Validating splice altering ‘variants of uncertain significance’ in genetically unsolved Leber congenital amaurosis patients using the RHCglo minigene

Abstract

Purpose : A confident molecular diagnosis is essential for patients that have the potential to be considered for personalized treatment. Many Mendelian diseases are often genetically heterogeneous which impedes the ability to confidently assign a molecular diagnosis to a significant portion of patients. Leber congenital amaurosis (LCA) is an inherited retinal disease in which ~30% of patients forgo a confident molecular diagnosis after sequencing associated disease genes. For our LCA patient cohort, we tested the hypothesis that variants of uncertain significance affect RNA splicing patterns by using computational splicing tools and an in vitro minigene assay.

Methods : Using exon-capture next generation sequencing data a member of our laboratory, Zachary Soens, identified splice disruption candidates within splice site regions by obtaining scores from the database of splicing consensus SNV’s (dbscSNV). For rare exonic variants outside splicing consensus regions, NNsplice was used to assess a variants ability to strengthen a cryptic splice site. These scores were then applied to our LCA cohort of 714 patients. Each variant that possesses a score and matches a genes inheritance pattern is then validated using a minigene vector termed “RHCglo.”

Results : We discovered 17 rare variants within 20 patients predicted to alter RNA splicing patterns. From these, 5 nonsynonymous variants were identified in 8 patients, 4 synonymous variants in 4 patients, and 8 intronic variants in 8 patients. All 17 variants caused a disruption in RNA splicing patterns by comparing to a patient/placenta wild-type sequence. 14 variants have caused exon loss, 2 variants have caused truncation of an exon, and 1 variant has caused intron retention.

Conclusions : Most of these splice altering variants have caused the loss of an entire exon thereby leading to the absence or truncation of a protein. We can therefore assign a confident molecular diagnosis to these patients. We suggest for other groups working with genetically heterogeneous Mendelian diseases use these splicing tools to identify splice-altering variants for patient cohorts to achieve a higher solving rate.

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