Beyond the Exon: Variants in regulatory and non-coding regions play a functional role in the pathogenesis of inherited retinal disease


Evan Jones1,2, Justin Branch1,2, Ruifang Sui3, Robert K Koenekoop4, Fernanda Belga Ottoni Porto5, Aleksandar Milosavljevic1, Yumei Li1,2, Rui Chen1,2


  1. Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
  2. Human Genome Sequencing Center, Baylor College of Medicine Houston, TX
  3. Department of Ophthalmology, Peking Union Medical College, Beijing, China
  4. Ocular Genetics Laboratory, McGill University, Montreal, Canada
  5. INRET – Clinica e Centro de Pesquisa, Belo Horizonte, Minas Gerais, Brazil



Purpose: Increasing evidence supports the hypothesis that non-coding variants may play a functional role in inherited retinal diseases, such as Stargardt disease (STGD). STGD is a recessive macular degenerative disease caused by mutations in the gene, ABCA4. The purpose of this work is to characterize the pathogenic role of non-coding variants in STGD patients. We hypothesize that STGD patients enriched for single exonic mutations in ABCA4 also harbor non-coding variants that play a functional role in contributing to disease.

Methods: Tissue-specific epigenetic profiling data was utilized to annotate functional non-coding regulatory regions, such as enhancers and suppressors in ABCA4. Targeted next-generation sequencing (NGS) was performed across the entire genomic locus of ABCA4 for 60 STGD individuals with a previously identified single exonic mutation in ABCA4. NGS data was aligned, processed and filtered against control datasets using an in-house custom pipeline. Non-coding rare variants identified in our annotated regulatory regions along with those predicted to affect splicing were tested for functional effects. These variants were functionally validated through in vitro cell line assays for their relative effects on gene expression and protein function.

Results: Analysis of tissue-specific epigenetic profiling data across the ABCA4 locus identified potential regulatory regions controlling ABCA4 expression. Through our NGS approach, targeting both introns and exons of ABCA4, we identified a number of recurrent variants within potential regulatory regions. We also identified a number of non-coding mutations predicted to disrupt splicing of ABCA4. We have functionally validated the role of non-coding splice-altering variants in ABCA4 and are validating the functional effects of additional recurrent variants within the characterized regulatory regions.

Conclusion: Through this work we show the role of non-coding variants in the functional dysregulation of ABCA4 in STGD patients. This research will help expand our understanding and ability to annotate and interpret non-coding variants that may contribute to disease and improve the rate of molecular diagnosis.