title: Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.
abstract: BACKGROUND
  Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested.
  
  
  METHODS
  We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G).
  
  
  RESULTS
  Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain.
  
  
  CONCLUSIONS
  This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. nmaciole@mcw.edu
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Maciolek
authors.forename: Wallace L M
authors.initials: WL
authors.lastname: Alward
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Murray
authors.forename: Elena V
authors.initials: EV
authors.lastname: Semina
authors.forename: Mark T
authors.initials: MT
authors.lastname: McNally
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D002460:Cell Line; D005124:Eye Abnormalities / Q000235:genetics*; D005260:Female; D018628:Gene Dosage; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007438:Introns; D008297:Male; D008957:Models, Genetic; D009154:Mutation; D010375:Pedigree; D022821:RNA Splice Sites; D012326:RNA Splicing; D013577:Syndrome; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000097577:Homeobox Protein PITX2
chemical_list: D018398:Homeodomain Proteins; D022821:RNA Splice Sites; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2