Abstract
Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.
Highlights
Leber congenital amaurosis (LCA; OMIM 204000) is a group of rare and severe inherited retinal dystrophies with a prevalence of ~1:50,000 individuals worldwide [1,2]
In order to generate a humanized knockin mouse model that would mimic the genotype and phenotype associated with the deep-intronic LCA-causing mutation, a recombination strategy was designed that consisted of the replacement of mouse exons and, as well as intron 25, by the human counterparts (Figure 1B)
In the field of vision science, they are useful because of their relatively short lifespan, which allows a quick read-out of the progression of retinal degeneration
Summary
Leber congenital amaurosis (LCA; OMIM 204000) is a group of rare and severe inherited retinal dystrophies with a prevalence of ~1:50,000 individuals worldwide [1,2]. The clinical characteristics of LCA include severe and early vision loss that appears in the first year of life, amaurotic pupils, sensory nystagmus and the absence of electrical signals on electroretinogram (ERG) [3]. Like other retinal disorders such as retinitis pigmentosa (RP; OMIM 268000), LCA shows a high genetic heterogeneity. Mutations in 19 different genes have been identified (RetNet: https://sph.uth.edu/retnet), mainly segregating in an autosomal recessive manner. The most frequently mutated LCA gene is CEP290 that encodes the centrosomal protein 290 kDa [3,4].
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