Identification of a Complex Allele in IMPG2 as a Cause of Adult-Onset Vitelliform Macular Dystrophy

Abstract

Purpose Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders with approximately 270 genes involved. IMPG2 is associated with adult-onset vitelliform macular dystrophy. Here, we investigated two unrelated patients with vitelliform macular dystrophy to identify the underlying genetic cause. Methods Whole-exome sequencing identified a putative causal complex allele consisting of c.3023-15T>A and c.3023G>A (p.(Gly1008Asp)) in IMPG2 in both individuals. To assess its effect, in vitro splice assays in HEK293T and further characterization in patient-derived photoreceptor precursor cells (PPCs) were conducted. Results The results of the midigene splice assays in HEK293T showed that the complex allele causes a variety of splicing defects ranging from a small deletion to (multiple-)exon skipping. This finding was further validated using patient-derived PPCs that showed a significant increase of out-of-frame transcripts lacking one or multiple exons compared to control-derived PPCs. Overall, control PPCs consistently showed low levels of aberrantly spliced IMPG2 transcripts that were highly elevated in patient-derived PPCs. These differences were even more obvious upon inhibition of nonsense-mediated decay with cycloheximide. Conclusions We report a heterozygous complex allele in IMPG2 causative for adult-onset vitelliform macular dystrophy in two unrelated individuals with mild visual loss and bilateral vitelliform lesions. The predicted causal missense mutation c.3023G>A, located in the consensus splice acceptor site, enhances the splicing effect of the upstream variant c.3023-15T>A, leading to the generation of aberrant transcripts that decrease the full-length IMPG2 levels. These results suggest a haploinsufficiency mechanism of action and highlight the complementarity of using different models to functionally assesses splicing defects.

Phenome- and genome-wide analyses of retinal optical coherence tomography images identify links between ocular and systemic health

“…IMPG2 encodes for the interphotoreceptor matrix proteoglycan 2 protein known to contribute to the organization of the interphotoreceptor matrix. IMPG2 defects are a cause of retinitis pigmentosa and IMPG2-related maculopathy ( 34 – 36 ). This gene was also a locus identified in the common variant genome-wide association analyses for PS thickness, further highlighting its important role, across both common and rare genomic variants, in PS thickness.…”

Analysis of the impact of DGAT1 p.M435L and p.K232A variants on pre-mRNA splicing in a full-length gene assay

“…The employment of a cell line originating from a tissue or a species different from those of the target gene is not really a matter since the splicing code is highly conserved between closely-related species 19 , as well as cell lines isolated from different organs showed most of the time comparable results when used to perform splicing reporter assays 20 . For instance, the HEK293T cells have been successfully used to analyse hundreds of variants in tens of genes expressed in many different tissues using minigene and midigene assays or FLGA (for examples, see references cited in introduction or 21 – 23 ). Also, we showed that plasmid contamination can be decreased to an acceptable rate by using DNase treatment.…”

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

“…In fact, some studies have highlighted inconsistencies between splicing aberrations detected using midigenes or simpler cellular models, as opposed to more sophisticated models derived from reprogrammed induced pluripotent stem cells, such as photoreceptor precursor cells or retinal organoids. As the genomic context of the complex models closely resembles that of the native retinal environment, these are more representative of the actual splicing process in the retina ( 29 , 32 , 39 , 40 ). These findings point to the limits imposed by the midigene system.…”