Purpose: To characterize the effect of variants identified in the 5’-untranslated region of the EYS gene in patients with autosomal recessive retinitis pigmentosa (ARRP).

Methods: Variant screening included gene panel, Sanger, exome, and genome sequencing. Functional validation included an electrophoretic mobility shift assay (EMSA) and various luciferase assays. Clinical examination included visual acuity testing, electroretinography (ERG) testing, and retinal imaging.

Results: Patients with RP from six EYS biallelic Arab-Muslim families harbored a 5’-noncoding EYS variant, c.-453G>T, and four harbored a structural variant affecting the 5’-noncoding exons. The EMSA analysis revealed an effect on the binding of transcription factors for c.-453G>T and a neighboring variant, c.-454G>T, which was reported previously in a patient with RP. Dual luciferase assays using the overexpression of various transcription factors showed distinct effects on expression. c.-453G>T was associated with higher luciferase expression with CRX overexpression, and c.-454G>C was associated with higher luciferase expression with OTX2 overexpression. In addition, these two variants were found to influence translation by affecting upstream initiation codons. Interestingly, the visual functions (including age of onset, visual acuity and ERG responses) of EYS RP patients who harbor c.-453G>T is better than that of those with biallelic null EYS mutations.

Conclusions: Our analysis revealed both single-nucleotide and structural variants in the EYS promoter as the cause of ARRP. These variants may affect EYS expression via a dual mechanism by altering transcription factor binding affinity at the EYS promoter and by affecting upstream open reading frames.