title: Identification of a MIP mutation that activates a cryptic acceptor splice site in the 3' untranslated region.
abstract: OBJECTIVE
  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.
authors.affiliation: Eye Center of the Second Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, China.
authors.forename: Chongfei
authors.initials: C
authors.lastname: Jin
authors.forename: Jin
authors.initials: J
authors.lastname: Jiang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Ke
authors.initials: K
authors.lastname: Yao
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D017398:Alternative Splicing / Q000235:genetics; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D040681:Structural Homology, Protein
chemical_list: D020413:3' Untranslated Regions; D020346:Aquaporins; D005136:Eye Proteins; D050505:Mutant Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; C030328:aquaporin 0