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Hoang Mai LE, Almudena Sacristan Reviriego, Jacqueline Van Der Spuy; Determination of AIPL1 variations as disease causing in Leber Congenital Amaurosis patients. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3083. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Leber Congenital Amaurosis (LCA) is the most severe form of inherited retinal dystrophy diagnosed in early childhood. Biallelic mutations in the photoreceptor-expressed aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) is responsible for around 7% of LCA cases. AIPL1 functions as a photoreceptor-specific co-chaperone that interacts with the molecular chaperone HSP90 to facilitate the stable assembly of the retinal cyclic GMP (cGMP) phosphodiesterase (PDE6) holoenzyme. We have recently established an in vitro system to analyse AIPL1 variations located in the coding and non coding regions of the gene that either alter the AIPL1 amino acid sequence or cause aberrant pre-mRNA splicing. The purpose of the study is to analyse the functional impact of previously uncharacterised AIPL1 variations in order to validate their potential disease-association with LCA and clarify the disease mechanism.
The disease-causing status of the AIPL1 variations was evaluated using in silico prediction tools. Variations predicted to affect AIPL1 splicing were analysed using an in vitro splice assay and the resultant transcripts identified by sequencing. The subcellular localisation and expression of AIPL1 variants encoded by missense and nonsense variations were investigated by immunocytochemistry and western blotting. A quantitative ELISA assay was used to investigate the impact of the AIPL1 variants on HSP90 interaction, and a competitive cGMP ELISA assay was used to determine the effect of the variants on the catalytic activity of the heterologously reconstituted PDE6 holoenzyme.
In vitro splice assays revealed that novel non-coding AIPL1 variations induced missplicing of AIPL1 pre-mRNA. The interaction of AIPL1 with HSP90 and the catalytic activity of the PDE6 holoenzyme were also compromised to various extents by mutations in both the FK506 Binding Protein (FKBP) - like domain and Tetratricopeptide Repeat (TPR) domain of AIPL1.
In our study, we validated the disease-causing status of previously uncharacterised AIPL1 variations. The mechanism of disease involved either aberrant pre-mRNA AIPL1 missplicing, AIPL1 misfolding, or a decreased interaction with HSP90 due to mutations in the FKBP-like domain or the TPR domain.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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