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Sari Tuupanen, Johanna Sistonen, Kati Kämpjärvi, Pauli Siivonen, Miika Mehine, Johanna Känsäkoski, Kirsty Wells, Jennifer Schleit, Miko Valori, Pertteli Salmenperä, Eeva-Marja Sankila, Eveliina Salminen, Tero-Pekka Alastalo, Juha Koskenvuo, Samuel Myllykangas; RPGR ORF15 sequencing improves diagnostic yield in patients with inherited retinal dystrophies.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2939.
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Pathogenic variants in the RPGR gene are the most common cause of X-linked retinitis pigmentosa (XLRP), accounting for 80% of cases. The C-terminal 567-aa exon ORF15 is a mutational hotspot for RPGR-associated RP, however, it generally performs poorly in standard sequencing -based assays due to a highly repetitive purine-rich sequence. To address the clinical importance of the RPGR ORF15 and the lack of high quality next generation sequencing (NGS) -based diagnostics, we aimed to develop a comprehensive high-throughput clinical test for inherited retinal dystrophies, and to specifically evaluate the performance of RPGR ORF15 sequencing in a clinical patient cohort.
We optimized a whole exome sequencing workflow with the Illumina NovaSeq 6000 platform to cover 266 retinal dystrophy-associated genes, including the difficult-to-sequence region in RPGR ORF15. We then evaluated the prevalence and characteristics of RPGR variants in a cohort of 1587 unselected patients with inherited retinal dystrophy. Additionally, a custom Sanger sequencing method was developed to confirm pathogenic and likely pathogenic RPGR variants.
In our clinical cohort, the overall diagnostic yield was 58%. A molecular diagnosis in RPGR was identified in 5.7% (90/1587) of the patients. The 90 pathogenic/likely pathogenic variants consisted of 63 frameshifts (70.0%), 21 nonsense (23.3%), three missense (3.3%), two consensus splice site (2.2%), and one gross deletion (1.1%). Seventy-one out of 90 (79%) pathogenic/likely pathogenic variants were detected in the ORF15, of which 28 (39%) were in the most difficult-to-sequence central region between amino acids p.824 and p.1077. Female patients accounted for 24% of the diagnostic cases. Furthermore, our data emphasizes the importance of RPGR in RP patients, as it accounts for approximately 9% of RP cases.
We have developed a high-quality diagnostic test for inherited retinal dystrophies including the difficult-to-sequence region in RPGR ORF15. Our results highlight the clinical importance of RPGR ORF15 sequencing in these patients. The high-quality NGS-based assay enables rapid and reliable molecular diagnostics of RPGR ORF15, and enhances the successful identification of patients for ongoing gene therapy trials.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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