Rhodopsin Screening in Greek Patients With Retinitis Pigmentosa and Identification of a Novel Rhodopsin Mutation

Abstract

Purpose:: Retinitis Pigmentosa is a heterogeneous group of retinal degenerations, with multiple genes identified in each mode of inheritance. For autosomal dominant retinitis pigmentosa (ADRP), the most common causative gene is the rhodopsin (RHO) gene, mutations in which contribute to about 25% of ADRP in Caucasians. RP patients in the Greek population have not been genetically characterised previously for sequence variations in rhodopsin or any other retinal disease gene. This is the first systematic study to investigate the frequency and pattern of disease-associated mutations and polymorphisms in the rhodopsin gene.

Methods:: 41 unrelated Greek RP probands with different types of inheritance (10 ADRP, 8 ARRP, 8 sporadic and 15 of uncertain mode) were screened for rhodopsin mutations by PCR and sequencing.

Results:: Two RHO mutations, G106R and a novel mutation F276V were identified each in one ADRP family and two common polymorphisms, IVS3+4C>T and -26A>G in 19 and 12/41 patients, respectively. In order to determine the pathogenicity of the novel amino acid substitution, 100 controls were tested for this sequence variation and shown to be negative which strongly suggests that this could be the disease-causing mutation. The frequency of rhodopsin mutations in the ADRP subgroup was thus found 20% which is similar to that in Europe and North America whereas the frequencies of the IVS3+4 C>T and -26A>G polymorphisms were 30% and 47.5%. By site directed mutagenesis, the amino acid change F276V was inserted in a rod opsin-GFP expression vector and mutant opsin processing compared to wild-type and P23H rod opsin in SK-N-SH cells. The novel amino acid change increased the amount of rod opsin retained within the ER of transfected cells and caused an increase in the incidence of intracellular inclusions.

Conclusions:: Our findings provide information for the first time about the distribution of rhodopsin sequence variants in Greek RP patients and normal controls. These data will be valuable for the effective genetic counselling of the patients and their families who have never received genetic advice in the past. Furthermore, the rod opsin amino acid change F276V appeared to be a Class II mutation which suggests that this change may lead to RP through rhodopsin misfolding.

Keywords: retinal degenerations: hereditary • gene screening • opsins