Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290 loss-of-function in individuals with unusually mild retinal disease

Abstract

CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base-pair deletion in exon 17, introducing a premature termination codon (PTC) in exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in exon 8 (c.508A>T, p.Lys170*) and exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing (NAS) alone (exon 8), or with BES (exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.

Targeted exon skipping of a CEP290 mutation rescues Joubert syndrome phenotypes in vitro and in a murine model

“…Following a report of a patient with a mild LCA phenotype associated with nonsense-mediated alternative splicing of CEP290 (11), an extensive study of endogenous (wild-type) splicing revealed widespread, low-level alternative splicing that could be modeled to predict genetic pleiotropy associated with CEP290 mutations (13). Indeed, confirming this hypothesis, endogenous basal exon skipping and nonsense-associated altered splicing has been documented in patient fibroblasts with nonsense mutations in CEP290 with mild retinal phenotypes (14). These observations are reminiscent of the Duchenne/Becker muscular dystrophy paradigm (15), which responds to targeted exon skipping therapies (16, 17).…”

A Mild Phenotype Caused by Two Novel Compound Heterozygous Mutations in CEP290

“…Two patients with biallelic CEP290 mutations described by Barny et al as having an unusually mild retinal disease in fact had macular involvement resulting in limited vision; one patient developed a chronic macular oedema refractory to treatment from the age of 10, and the other showed nystagmus and a poor visual acuity of 0.05 in RE and 0.2 in LE. Both had a non-recordable photopic response to the ERG [ 16 ].…”

Aminoglycoside drugs induce efficient read-through ofCDKL5nonsense mutations, slightly restoring its kinase activity

“…We are aware that our in vitro assays have limitations. Indeed, PTCs can affect splicing, possibly inducing exon skipping [35,36]. Further, it is well known that the level of nonsense transcripts can significantly affect read-through efficiency.…”