Abstract
Abstract: :
Purpose: Mutations in AIPL1 cause a severe form of Leber congenital amaurosis (LCA), while in heterozygous state, these mutations can cause selective rod ERG abnormalities, which correlates with the exclusive expression of AIPL1 in adult rod photoreceptors. We found different ERG abnormalities for two disparate AIPL1 mutations (a nonsense and an in frame deletion), and wanted to test the hypothesis that these mutations have diverging biochemical effects in an in vitro expression system. Methods: A W278X nonsense mutation was found in a father, and a Leu257del9bp in–frame deletion was found in the mother of a child with LCA, who carried both mutations. These mutations were detected by dHPLC wave analysis and automated sequencing. Scotopic and photopic electroretinograms (ERGs) were done on both eyes of both parents according to ISCEV standards. The mutations were introduced into a mammalian expression vector containing the complete AIPL1 sequence, and each mutant construct was transiently transfected into 293T cells along with the molecular partner of AIPL1, NUB1. Protein stability was assayed by Western blot, and interactions of the proteins were assayed by co–immunoprecipitation (Co–IP). Results: Photopic ERGs were within normal limits for both parents. Scotopic ERGs, however, showed a marked decrease in amplitude of the b–wave (144 µV, while average normal (N=10) is 210 µV) (t = 1.93, p< 0.05) for the father with the W278X mutation, while the b–wave of the mother with the Leu257del9bp mutation was 243 µV and within normal range. Expression experiments showed protein instability of the W278X protein, with levels comparable to that of the empty vector control. The Leu257del9bp mutant protein was stable and able to interact with NUB1. Conclusions: We have determined by Co–IP that the L257del9bp protein is stable and does interact with the NUB1 protein. However, the W278X protein is unstable, and the resulting elimination of the AIPL1/ NUB1 interaction may be responsible for the significant rod ERG phenotype in the heterozygous carrier of this mutation.
Keywords: retinal degenerations: hereditary • electroretinography: clinical • mutations