EPOS

European Paediatric Ophthalmological Society

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11 Mutational analysis in Leber congenital amaurosis

Sitorus, R.¹, Rosenberg, T.², Kellner, U.³, Preising, M.N.¹, Lorenz, B.¹
Dept. of Paediatric Ophthalmology, Strabismology and Ophthalmogenetics, University of Regensburg, Germany, National Eye Clinic for the Visual Impaired, Hellerup, Denmark, Augenklinik, Universitätsklinikum Benjamin-Franklin, Berlin, Germany

Objectives: To analyse the genes for RPE65, GUCY2D and AIPL1 in patients diagnosed with Leber Congenital Amaurosis (LCA), and to define their clinical correlation.

Patients and Methods: DNAs of 58 index patients with an initial diagnosis of LCA have been collected since 1992 at the Department of Paediatric Ophthalmology, Strabismology and Ophthalmogenetics.

Patients have been screened for mutations of the genes for RPE65, RetGC1 and AIPL1 by Single-Strand Conformational Polymorphism analysis.

PCR-products showing aberrant banding patterns were subjected to direct sequencing. DNA sequence variations were confirmed by restriction enzyme digestion and/or direct sequencing of the complementary DNA strand.

Clinical diagnosis was made based on age at onset, severity of visual dysfunction, nystagmus, ERG and fundus photography.

Results: We have completed our screen in a large subset of index patients (RPE65: 37/58, RetGC1: 22/54, AIPL1: 38/53). Mutations in both alleles were found in 5/37 (13,5%) index patients for RPE65, 3/22 (13,6%) index patients for RetGC1, and 1/37 (2,7%) index patients for AIPL1. Mutations in only one allele have been identified in one index patient for RPE65 (IVS5+1ins g/?), 2 index patients for RetGC1 (P701S/?, A52S/?), and 2 index patients for AIPL1 (V96I/?, I206N/?).

Three of the index patients carrying mutations on both alleles in the RPE65 gene have been reported previously. One index patient carried the R91W mutation in the homozygous state, and one index patient carried the W458X/IVS5+1ins g mutations in a compound heterozygous state.

Mutations on both alleles in the RetGC1 gene were found in the homozygous state in one index patient (A52S) from a consanguineous marriage, and in the compound heterozygous state in two index patients (P701S/A950V and G653R/2234delC).

For the AIPL1 gene one index patient was identified to carry two compound heterozygous mutations (R53W/N90H). The severely affected father was homozygous for the N90H mutation.

Additionally, intronic and exonic polymorphisms were identified in all three genes. The polymorphism frequency was pronounced in the AIPL1 gene which showed the most polymorphisms per coding basepairs. As previously reported, the IVS6-33g/t polymorphism in the RPE65-gene, the A703A and 3528c/t polymorphism in the RetGC1-gene, and the P217P polymorphism in the AIPL1-gene showed a frequency high enough to be used in linkage studies.

Clinical data revealed an early onset of severe visual impairment in RetGC1 patients without clinical signs in the fundus in childhood. Obvious fundus changes appeared during childhood in patients carrying mutations in the RPE65 and AIPL1 genes. The age of onset of these fundus changes was after 1 or 2 years of age. ERGs were flat in all patients but visual acuity was preserved at least during the first decade in patients carrying mutations in the RPE65 gene.

Conclusions : Patients carrying mutations in the RetGC1, RPE65, and AIPL1 genes could be differentiated by their clinical features prior to genetics testing. While RetGC1 mutations cause severe visual impairment with very early onset but unremarkable fundus over a long time, RPE65 mutations cause an early onset of visual impairment but still measurable visual function and early fundus changes. Mutations in the AIPL1 gene are associated with severe visual impairment and distinct fundus changes during infancy.