Duped by a duplication: Heterozygous duplication in PRPF31 causes Retinitis pigmentosa in elusive gene family

Inherited retinal degenerations (IRD) are an important cause of blindness affecting 1/4000 people worldwide. Presently, over 270 genes have been implicated in IRDs, yet 30-40% cases remain undiagnosed. The goal of this study was to determine genetic causality in a family with apparent autosomal recessive RP. Subjects were given an ophthalmic exam at the Retinal Service at MEE and consented to genetic testing. Whole exome sequencing(WES) was performed on the parents and affected children. The identified variants were Sanger validated and found to segregate with disease. Functional assays were conducted in zebrafish, ciliated cell lines(mIMCD3) and mice retinae, with splicing assays in relevant cell lines. WES identified rare variants in two potentially novel IRD genes; GNL3 (G Protein Nucleolar 3) c.1187+3A>C, c.1568-8C>A and PDE4DIP (Phosphodiester 4D Interacting Protein) c.3868G>A (p.Glu1290Lys), c.4603G>A (p.Ala1535Thr). Morpholino (MO) knockdown of gnl3 in zebrafish embryos led to an unspecific phenotype and minigene splicing assays of GNL3 variants showed that c.1187+3A>C affected splicing while c.1568-8C>A had no effect eliminating GNL3 as a candidate. MO knockdown of pde4dip in zebrafish led to a specific retinal phenotype. Since PDE4DIP has been shown to interact with Usher interactome, we further studied the functional of PDE4DIP missense variants to determine their effects on ciliogenesis. No significant difference in localization was found between wild type and variant PDE4DIP in both mice and mIMCD3 ciliated cell line. In addition, the wild-type and mutant PDE4DIP mRNA was able to rescue the MO-induced phenotype to similar extent, excluding this gene as a candidate. Subsequently, samples were reanalyzed with updated bioinformatics pipeline which led to the detection of a 94bp duplication in PRPF31 (pre-mRNA Processing Factor 31) c.73_166dup (p.Asp56GlyfsTer33) believed to be the cause of disease in this family. Missing genetic causality in inherited retinal disease is more likely present in known genes currently missed with existing sequencing and analytical strategies. Reanalysis of sequencing with updated bioinformatics pipelines led to the discovery of a 94bp duplication in PRPF31 resulting in a null allele, most likely cause of disease given haploinsufficiency of PRPF31 has been shown to cause retinal degeneration. This is a 2020 ARVO Annual Meeting abstract.