Biology subjects

Biar, C. G.

Publications and source records attributed to Biar, C. G..

5 recordsLinked to original sources

bioRxiv2026

An integrated, scaled approach to resolve TSC2 variants of uncertain significance

Obtaining a precise genetic tuberous sclerosis diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance (VUS). VUS in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3,634 TSC2 missense VUS observed so far. To address this challenge, we used massively parallel sequencing to measure the steady-state abundance of almost 9,000 TSC2 missense variants and developed an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. 1,288 of 8,891 (14.49%) missense variants assayed had altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed had altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassified 212 of 276 (76.8%) TSC2 missense VUS. These datasets will lead to improved genetic diagnosis of tuberous sclerosis with potential positive impacts on the clinical management of patients and their families.

genetics↗

bioRxiv2025

Principles and functional consequences of plasmid chromatinization in mammalian cells

Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, although chromatin plays an integral role in regulating the function of regulatory elements along the nuclear genome, our understanding of how, or whether, similar chromatin structures form on plasmids transfected into mammalian cells remains limited. We demonstrate that plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) can accurately map chromatin architectures along individual, full-length transfected plasmid molecules at near single-nucleotide resolution. Application of plasmid Fiber-seq to diverse plasmids and cell lines demonstrates that plasmids are chromatinized in a sequence-dependent organized manner and adopt a heterogeneous and incomplete chromatin architecture relative to nuclear-encoded chromatin fibers. We show that the focal occupancy of nucleosomes and transcription factors along transfected plasmids is central to their transcriptional activity within mammalian cells, and demonstrate that plasmids indeed are capable of recapitulating nuclear genome-encoded chromatin architectures, although not always. Finally, we demonstrate that combining plasmid Fiber-seq with high-throughput reporter assays can establish the molecular mechanisms underlying pathogenic non-coding variants, including disentangling the effects of transcriptional activators and repressors with near-single-nucleotide resolution. Overall, our findings reveal the principles by which plasmid-based assays can be used for accurate fine-scale mapping of chromatin-dependent regulatory grammar.

genomics↗

bioRxiv2025

Analysis of 14q12 microdeletions reveals novel regulatory loci for the neurodevelopmental disorder-related gene, FOXG1

Up to 17% of neurodevelopmental disorders (NDDs) can be explained by pathogenic structural variants (SVs) that disrupt coding regions and elicit gene dosage defects. However, noncoding SVs which can perturb cis-regulatory elements (CREs) and downstream gene expression are understudied. In this study, we describe multiple 14q12 deletions downstream of NDD-related gene FOXG1 in individuals with overlapping phenotypes of FOXG1 haploinsufficiency. We show that deletion of a minimum region of overlap (MRO) reduced FOXG1 expression, disrupted CREs and altered FOXG1s native genomic interactions. Deleting the MRO did not fully eliminate FOXG1 expression, indicating that multiple CREs likely cooperate to regulate FOXG1 and would need to be deleted to completely prevent expression. The transcriptomic profiles of MRO loss overlap in part with FOXG1 loss, including direct FOXG1 targets, indicating converging molecular pathways. These findings expand the scope of FOXG1s complex regulatory region, and more broadly, of regulatory SVs in NDD susceptibility.

genetics↗

bioRxiv2024

Multimodal framework to resolve variants of uncertain significance in TSC2

Efforts to resolve the functional impact of variants of uncertain significance (VUS) have lagged behind the identification of new VUS; as such, there is a critical need for scalable VUS resolution technologies. Computational variant effect predictors (VEPs), once trained, can predict pathogenicity for all missense variants in a gene, set of genes, or the exome. Existing tools have employed information on known pathogenic and benign variants throughout the genome to predict pathogenicity of VUS. We hypothesize that taking a gene-specific approach will improve pathogenicity prediction over globally-trained VEPs. We tested this hypothesis using the gene TSC2, whose loss of function results in tuberous sclerosis, a multisystem mTORopathy affecting about 1 in 6,000 individuals born in the United States. TSC2 has been identified as a high-priority target for VUS resolution, with (1) well-characterized molecular and patient phenotypes associated with loss-of-function variants, and (2) more than 2,700 VUS already documented in ClinVar. We developed Tuberous sclerosis classifier to Resolve variants of Uncertain Significance in TSC2 (TRUST), a machine learning model to predict pathogenicity of TSC2 missense VUS. To test whether these predictions are accurate, we further introduce curated loci prime editing (cliPE) as an accessible strategy for performing scalable multiplexed assays of variant effect (MAVEs). Using cliPE, we tested the effects of more than 200 TSC2 variants, including 106 VUS. It is highly likely this functional data alone would be sufficient to reclassify 92 VUS with most being reclassified as likely benign. We found that TRUSTs classifications were correlated with the functional data, providing additional validation for the in silico predictions. We provide our pathogenicity predictions and MAVE data to aid with VUS resolution. In the near future, we plan to host these data on a public website and deposit into relevant databases such as MAVEdb as a community resource. Ultimately, this study provides a framework to complete variant effect maps of TSC1 and TSC2 and adapt this approach to other mTORopathy genes.

genetics↗

bioRxiv2023

Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an SCN1A poison exon in epilepsy

Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A-related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Nav1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Nav1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Nav1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A-related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.

genetics↗