Structural and splicing RPGRIP1 mutations in inherited retinal degenerations

Abstract

Purpose : The focus of clinical diagnostic sequencing panels for inherited retinal degenerations (IRDs) has been on coding sequence mutations. We tested the hypothesis that non-coding splicing and structural mutations contribute significantly to RPGRIP1 associated disease. We analyzed 7 families with a single loss of function (LoF) mutation in RPGRIP1 for non-coding second-hit mutations.

Methods : Patients provided informed consent and were followed at the Massachusetts Eye and Ear. The study was approved by the institutional review board and adhered to the Declaration of Helsinki. All sequencing was done on DNA extracted from blood. From 1722 families evaluated by panel sequencing, 7 families with a single LoF mutation in RPGRIP1 underwent whole exome (WES) and whole genome sequencing (WGS). In additional to nucleotide variant calling, genome STRiP structural analysis was performed. Variants with a frequency of less than 0.003 in gnomaD that exhibited proper segregation patterns were evaluated in vitro via mini-gene assays in two cell lines (HEK293T and WERI-Rb1), RT-PCR, qPCR, and Sanger sequencing.

Results : Among 1722 IRD families, 3 had biallelic LoF coding mutations corresponding to the reported rate of mutations in this gene in the literature. 7 additional families had a single LoF mutation in RPGRIP1 and were the focus of this study. WES and WGS identified non-coding variants in 7/7 families, including 3 deep intronic mutations, 3 structural aberrations and 1 variant of unclear significance. 1/3 intronic mutations led to splicing aberrations in vitro. The 3 structural mutations were mapped and shown to lead to LoF alleles. Overall, we identified 10 novel RPGRIP1 mutations: 6 novel coding and 4 validated non-coding. Additionally, comparison of splicing assays in a cell line of non-retinal origin vs. a cell line of retinal origin will be presented.

Conclusions : Our results are consistent with the hypothesis that non-coding mutations significantly contribute to RPGRIP1 mediated IRD. We identified 7 potentially significant non-coding mutations and validated 4 in vitro. These results increase the prevalence of RPGRIP1-associated cases by two-fold or more. Thus we believe the previously reported frequency of RPGRIP1 mediated IRD is an underestimate. Consideration of non-coding RPGRIP1 mutations will be important for clinical diagnostics and future therapeutic trials.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.