Purpose: To investigate the consequence of a major intrinsic protein MIP splice-site mutation (c.607-1G>A) in a four-generation Chinese pedigree afflicted with autosomal dominant congenital cataracts (ADCC).
Methods: Both a mutated minigene with c.607-1G>A, and a wild-type minigene were constructed using the pTARGET mammalian expression vector. They were transiently transfected into HeLa cells and human lens epithelial cells, respectively. After 48 h incubation, RNA extraction and RT-PCR analysis were performed and PCR products were separated and confirmed by sequencing. Structural models of the wild-type and the mutant aquaporin 0 (AQP0) were generated and analyzed using SWISS-MODEL.
Results: The G>A transition activated a cryptic acceptor splice site (c.965-966) in the 3' untranslated region (3' UTR), resulting in the absence of the coding region and most of the 3'UTR in exon 4 of the mature mRNA. Moreover, homology modeling of the mutant protein suggested that the sixth transmembrane helix and carboxyl terminus were replaced with the Leu-His-Ser tripeptide (AQP0-LHS).
Conclusions: The MIP splice-site mutation (c.607-1G>A) activates a cryptic acceptor splice site in the 3' UTR, which may result in substitution of the sixth transmembrane helix and carboxyl terminus for AQP0-LHS. To our knowledge, this is the first report of activation of a cryptic splice site in the 3' UTR in a human disease gene.