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Keri Allen, DanYi Wang, Maria Janessian, Daniel Navarro-Gomez, Joseph White, Eun Young Choi, Louis R Pasquale, Mark Consugar, Xiaowu Gai, Janey L Wiggs; Panel-based genetic testing of Juvenile-Open Angle Glaucoma probands in the United States. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2544.
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© ARVO (1962-2015); The Authors (2016-present)
Panel-based genetic testing utilizing targeted enrichment and next generation sequencing (NGS) has been shown to be highly accurate and reproducible for gene variant detection in inherited eye disorders (Consugar et al., 2014). The purpose of this study was to use the Genetics and Eye Disease of Optic Atrophy and Glaucoma (GEDi-O) panel and a customized comparative genomics hybridization (aCGH) microarray to screen juvenile open angle glaucoma (JOAG) probands for mutations in six known early-onset glaucoma genes.
Genomic DNA samples extracted from blood and mouthwash samples of fifty probands diagnosed with JOAG, before the age of 40, were submitted for GEDi-O and aCGH panel screening. The custom SureSelect targeted enrichment GEDi capture kit (Agilent Technologies, Santa Clara, CA) was used to capture coding regions of six glaucoma genes, including: CYP1B1, FOXC1, LTBP2, MYOC, PAX6, and PITX2. GEDi-O targeted enrichment samples were 2 x 121bp paired-end sequenced using an Illumina MiSeq NGS platform (Illumina, San Diego, CA). Copy number variation (CNV) analysis was performed using a custom aCGH microarray slides scanned on an Agilent Technologies SureScan microarray scanner. All mutations identified with the GEDi-O panel were confirmed using PCR and Sanger sequencing. All CNVs detected using aCGH were confirmed with Multiplex Ligation Probe Amplification (MLPA).
Disease-causing mutations were identified in 10 of the 50 JOAG probands (20%): one MYOC mutation (I477N); four FOXC1 mutations ( E54X, c.81_89delCCGCGGCG, c.67_68insAGAG, two whole gene duplications, two whole gene deletions) and three probands with homozygous or compound heterozygous CYP1B1 mutations (R390H/R390H, R368H/A106D, and R368H/E229K). Only one mutant allele in either CYP1B1 or LTBP2 was found in 5 probands. Disease-causing mutations were not detected in PAX6 or PITX2.
GEDi-O and aCGH panel-based testing are reliable methods for screening for mutations in the six early-onset glaucoma genes. In the United States, JOAG is caused by a spectrum of glaucoma genes including MYOC, FOXC1 and CYP1B1. The genetic etiology for the majority of JOAG probands is still unknown. These results suggest that other novel genes are responsible for the majority of juvenile glaucoma cases in the United States.
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