Assessing Variant Causality and Severity Using Retinal Pigment Epithelial Cells Derived from Stargardt Disease Patients

Abstract

Purpose Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance This “Disease in a Dish” approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.

Human pluripotent stem cells for the modelling of retinal pigment epithelium homeostasis and disease: A review

“…43 In comparing patient phenotypes, the higher the missense variant expression, the more deleterious outcomes for the patient, and iPSC studies confirmed this genotype-phenotype correlation in the severity of ABCA4 variants. 38,43 Consistent with STGD1 disease phenotypes, the transcriptomic analysis of the patient iPSC-derived RPE cells, links oxidative and endoplasmic reticulum stress, likely symptoms of reactive responses rather than primary protein alterations due to ABCA4 variants. 38,43 This study highlights the benefits of using iPSC-derived RPE to assess casual variants in difficult to detect genomic regions.…”

“…38,43 Consistent with STGD1 disease phenotypes, the transcriptomic analysis of the patient iPSC-derived RPE cells, links oxidative and endoplasmic reticulum stress, likely symptoms of reactive responses rather than primary protein alterations due to ABCA4 variants. 38,43 This study highlights the benefits of using iPSC-derived RPE to assess casual variants in difficult to detect genomic regions.…”

“…43 The allele-specific expression results showed normal ABCA4 transcripts were present across splice variants, however, the corresponding protein was not detected. 43 In this study, all patient lines showed upregulation in the unfolded protein response pathway implying membrane mislocalization and protein misfolding, indiscriminate between missense and splice variants. 43 In comparing patient phenotypes, the higher the missense variant expression, the more deleterious outcomes for the patient, and iPSC studies confirmed this genotype-phenotype correlation in the severity of ABCA4 variants.…”

“…43 In a patient line containing two ABCA4 splice variants, aberrantly produced polypeptides were shortlived in the endoplasmic reticulum. 43 The allele-specific expression results showed normal ABCA4 transcripts were present across splice variants, however, the corresponding protein was not detected. 43 In this study, all patient lines showed upregulation in the unfolded protein response pathway implying membrane mislocalization and protein misfolding, indiscriminate between missense and splice variants.…”

“…Using iPSC‐derived tissue to model a disease such as STGD1 is useful in attempts to link causality and severity with expression levels, since patient lines retain the complex genetic signatures created via natural splicing and editing that occurs in vivo. In one study, eight iPSC lines from patients harbouring a single pathogenic ABCA4 variant and normal controls were differentiated into RPE and subjected to RNA equencing and protein analysis 43 . The resulting principal component analysis of the iPSC‐derived RPE transcriptomes showed the controls clustering distinctly from the patient samples, indicating the differentially expressed genes are driven by ABCA4 mutations 43 .…”