Custom Ampliseq Targeted Sequencing Panel For Orphan Pediatric Retinal Diseases: Norrie Disease, FEVR, and Retinoschisis

Abstract

Purpose : DNA-sequencing is not readily available in countries with little research resources or where health insurance does not cover the costs even for those who have it. This is especially true for very rare (orphan) inheritable retinal diseases. We wanted to develop a rapid targeted-sequencing protocol for eight genes involved in Familial Exudative Vitreo-Retinopathy (FEVR), Norrie Disease, and Retinoschisis, at greatly reduced cost. Furthermore, we desired a workflow which could be deployed locally and support training in human genomics for science and medical students

Methods : DNA is extracted from 100 uL samples of whole frozen blood. An Ampliseq targeted-panel (180 amplicons) for 8 genes was designed with illumina's DesignStudio Sequencing Assay Designer, 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), ZNF408 (Chr11). Ampliseq libraries were quality controlled by capillary electrophoresis (Agilent Bioanalyzer) and several sample pool sizes were tested for capacity and coverage using sequencing and variant calling on the Illumina iSeq-100 platform.

Results : An average 2500-times read coverage was obtained for a pool of 16 patient libraries and 800-times for a pool of 48 patient libraries. Numerous potential disease-associated variants were detected in targeted libraries from patients diagnosed with Norrie Disease, FEVR, and Retinoschisis. Average numbers of variants per patient for the 48 patient pool were: 19.5 ± 1.6 SNVs, 0.6 ± 0.2 Inserts, and 0.6 ± 0.2 Deletions.

Conclusions : We developed a targeted exome-sequencing protocol using Illumina Ampliseq reagents and the iSeq-100 platform for three rare pediatric retinal diseases. Coverage validated the ability to pool 40-50 patients per run for eight genes and provides for excellent base call accuracy (>Q30). Over 90 patient samples were successfully sequenced during validation. Potential mono and digenic variant contributions in FEVR patients are detectable by testing multiple genes. Research analysis costs were reduced to $250 per patient and now involve an academic eye research institute / retinal clinic research partnership.

This is a 2021 ARVO Annual Meeting abstract.