Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

Abstract

Purpose: Many patients with clinical features of ABCA4-associated retinal disease have one or no disease-causing mutations found after screening the entire ABCA4 coding sequence. The purpose of this experiment was to identify as many non-exomic disease-causing ABCA4 mutations as possible.

Methods: We hypothesized that mutations near minor splice junctions in ABCA4 might increase the probability of missplicing at these sites. We performed next generation sequencing of RNA extracted from normal eye bank eyes to identify and characterize minor ABCA4 transcripts. We screened 15 patients who had clear clinical evidence of recessive Stargardt disease but only one coding sequence mutation in ABCA4 for mutations at 15 of the most convincing alternate splice junctions suggested by the RNA sequencing data. Stable cell lines were derived from patients who harbored novel variants at these junctions and RNA from these cells was used to assess splicing of ABCA4. A second cohort of 79 Stargardt patients was used to validate these results.

Results: Four of 15 individuals in the initial cohort (and none of 412 controls) were found to harbor a single novel intronic mutation that increased the predicted strength of a cryptic splice recognition sequence. Analysis of RNA obtained from stable cell lines prepared from 2 of these patients revealed improper splicing at this site. Screening of the second Stargardt cohort revealed 4 additional patients with this mutation. An additional intronic variant affecting splicing of the same exon was also identified and confirmed by RNA analysis. Finally, we noted that one of the most abundant minor ABCA4 transcripts in human retina contains an improperly spliced exon 46. A rare synonymous codon variant at this position (Val2114Val), previously thought to be a non-disease-causing polymorphism, accentuates missplicing at this locus and inactivates the allele. Collectively, these three new mutations account for 12 of 94 (12.7%) previously undetectable disease-causing ABCA4 variants.

Conclusions: Each new disease-causing ABCA4 variant that is discovered improves the sensitivity of clinical testing for Stargardt disease and other ABCA4-associated retinal diseases. It also increases the accuracy of genetic counseling that can be given to patients and increases the number of individuals eligible for clinical trials of gene-replacement and other therapies for these diseases.

Keywords: 688 retina • 539 genetics • 721 stem cells