Development of in vitro and ex vivo tools for functional characterization of variants of uncertain significance in aniridia

Abstract

Purpose : PAX6 mutations lead to aniridia, a panocular disorder that usually involves iris and foveal hypoplasia, cataract, corneal dystrophy, as well as abnormalities of the optic nerve.
Here, we describe the development of two molecular approaches for the functional characterization of coding and non-coding variants with uncertain significance (VUS) and their impact on the canonical PAX6 splicing in aniridia patients.

Methods : For in vitro splice assays, a genomic segment encompassing the region of interest of PAX6 along with flanking sequences was amplified by PCR from genomic DNA and was cloned into an expression vector. VUS were introduced in the wild-type minigene constructs by site-directed mutagenesis. The minigene assays were performed by transient transfection into kidney HEK-293 and/or retinal ARPE-19 cell lines.
For ex vivo splice assays, lymphoblastoid cell lines (LCL) were established by Epstein Barr virus transformation of blood lymphocytes from patients carrying VUS and control healthy individuals.
Both HEK-293 transfected cells and lymphoblastoid cells were harvested and total RNA was extracted and reverse transcribed. The splicing patterns of mRNA transcripts were compared by semiquantitative PCR and sequencing.

Results : We have developed four minigene wild-type constructs involving the full exonic and intronic sequences of PAX6: i) the 5’UTR region (exons 1 to 4), ii) exons 5a and 6, iii) exons 5 to 7 and iv) exons 8 to 11, respectively. To date, a total of 13 variants have been assayed. 11 out 13 showed aberrant splicing patterns that include different mechanisms such as single or multiple exon skipping, exon elongations, intron retentions and new pseudoexon inclusions by the alternative use of cryptic splicing sites.
Ex-vivo expression analysis in LCL from 5 carrier patients showed alterations on splicing patterns. Two out five were also tested by in vitro splice assays and the findings on splicing patterns correlate with those observed in minigene assays.

Conclusions : Minigene PAX6 assays carrying multiple exons and LCL studies are useful strategies to gain insight into the pathogenicity of the poorly explored non-canonical splice PAX6 variants. These methods along with in silico analysis are easy-to-carry approaches for robust splice variant analysis and to help bring molecular diagnosis to aniridia patients.

This is a 2021 ARVO Annual Meeting abstract.