title: Splicing factor polymorphisms, the control of VEGF isoforms and association with angiogenic eye disease.
abstract: OBJECTIVE
  Alternative splicing of the last exon (exon 8) of vascular endothelial growth factor (VEGF) pre-mRNA is a key element in the balance of pro- and anti-angiogenic VEGF isoforms in exudative age-related macular degeneration (exAMD) and proliferative diabetic retinopathy (PDR). Three splicing factors, SRp40, ASF/SF2, and SRp55 are predicted to control alternative splicing by binding to exonic splice enhancers (ESE) in VEGF exon 8. This pilot study examines whether there is an association between angiogenic eye disease and splicing factor polymorphisms, and whether there are sequence variations in the alternative splice sites of the VEGF gene.
  
  
  METHODS
  A case:control pilot study comparing 163 individuals with angiogenic eye disease (94 exAMD and 69 PDR patients) with 95 age-matched controls. Splicing factor polymorphisms were genotyped by Restriction Fragment Length Polymorphism (RFLP) and sequencing, and the VEGF alternatively spliced region was assessed by denaturing High Performance Liquid Chromatography (dHPLC) using a transgenomic WAVE heteroduplex analyzer.
  
  
  RESULTS
  No variations were observed in the alternatively spliced region of VEGF exon 8. ASF/SF2 polymorphisms showed no association with exAMD or PDR. For PDR, we observed a trend in SRp40 (rs6573908) where the 5136CC genotype was more frequent in controls (p = 0.0517) and a significant association of the SRp55 (rs2235611), where the 2994C allele was more common in the PDR group (p = 0.03). This remained strong, but not significant, after logistic regression for age, sex, disease type, and duration (p = 0.06).
  
  
  CONCLUSIONS
  The lack of variation in the VEGF alternatively spliced region suggests the importance of sequence conservation in this area in maintaining the balance of pro- and anti-angiogenic VEGF isoforms. The link between PDR and the SRp55 2994 polymorphism suggests a disease-specific association between factors controlling VEGF splicing and ocular angiogenesis.
authors.affiliation: Unit of Ophthalmology, University of Bristol, Bristol, UK.
authors.forename: J G
authors.initials: JG
authors.lastname: Carter
authors.forename: J
authors.initials: J
authors.lastname: Cherry
authors.forename: K
authors.initials: K
authors.lastname: Williams
authors.forename: S
authors.initials: S
authors.lastname: Turner
authors.forename: D O
authors.initials: DO
authors.lastname: Bates
authors.forename: A J
authors.initials: AJ
authors.lastname: Churchill
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing / Q000235:genetics*; D016022:Case-Control Studies; D020256:Choroidal Neovascularization / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003930:Diabetic Retinopathy / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D005838:Genotype; D020180:Heteroduplex Analysis; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics; D010865:Pilot Projects; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D015861:Retinal Neovascularization / Q000235:genetics*; D000068103:Serine-Arginine Splicing Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D055815:Young Adult
chemical_list: D009687:Nuclear Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; C489470:SRSF6 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068103:Serine-Arginine Splicing Factors