Utilizing a Custom Targeted-Sequencing Panel to Identify Gene Variants in FEVR and Possible FEVR Patients

Document Type

Conference Proceeding

Publication Date


Publication Title

Investigative Ophthalmology and Visual Science


urpose : To identify gene variants in pediatric vitreoretinal disease patients and their family members using a custom Illumina AmpliSeq orphan pediatric retinal disease gene panel. Our panel and sequencing protocol can sequence eight genes commonly associated with various inherited retinal diseases (IRDs) at greatly reduced cost and with a more streamlined sequencing approach.

Methods : Participants for DNA-sequencing analysis were referred to Associated Retinal Consultants, LLC, (ARC) of Royal Oak, Michigan, USA. Ten participants were consented under IRB approval for donation of blood to the ARC Eye Biobank, and for genetic analysis at Oakland University. A targeted 8-gene panel included 7 genes required for normal function of retinal vascular endothelial cells: NDP (ChrX), CTNNB1 (Chr3), TSPAN12 (Chr7), KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11), and RS1 (ChrX). 180 amplicons, averaging 250 base pairs (bp), with overlapping sequence, targeted all exons and at 25 bp of adjacent intron sequence, totalling 32,000 bp. Genomic DNA was extracted from 200 uL of frozen whole blood, quantified, Illumina Ampliseq libraries prepared, diluted, pooled, and sequenced with the Illumina iSeq-100. VCF files were analyzed with the Ensembl Variant Effect Predictor database.

Results : Seven of the ten samples had coding consequence variants, in genes including LRP5, CTNNB1, TSPAN-12, ZNF408, and FZD4. The variants included missense, in-frame insertion, and in-frame deletion variants. In the NCBI ClinVar database, two variants were identified, one of these samples was successfully identified as pathogenic with an amino acid change of Methionine to Valine, and another one was identified as likely pathogenic, with an amino acid change of Threonine to Methionine.

Conclusions : Our panel’s sequencing coverage was of sufficient depth to detect protein-altering variants of interest in pediatric vitreoretinal disease patients. This custom targeted-sequencing approach has promising potential in broadening accessibility, adoptability, and advancement of more widespread gene sequencing in the pediatric retinal disease population. Identifying known variants allows us to give much-needed and appreciated updates to the patients and their families affected by genetic pediatric vitreoretinal diseases.





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Annual Meeting Association for Research in Vision and Ophthalmology, ARVO 2023, April 23-27, 2023, New Orleans, LA