iSyTE: integrated Systems Tool for Eye gene discovery

Abstract

Purpose: To facilitate the identification of genes associated with cataract and other ocular defects, the authors developed and validated a computational tool termed iSyTE (integrated Systems Tool for Eye gene discovery; http://bioinformatics.udel.edu/Research/iSyTE). iSyTE uses a mouse embryonic lens gene expression data set as a bioinformatics filter to select candidate genes from human or mouse genomic regions implicated in disease and to prioritize them for further mutational and functional analyses.

Methods: Microarray gene expression profiles were obtained for microdissected embryonic mouse lens at three key developmental time points in the transition from the embryonic day (E)10.5 stage of lens placode invagination to E12.5 lens primary fiber cell differentiation. Differentially regulated genes were identified by in silico comparison of lens gene expression profiles with those of whole embryo body (WB) lacking ocular tissue.

Results: Gene set analysis demonstrated that this strategy effectively removes highly expressed but nonspecific housekeeping genes from lens tissue expression profiles, allowing identification of less highly expressed lens disease-associated genes. Among 24 previously mapped human genomic intervals containing genes associated with isolated congenital cataract, the mutant gene is ranked within the top two iSyTE-selected candidates in approximately 88% of cases. Finally, in situ hybridization confirmed lens expression of several novel iSyTE-identified genes.

Conclusions: iSyTE is a publicly available Web resource that can be used to prioritize candidate genes within mapped genomic intervals associated with congenital cataract for further investigation. Extension of this approach to other ocular tissue components will facilitate eye disease gene discovery.

Figure 1.

Strategy for building iSyTE. To identify genes that are specifically expressed in the lens during embryonic development, mouse lens tissue at E10.5, E11.5, and E12.5 was profiled using microarrays. Several hundred lenses at stages E10.5, E11.5, and E12.5 were pooled for generating total RNA for each biological replicate in microarrays conducted in triplicate. We also obtained the microarray gene expression profile for pooled embryonic WB tissue at each stage; the ocular region was removed from the whole embryonic tissue before profiling. Lens-specific profiles are “subtracted” from the WB control using a moderated t-test. A lens enrichment P value was assigned to each gene for each embryonic stage, and a false-discovery rate (FDR) was calculated based on the P value. t-Statistics were used to rank the genes for lens enrichment. The green of the lenses represents fluorescence caused by the use of lens tissue carrying a Pax6P03.9-GFP reporter, included in pilot experiments as a quality control for the fidelity of lens collection.

Figure 2.

In silico subtraction is an effective tool to identify lens-enriched genes. (A) The 200 most highly ranked genes with WB subtraction and without WB subtraction (No WB) at E10.5, E11.5, and E12.5 were tested against many functional biological gene categories to identify statistically significantly enriched gene sets (Fisher's exact test, Bonferroni corrected P < 0.05; odds ratio of gene set overlap > 20). Significantly enriched genes sets are visualized in the heat map. (B) Heat maps representing expression levels and lens enrichment P values of all nonsyndromic human cataract genes cataloged at CatMap. (C) A rank list showing the distribution of known genes related to human cataract and embryonic lens development based on the lens enrichment t-statistics (with WB) or microarray expression (without WB). The ranked list of the 200 most highly ranked genes is expanded and shown under the full ranked list.

Figure 3.

iSyTE predicts potential candidate genes in mapped cataract loci in human and mouse. Section in situ hybridization on E11.5 to E12.0 mouse embryonic tissue confirms lens expression for Sipa1l3 (human locus 19q13.13, SIPA1L3), Ptpru (human locus 1p35.3, PTPRU), Ng23 (human locus 6p21.33, C6orf26), Fam198b (human locus 4q32.1, FAM198B), Rbm24 (human locus, 6p22.3, RBM24) Ypel2 (human locus 17q22, YPEL2), Gje1 (human locus 6q24.1, GJE1), and Vit (human locus, 2p22.2, VIT).

Figure 4.

In silico subtraction strategy is robust against use of different WB controls. After swapping WB control profiles generated for separate lens and tooth analyses, the in silico subtraction strategy still robustly identifies genes that are specific to (A) lens and (B) tooth. Thus, the in silico subtraction strategy is robust against the use of different WB. This supports the idea that the WB generated in this study can be used as a public resource for comparison with gene expression profiles of other embryonic tissues at similar stages.

Figure 5.

iSyTE tracks on the UCSC Genome Browser represent a publicly available resource for cataract gene identification. iSyTE custom tracks (accessible at http://bioinformatics.udel.edu/Research/iSyTE) visualize the ranking of the lens enrichment for E10.5, E11.5, and E12.5 mouse lens. iSyTE tracks on the UCSC Genome Browser identify genes associated with primary congenital cataract in human and mouse. This figure visualizes a 10-Mb locus on human chromosome 9 with iSyTE tracks, which confirms a nonsyndromic cataract–associated gene, TDRD7, as the most promising candidate in this interval. Color of the iSyTE tracks is based on the rank of the individual genes after WB subtraction.