Abstract
Purpose :
To confirm the usefulness of a novel orphan pediatric retinal disease panel for detecting variants in the NDP (Norrie Disease Protein) gene. Familial Exudative Vitreo Retinopathy (FEVR) and Norrie Disease (ND) can be caused by variants of the NDP gene (Norrin). A cohort of 76 subjects diagnosed with FEVR/ND and close relatives were sequenced using a custom Ampliseq panel that includes NDP and seven other genes linked to FEVR/ND and Retinoschisis (CTNNB1, TSPAN12, KIF11, FZD4, LRP5, ZNF408, RS1).
Methods :
A custom Ampliseq targeted panel (180 amplicons) for 8 genes was designed with Illumina's DesignStudio Sequencing Assay Designer. The targeted panel was distributed into three pools (PCR reactions) per patient sample for complete coverage of 83 exons with 25 bp adjacent intron sequence. Target Genes were: NDP (ChrX), RS1 (Chr10); CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), and ZNF408 (Chr11). Ampliseq libraries were quality controlled by capillary electrophoresis and sequenced on the Illumina iSeq-100 platform at a scale of up to 50 samples per run. Variant impacts and allele frequency data were determined from ClinVar and The Genome Aggregation Databases (gnomAD).
Results :
A total of 33 protein-altering variants were found in six FEVR/ND-related genes. Of note, 1/33 (3%) of the variants was present in the NDP gene. This patient was heterozygous for the NDP: His42Arg variant, pathogenic for ND. This mutation was found in a patient with a clinical FEVR grade of 4, indicating the diagnosis being ND rather than FEVR. The sequencing data revealed 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 :
The custom Ampliseq targeted-sequencing Orphan Pediatric Retinal Disease panel was developed with the intention to detect genes associated with FEVR/ND and Retinoschisis. The panel’s sequencing coverage was of sufficient depth to detect protein-altering variants in the NDP gene, the primary gene for ND.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.