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. 2026 Aug 3;67(10):1. doi: 10.1167/iovs.67.10.1

Identification of a Duplication in the RP17 Locus in an Individual With Pathogenic CEP290 Variants: Implications for RP17 Variant Classification

Lara K Holtes 1, Di Chen 2, Siobhan Guilfoyle 2, Harriet O'Leary 2, Julio C Corral-Serrano 2, Erica G M Boonen 1,3, Lonneke Haer-Wigman 1, Sara E Nieuwenhuis 2, Rosellina Guarascio 2, Vasileios Toulis 2,4, Danilo Andrade de Jesus 3,5,6, Frans P M Cremers 1, Alberta A H J Thiadens 6, Michael E Cheetham 2, Suzanne E de Bruijn 1, Alison J Hardcastle 2, Susanne Roosing 1,✉
PMCID: PMC13440643  PMID: 42545071

Abstract

Purpose

To assess the pathogenicity of a novel duplication in the RP17 locus identified in a cone dystrophy proband with biallelic CEP290 variants. Structural variants (SVs) in this locus have previously been associated with dominant retinitis pigmentosa.

Methods

Inheritance of the duplication was assessed by breakpoint polymerase chain reaction (PCR). Ophthalmic evaluation included fundus examination, multimodal retinal imaging, and full-field electroretinogram (ERG). A proband-derived pluripotent stem cell line was differentiated into photoreceptor precursor cells (PPCs) and retinal organoids (ROs). Variant-induced mis-splicing of CEP290 was assessed by reverse-transcription PCR (RT-PCR) and long-read cDNA sequencing, and immunohistochemistry was used to assess photoreceptor morphology. Expression of GDPD1 was quantified by quantitative RT-PCR.

Results

The proband and father carried the 324-kb duplication in the RP17 locus. The father was clinically unaffected, but the proband showed features of cone dystrophy, including reduced visual acuity, foveal abnormalities, diminished cone density, and preserved dark-adapted but absent light-adapted ERG responses. The compound heterozygous variants in CEP290 resulted in pseudoexon inclusion and exon 36 skipping in patient-derived retinal cells. Immunohistochemistry revealed altered ciliation and reduced trafficking of L/M opsin and rhodopsin in ROs. In silico modeling predicted that the novel RP17 duplication does not disrupt chromatin looping, and GDPD1 expression was not upregulated in patient ROs, in contrast to pathogenic RP17-SVs.

Conclusions

The cone dystrophy phenotype of the proband can be attributed to the CEP290 variants, whereas the novel RP17 duplication can be classified as likely benign based on the integrated evidence. These findings emphasize the importance of modeling and functional studies for accurately classifying RP17-SVs and preventing misinterpretation in clinical diagnostics.

Keywords: retinitis pigmentosa, RP17, topologically associating domain, likely benign duplication

Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with a prevalence of one in 4000 people.1 Clinical features of RP include night blindness starting in adolescence and progressive loss of the peripheral visual field which can result in legal blindness in adulthood. Characteristic retinal findings include bone-spicule pigmentation, arterial attenuation, structural changes observed by optical coherence tomography (OCT), and dark-adapted abnormalities detected on ERG examination.1,2 RP is exceptionally heterogeneous, with multiple modes of inheritance and more than 110 associated genes (https://retigene.erdc.info/).3 Approximately 25% to 30% of RP cases are autosomal dominant RP (adRP) cases, and pathogenic variants are identified in 50% to 75% of these cases.1 To date, variants in 28 genes and one locus, RP17 (MIM: 600852), are known to be causative for adRP.3

The RP17 locus (chr17q22-q23.1) was identified in 1995 by linkage analysis in a large South African adRP pedigree.4 Subsequently, adRP was found to segregate with this locus in additional families from South Africa, the Netherlands, and the United Kingdom; however, a compelling cause of disease in all families was elusive for several decades.5–8

In 2020, complex structural variants (SVs) were identified in the RP17 locus in 22 unrelated adRP families with more than 300 affected individuals.8 These SVs consist of duplications, paired duplicated inversions, and more complex events that affect multiple genes in the locus including GDPD1 and YPEL2. Although all of the SVs have unique breakpoints, it was proposed that the unifying feature is the reorganization of the three-dimensional (3D) chromatin architecture of the RP17 locus.8 Topologically associating domains (TADs) are self-interacting chromatin domains that can control the interactions between regulatory elements such as enhancers and their cognate genes.9,10 The boundaries of these chromatin domains are delineated by architectural proteins such as CCCTC-binding factor (CTCF), cohesion complexes, transposable elements, and housekeeping genes.9 The formation of TADs, and their substructures, are best described by the loop extrusion model, where DNA is dynamically extruded through a cohesion ring but halted by pairs of CTCF proteins in the convergent orientation.10–12 In the normal RP17 locus, a TAD structure facilitates the expression of YPEL2 in the retina as the YPEL2 promoter is brought into contact with distal, active retinal enhancers.8,13 In individuals with a pathogenic RP17-SV, this TAD structure is predicted to be disrupted and causes the formation of new active domains (neo-TADs) with ectopic contacts occurring between the retinal enhancers and the gene GDPD1. In healthy photoreceptors, GDPD1 is lowly expressed, and the upregulation of GDPD1 is the lead hypothesis for a convergent disease mechanism underlying RP17.8

RP17-SVs may be an underrecognized cause of adRP, as they are difficult to detect and were previously only identified through genome sequencing. Therefore, we created a high-throughput and cost-effective workflow for screening for RP17-SVs and identified previously described and novel SVs in seven adRP probands from Germany, Australia, and the United States.14 One entry point into the workflow is to extract SVs in the RP17 locus from existing genome sequencing data of IRD cases. We performed a reanalysis of genome sequencing data of almost 500 IRD probands. Through this approach, we identified a novel tandem duplication in the RP17 locus in an isolated cone dystrophy proband, who was considered genetically resolved due to compound heterozygous variants in CEP290. The aim of this study was to investigate the effect of the CEP290 variants and the novel duplication in the RP17 locus, to inform future studies that identify RP17-SVs in IRD cases and to define pathogenic versus benign criteria of an RP17-SV.

Methods

Ethics Approval

The study adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee of the Radboud University Medical Center (Nijmegen, the Netherlands). Written informed consent was obtained from the proband before examination and inclusion in this study.

Genome Sequencing Reanalysis

Genome sequencing had been performed by BGI (Beijing, China) as previously described by de Bruijn et al.15 The cohort was comprised of 496 genetically explained and unexplained samples with a variety of IRD phenotypes from the Radboud University Medical Center. This cohort was assessed for SVs in the RP17 locus. Briefly, SVs were called using Canvas Copy Number Variant Caller16 and Manta Structural Variant Caller v.1.1.0.17 Calls between chr17:59,000,000 and 61,000,000 (hg38) were extracted from all genomes and visualized using Integrative Genomics Viewer (IGV) 2.17.0.18 SVs were assessed for putative pathogenicity by predictive TAD modeling of the RP17 locus as previously described by de Bruijn et al.8

Breakpoint Polymerase Chain Reaction and Sanger Sequencing

The SV breakpoint junction obtained from genome sequencing data was polymerase chain reaction (PCR) amplified and validated with Sanger sequencing. Primer sequences and coordinates of the breakpoint are provided in Supplementary Table S1. Parental testing was performed by using this mutation-specific breakpoint PCR.

Clinical Assessment

The patient and her father were (re-)evaluated at the ophthalmologic clinic where data on their medical history were obtained. A complete ophthalmic examination was performed, including best-corrected visual acuity (BCVA) using Snellen vision, slit-lamp examination, and fundoscopy. Additional examination included OCT images, fundus autofluorescence (FAF), and infrared (IR) images were acquired using the SPECTRALIS HRA system (Heidelberg Engineering, Heidelberg, Germany). OCT images were evaluated for the presence of a structurally intact foveal outer nuclear layer (ONL) and an ellipsoid zone.

To enable high-resolution assessment of the cone photoreceptor mosaic, adaptive optics flood illumination ophthalmoscopy (AO-FIO) was performed using the rtx1 retinal camera (Imagine Eyes, Orsay, France). The AO-FIO protocol consisted of 15 images per eye, each covering a 4° × 4° field of view and up to 2° overlap to facilitate accurate montage reconstruction. Fixation was centered on the fovea (0°, 0°), and images were captured up to 4° nasally and 12° temporally, as well as up to 3° superiorly and inferiorly, ensuring continuous coverage of the macular region. The 15 AO-FIO images were aligned and combined into high-resolution retinal montages using i2K Retina AO software (DualAlign, Clifton Park, NY, USA). Cone density was automatically quantified using AO-FIO ConeDetect,19,20 and cone density maps were generated. Quantitative comparisons were performed across defined eccentricity ranges at 1° intervals from 1.5° to 10.5°. Due to the limited resolving power of the rtx1 system in the central fovea, the central 3° region (1.5° radius) was masked in all analyses.

Full‑field electroretinogram (ERG) responses were recorded using the center's standard extended protocol, incorporating International Society for Clinical Electrophysiology of Vision (ISCEV)‑recommended dark‑adapted and light‑adapted stimuli. Dark‑adapted recordings included an intensity series from 0.001 to 3.0 cd·s/m2, strong flashes of 10 and 30 cd·s/m2, and oscillatory potentials. Light‑adapted testing was comprised of single‑flash 3.0 cd·s/m2 ERG, a 30-Hz flicker, and oscillatory potentials. Interpretation of the implicit times and amplitudes used normative reference ranges derived from Jacobi et al.21 Recordings were obtained using ERG‑Jet corneal contact lens electrodes (Fabrinal SA, La Chaux-de-Fonds, Switzerland), a ColorDome Desktop Ganzfeld Stimulator (Diagnosys, Lowell, MA, USA), and a Nicolet Viking EDX acquisition system (Natus Neuro, Middleton, WI, USA).

Generation and Culturing of Induced Pluripotent Stem Cell Lines

The proband and control induced pluripotent stem cells (iPSCs) were derived, expanded, and characterized by the Radboudumc Stem Cell Technology Center using peripheral blood mononuclear cells (PBMCs) and episomal vectors. Another control iPSC line was reprogrammed from healthy human dermal fibroblasts of neonatal origin (HDFn) that had been previously characterized and reported.22,23

The iPSCs were cultured at 37°C and 5% CO2 in Gibco Essential 8 Flex Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 µg/mL Primocin (InvivoGen, San Diego, CA, USA). Maintenance of iPSC lines was conducted on six-well plates coated with 1:50 Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, Corning, NY, USA) diluted in Gibco Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; (Thermo Fisher Scientific). Colonies were split with 0.5-mM ethylenediaminetetraacetic acid (EDTA; InvivoGen) when they reached ∼80% confluency.

Differentiation of Photoreceptor Precursor Cells and Retinal Organoids

Patient and control iPSC lines were used to produce photoreceptor precursor cells (PPCs) and retinal organoids (ROs). The PBMC control iPSC line was used for the PPCs, and the control HDFn iPSC line was used for the RO differentiations. The iPSCs were differentiated to PPCs in 30 days as described previously,24 except that the cells were detached with Gibco TrypLE Select (Thermo Fisher Scientific), and the 12-well plates were coated with Matrigel GFR Basement Membrane Matrix at 1:50 dilution. The iPSCs were differentiated into ROs (technical replicates from one differentiation batch) as previously described.25–27 Quality control of the RO differentiation was based primarily on morphology and development of appropriate lamination and brush borders of the inner segment (IS) and the outer segment (OS). Differentiation of photoreceptors was confirmed by immunostaining for recoverin as a photoreceptor marker (Supplementary Table S2). Representative images of RO appearance and immunostaining are shown in Supplementary Figure S1.

RNA Extraction

RNA from the PPCs was isolated using the NucleoSpin RNA Clean-Up Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's protocols. RNA quantification was performed using nano ultraviolet–visible (UV-Vis) spectrometry on a DS-11 FX+ (DeNovix, Wilmington, DE, USA). Total RNA was extracted from the ROs at 180 ± 5 days using the PicoPure RNA Isolation Kit (Thermo Fisher Scientific).

cDNA Synthesis and Evaluation of CEP290 Mis-Splicing

RNA from PPCs was used for PacBio cDNA amplicon sequencing of CEP290. cDNA synthesis, reverse-transcription PCR (RT-PCR), and targeted long-read cDNA sequencing were performed as described by Capasso et al.28 The primers were designed to capture both CEP290 splicing variants in the same amplicon (exons 25–37), and the primer sequences are detailed in Supplementary Table S1.

The cDNA was synthesized from the RNA isolated from the ROs with the Tetro cDNA Synthesis Kit (Meridian Bioscience, Cincinnati, OH, USA) according to the manufacturer's instructions, using 100 ng of RNA as input. The reverse-transcription reaction was performed at 45°C for 1 hour followed by 85°C for 5 minutes. PCR amplification was performed using 2× GoTaq Green Master Mix (Promega, Madison, WI, USA) with forward and reverse primers (Supplementary Table S1) under standard cycling conditions, using cDNA as the template. PCR products were separated on a 3% (w/v) agarose gel (Eurogentec, Seraing, Belgium) and visualized using a ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Quantitative Reverse-Transcription PCR

Quantitative PCR (qPCR) was performed on a QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using LabTaq Green Hi Rox (Labtech, Hopkinton, MA, USA). Relative gene expression levels were calculated using the ΔΔCt method, with GUSB as the reference gene. Primer sequences are listed in Supplementary Table S1.

Cryopreservation and Immunohistochemistry of ROs

ROs were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde supplemented with 5% sucrose in PBS for 40 minutes at 4°C. The ROs were cryopreserved by sequential incubation in 6.25%, 12.5%, and 25% sucrose in PBS for 1 hour per step at 4°C, followed by embedding in optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA, USA) for storage at −80°C. Cryosections were cut at 10-µm thickness using a cryostat and mounted onto SuperFrost Plus Adhesion slides (Epredia, Kalamazoo, MI, USA), for storage at −20°C.

Cryosections were washed once with PBS, permeabilized, and blocked in a solution containing 10% Gibco fetal bovine serum (Thermo Fisher Scientific) and 0.025% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature. Primary and secondary antibody solutions were diluted in blocking buffer (1:2 in PBS). Cryosections were incubated with primary antibodies listed in Supplementary Table S2 for 1 hour at room temperature. Slides were incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour at room temperature, followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; 1:5000; Thermo Fisher Scientific) for 5 minutes. Slides were mounted using Dako Fluorescent Mounting Media (Agilent Technologies, Santa Clara, CA, USA).

Confocal Microscopy and Analysis

Images were acquired using a STELLARIS 8 confocal microscope (Leica Microsystems, Wetzlar, Germany). At least two representative regions were imaged per RO section. Image analysis was performed using ImageJ Fiji software (National Institutes of Health, Bethesda, MD, USA). Ciliation was quantified by manually counting polyglutamylated tubulin GT335-positive basal bodies stained with pericentrin (PCN) and expressing this as a percentage of the total number of basal bodies per image. Localization of L/M opsin and rhodopsin was assessed by measuring mean fluorescence intensity across defined regions of interest using Fiji. Statistical analysis was conducted using Prism software (GraphPad, Boston, MA, USA). Specific statistical tests used are detailed in the corresponding figure legends.

Results

A Novel Structural Variant in the RP17 Locus Was Identified in a Cone Dystrophy Proband

With the aim to identify more RP17-SVs in IRD cases, we extracted all SVs in the locus from a cohort of almost 500 probands, which were a mix of genetically explained and unexplained samples. Through this genome sequencing reanalysis, a novel 324-kb duplication in the RP17 locus (chr17:g.59220482_59544622dup [V3]) was identified in an isolated cone dystrophy proband that was considered genetically resolved with compound heterozygous variants in CEP290 (c.4705-1G>T [V1] and c.2991+1655A>G [V2]) (Fig. 1A).29 The tandem duplication is absent from gnomAD SV v.4.1.0,30 and the breakpoint was confirmed by mutation-specific PCR (Supplementary Fig. S2A). Parental DNA was tested via breakpoint PCR to determine if the duplication was inherited or a de novo event. The father was also found to carry the novel duplication (Fig. 1A; Supplementary Fig. S2B).

Figure 1.

Figure 1.

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Ophthalmic examination shows a typical cone dystrophy phenotype in the proband with biallelic CEP290 variants. (A) Pedigree of the family, which shows that the proband (II.1), indicated with an arrow, carries biallelic variants in CEP290 V1 and V2, both of which are predicted to cause mis-splicing. In addition, the affected proband and unaffected father (I.1) carry a novel 324-kb tandem duplication in the RP17 locus (V3). (B) Images of the proband retina (II.1). (B, i) OCT image of the retina, showing thinning of the ONL and interruption of the ellipsoid zone in the macula. (B, ii) FAF showing a hyper-autofluorescent ring in the macula region. (B, iii) Infrared imaging reveals a hyperreflective ring surrounding a hyporeflective center in the macula. (C) Images of the father's retina (I.1) with no evidence of retinal degeneration. (C, i) OCT image of the retina reveals a typical retina structure. (C, ii) FAF shows a normal appearing fundus. OD, right eye.

Clinical Assessment of the Proband and Father Carrying the Novel Duplication in the RP17 Locus

At the age of 23, the patient's clinical image resembled cone dystrophy with a well-preserved visual acuity of 0.40/0.50. The OCT showed abnormalities in the foveal region with thinned maculae and a disrupted ellipsoid zone (Fig. 1B). A bull's-eye maculopathy was apparent on FAF and IR imaging (Fig. 1B). AO of the parafoveal retina revealed a progressive enlargement of atrophic regions and reduced cone density in 1 year (Supplementary Figs. S3A, S3C). The full-field ERG (age 20 years) showed normal dark-adapted responses across all flash strengths, and dark-adapted oscillatory potentials were present (Supplementary Figs. S4A–S4C; Supplementary Table S3). In contrast, the light-adapted single-flash response, the 30-Hz flicker, and the light-adapted oscillatory potentials were not detectable, consistent with generalized cone photoreceptor dysfunction (Supplementary Figs. S4D–S4F).

Neither parent had reported any ophthalmic complaints. Nevertheless, the 53-year-old father was clinically assessed to examine for any signs of retinal degeneration, as he was the only other available carrier of the novel duplication in the RP17 locus. Visual acuity was 1.2/1.2; he had a normal appearing fundus, typical retina structure on OCT, and normal cone mosaic upon AO imaging (Fig. 1C; Supplementary Figs. S3B, S3C). No typical signs of RP such as bone spicules, attenuated arterial vessels, or pale optic disc were observed upon fundoscopy examination of the retina.

Compound Heterozygous Variants in CEP290 Lead to Mis-Splicing Events in Human Photoreceptors

An iPSC line was established from the proband and subsequently differentiated to both PPCs and ROs to study the molecular impact of the biallelic CEP290 variants. Long-read cDNA amplicon sequencing was performed on RNA extracted from PPCs, and primers were designed to capture both mis-splicing effects in one amplicon (Fig. 2A). However, no single nucleotide variants were present within the targeted exons to distinguish which allele the correctly spliced transcripts derived from and to allow for the quantification of the aberrant splicing for each allele. The aberrant splicing events were also assessed in 180-day patient-derived ROs by RT-PCR (Fig. 2B). These results confirmed that the canonical splice variant, c.4705-1G>T (V1), leads to in-frame exon 36 skipping (p.[Glu1569_Trp1604del,=]), and the deep intronic variant, c.2991+1655A>G (V2), leads to pseudoexon inclusion (p.[Cys998*,=]). This combination of variants does not lead to complete mis-splicing of CEP290 transcripts, and the correctly spliced transcript was also present (Fig. 2).

Figure 2.

Figure 2.

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Compound heterozygous CEP290 variants cause different mis-splicing events in both PPCs and ROs. (A) Long-read amplicon cDNA sequencing of CEP290 exons 25 to 37 from RNA derived from PPCs. The aberrant splicing events in the proband are highlighted by the red boxes. The drop in coverage over exon 36 is due to in-frame exon skipping caused by the canonical splice variant (V1), and the pseudoexon (PE) inclusion is driven by the deep intronic variant (V2). (B) RT-PCR of CEP290 transcripts in patient and control ROs (day 180 ± 5 days) reveals in-frame exon 36 skipping and PE inclusion. ACTB was used as a reference gene.

Altered Ciliation and Opsin Mislocalization in Patient-Derived ROs

The ROs were immunostained for polyglutamylated tubulin and PCN to highlight features of the connecting cilium and study the impact of the CEP290 splicing variants on ciliation. Polyglutamylated tubulin, a component of the connecting cilium, was used to assess the integrity of the cilia, and PCN was used as a marker for the basal body of the photoreceptors. The patient-derived ROs had significantly reduced basal body ciliation compared to the control ROs (Figs. 3A, 3B). In addition, the trafficking of L/M opsin and rhodopsin was investigated, and the patient-derived ROs displayed significant L/M opsin retention in the photoreceptor IS and ONL and rhodopsin retention in the ONL compared to control ROs (Figs. 3C–F).

Figure 3.

Figure 3.

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CEP290 mis-splicing variants alter ciliation and opsin trafficking in ROs. (A) Representative images of control and proband-derived ROs (day 180 ± 5 days) immunostained with GT335 (green), DAPI (blue), and PCN (magenta). Scale bar: 10 µm. (B) Proband RO photoreceptors have reduced ciliation quantified from GT335 elongation from the PCN-stained basal body when compared to control ROs (n = 2 ROs from one differentiation for both conditions, four images per RO; n = 937 and n = 867 basal bodies analyzed for control and proband ROs, respectively; Welch's unpaired t-test). (C) Cryosections immunostained for L/M opsin (grayscale in the top panel, magenta in the bottom panel) and TOMM20 (cyan). Scale bars: 50 µm (top panel) and 20 µm (bottom panel). (D) Proband ROs reveal significant L/M opsin retention in photoreceptor ISs and the ONL and reduced trafficking to the photoreceptor OS (n = 4 control ROs and n = 3 proband ROs from one differentiation, two to four images per RO; Mann–Whitney test). (E) Cryosections were stained for rhodopsin (gray). Scale bar: 50 µm. (F) Proband ROs show rhodopsin retention in the ONL and reduced trafficking to the IS and OS (n = 3 ROs for both conditions from one differentiation, four images per RO; Mann–Whitney test).

Novel Duplication Identified in the RP17 Locus Does Not Lead to Overexpression of GDPD1

In silico modeling was performed to determine the impact of the novel duplication on the TAD structure of the RP17 locus based on retina-specific epigenetic datasets (Fig. 4A). The duplication is not predicted to interrupt the YPEL2 TAD structure, as the breakpoints of the SV are not within the TAD; rather, the entire TAD structure is part of the duplication. Therefore, this SV does not fit the current model of neo-TAD formation allowing for an ectopic contact between the retinal enhancer elements and the GDPD1 promoter. GDPD1 expression was quantified by quantitative RT-PCR in patient-derived 180-day ROs. There was no upregulation of GDPD1 in the patient ROs compared to control ROs, further supporting the in silico modeling (Fig. 4B). Based on this study, previously reported pathogenic RP17 duplications, and duplications present in the general population, a framework to assess the pathogenicity of tandem duplications in the RP17 locus is proposed (Fig. 5). This framework specifies the smallest possible duplication and the critical region for breakpoints that would disrupt the YPEL2 TAD structure and bring GDPD1 into contact with the retinal enhancer elements.

Figure 4.

Figure 4.

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TAD modeling and gene expression analysis confirmed that the novel duplication in the RP17 locus does not upregulate GDPD1. (A) Schematic TAD modeling of the RP17 locus is based on publicly available retina-specific epigenetic datasets as previously described.8 NL-SV1 and NL-SV5 are included as examples of previously described pathogenic RP17 duplications. The red arrow indicates the predicted creation of ectopic contacts between the retinal enhancer elements and GDPD1 promoter. For the novel tandem duplication identified in the proband in this study, the TAD structure around YPEL2 was not disrupted by the SV; therefore, no ectopic contact was predicted. The duplicated region is highlighted in orange for the pathogenic duplications and purple for the novel, likely benign duplication. (B) RT-qPCR of control and proband ROs (day 180 ± 5 days, n = 3 ROs from one differentiation) shows that GDPD1 is not upregulated in the proband and is comparable to control RO levels. Welch's unpaired t-test; ns, non-significant. Schematics show the possible loops that could form, focused on YPEL2, which is the normal target of the retinal enhancer, and GDPD1. The novel tandem duplication in the proband would not result in the inclusion of a complete copy of GDPD1 within an active loop.

Figure 5.

Figure 5.

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Provisional framework for the pathogenicity of tandem duplications in the RP17 locus. The tandem duplication identified in the cone dystrophy proband (chr17:59,220,482–59,544,622) is shown in relation to two previously reported, pathogenic RP17 duplications: NL-SV1 (chr17:59,214,554–59,440,776) and NL-SV5 (chr17:59,183,160–59,438,501), duplications from gnomAD SV v.4.1.0,30 and key regulatory features extracted from the RegRet dataset derived from human retina samples.52 The TAD around the YPEL2 gene is delineated by the forward (chr17:59,310,800–59,330,800) and reverse (chr17:59,440,900–59,441,100) CTCF boundaries. Distal retinal enhancer elements reside within the TAD structure (chr17:59,422,400–59,427,200 and chr17:59,437,700–59,438,400); they are indicated by H3K27ac marks and contain binding sites for CRX, NRL, and OTX2, which promote the expression of YPEL2 in the human retina. The promoter region of GDPD1 was defined based on DNase-sequencing data from human embryonic retina (day 125), assay for transposase-accessible chromatin (ATAC)-sequencing data from adult human retina, H3K27ac signatures, Encyclopedia of DNA Elements (ENCODE) data for RNA polymerase II binding and cis-regulatory elements, and FANTOM5 transcription start sites. The shaded orange box indicates the current hypothesis for a minimum size of a pathogenic duplication, which includes the entire GDPD1 gene and its promoter region to the end of the first retinal enhancer element (chr17:59,220,327–59,427,200). The critical region, depicted by the dotted box, (chr17:59,427,200–59,440,900) is where the 3′ breakpoint of the tandem duplication would have to be located to be considered pathogenic, which is before the reverse CTCF boundary element but after at least one of the two retinal enhancer elements.

Discussion

RP and cone dystrophies are both IRDs; however, they represent a different profile of initial symptoms. Cone dystrophies are defined by the primary dysfunction and degeneration of cone photoreceptors, which cause decreased visual acuity and decreased sensitivity in the central visual field. By contrast, RP results from the primary degeneration of the rod photoreceptors, which results in night blindness and loss of peripheral vision.31 RP has been associated with a total of 113 genes and loci, and cone dystrophy/cone–rod dystrophy has been associated with 38 genes, with 17 genes reported to cause both phenotypes (https://retigene.erdc.info/).3 The 23-year-old proband described in this study presented with a typical cone dystrophy phenotype, including reduced BCVA, foveal abnormalities, reduced cone density, normal dark-adapted responses, and absent light-adapted responses (Fig. 1B; Supplementary Figs. S3A, S3C, S4). The cone dystrophy phenotype had already been attributed to the biallelic variants in CEP290,29 and in this study we sought to investigate the pathogenicity of a novel tandem duplication in the RP17 locus and whether this could impact the phenotype.

The tandem duplication was inherited from the father, who had no previous ophthalmic complaints. As pathogenic RP17-SVs cause dominant RP, the father was clinically assessed and showed no signs of retinal degeneration at age 53 years (Fig. 1C). The age of onset of RP17 has been reported to be as early as 12 years and as late as the mid-50s, but most patients start to develop symptoms by their 30s.8 A limitation of our clinical assessment of the father was that no electrophysiological testing was performed so subclinical or late-onset functional abnormalities may have been overlooked. Therefore, based on the supportive clinical and familial evidence, the novel RP17 duplication seemed unlikely to be causative for RP, but because of the range in age of onset for RP17 and the lack of electrophysiological testing in the father, additional functional studies were conducted.

To further investigate and functionally validate the pathogenicity of the variants, an iPSC line was established and differentiated to both PPCs and ROs. The proband carries biallelic variants in CEP290, c.2991+1655A>G and c.4705-1G>T. The deep intronic variant is a well-known pathogenic variant that creates a donor splice site and the insertion of a pseudoexon.32 The aberrant splicing is a partial effect and has been studied in patient immortalized lymphoblastoid cell lines, fibroblasts, and iPSC-derived retinal pigment epithelium and ROs.22,32,33 Of particular interest, the aberrant splicing has been reported to be more severe in iPSC-derived ROs, than in iPSC-derived retinal pigment epithelium and fibroblasts, with only 10% to 20% residual wild-type CEP290 transcript present.22 The canonical splice site variant c.4705-1G>T is predicted to lead to the loss of the acceptor and donor splice sites of exon 36 (1.0 and 0.71 SpliceAI delta scores, respectively34) and has been reported previously in compound heterozygous cases and one homozygous case.35–37 The RNA isolated from the patient-derived PPCs and ROs, confirms CEP290 transcripts with both the pseudoexon inclusion and exon 36 skipping (Fig. 2). However, this could not be accurately quantified because phasing of the transcripts by allele was not possible, and nonsense-mediated decay would act on the pseudoexon-containing transcript.

CEP290 encodes a centrosomal protein that has a crucial role in the development, maintenance, and function of cilia, and in photoreceptor cells it is involved in ciliary transport processes. Variants in CEP290 are involved in a spectrum of ciliopathies, ranging from isolated retinal dystrophy, to Leber congenital amaurosis, to syndromic ciliopathies such as Joubert syndrome and Senior-Løken syndrome (MIM: 610142). CEP290 pleiotropy can be partially attributed to how different variants influence the amount of protein retaining all or some CEP290 functionality across various tissues.38 A process called nonsense-associated splicing has been implicated in attenuating the impact of nonsense variants and accounting for atypical CEP290-related IRDs.39–41 Alternative splicing is upregulated to skip exons containing premature stop codons, and, if the exon is in frame, which is the case for almost half of the coding exons of CEP290, an intact reading frame is maintained and a shorter CEP290 protein can be translated. Exon 36 is an in-frame exon that encodes a linker region between two predicted coiled coil domains and is a hotspot for nonsense variants, particularly those that result in non-syndromic retinal disease and are predicted to significantly alter splicing.42 A study of nonsense variants in exon 36 using ROs and kidney organoids showed that the nonsense-associated splicing of exon 36 is tissue specific.42 The skipping of exon 36 was shown to improve cellular disease phenotypes and has therefore been proposed as a potential therapeutic strategy.42 This would support the hypothesis that the canonical splice variant, c.4705-1G>T, could lead to a protein with residual function.

The cone dystrophy phenotype of the proband in this study is on the mild end of the CEP290 ciliopathy spectrum. The two CEP290 splicing variants result in residual full-length wild-type transcripts and transcripts lacking exon 36 in patient-derived ROs. The patient-derived ROs show reduced basal body ciliation resulting in trafficking defects of L/M opsin and rhodopsin compared to control ROs (Fig. 3). The data are consistent with previously described CEP290-related defects in RO models that show an impact on the formation of the OS and thus photoreceptor function.22,25 Given that these findings were observed in ROs from a single differentiation and without comparison to an additional CEP290 control line, they support rather than definitively establish the contribution of the CEP290 variants to the cellular phenotype. The specific combination of CEP290 variants, a retina-enhanced effect of the deep intronic variant and a disease-ameliorating exon skipping event, could account for both the non-syndromic nature and milder CEP290-associated cone dystrophy observed in the proband.

Complex structural variants in the RP17 locus were proposed to cause RP when they disrupt the TAD structure around the YPEL2 gene and lead to ectopic GDPD1 expression in the retina.8,14 With in silico TAD modeling based on retina-specific epigenetic datasets, we predicted that the TAD structure around YPEL2 would not be disrupted by the novel tandem duplication. Although we did not perform chromatin conformation assays to prove an undisrupted TAD structure, the expected downstream consequence of TAD disruption in RP17 (ectopic GDPD1 expression) was absent in patient-derived ROs (Fig. 4). Thus, although the in silico TAD modeling remains predictive, the outcome is aligned with the experimental expression data in ROs, supporting the interpretation that the novel duplication does not create pathogenic retinal enhancer-GDPD1 contact and GDPD1 overexpression.

We propose a provisional framework for interpretating the pathogenicity of tandem duplications in the RP17 locus (Fig. 5). The smallest, pathogenic duplication and the critical region for the 3′ breakpoint of tandem duplications is indicated. These requirements specify that in a pathogenic SV allele a complete copy of the GDPD1 gene is (1) adjacent to at least one distal retinal enhancer element and (2) not obstructed by a CTCF boundary element. The tandem duplication (V3) does not meet these criteria as the 5′ breakpoint is within the promoter region of GDPD1 and the 3′ breakpoint is located after the reverse CTCF boundary. In the 63,046 genomes in gnomAD SV v.4.1.0,30 none of the tandem duplication calls in the RP17 locus would fulfill these two criteria to be pathogenic. In contrast, the two previously reported duplications (NL-SV1 and NL-SV5) have (1) the whole of the GDPD1 gene included in the duplication and (2) their 3′ breakpoint within the critical region. The framework requires further refinement in terms of how far a pathogenic duplication can extend upstream of GDPD1. It is currently unclear how the linear distance between the GDPD1 promoter and the retinal enhancer elements in the altered SV allele could affect the level of ectopic GDPD1 expression (Supplementary Fig. S5). Several recent studies have demonstrated that transcriptional activity and stability at a gene promoter decrease with increasing genomic distance from an enhancer and are influenced by the strength and cooperativity between enhancers.43–46 Moreover, the precise spatiotemporal control of the ectopic expression of GDPD1 remains to be defined. In the gnomAD SV population data, several tandem duplications encompass the entire GDPD1 gene, supporting the hypothesis that increased gene dosage of GDPD1 alone is insufficient to induce an RP phenotype. Instead, the orientation and location of the genes and key regulatory features in the mutated SV allele appear to be crucial for driving GDPD1 overexpression in the retina and leading to an RP phenotype. This framework is provisional and intended as a working, model-based interpretation that can be refined as additional RP17 duplications are reported. The development of this framework is aligned with previous studies on chromosomal rearrangements affecting TADs which call for understanding locus-specific gene regulation to develop guidelines for the interpretation of clinical variants.47,48

The American College of Medical Genetics and Genomics (ACMG) has provided a quantitative scoring framework for the interpretation of copy number variants.49 The existing criteria mainly address gene dosage effects of SVs but do not account for alternative disease mechanisms such as TAD disruptions or regulatory pertubations.49,50 Strictly using the ACMG framework, the novel RP17 duplication is formally classified as a variant of uncertain significance (Supplementary Table S4). However, there are no options to incorporate evidence from in silico modeling (PP3/BP4), functional studies (PS3/BS3), and an alternative molecular basis of disease (BP5), which are criteria included in the framework for short-sequence variants.51 Future revisions of the ACMG guidelines should address this limitation in interpretating the clinical relevance of candidate SVs working via alternative disease mechanisms, such as incorporating the workflow proposed by Sreenivasan et al.50

Our study highlights the possible diverse consequences of SVs affecting the RP17 locus. As the ACMG copy number variant framework does not accommodate a TAD disruption disease mechanism, we classify the novel RP17 duplication as likely benign based on the integrated evidence from the supportive clinical findings, in silico TAD modeling, and functional assays presented in this study. This emphasizes the need to establish pathogenicity criteria for different types of RP17-SVs based on their breakpoints to prevent a misdiagnosis of RP17.

Supplementary Material

Supplement 1
iovs-67-10-1_s001.docx (5.5MB, docx)

Acknowledgments

The authors thank the Radboudumc Stem Cell Technology Center for reprogramming and characterizing the iPSC cell line from the cone dystrophy proband. We thank the Department of Human Genetics and the Radboud Genome Technology Center for infrastructural and computational support. We thank Saskia D. van der Velde-Visser, Marlie Jacobs-Camps, and Ellen Blokland for handling DNA samples and administration. We thank Dirk Straver for his technical expertise for the ERG assessment. Parts of this manuscript were edited from July to November 2025 with Microsoft Copilot, a generative AI technology based on the OpenAI GPT-4 model, to assist with language refinement, clarity, flow, and formatting suggestions. All content edited by Copilot was reviewed and verified by the authors.

Supported by a Foundation Fighting Blindness Program Project Award (PPA-0622-0841-UCL to AJH, MEC, SR, SEdB); Rotterdamse Stichting Blindenbelangen; Stichting Blinden-Penning; Ooglijders; Stichting tot Verbetering van het Lot der Blinden; Stichting Blindenhulp; Stichting Oogfonds Nederland; Landelijke Stichting voor Blinden en Slechtzienden (SEdB, SR, FPMC); Foundation Fighting Blindness Career Development Award (CDGE-0621-0809-RAD to SR); Fight for Sight and National Institute for Health and Care Research Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology (AJH, MEC); the Wellcome Trust (205041/Z/16/Z to MEC); and a Moorfields Eye Charity Career Development Award (JCCS).

Author Contributions: Conceptualization: F.P.M.C., M.E.C., S.E.dB., A.J.H., and S.R.; Formal analysis: L.K.H., D.C., and S.G.; Investigation: L.K.H., D.C., S.G., H.O., J.C.C.-S., E.G.M.B., L.H.-W., D.A.dJ., A.A.H.J.T., S.E.dB., R.G., V.T., and S.E.N.; Resources: A.A.H.J.T.; Funding acquisition: M.E.C., S.E.dB., A.J.H., and S.R.; Project administration: M.E.C., S.E.dB., A.J.H., and S.R.; Supervision: M.E.C., S.E.dB., A.J.H., and S.R.; Visualization: L.K.H., D.C., and S.G.; Writing–original draft: L.K.H. Writing–review and editing: all authors.

Data and Code Availability: Data are available upon reasonable request. The genetic variants from this study have been submitted to the Leiden Open (source) Variation Database (LOVD) (curated by J.T. den Dunnen and S.E. de Bruijn). The genome sequencing data is subject to controlled access because it may compromise the privacy of the research participant.

Disclosure: L.K. Holtes, None; D. Chen, None; S. Guilfoyle, None; H. O'Leary, None; J.C. Corral-Serrano, None; E.G.M. Boonen, None; L. Haer-Wigman, None; S.E. Nieuwenhuis, None; R. Guarascio, None; V. Toulis, None; D. Andrade de Jesus, None; F.P.M. Cremers, None; A.A.H.J Thiadens, None; M.E. Cheetham, None; S.E. de Bruijn, None; A.J. Hardcastle, None; S. Roosing, None

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