Identification of a Duplication in the RP17 Locus in an Individual With Pathogenic <i>CEP290</i> Variants: Implications for RP17 Variant Classification

Purpose: To assess the pathogenicity of a novel duplication in the RP17 locus identified in a cone dystrophy proband with biallelic CEP290 variants. Structural variants (SVs) in this locus have previously been associated with dominant retinitis pigmentosa. Methods: Inheritance of the duplication was assessed by breakpoint polymerase chain reaction (PCR). Ophthalmic evaluation included fundus examination, multimodal retinal imaging, and full-field electroretinogram (ERG). A proband-derived pluripotent stem cell line was differentiated into photoreceptor precursor cells (PPCs) and retinal organoids (ROs). Variant-induced mis-splicing of CEP290 was assessed by reverse-transcription PCR (RT-PCR) and long-read cDNA sequencing, and immunohistochemistry was used to assess photoreceptor morphology. Expression of GDPD1 was quantified by quantitative RT-PCR. Results: The proband and father carried the 324-kb duplication in the RP17 locus. The father was clinically unaffected, but the proband showed features of cone dystrophy, including reduced visual acuity, foveal abnormalities, diminished cone density, and preserved dark-adapted but absent light-adapted ERG responses. The compound heterozygous variants in CEP290 resulted in pseudoexon inclusion and exon 36 skipping in patient-derived retinal cells. Immunohistochemistry revealed altered ciliation and reduced trafficking of L/M opsin and rhodopsin in ROs. In silico modeling predicted that the novel RP17 duplication does not disrupt chromatin looping, and GDPD1 expression was not upregulated in patient ROs, in contrast to pathogenic RP17-SVs. Conclusions: The cone dystrophy phenotype of the proband can be attributed to the CEP290 variants, whereas the novel RP17 duplication can be classified as likely benign based on the integrated evidence. These findings emphasize the importance of modeling and functional studies for accurately classifying RP17-SVs and preventing misinterpretation in clinical diagnostics.