July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Reduced expression of “normal” ABCA4 alleles in iPS-derived RPE cells from Stargardt Disease patients
Author Affiliations & Notes
  • Anna Matynia
    Ophthalmology, Stein Eye Institute, Los Angeles, California, United States
  • Saravanan Karumbayaram
    Eli and Edythe Broad Stem Cell Research, California, United States
    Microbiology, Immunology and Molecular Genetics, UCLA, California, United States
  • Samuel Strom
    Fulgent Genetics, California, United States
  • Jane Hu
    Ophthalmology, Stein Eye Institute, Los Angeles, California, United States
  • Nermin Kady
    Ophthalmology, Stein Eye Institute, Los Angeles, California, United States
  • Roxana A Radu
    Ophthalmology, Stein Eye Institute, Los Angeles, California, United States
  • Michael B Gorin
    Ophthalmology, Stein Eye Institute, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Anna Matynia, None; Saravanan Karumbayaram, None; Samuel Strom, None; Jane Hu, None; Nermin Kady, None; Roxana Radu, None; Michael Gorin, None
  • Footnotes
    Support  Dalit S. and Elaine Sarkaria Charitable Foundation
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 388. doi:
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      Anna Matynia, Saravanan Karumbayaram, Samuel Strom, Jane Hu, Nermin Kady, Roxana A Radu, Michael B Gorin; Reduced expression of “normal” ABCA4 alleles in iPS-derived RPE cells from Stargardt Disease patients. Invest. Ophthalmol. Vis. Sci. 2019;60(9):388.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Despite the identification of over 900 exonic or intronic ABCA4 variants that range from unknown significance to pathogenic, many Stargardt Disease patients still do not have a complete molecular genetics diagnosis. ABCA4 is expressed only in photoreceptors and retinal pigment epithelium (RPE), limiting our ability to assess molecular phenotypes by any technique other than genomic sequencing. The fate of the “normal” allele in patients with a single identified variant was investigated using ABCA4 gene expression in retinal pigment epithelium derived from skin biopsies via induced pluripotent stem (iPS) cells in three such patients (c.3386G>T; c.3364G>A and c.570+1978A>G).

Methods : Three independent iPS cell lines were generated from skin biopsies of three patients, each with a single identified ABCA4 mutation, and the non-integrating SimpliconTMRNA Reprogramming method. All patient iPS cell lines and a normal control iPS cell line, were differentiated into mature retinal pigment epithelium, assessed by characteristic RPE morphology, pigmentation, specific marker expression and transepithelial resistance. The known mutations were confirmed using genomic sequencing from the same culture used to extract total RNA. RNASeq was performed, sequences aligned using STAR and analyzed by Cufflinks. Allele frequencies were assessed using the Integrative Genomics Viewer.

Results : Differentiation and maturation for each RPE cell line occurred at similar rates and ABCA4 transcripts were detected in all cell lines. The known mutations were identified by RNASeq. “Normal” allele read frequencies were approximately 20%G for RPE lines with c.3386G>T variant, 31%G for RPE lines with the c.3367G>A variant. The RPE lines with the splice variant (c570+1978A>G) had reduced levels of ABCA4 expression.

Conclusions : Gene integration free patient-specific iPSC derived RPE cells provide an opportunity to identify the fate of an otherwise normal allele in confirmed cases of retinal disease in which gene expression patterns limit the ability to perform molecular genetic analyses beyond genomic sequencing. Both the mutated and “normal” ABCA4 alleles are expressed in RPE cells from Stargardt Disease patients with a single pathogenic variant, although expression of the “normal” allele is significantly reduced. This reduced expression indicates a cis-acting factor that either result in a hypomorphic or non-functional allele.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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