title: A comprehensive clinical and biochemical functional study of a novel RPE65 hypomorphic mutation.
abstract: OBJECTIVE
  Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. The functional consequences of the novel P25L RPE65 mutation was correlated with its early-childhood phenotype and compared with other pathogenic missense mutations.
  
  
  METHODS
  In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and two-color threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids.
  
  
  RESULTS
  A very mild phenotype was detected in a now 7-year-old boy homozygous for the P25L mutation in RPE65. Although abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5 years and 20/30 at age 7 years. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated responses in scotopic conditions, and light-adapted cone responses were approximately 1.5 log units below normal. High-resolution spectral domain OCT revealed morphologic changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wild-type RPE65-transfected cells, whereas RPE65/L22P-transfected cells had 13.5%.
  
  
  CONCLUSIONS
  The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to <10% of wild-type RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. Functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy.
authors.affiliation: Department of Ophthalmology, Justus-Liebig-University, Universitaetsklinikum Giessen und Marburg GmbH, Giessen Campus, Giessen, Germany. birgit.lorenz@uniklinikum-giessen.de
authors.forename: Birgit
authors.initials: B
authors.lastname: Lorenz
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.forename: Maria
authors.initials: M
authors.lastname: Schambeck
authors.forename: Christoph
authors.initials: C
authors.lastname: Friedburg
authors.forename: Markus N
authors.initials: MN
authors.lastname: Preising
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
mesh_terms: D000328:Adult; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002648:Child; D003117:Color Vision Defects / Q000503:physiopathology*; D003241:Consanguinity; D017931:DNA Primers / Q000737:chemistry; D004596:Electroretinography; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D005838:Genotype; D006801:Humans; D015151:Immunoblotting; D007535:Isomerases / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense / Q000502:physiology*; D020419:Photoreceptor Cells, Vertebrate / Q000502:physiology*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012162:Retinal Degeneration / Q000503:physiopathology*; D041623:Tomography, Optical Coherence; D014162:Transfection; D014792:Visual Acuity / Q000502:physiology; D058609:Visual Field Tests; D014794:Visual Fields / Q000502:physiology; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D005136:Eye Proteins; D007535:Isomerases; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase