title: Investigation of Aberrant Splicing Induced by AIPL1 Variations as a Cause of Leber Congenital Amaurosis.
abstract: OBJECTIVE
  Biallelic mutations in AIPL1 cause Leber congenital amaurosis (LCA), a devastating retinal degeneration characterized by the loss or severe impairment of vision within the first few years of life. AIPL1 is highly polymorphic with more than 50 mutations and many more polymorphisms of uncertain pathogenicity identified. As such, it can be difficult to assign disease association of AIPL1 variations. In this study, we investigate suspected disease-associated AIPL1 variations, including nonsynonymous missense and intronic variants to validate their pathogenicity.
  
  
  METHODS
  AIPL1 minigenes harboring missense and intronic variations were constructed by amplification of genomic fragments of the human AIPL1 gene. In vitro splice assays were performed to identify the resultant AIPL1 transcripts.
  
  
  RESULTS
  We show that all nine of the suspected disease-associated AIPL1 variations investigated induced aberrant pre-mRNA splicing of the AIPL1 gene, and our study is the first to show that AIPL1 missense mutations alter AIPL1 splicing. We reveal that the presumed rare benign variant c.784G>A [p.(G262S)] alters in vitro AIPL1 splicing, thereby validating the disease-association and clarifying the underlying disease mechanism. We also reveal that in-phase exon skipping occurs normally at a low frequency in the retina, but arises abundantly as a consequence of specific AIPL1 variations, suggesting a tolerance threshold for the expression of these alternative transcripts in the retina normally, which is exceeded in LCA.
  
  
  CONCLUSIONS
  Our data confirm the disease-association of the AIPL1 variations investigated and reveal for the first time that aberrant splicing of AIPL1 is an underlying mechanism of disease in LCA.
authors.affiliation: UCL Institute of Ophthalmology London, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Bellingham
authors.affiliation: UCL Institute of Ophthalmology London, United Kingdom.
authors.forename: Alice E
authors.initials: AE
authors.lastname: Davidson
authors.affiliation: UCL Institute of Ophthalmology London, United Kingdom 2Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Aboshiha
authors.affiliation: Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Second University of Naples, Naples, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Simonelli
authors.affiliation: UCL Institute of Ophthalmology London, United Kingdom 2Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom.
authors.forename: James W
authors.initials: JW
authors.lastname: Bainbridge
authors.affiliation: UCL Institute of Ophthalmology London, United Kingdom 2Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom.
authors.forename: Michel
authors.initials: M
authors.lastname: Michaelides
authors.affiliation: UCL Institute of Ophthalmology London, United Kingdom.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: van der Spuy