Gene Correction Recovers Phagocytosis in Retinal Pigment Epithelium Derived from Retinitis Pigmentosa-Human-Induced Pluripotent Stem Cells.

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone–rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants.Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases.Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis

Generation of gene-corrected human induced pluripotent stem cell lines derived from retinitis pigmentosa patient with Ser331Cysfs*5 mutation in MERTK

The human induced pluripotent stem cell (hiPSC) line RP1-FiPS4F1 generated from the patient with autosomal recessive retinitis pigmentosa (arRP) caused by homozygous Ser331Cysfs*5 mutation in Mer tyrosine kinase receptor (MERTK) was genetically corrected using CRISPR/Cas9 system. Two isogenic hiPSCs lines, with heterozygous and homozygous correction of c.992_993delCA mutation in the MERTK gene were generated. These cell lines demonstrate normal karyotype, maintain a pluripotent state, and can differentiate toward three germ layers in vitro. These genetically corrected hiPSCs represent accurate controls to study the contribution of the specific genetic change to the disease, and potentially therapeutic material for cell-replacement therapy

Gene Correction Recovers Phagocytosis in Retinal Pigment Epithelium Derived from Retinitis Pigmentosa-Human-Induced Pluripotent Stem Cells

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders

Aggregated Genomic Data as Cohort-Specific Allelic Frequencies can Boost Variants and Genes Prioritization in Non-Solved Cases of Inherited Retinal Dystrophies

The introduction of NGS in genetic diagnosis has increased the repertoire of variants and genes involved and the amount of genomic information produced. We built an allelic-frequency (AF) database for a heterogeneous cohort of genetic diseases to explore the aggregated genomic information and boost diagnosis in inherited retinal dystrophies (IRD). We retrospectively selected 5683 index-cases with clinical exome sequencing tests available, 1766 with IRD and the rest with diverse genetic diseases. We calculated a subcohort’s IRD-specific AF and compared it with suitable pseudocontrols. For non-solved IRD cases, we prioritized variants with a significant increment of frequencies, with eight variants that may help to explain the phenotype, and 10/11 of uncertain significance that were reclassified as probably pathogenic according to ACMG. Moreover, we developed a method to highlight genes with more frequent pathogenic variants in IRD cases than in pseudocontrols weighted by the increment of benign variants in the same comparison. We identified 18 genes for further studies that provided new insights in five cases. This resource can also help one to calculate the carrier frequency in IRD genes. A cohort-specific AF database assists with variants and genes prioritization and operates as an engine that provides a new hypothesis in non-solved cases, augmenting the diagnosis rate

Image1_ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease.tif

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone–rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants.Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48.Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases.Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.</p

Table1_ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease.pdf

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone–rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants.Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48.Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases.Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.</p