USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids.

There is an emblematic clinical and genetic heterogeneity associated with inherited retinal diseases (IRDs). The most common form is retinitis pigmentosa (RP), a rod-cone dystrophy caused by pathogenic variants in over 80 different genes. Further complexifying diagnosis, different variants in individual RP genes can also alter the clinical phenotype. USH2A is the most prevalent gene for autosomal-recessive RP and one of the most challenging because of its large size and, hence, large number of variants. Moreover, USH2A variants give rise to non-syndromic and syndromic RP, known as Usher syndrome (USH) type 2, which is associated with vision and hearing loss. The lack of a clear genotype-phenotype correlation or prognostic models renders diagnosis highly challenging. We report here a long-awaited differential non-syndromic RP and USH phenotype in three human disease-specific models: fibroblasts, induced pluripotent stem cells (iPSCs), and mature iPSC-derived retinal organoids. Moreover, we identified distinct retinal phenotypes in organoids from multiple RP and USH individuals, which were validated by isogenic-corrected controls. Non-syndromic RP organoids showed compromised photoreceptor differentiation, whereas USH organoids showed a striking and unexpected cone phenotype. Furthermore, complementary clinical investigations identified macular atrophy in a high proportion of USH compared with RP individuals, further validating our observations that USH2A variants differentially affect cones. Overall, identification of distinct non-syndromic RP and USH phenotypes in multiple models provides valuable and robust readouts for testing the pathogenicity of USH2A variants as well as the efficacy of therapeutic approaches in complementary cell types.

TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.

Identification and in vivo functional investigation of a HOMER2 nonstop variant causing hearing loss.

DFNA68 is a rare subtype of autosomal dominant nonsyndromic hearing impairment caused by heterozygous alterations in the HOMER2 gene. To date, only 5 pathogenic or likely pathogenic coding variants, including two missense substitutions (c.188 C > T and c.587 G > C), a single base pair duplication (c.840dupC) and two short deletions (c.592_597delACCACA and c.832_836delCCTCA) have been described in 5 families. In this study, we report a novel HOMER2 variation, identified by massively parallel sequencing, in a Sicilian family suffering from progressive dominant hearing loss over 3 generations. This novel alteration is a nonstop substitution (c.1064 A > G) that converts the translational termination codon (TAG) of the gene into a tryptophan codon (TGG) and is predicted to extend the HOMER2 protein by 10 amino acids. RNA analyses from the proband suggested that HOMER2 transcripts carrying the nonstop variant escaped the non-stop decay pathway. Finally, in vivo studies using a zebrafish animal model and behavioral tests clearly established the deleterious impact of this novel HOMER2 alteration on hearing function. This study identifies the fourth causal variation responsible for DFNA68 and describes a simple in vivo approach to assess the pathogenicity of candidate HOMER2 variants.

SpliceAI-visual: a free online tool to improve SpliceAI splicing variant interpretation.

SpliceAI is an open-source deep learning splicing prediction algorithm that has demonstrated in the past few years its high ability to predict splicing defects caused by DNA variations. However, its outputs present several drawbacks: (1) although the numerical values are very convenient for batch filtering, their precise interpretation can be difficult, (2) the outputs are delta scores which can sometimes mask a severe consequence, and (3) complex delins are most often not handled. We present here SpliceAI-visual, a free online tool based on the SpliceAI algorithm, and show how it complements the traditional SpliceAI analysis. First, SpliceAI-visual manipulates raw scores and not delta scores, as the latter can be misleading in certain circumstances. Second, the outcome of SpliceAI-visual is user-friendly thanks to the graphical presentation. Third, SpliceAI-visual is currently one of the only SpliceAI-derived implementations able to annotate complex variants (e.g., complex delins). We report here the benefits of using SpliceAI-visual and demonstrate its relevance in the assessment/modulation of the PVS1 classification criteria. We also show how SpliceAI-visual can elucidate several complex splicing defects taken from the literature but also from unpublished cases. SpliceAI-visual is available as a Google Colab notebook and has also been fully integrated in a free online variant interpretation tool, MobiDetails ( https://mobidetails.iurc.montp.inserm.fr/MD ).

Retinoic acid delays initial photoreceptor differentiation and results in a highly structured mature retinal organoid.

BACKGROUND: Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. METHODS: A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. RESULTS: Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. CONCLUSIONS: This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.

SPACR Encoded by IMPG1 Is Essential for Photoreceptor Survival by Interplaying between the Interphotoreceptor Matrix and the Retinal Pigment Epithelium.

Several pathogenic variants have been reported in the IMPG1 gene associated with the inherited retinal disorders vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). IMPG1 and its paralog IMPG2 encode for two proteoglycans, SPACR and SPACRCAN, respectively, which are the main components of the interphotoreceptor matrix (IPM), the extracellular matrix surrounding the photoreceptor cells. To determine the role of SPACR in the pathological mechanisms leading to RP and VMD, we generated a knockout mouse model lacking Impg1, the mouse ortholog. Impg1-deficient mice show abnormal accumulation of autofluorescent deposits visible by fundus imaging and spectral-domain optical coherence tomography (SD-OCT) and attenuated electroretinogram responses from 9 months of age. Furthermore, SD-OCT of Impg1-/- mice shows a degeneration of the photoreceptor layer, and transmission electron microscopy shows a disruption of the IPM and the retinal pigment epithelial cells. The decrease in the concentration of the chromophore 11-cis-retinal supports this loss of photoreceptors. In conclusion, our results demonstrate the essential role of SPACR in maintaining photoreceptors. Impg1-/- mice provide a novel model for mechanistic investigations and the development of therapies for VMD and RP caused by IMPG1 pathogenic variants.

Generation of a human iPSC line, INMi005-A, from a patient with non-syndromic USH2A-associated retinitis pigmentosa.

We report here the generation of the human iPSC line INMi005-A from a patient with non-syndromic autosomal recessive retinitis pigmentosa caused by compound heterozygous mutations in the USH2A gene. The reprogramming of primary human dermal fibroblasts was performed using the non-integrative Sendai virus method and the OSKM transcription factor cocktail. The generated INMi005-A iPSC line is pluripotent and genetically stable, and will represent a valuable tool for understanding the pathophysiology associated with USH2A mutations.

Identification of the First Single GSDME Exon 8 Structural Variants Associated with Autosomal Dominant Hearing Loss.

GSDME, also known as DFNA5, is a gene implicated in autosomal dominant nonsyndromic hearing loss (ADNSHL), affecting, at first, the high frequencies with a subsequent progression over all frequencies. To date, all the GSDME pathogenic variants associated with deafness lead to skipping of exon 8. In two families with apparent ADNSHL, massively parallel sequencing (MPS) integrating a coverage-based method for detection of copy number variations (CNVs) was applied, and it identified the first two causal GSDME structural variants affecting exon 8. The deleterious impact of the c.991-60_1095del variant, which includes the acceptor splice site sequence of exon 8, was confirmed by the study of the proband's transcripts. The second mutational event is a complex rearrangement that deletes almost all of the exon 8 sequence. This study increases the mutational spectrum of the GSDME gene and highlights the crucial importance of MPS data for the detection of GSDME exon 8 deletions, even though the identification of a causal single-exon CNV by MPS analysis is still challenging.

Reclassification of a TMC1 synonymous substitution as a variant disrupting splicing regulatory elements associated with recessive hearing loss.

Alterations of the transmembrane channel-like 1 gene (TMC1) are involved in autosomal recessive and dominant nonsyndromic hearing loss (NSHL). To date, up to 117 causal variants including substitutions, insertions and splice variants have been reported in families from different populations. In a patient suffering from severe prelingual NSHL, we identified, in the homozygous state, the previously considered likely benign synonymous c.627C>T; p.(Leu209=) substitution. We used in silico tools predicting variant-induced alterations of splicing regulatory elements (SREs) and pinpointed this transition as a candidate splice-altering variation. Functional splicing analysis, using a minigene assay, confirmed that the variant altered a critical regulatory sequence which is essential for the exon 11 inclusion in the TMC1 transcripts. This result was reinforced by the analysis of orthologous TMC1 mammalian sequences for which the deleterious effect on the mRNA processing of a native thymidine was always counteracted by the presence of a stronger donor splice site or additional enhancer motifs. This study demonstrates, for the first time, the pathogenicity of the c.627C>T alteration leading to its reclassification as a causal variant impacting SREs and highlights the major importance of exhaustive studies to accurately evaluate the pathogenicity of a variant, regardless of the variation type.

CRISPR/Cas9-Mediated Genome Editing to Generate Clonal iPSC Lines.

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian cells. The coupling of both human (h)iPSCs and CRISPR/Cas9 technology offers great promise for cell therapy and regenerative medicine. However, several limitations including time and labor consumption, efficiency and efficacy of the system, and the potential off-targets effects induced by the Cas9 nuclease still need to be addressed. Here, we describe a detailed method for easily engineering genetic changes in hiPSCs, using a nucleofection-mediated protocol to deliver the CRISPR/Cas9 components into the cells, and discuss key points to be considered when designing your experiment. The clonal, genome-edited hiPSC line generated via our method can be directly used for downstream applications.

The Study of a 231 French Patient Cohort Significantly Extends the Mutational Spectrum of the Two Major Usher Genes MYO7A and USH2A.

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing loss combined with retinitis pigmentosa, and in some cases, vestibular areflexia. Three clinical subtypes are distinguished, and MYO7A and USH2A represent the two major causal genes involved in Usher type I, the most severe form, and type II, the most frequent form, respectively. Massively parallel sequencing was performed on a cohort of patients in the context of a molecular diagnosis to confirm clinical suspicion of Usher syndrome. We report here 231 pathogenic MYO7A and USH2A genotypes identified in 73 Usher type I and 158 Usher type II patients. Furthermore, we present the ACMG classification of the variants, which comprise all types. Among them, 68 have not been previously reported in the literature, including 12 missense and 16 splice variants. We also report a new deep intronic variant in USH2A. Despite the important number of molecular studies published on these two genes, we show that during the course of routine genetic diagnosis, undescribed variants continue to be identified at a high rate. This is particularly pertinent in the current era, where therapeutic strategies based on DNA or RNA technologies are being developed.

CRB1-Related Retinal Dystrophies in a Cohort of 50 Patients: A Reappraisal in the Light of Specific Müller Cell and Photoreceptor CRB1 Isoforms.

Pathogenic variants in CRB1 lead to diverse recessive retinal disorders from severe Leber congenital amaurosis to isolated macular dystrophy. Until recently, no clear phenotype-genotype correlation and no appropriate mouse models existed. Herein, we reappraise the phenotype-genotype correlation of 50 patients with regards to the recently identified CRB1 isoforms: a canonical long isoform A localized in Müller cells (12 exons) and a short isoform B predominant in photoreceptors (7 exons). Twenty-eight patients with early onset retinal dystrophy (EORD) consistently had a severe Müller impairment, with variable impact on the photoreceptors, regardless of isoform B expression. Among them, two patients expressing wild type isoform B carried one variant in exon 12, which specifically damaged intracellular protein interactions in Müller cells. Thirteen retinitis pigmentosa patients had mainly missense variants in laminin G-like domains and expressed at least 50% of isoform A. Eight patients with the c.498_506del variant had macular dystrophy. In one family homozygous for the c.1562C>T variant, the brother had EORD and the sister macular dystrophy. In contrast with the mouse model, these data highlight the key role of Müller cells in the severity of CRB1-related dystrophies in humans, which should be taken into consideration for future clinical trials.

Molecular Therapy for Choroideremia: Pre-clinical and Clinical Progress to Date.

Choroideremia is an inherited retinal disease characterised by a degeneration of the light-sensing photoreceptors, supporting retinal pigment epithelium and underlying choroid. Patients present with the same symptoms as those with classic rod-cone dystrophy: (1) night blindness early in life; (2) progressive peripheral visual field loss, and (3) central vision decline with a slow progression to legal blindness. Choroideremia is monogenic and caused by mutations in CHM. Eight clinical trials (three phase 1/2, four phase 2, and one phase 3) have started (four of which are already finished) to evaluate the therapeutic efficacy of gene supplementation mediated by subretinal delivery of an adeno-associated virus serotype 2 (AAV2/2) vector expressing CHM. Furthermore, one phase 1 clinical trial has been initiated to evaluate the efficiency of a novel AAV variant to deliver CHM to the outer retina following intravitreal delivery. Lastly, a non-viral-mediated CHM replacement strategy is currently under development, which could lead to a future clinical trial. Here, we summarise the rationale behind these various studies, as well as any results published to date. The diversity of these trials currently places choroideremia at the forefront of the retinal gene therapy field. As a consequence, the trial outcomes, regardless of the results, have the potential to change the landscape of gene supplementation for inherited retinal diseases.

Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.

Novel roles for voltage-gated T-type Ca2+ and ClC-2 channels in phagocytosis and angiogenic factor balance identified in human iPSC-derived RPE.

Human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) is a powerful tool for pathophysiological studies and preclinical therapeutic screening, as well as a source for clinical cell transplantation. Thus, it must be validated for maturity and functionality to ensure correct data readouts and clinical safety. Previous studies have validated hiPSC-derived RPE as morphologically characteristic of the tissue in the human eye. However, information concerning the expression and functionality of ion channels is still limited. We screened hiPSC-derived RPE for the polarized expression of a panel of L-type (CaV 1.1, CaV 1.3) and T-type (CaV 3.1, CaV 3.3) Ca2+ channels, K+ channels (Maxi-K, Kir4.1, Kir7.1), and the Cl- channel ClC-2 known to be expressed in native RPE. We also tested the roles of these channels in key RPE functions using specific inhibitors. In addition to confirming the native expression profiles and function of certain channels, such as L-type Ca2+ channels, we show for the first time that T-type Ca2+ channels play a role in both phagocytosis and vascular endothelial growth factor (VEGF) secretion. Moreover, we demonstrate that Maxi-K and Kir7.1 channels are involved in the polarized secretion of VEGF and pigment epithelium-derived factor (PEDF). Furthermore, we show a novel localization for ClC-2 channel on the apical side of hiPSC-derived RPE, with an overexpression at the level of fluid-filled domes, and demonstrate that it plays an important role in phagocytosis, as well as VEGF and PEDF secretion. Taken together, hiPSC-derived RPE is a powerful model for advancing fundamental knowledge of RPE functions.

Retinitis Punctata Albescens and RLBP1-Allied Phenotypes: Phenotype-Genotype Correlation and Natural History in the Aim of Gene Therapy.

Purpose: To identify relevant criteria for gene therapy based on clinical and genetic characteristics of rod-cone dystrophy associated with RLBP1 pathogenic variants in a large cohort comprising children and adults. Design: Retrospective cohort study. Participants: Patients with pathogenic variants in RLBP1 registered in a single French reference center specialized in inherited retinal dystrophies. Methods: Clinical, multimodal imaging, and genetic findings were reviewed. Main Outcome Measures: Age of onset; visual acuity; ellipsoid line length; nasal, temporal, and foveal retinal thickness; and pathogenic variants and related phenotypes, including Newfoundland rod-cone and Bothnia dystrophies (NFRCDs), were reappraised. Results: Twenty-one patients (15 families) were included. The most frequent form was NFRCD with 12 patients (8 families) homozygous for the recurrent deletion of exons 7 through 9 in RLBP1 and 5 patients (4 families) with biallelic protein-truncating variants (2 novel: p.Gln16∗ and p.Tyr251∗). A novel combination of the p.Arg234Trp Bothnia variant with a nonsense variant in trans led to Bothnia dystrophy in 2 sisters. One proband carrying the p.Met266Lys Bothnia variant and in trans p.Arg121Trp and a second, with the p.Arg9Cys and p.Tyr111∗ combination, both demonstrated mild retinitis punctata albescens. Independently of genotype, all patients showed a visual acuity of worse than 20/200, an ellipsoid line width of less than 1000 µm, and a mean foveal thickness of less than 130 to 150 µm, with loss of both the interdigitation and ellipsoid lines. Conclusions: The eligibility for RLBP1 gene therapy first should be determined according to the biallelic variant combination using a robust classification as proposed herein. An ellipsoid line width of more than 1200 µm and a central thickness of more than 130 to 150 µm with detectable ellipsoid and interdigitation lines should be 2 prerequisite imaging indicators for gene therapy.

Preclinical pharmacology of a lipophenol in a mouse model of light-induced retinopathy.

Environmental light has deleterious effects on the outer retina in human retinopathies, such as ABCA4-related Stargardt's disease and dry age-related macular degeneration. These effects involve carbonyl and oxidative stress, which contribute to retinal cell death and vision loss. Here, we used an albino Abca4-/- mouse model, the outer retina of which shows susceptibility to acute photodamage, to test the protective efficacy of a new polyunsaturated fatty acid lipophenol derivative. Anatomical and functional analyses demonstrated that a single intravenous injection of isopropyl-phloroglucinol-DHA, termed IP-DHA, dose-dependently decreased light-induced photoreceptor degeneration and preserved visual sensitivity. This protective effect persisted for 3 months. IP-DHA did not affect the kinetics of the visual cycle in vivo or the activity of the RPE65 isomerase in vitro. Moreover, IP-DHA administered by oral gavage showed significant protection of photoreceptors against acute light damage. In conclusion, short-term tests in Abca4-deficient mice, following single-dose administration and light exposure, identify IP-DHA as a therapeutic agent for the prevention of retinal degeneration.

Study of Usutu virus neuropathogenicity in mice and human cellular models.

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.

Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles.

Inherited retinal dystrophies (IRDs) are characterized by progressive photoreceptor degeneration and vision loss. Usher syndrome (USH) is a syndromic IRD characterized by retinitis pigmentosa (RP) and hearing loss. USH is clinically and genetically heterogeneous, and the most prevalent causative gene is USH2A. USH2A mutations also account for a large number of isolated autosomal recessive RP (arRP) cases. This high prevalence is due to two recurrent USH2A mutations, c.2276G>T and c.2299delG. Due to the large size of the USH2A cDNA, gene augmentation therapy is inaccessible. However, CRISPR/Cas9-mediated genome editing is a viable alternative. We used enhanced specificity Cas9 of Streptococcus pyogenes (eSpCas9) to successfully achieve seamless correction of the two most prevalent USH2A mutations in induced pluripotent stem cells (iPSCs) of patients with USH or arRP. Our results highlight features that promote high target efficacy and specificity of eSpCas9. Consistently, we did not identify any off-target mutagenesis in the corrected iPSCs, which also retained pluripotency and genetic stability. Furthermore, analysis of USH2A expression unexpectedly identified aberrant mRNA levels associated with the c.2276G>T and c.2299delG mutations that were reverted following correction. Taken together, our efficient CRISPR/Cas9-mediated strategy for USH2A mutation correction brings hope for a potential treatment for USH and arRP patients.

A Novel Chromosomal Translocation Identified due to Complex Genetic Instability in iPSC Generated for Choroideremia.

Induced pluripotent stem cells (iPSCs) have revolutionized the study of human diseases as they can renew indefinitely, undergo multi-lineage differentiation, and generate disease-specific models. However, the difficulty of working with iPSCs is that they are prone to genetic instability. Furthermore, genetically unstable iPSCs are often discarded, as they can have unforeseen consequences on pathophysiological or therapeutic read-outs. We generated iPSCs from two brothers of a previously unstudied family affected with the inherited retinal dystrophy choroideremia. We detected complex rearrangements involving chromosomes 12, 20 and/or 5 in the generated iPSCs. Suspecting an underlying chromosomal aberration, we performed karyotype analysis of the original fibroblasts, and of blood cells from additional family members. We identified a novel chromosomal translocation t(12;20)(q24.3;q11.2) segregating in this family. We determined that the translocation was balanced and did not impact subsequent retinal differentiation. We show for the first time that an undetected genetic instability in somatic cells can breed further instability upon reprogramming. Therefore, the detection of chromosomal aberrations in iPSCs should not be disregarded, as they may reveal rearrangements segregating in families. Furthermore, as such rearrangements are often associated with reproductive failure or birth defects, this in turn has important consequences for genetic counseling of family members.