11.2. Analysis of differentially expressed genes (DEGs) in mouse cortex, striatum, cerebellum and three non-brain tissues, as well as in human neural stem cells and induced glutamatergic neurons revealed that the strongest and most consistent effects occurred within the CNV sequence, with notable instances of differential expression of genes in the immediate vicinity that could reflect position effect. While differential expression of genes outside of chromosome 16p11.2 was largely region, tissue, and cell type-specific, a small but significant minority of such DEGs was shared between brain regions or human cell types. Gene Ontology (GO) enrichment analyses to identify cellular processes dysregulated due to these CNVs found support in select circumstances for terms related to energy metabolism, RNA metabolism, and translation but did not reveal a single universally affected process. Weighted gene co-expression network analysis identified modules that showed significant correlation with reciprocal or individual CNV genotype and better captured shared effects, indicating that energy metabolism, RNA metabolism, translation and protein targeting were disrupted across all three brain regions. The first two of these processes also emerged in the human neural stem cell (NSC) data. A subset of co-expression modules that correlated with CNV genotype revealed significant enrichments for known neurodevelopmental disorder genes, loss-of-function constrained genes, FMRP targets, and chromatin modifiers. Intriguingly, neuronal differentiation of the hiPSCs revealed that both the deletion and duplication CNV resulted in similar deficits in neurite extension and branching and alterations in electrical activity. Finally, generation of cerebral organoid derivatives indicated that the CNVs reciprocally altered the ratio of excitatory and inhibitory GABAergic neurons generated during in vitro neurodevelopment, consistent with a major mechanistic hypothesis for ASD. Collectively, our data suggest that the 16p11.2 RGD involves disruption of multiple biological processes, with a relative impact that is context-specific. Perturbation of individual and multiple genes within the CNV region will be required to dissect single-gene effects, uncover regulatory interactions, and define how each contributes to abnormal neurodevelopment.

genomics↗

bioRxiv2022

Transcriptional and functional consequences of alterations to MEF2C and its topological organization in neuronal models

Point mutations and structural variants directly disrupting the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs), while recent studies have also implicated altered noncoding regulation of MEF2C expression in NDDs. However, the impact of haploinsufficiency of MEF2C on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern regulation of MEF2C. To explore the transcriptional and functional changes associated with coding and noncoding structural variants, we generated an allelic series of 204 isogenic iPSC-derived neuronal cell lines harboring CRISPR-engineered mutations that directly delete predominant isoforms of MEF2C, as well as deletions to the boundaries of topologically associating domains (TADs) and chromatin loops encompassing MEF2C. We then performed systematic profiling of mutation-specific alterations to transcriptional signatures, regulatory interactions, chromatin contacts, and electrophysiological effects. Our analyses reveal that direct deletion of MEF2C causes differential expression of genes enriched for neurodevelopmental and synaptic-associated pathways, accompanied by a significant reduction in synaptic firing and synchrony in neurons. By contrast, we observe robust buffering against MEF2C regulatory disruption upon deletion of a distal 5q14.3 TAD and loop boundary; however, homozygous loss of proximal loop boundary resulted in significant down-regulation of MEF2C expression and significantly reduced electrophysiological activity that was comparable to direct MEF2C disruption. Collectively, our findings demonstrate the functional impact of MEF2C haploinsufficiency in human-derived neural models and highlight the complex interactions of gene regulation and chromatin topology that challenge a priori regulatory predictions of structural variant disruption to three-dimensional genome organization.

genomics↗

bioRxiv2021

FAN1 nuclease activity affects CAG expansion and age at onset of Huntington's disease

The age at onset of motor symptoms in Huntingtons disease (HD) is driven by HTT CAG repeat length but modified by other genes. We used exome sequencing of 683 HD patients with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We identified damaging coding variants in candidate modifier genes from prior genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier onset HD. Nuclease activities of these variants correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion comparable to those observed with complete FAN1 knock out. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.

neuroscience↗

bioRxiv2020

Systematic evaluation of genome sequencing as a first-tier diagnostic test for prenatal and pediatric disorders

Current clinical guidelines recommend three genetic tests for the assessment of fetal structural anomalies: karyotype to detect microscopically-visible balanced and unbalanced chromosomal rearrangements, chromosomal microarray (CMA) to detect sub-microscopic copy number variants (CNVs), and exome sequencing (ES) to identify individual nucleotide changes in coding sequence. Advances in genome sequencing (GS) analysis suggest that it is poised to displace the sequential application of all three conventional tests to become a single diagnostic approach for the assessment of fetal structural anomalies. However, systematic benchmarking is required to assure that GS can capture the full mutational spectrum associated with fetal structural anomalies and to accurately quantify the added diagnostic yield of GS. We applied a novel GS analytic framework that included the discovery, filtration, and interpretation of nine classes of genomic variation to 7,195 individuals. We assessed the sensitivity of GS to detect diagnostic variants (pathogenic or likely pathogenic) from three standard-of-care tests using 1,612 autism spectrum disorder quartet families (ASD; n=6,448) with matched GS, ES, and CMA data, and validated these findings in 46 fetuses with a clinically reportable variant originally identified by karyotype, CMA, or ES. We then assessed the added diagnostic yield of GS in 249 trios (n=747) comprising a fetus with a structural anomaly detected by ultrasound and two unaffected parents that were pre-screened with a combination of all three standard-of-care tests. Across both cohorts, our GS analytic framework identified 98.2% of all diagnostic variants detected by standard-of-care tests, including 100% of those originally detected by CMA (n=88) and ES (n=61), as well as 78.6% (n=11/14) of the chromosomal rearrangements identified by karyotype. The diagnostic yield from GS was 7.8% across all 1,612 ASD probands, almost two-fold more than CMA (4.4%) and three-fold more than ES (3.0%). We also demonstrated that the yield of ES can approach that of GS when CNVs are captured with high sensitivity from exome data (7.4% vs. 7.8%, respectively). In 249 pre-screened fetuses with structural anomalies, GS provided an additional diagnostic yield of 0.4% beyond the combination of all three tests (karyotype, CMA, and ES). Applying our benchmarking results to existing data indicates that GS can achieve an overall diagnostic yield of 46.1% in unselected fetuses with fetal structural anomalies, providing an estimated 17.2% increase in diagnostic yield over karyotype, 14.1% over CMA, and 36.1% over ES when sequence variants are assessed, and 4.1% when CNVs are also identified from exome data. In this study we demonstrate that GS is sensitive to the detection of almost all pathogenic variation captured by karyotype, CMA, and ES, provides a superior diagnostic yield than any individual test by a wide margin, and contributes a modest increase in diagnostic yield beyond the combination of all three tests. We also outline several strategies to aid the interpretation of GS variants that are cryptic to conventional technologies, which we anticipate will be increasingly encountered as comprehensive variant identification from GS is performed. Taken together, these data suggest GS warrants consideration as a first-tier diagnostic approach for fetal structural anomalies.

genomics↗

bioRxiv2019

Genetic overlap between psychiatric disorders and neuropsychiatric symptoms in HD

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene. It is diagnosed following a standardized exam of motor control and often presents with cognitive decline and psychiatric symptoms. Recent studies have detected genetic loci modifying the age at onset of motor symptoms in HD, but genetic factors influencing cognitive and psychiatric presentations are unknown. We tested the hypothesis that psychiatric and cognitive symptoms in HD are influenced by the same common genetic variation as in the general population by constructing polygenic risk scores from large genome-wide association studies of psychiatric and neurodegenerative disorders, and of intelligence, and testing for correlation with the presence of psychiatric and cognitive symptoms in a large sample (n=5160) of HD patients. Polygenic risk score for major depression was associated specifically with increased risk of depression in HD, as was schizophrenia risk score with psychosis and irritability. Cognitive impairment and apathy were associated with reduced polygenic risk score for intelligence. In general, polygenic risk scores for psychiatric disorders, particularly depression and schizophrenia, are associated with increased risk of the corresponding psychiatric symptoms in HD, suggesting a common genetic liability. However, the genetic liability to cognitive impairment and apathy appears to be distinct from other psychiatric symptoms in HD. No associations were observed between HD symptoms and risk scores for other neurodegenerative disorders. These data provide a rationale for treatments effective in depression and schizophrenia to be used to treat depression and psychotic symptoms in HD.

genetics↗

bioRxiv2019

Huntington's disease onset is determined by length of uninterrupted CAG, not encoded polyglutamine, and is modified by DNA maintenance mechanisms

The effects of variable, glutamine-encoding, CAA interruptions indicate that a property of the uninterrupted HTT CAG repeat sequence, distinct from huntingtins polyglutamine segment, dictates the rate at which HD develops. The timing of onset shows no significant association with HTT cis-eQTLs but is influenced, sometimes in a sex-specific manner, by polymorphic variation at multiple DNA maintenance genes, suggesting that the special onset-determining property of the uninterrupted CAG repeat is a propensity for length instability that leads to its somatic expansion. Additional naturally-occurring genetic modifier loci, defined by GWAS, may influence HD pathogenesis through other mechanisms. These findings have profound implications for the pathogenesis of HD and other repeat diseases and question a fundamental premise of the "polyglutamine disorders".

genetics↗