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B. A. Lorenz, S. Gentleman, M. Schambeck, C. Friedburg, M. N. Preising, T. M. Redmond; A Novel RPE65 Hypomorph Expands the Clinical Phenotype of RPE65 Mutations. A Comprehensive Clinical and Biochemical Functional Study. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3104.
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© ARVO (1962-2015); The Authors (2016-present)
To correlate the functional consequences of the P25L missense mutation in RPE65 with the phenotype in early childhood, and to compare this phenotype with the L22P missense mutation.
Systematic clinical and molecular genetic screen of patients with early onset retinal dystrophies (EOSRD). Clinical work-up including best corrected visual acuity (BCVA) with age-adapted tests, orthoptic examination, color vision testing, slit-lamp biomicroscopy, funduscopy, fundus photography, fundus autofluorescence (FAF), optical coherence tomography (OCT), Fullfield-ERG, kinetic and static perimetry (KP, SP), 2-color-threshold perimetry (2CTP). Molecular genetic screening was done by SSCP and direct sequencing of RPE65.Site-directed mutagenesis was used to make RPE65 mutants in pVitro2 expression vector expressing RPE65 and CRALBP. Wildtype and mutant pVitro2 constructs were transfected into 293-F cells along with pVitro3-LRAT+RDH5. RPE65 and CRALBP expression was analyzed by immunoblot. Isomerase activity was quantified by HPLC analysis of retinoids.
The mildest phenotype was detected in a now 6 y old boy followed by us since age 3 y. Difficulties with dark adaptation were noticed from early on. He was homozygous for the P25L mutation in RPE65. At age 5y, BCVA was still 20/20, and had declined to 20/30 at age 6 y. Nystagmus was not detected. Cone ERG was measurable, rod ERG severely reduced to absent, and fundus autofluorescence FAF very low. 2 CT perimetry detected mainly cone mediated answers under scotopic conditions, light adapted cone answers were about 1.5 log units below normal. RPE65 expression in 293-F cells transfected with RPE65/P25L was reduced compared to that of cells transfected with wild type. Cells transfected with RPE65/P25L showed 7% of isomerase activity of cells.
This is the first documented case of a missense mutation in RPE65 in conjunction with full visual acuity in childhood. Reduction to < 10% of wildtype RPE65 activity by homozygous P25L was associated with an almost complete loss of rod function both in dark adapted fullfield ERG and in 2 CT perimetry and with a reduction of cone amplitude to 30% in fullfield ERG and 1.5 log units cone sensitivity loss, providing a relatively low datum point in the dynamic range of visual pigment regeneration. This residual RPE65 activity is consistent with the low FAF. This patient should profit most from subretinal gene therapy.
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