Difficult-to-sequence genes in ophthalmology: improved sequencing coverage and mapping quality in ORF15 enable unmatched diagnostic yield in XLRP

Blueprint Genetics’ updated technology enables improved coverage and sensitivity in clinically relevant and challenging genes such as RPGR and especially the ORF15 region of this gene. Current next generations sequencing (NGS) strategies and standard Sanger sequencing do not adequately cover the RPGR ORF15 region, leading to poor sensitivity and decreased diagnostic yield.

Mutations in RPGR are associated with a more severe form of retinal degeneration. Blueprint Genetics’ new laboratory assay, powered by customized oligo design sequencing with Illumina NovaSeq and combined with an updated proprietary analysis pipeline, provides a high diagnostic rate in internal clinical validation studies, even with challenging regions such as the repetitive central region of the ORF15 exon.

“We included several cases of X-linked retinitis pigmentosa (XLRP) in our clinical validation and successfully detected all pathogenic or likely pathogenic RPGR ORF15 variants. RPGR mutations explain over 70% of XLRP cases and about 8% of all RP cases. Because of the significance of this gene, it is essential to develop tools and strategies to maximize quality and performance within the ORF15 region,” says Blueprint Genetics’ Chief Medical Officer, Tero-Pekka Alastalo.

RPGR is an important gene in the diagnosis of XLRP. The ORF15 region is a unique C-terminal exon, which encodes 567 amino acids. Its repetitive glutamic acid/glycine-rich domain is a mutation hotspot for XLRP.

“Technically, the ORF15 region of the RPGR gene is very challenging to sequence. With our updated technology the entire RPGR gene is now covered with at least 20x with mapping quality over 20. Additionally, we have developed a custom Sanger sequencing method to confirm variants specifically in the ORF15 region. The combination of these sequencing and bioinformatic innovations represents one of the best strategies for RPGR gene diagnostics available on the market,” Alastalo continues.

Generally, the challenges in inherited retinal diseases, in addition to difficult-to-sequence genes, is the large number of overlapping disease-causing genes and phenotypes.

“We see significant number of patients with disease-causing copy number variants and non-coding variants. An optimal testing strategy does not only include the coverage of significant genes and coverage of RPGR ORF15, but also provides high resolution CNV detection and covers the established non-coding variants,” says Alastalo.

In inherited retinal diseases, genetic testing facilitates a more accurate diagnosis, helps in determining the inheritance pattern and the risk to family members, enables better prognostics and can even help in selecting personalized management for the patient.

“Inherited retinal diseases have decades of research that have revealed the major underlying genetic causes for the diseases. This information has been used in diagnostics and developing therapies. Already 50-76% of patients undergoing high-quality genetic testing will receive a molecular diagnosis or very likely diagnosis. The molecular diagnosis is extremely important as, in this field, there is a growing number of emerging therapies targeted to specific genes,” Alastalo says.

Further information
Chief Medical Officer Tero-Pekka Alastalo, tero-pekka.alastalo(at)blueprintgenetics.com
Communication Manager Juulia Simonen, tel. +35850 305 9018, juulia.simonen(at)blueprintgenetics.com