Non-coding single nucleotide and structural variants affecting the EYS putative promoter cause autosomal recessive retinitis pigmentosa.

Abstract

Introduction: Variants in untranslated genomic regions are difficult to identify as pathogenic but are capable of causing disease by interfering with gene expression. This study aimed to characterize the effect of variants identified in the 5'-untranslated region of EYS in patients with autosomal recessive retinitis pigmentosa (ARRP).

Methods: Variant screening included gene panels, Sanger, exome and genome sequencing. Functional validation included an electrophoretic mobility shift assay (EMSA) and various luciferase assays.

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. EMSA analysis revealed an effect on binding of transcription factors for c.-453G>T and a neighboring variant c.-454G>T. Dual luciferase assays using 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 with OTX2 overexpression. In addition, the two variants were found to influence translation by affecting upstream initiation codons. Interestingly, visual function of EYS RP patients who harbor c.-453G>T are better than those with biallelic null EYS variants.

Discussion: 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.