Abstract
Purpose :
To test the custom orphan pediatric retinal disease panel for its detection in variants in the LRP5 gene. Familial Exudative Vitreo Retinopathy (FEVR) is due to variants that affect various genes including the LRP5 gene. LRP5 (LDL Receptor Related Protein 5) is an essential coreceptor, along with TSPAN12 and FZD4, which makes retinal endothelial cells uniquely responsive to Norrin WNT-signaling. 76 subjects diagnosed with FEVR, and near relatives were sequenced through a custom Ampliseq panel that includes eight genes related to FEVR/Norrie Disease and Retinoschisis (NDP, CTNNB1, TSPAN12, KIF11, FZD4, LRP5, ZNF408, RS1).
Methods :
8 genes were run through a custom Ampliseq targeted panel (180 amplicons) which was designed with the Illumina’s DesignStudio Sequencing Assay Designer. The targeted panel was put into three pools (PCR reactions) per patient sample for complete coverage of 83 exons with 25 bp adjacent intron sequence. The targeted genes included: NDP (ChrX), RS1 (Chr10); CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), and ZNF408 (Chr11). Then the Ampliseq libraries were sequenced on the Illumina iSeq-100 platform. Through the use of ClinVar and The Genome Aggregation Databases (gnomAD), the variant impacts and allele frequency data was collected.
Results :
33 protein-altering variants were found in six FEVR/ND related genes. Of note, 13/33 (39.4%) of the variants were present in the LRP5 gene. The LRP5 gene is involved in the Wnt signaling pathway associated with FEVR with mutations showing a lack of vascularization to the retina. For the variants, two are found to be pathogenic, 1 novel likely pathogenic and 1 novel variant of unknown significance. 95.5% of the base reads were > Q30 quality, the percent on-target bases passing filer was 92.2%, and the average sequencing depth coverage was 978.
Conclusions :
Our custom targeted-sequencing panel was developed in order to detect variants in seven FEVR / Norrie Disease genes. We conclude that this panel provides sufficient depth of sequencing to detect variants in the LRP5 gene (exons and splice sites) and to determine zygosity of variants.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.