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Connecting cilium, stress response and proteostasis abnormalities inform variant and therapy assessment in RPGRIP1 retinal organoid [scRNA-seq]

ABSTRACT: RPGRIP1 encodes a connecting cilium (CC) protein essential for normal photoreceptor cell development and maintenance. Damaging variants in RPGRIP1 cause severe inherited retinal degeneration (IRD) and currently incurable vision loss, with mice studies showing promising preclinical gene augmentation therapy results. Almost one-half of variants in RPGRIP1 in the ClinVar database are variants of uncertain significance (VUS), hindering genetic diagnosis for affected individuals, and hence access to clinical trials of novel therapies and other management options. Here, we use human induced pluripotent stem cell (iPSC)-derived retinal organoids to model RPGRIP1-associated IRD, detecting biomarkers of disease including CC interactome dysfunction, stress response and proteostasis abnormalities. In parallel, utilising these novel disease biomarkers we demonstrate the pathogenicity of a missense VUS, RPGRIP1 c.2108 T>C p.(Ile703Thr). Finally, RPGRIP1 gene augmentation therapy rescued disease phenotypes, further supporting the utility of these biomarkers of RPGRIP1-LCA disease for reclassifying VUS and testing response to therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE293984 | GEO | 2025/11/18

REPOSITORIES: GEO

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Connecting cilium, stress response and proteostasis abnormalities inform variant and therapy assessment in RPGRIP1 retinal organoids [RNA-Seq]

Project description:RPGRIP1 encodes a connecting cilium (CC) protein essential for normal photoreceptor cell development and maintenance. Damaging variants in RPGRIP1 cause severe inherited retinal degeneration (IRD) and currently incurable vision loss, with mice studies showing promising preclinical gene augmentation therapy results. Almost one-half of variants in RPGRIP1 in the ClinVar database are variants of uncertain significance (VUS), hindering genetic diagnosis for affected individuals, and hence access to clinical trials of novel therapies and other management options. Here, we use human induced pluripotent stem cell (iPSC)-derived retinal organoids to model RPGRIP1-associated IRD, detecting biomarkers of disease including CC interactome dysfunction, stress response and proteostasis abnormalities. In parallel, utilising these novel disease biomarkers we demonstrate the pathogenicity of a missense VUS, RPGRIP1 c.2108 T>C p.(Ile703Thr). Finally, RPGRIP1 gene augmentation therapy rescued disease phenotypes, further supporting the utility of these biomarkers of RPGRIP1-LCA disease for reclassifying VUS and testing response to therapy.

2025-11-18 | GSE293982 | GEO

Saturation genome Editing of BRCA2 variants from C-terminal DNA binding domain

Project description:Accurate interpretation of genetic variation is a critical step towards realizing the potential of precision medicine. Sequencing-based genetic tests have uncovered a vast array ofBRCA2sequence variants. Due to limited clinical, familial and/or epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). Here we have utilized CRISPR-Cas9-based saturation genome editing (SGE) in a humanized-mouse embryonic stem cell line. We have categorized nearly all possible missense single nucleotide variants (SNVs) encompassing the C-terminal DNA binding domain ofBRCA2.We have generated the function scores for 6270 SNVs, covering 95.5% of possible SNVs in exons 15-26 spanning residues 2479-3216, including 1069 unique missense VUS, with 81% functional and 14% found to be nonfunctional. Our classification aligns strongly with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. Our statistical classifier exhibits 92.2% sensitivity and 96% specificity in distinguishing clinically benign and pathogenic variants recorded in ClinVar. Furthermore, we offer proactive evidence for 617 SNVs being non-functional and 3396 SNVs being functional demonstrated by impact on cell growth and response to DNA-damaging drugs like cisplatin and olaparib. This classification serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counselors assessingBRCA2VUSs in patients.

2024-09-27 | GSE248438 | GEO

Sequencing-based Functional Assays for Classification of BRCA2 Variants in Mouse ES Cells

Project description:Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer, or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 6000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUS). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. While the assays are reliable, the experimental process is time-consuming. This has impeded the number of variants that can be examined at a time. To overcome this, we have now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants. In this approach, instead of using clonogenic or XTT-based cell survival assays, pools of mESC expressing 25-30 BRCA2 variants from a given exon are analyzed by NGS. We have also developed a statistical model that utilizes the NGS data to determine the probability of impact on function (PIF) for these variants. While CRISPR/Cas9-based high throughput methods have resulted in the functional characterization of thousands of variants, only a fraction of the variants are included in ClinVar. In contrast, our targeted approach, which involves generating specific variants, resulted in the functional evaluation of 223 variants that are all listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or reported by other well-established assays.

2023-11-20 | GSE244578 | GEO

Comparative 3D genome analysis between neural retina and RPE reveals differential cis-regulatory interactions at retinal disease loci [Hi-C]

Project description:Vision depends on the functional interplay between the photoreceptor cells of the neural retina and the supporting cells of the underlying retinal pigment epithelium (RPE). Many genes involved in inherited retinal diseases (IRD) display highly specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. To understand the role of differential chromatin architecture in establishing tissue-specific expression patterns at IRD loci in the human neural retina and the RPE, we mapped genome-wide chromatin interactions by applying in situ Hi-C and H3K4me3 HiChIP to human adult post-mortem donor retinas. A comparative 3D genome analysis between neural retina and RPE revealed that almost 60% of known IRD genes were marked by differential cis-regulatory interactions or 3D genome structure. Furthermore, we zoomed in on tissue-specific chromatin interactions at the ABCA4 locus, which is implicated in the most common autosomal recessive IRD. We constructed high-resolution ABCA4 interaction profiles using UMI-4C, which, upon integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish, revealed tissue-specific CREs for ABCA4. In summary, through extensive comparative 3D genome mapping, based on genome-wide (Hi-C), promoter-centric (HiChIP) and locus-specific (UMI-4C) assays of human neural retina and RPE, we have shown that gene regulation at key IRD loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for genomic variation underlying unsolved IRD.

2024-04-08 | GSE235726 | GEO

Comparative 3D genome analysis between neural retina and RPE reveals differential cis-regulatory interactions at retinal disease loci [HiChIP]

2024-04-08 | GSE236021 | GEO

Comparative 3D genome analysis between neural retina and RPE reveals differential cis-regulatory interactions at retinal disease loci [UMI4C]

2024-04-08 | GSE235728 | GEO

Saturation genome editing of 11 codons and exon 13 of BRCA2 determines pathogenicity of variants

Project description:The pathogenicity of majority of variants identified in cancer-causing genes is unknown due to limited epidemiological data, hence they are considered to be variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate that variants with partial loss of BRCA2 function are sensitive to the DNA-damaging agents, cisplatin and olaparib, allowing us to discriminate between functional and intermediate variants. We have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional, 60 non-functional and 4 intermediate variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

2023-08-30 | GSE238143 | GEO

Retinal organoids with X-linked retinoschisis RS1 (E72K) mutation exhibit a photoreceptor developmental delay and are rescued by gene augmentation therapy

Project description:Background X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. Methods hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. Results ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation. Conclusions The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.

2024-05-17 | GSE267405 | GEO

An in vitro high-content screening unveils miR-429 as a protective molecule in photoreceptor degeneration models

Project description:Transcriptome profile of optic cups from RhoP23H/+ mice following AAV-mediated subretinal delivery of pre-miR-429 or GFP (control vector) to study the molecular changes underlying the neuroprotective effect of miR-429 overexpression in an Inherited Retinal Disease (IRD) model.

2025-01-07 | GSE272674 | GEO

DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus

Project description:Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1.

2023-06-12 | GSE234482 | GEO

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