Hidden genetic variation in Stargardt disease: novel copy number variations, cis-regulatory and deep-intronic splice variants within the ABCA4 locus

Abstract

Purpose : A large proportion of Stargardt disease (STGD1) patients have single coding variants in the disease gene ABCA4, suggestive of hidden genetic variations in non-coding regions. We aimed to assess the contribution of copy number variations (CNVs) and non-coding sequence variations in mono-allelic STGD1 patients. Moreover, we attempted to gain more insight into the cis-regulatory landscape of ABCA4.

Methods : A total of 116 monoallelic STGD1 patients underwent targeted resequencing of a conserved block of synteny encompassing the ABCA4 gene (HaloPlex enrichment). Filtering of variants was based on criteria such as publicly available minor allele frequencies and frequency in the total cohort of sequenced patients. Altered splice predictions, presence in cis-regulatory regions of ABCA4 and RegulomeDB data served as a secondary filter. Candidate splice variants were tested by mini-gene assays while putative cis-regulatory variants were investigated by ex vivo electroporation in mouse retinas. 4C-seq was performed on human retinal cells, using an anchor in the ABCA4 promoter region. Customized arrayCGH (arrEYE) was used for CNV analysis of 5 patients.

Results : Mini-gene assays were performed for a subset of 12 intronic variants, confirming a splice effect for 3 variants. The cis-regulatory effect of 3 promoter and 2 deep intronic variants were tested in mouse retinal explants, revealing a significant effect on regulation for 2 of the promoter variants. A chromatin interaction map of the ABCA4 region was generated by 4C-seq in human adult retinal cells. 11 putative cis-regulatory variants are being tested by luciferase assays in Y79 cells and nine putative splice variants are being assessed by mini-gene assays. CNV analysis revealed a novel ABCA4 deletion (ex 40-50) and a duplication (ex 2-6) in 2/5 patients without candidate non-coding variants.

Conclusions : Resequencing of the whole ABCA4 locus uncovered novel deep intronic splice variants and cis-acting regulatory variants in unsolved cases of STGD1, representing the first report of non-coding regulatory ABCA4 variants. In addition, a chromatin interaction dataset for the ABCA4 locus was generated in retinal cells. Finally, apart from a novel deletion, the first duplication was identified in ABCA4, expanding the CNV spectrum which represents a very small fraction of the known mutational load in ABCA4.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.