Biology subjects

Place, E.

Publications and source records attributed to Place, E..

6 recordsLinked to original sources

bioRxiv2026

In vitro pathogenicity evaluation of deep intronic variants for recessive genetic retinal diseases

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

genetics↗

bioRxiv2025

Resolving forebrain developmental organisation by analysis of differential growth patterns

The forebrain is the most complex region of the vertebrate CNS, and its developmental organisation is controversial. We fate-mapped the embryonic chick forebrain using lipophilic dyes and Cre-recombination lineage tracing, and built a 4D model of brain growth. We reveal modular patterns of anisotropic growth, ascribed to progenitor regions through multiplex HCR. Morphogenesis is dominated by directional growth towards the eye, more isometric expansion of the prethalamus and dorsal telencephalon, and anterior movement of ventral cells into the hypothalamus. Fate conversion experiments in chick and comparative gene expression analysis in chick and mouse support placement of the hypothalamus ventral to structures extending from the telencephalon up to and including the zona limitans intrathalamica (ZLI), with the dorsoventral axis becoming distorted at the base of the ZLI. Our findings challenge the widely accepted prosomere model of forebrain organisation, and we propose an alternative tripartite hypothalamus model.

developmental biology↗

bioRxiv2022

A neuroepithelial wave of BMP signalling drives anteroposterior specification of the tuberal hypothalamus

The tuberal hypothalamus houses several major hypothalamic nuclei, dozens of transcriptionally distinct cell types, and clinically relevant cell populations implicated in obesity and related metabolic disorders. Building on recent advances in the field, here we draw upon transcriptional, signalling, and fate mapping analyses of chicken embryos and neuroepithelial explants to analyze tuberal hypothalamic development. We show that a wave of BMP signalling sweeps through early floor plate-like progenitors overlying prospective Rathkes pouch as they track anteriorly. The timing of BMP signalling correlates with cell fate, with anterior tuberal specification complete by Hamilton-Hamburger (HH) stage 10 but posterior tuberal progenitors requiring BMPs after this point. scRNA-Seq profiling of FGF10-expressing cells, a proxy for cells with active BMP signalling, through HH8-21 reveals transcriptional differences that may underlie their differing response to BMPs, and the switch from neuroepithelial progenitors to stem-like radial glial cells. This study provides an integrated account of the development of the tuberal hypothalamus.

developmental biology↗

bioRxiv2021

Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

The hypothalamus is an evolutionarily ancient brain region that regulates many innate behaviors, but its development is still poorly understood. To identify molecular mechanisms controlling hypothalamic specification and patterning, we used single-cell RNA-Seq to profile multiple stages of early hypothalamic development in the chick. We observe that hypothalamic neuroepithelial cells are initially induced from prethalamic-like cells. Two distinct hypothalamic progenitor populations emerge later, which give rise to paraventricular/mammillary and tuberal hypothalamus. At later developmental stages, the regional organization of the chick and mouse hypothalamus closely resembles one another. This study identifies selective markers for major subdivisions of the developing chick hypothalamus and many uncharacterized candidate regulators of hypothalamic patterning and neurogenesis. As proof of concept for the power of the dataset, we demonstrate that follistatin, a novel prethalamic progenitor-like marker, inhibits hypothalamic induction. This study both clarifies the organization of the early developing hypothalamus and identifies novel molecular mechanisms controlling hypothalamic induction, regionalization, and neurogenesis. HighlightsO_LIEarly hypothalamic development was profiled in chick using scRNA-Seq and multiplexed HCR. C_LIO_LIHypothalamic cells are induced from prethalamic-like neuroepithelial cells. C_LIO_LIDistinct paraventricular/mammillary and tuberal progenitor populations emerge later, and hypothalamic organization is evolutionarily conserved. C_LIO_LIPrethalamic progenitor-derived follistatin inhibits hypothalamic specification. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/438683v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@18ac4c2org.highwire.dtl.DTLVardef@17bb90aorg.highwire.dtl.DTLVardef@1b17cceorg.highwire.dtl.DTLVardef@178e769_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families.

Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient induced pluripotent stem cell (iPSC) derived retinal organoids (ROs) transcriptome analysis. With this approach we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.

genetics↗

bioRxiv2019

Characterizing variants of unknown significance inrhodopsin: a functional genomics approach

Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing was created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next generation sequencing (NGS)-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized surface expression of every RHO library variant with a high degree of reproducibility (Z=0.94, R2=0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (G18D, G101V, P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in rhodopsin, and can be applied more broadly to other inherited retinal disease genes and other disorders.

genetics↗