Abstract
Purpose :
Glaucoma is a blinding multifactorial genetic disease for which the entire genetic landscape is still unclear. This study explores novel genes using whole-exome sequencing as an unbiased approach in familial glaucoma.
Methods :
Five families of patients (mean age 45±16.8 years) with primary open angle glaucoma, POAG (n=2) or angle closure glaucoma (n=3), PACG, underwent whole exome sequencing from blood representing 16 affected members/cases (disc and field changes with raised intraocular pressure) and 5 unaffected or controls,Figure 1. After whole exome capture using Illumina Nextera chemistry,sequencing was performed on Hi-Seq2000 after excluding Myocilin mutations. The 100 base-pair paired end reads were generated and variants were called using the Genome Analysis Tool Kit (GATK2) after quality checks and alignment to reference genome. Variant annotation was done using ANNOVAR and GEMINI. Only variants that segregated with the disease under the autosomal dominant model were selected. The probable mutations were prioritized based on protein prediction tools (PolyPhen2, SIFT prediction, CADD and GERP scores) followed by validation using Sanger sequencing. Pathway enrichment analysis was done using ENRICHR.
Results :
The average depth of sequencing was 38x (27x -72x). Around 47,500 single nucleotide variants were identified for each sample that differed with the reference genome. After the segregation analysis, we identified 261 variants likely to be contributing to glaucoma, representing195 genes significantly enriched for extracellular matrix (ECM) remodelling and glutathione metabolism (p value < 0.05). Interestingly, 3 members in family 5 had angle closure with hypercholestrelemia (Figure 2) with a heterozygous deletion in LOXL2 and heterozygous SNV in NEB. The single nucleotide deletion in exon-5 would lead to a frame-shift and premature stop codon while the NEB variant would cause a missense change (p.288Lys>Arg). The unaffected parent had the reference genotypes for both variants while the unaffected child inherited the deletion allele in LOXL2.
Conclusions :
An overall enrichment for genes involved in ECM remodeling was observed in this cohort of familial primary glaucoma. Of interest are variants identified in LOXL2 and NEB genes reportedly implicated in choroidal neovascularization and dog model of angle-closure glaucoma, respectively. Further studies exploring their role in familial glaucoma are required.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.