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Shiming Li, Baojian Fan, Anna Larson, Janey L Wiggs; Whole exome sequencing identifies GPC6 as a candidate early-onset glaucoma gene. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1609. doi: https://doi.org/.
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Glaucoma developing before age 40 is defined as early-onset glaucoma (EOG). Currently 9 genes are known to cause EOG and using these genes for genetic testing can inform genetic counseling and risk assessment. However, the diagnostic yield for these genes is only 25% in families from the United States (US) implicating the contributions of additional genes. The purpose of this study is to identify novel EOG genes using whole exome sequencing (WES) following by functional screening in zebrafish.
DNA samples were obtained for all 8 members of an European ancestry Caucasian family with EOG in the US. WES was performed using the Agilent SureSelect Human All Exon v6+UTR+Mito kit and the Illumina HiSeq 2000 for all 8 individuals. WES data was analyzed using GATK and annotated using a customized pipeline. Zebrafish screening for functional effects used standard and splice-site blocking morpholinos (Gene Tools) injected into 1-4 cell stage ABTL embryos. Zebrafish morphants were characterized using the visual motor response (VMR) and light microscopy.
High quality WES data was obtained for all samples (average coverage 105× for 99% of coding sequences), and was filtered for qualities expected for disease-causing mutations: predicted to disrupt protein function, rare (< 1% in population databases), and high estimates of pathogenicity. Of 15 variants meeting these criteria, two variants (GPC6 p.P344S and DHX34 p.R956W) remained in consideration after removing variants also present in unaffected family members. To further prioritize these candidates each gene was knocked down using morpholinos in zebrafish. The VMR was significantly reduced in the GPC6 morphants compared with controls (P < 0.001), while the DHX34 morphants had normal visual response (P > 0.05). Light microscopy showed anterior segment abnormalities in the GPC6 morphants, particularly thick and irregular corneas.
WES is a useful approach for gene discovery particularly in smaller families. However, prioritization of disease-causing candidates remains a challenge. Zebrafish knock-down using morpholinos provides a rapid and efficient method to identify candidates influencing vision and eye development. Using this approach we identified GPC6 as a candidate disease gene for EOG. GPC6 can influence hedgehog signaling, a pathway known to be involved in ocular development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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